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Third Inter-American Congress ON

Brucellosis Held under the joint auspices of: The Inter-American Committee on Brucellosis The United States Committee on Brucellosis of the National Research Council The Pan American Sanitary Bureau Regional Office, World Health Organization

WASHINGTON, D. C. November 6-10, 1950

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Third Inter-American Congress ON

Brucellosis Held under the joint auspices of: The Inter-American Committee on Brucellosis The United States Committee on Brucellosis of the National Research Council The Pan American Sanitary Bureau Regional Office, World Health Organizatio

WASHINGTON, D. C.

November 6-10, 1950 j "`

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Prepared for publication by the Editorial Section -Pan American Sanitary Bureau Regional Office of the World Health Organization 1501 New Hampshire Avenue, N. W. Washington, D. C., U. S. A.

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Held in Washington, D. C., Nov. 6-10, 1950

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Dr. Alice C. Evans, President, Inter-American Committee on Brucellosis, addressing the Congress.

Dr. Weslev W. Spink, President of the Third Inter-American Congress on Brucellosis and of the United States Committee on Brucellosis.

9

Group of Delegates. on their visit to the United States Animal Industry Laboratories at Beltsville, Md. accompanied by members of the laboratory.

FOREWORD The Third Inter-American Congress on Brucellosis represented a continuation of the effort to bring about a better understanding of animal and human brucellosis in the Western Hemisphere. The First Congress was held in 1946 in Mexico City, and the Second in 1948 in Argentina. When workers in the field of brucellosis gathered in Washington, D. C. in November 1950 for the Third Inter-American Congress on Brucellosis, a worldwide interest in the problems of brucellosis was expressed. Through the World Health Organization and the Food and Agriculture Organization, which are agencies of the United Nations, official representatives were present from Great Britain, several European countries and from Japan. Although the clouds of war hovered on the horizon, and bitter fighting was engaging armies in Korea, these scientists met in Washington to discuss the problems of brucellosis. Misunderstanding between individuals and nations often stems from ignorance of each other's problems. Though there remain many problems on brucellosis to be solved, the Third Inter-American Congress permitted a review of the disease as it exists in different countries, and a recital of advances that had been made in various clinics and laboratories. As the scientific sessions proceeded, it became quite apparent that the problems of brucellosis varies from country to country. A failure to appreciate these variations had accounted for much of the misunderstanding and disagreement that had existed prior to the Congress. As a result of the Congress, a more comprehensive picture of brucellosis as it exists throughout the world was crystallized. Equally important was the opportunity scientists had to meet in person and create friendships that are so essential in international relationships today. The following papers, with a few exceptions, constitute the program of the Third Inter-American Congress on Brucellosis. There has been no editorial revision of these papers; they are printed as submitted by the authors, and in order to publish the manuscripts at a minimum of expense and without too much delay, the authors have had no opportunity to proofread their papers. No discussions are included and all illustrations have been omitted. The members of the Congress are sincerely appreciative of the Pan American Sanitary Bureau's willingness to publish, at cost, this volume containing a compilation of the papers presented. WESLEY W. SPINr, M.D. President, Third Inter-American Congress on Brucellosis

ACKNOWLEDGMENTS

The scientific meetings of the Third Inter-American Congress on Brucellosis were made possible through the generous financial support of the following organizations in the United States: Abbott Laboratories Merck and Co., Inc. The Borden Company National Dairy Products Corp. Burroughs Wellcome and Co. Parke, Davis and Co. Pet Milk Co. Lederle Laboratories Division, American Cyanamid Co. Eli Lilly and Co. Chas. Pfizer and Co., Inc. E. R. Squibb & Sons The Pan American Sanitary Bureau provided the generous assistance of its staff, especially the services of the Veterinary Public Health Section Secretary General's Office Conference Section Public Health Division Office Services Section The National Research Council contributed to the success of the Congress, especially Dr. LeRoy Voris, Executive Secretary of the Agriculture Board; and the United States Department of Commerce by making available the Departmental Auditorium for the meetings.

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TABLE OF CONTENTS Page

Foreword . ............................................................... Acknowledgments......................... Alphabetical list of authors ............................................... Committees: Inter-American Committee on Brucellosis ................................ United States Committee on Brucellosis ................................. Pan American Sanitary Bureau ......................................... Alphabetical list of participants ........................................... Addresses: Address of Welcome Dr. Alice C. Evans .................................................... Welcome address Dr. Miguel E. Bustamante ............................................. Opening remarks Wesley W. Spink, M.D ................................................. Papers presented: Brucellosis-a World Problem Martin M. Kaplan, V.M.D., M.P.H.................................... Epidemiología de la Brucelosis Humana en la República Argentina; la Brucelosis del Este y del Oeste E. A. Molinelli, D. Ithurralde,G. Basso, S. Miyara, y C. P. Pandolfo...... Brucellosis Incidence in the United States James H. Steele and L. Otis Emik ...................................... La Brucelosis Humana en Cuba. Resumen del Estado Actual Arturo Curbelo y Viola Márquez ........................................ Estado Actual de la Brucelosis Bovina en Cuba Ana J. Pelaiz ......................................................... Animal Brucellosis in Brazil Milton Thiago de Mello ................................................ La Brucelosis Animal en el Uruguay B. Szyfres, J. L. Stella, W. Erradonea, H. Trenchi, D. Abaracón, J. C. Piñón, y J. M. Infantozzi .......................................... Brucellosis, A Packing Plant Problem Norman B. McCullough................................................ Variations as a Tool in Brucellosis Research W. Braun, A. Gorelick, M. Kraft and D. Mead ........................... Bactericidal Action of Human Blood Against Brucella and Its Specific Inhibition Wendell H. Hall ....................................................... The Clinical Course of Human Brucellosis in Minnesota Wesley W. Spink and Robert L. Magoifin ................................ The Natural Course of Bovine Brucellosis David T. Berman ...................................................... vii

v vi ix xiii xiii xiii xiii

1 3 5

6

15 28 37 48 59

69 76 83

87 94 104

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Page

The Natural Course of Swine Brucellosis L. M. Hutchings....................................................... Studies of the Physical Properties and Agglutinability of Brucella Antigens Used in the Americas Fred Murdock, Martin H. Roepke and B.D. Blood ........................ *Field Studies on the Diagnosis of Animal Brucellosis with Special Emphasis on the Ring Test F. C. Driver and M. H. Roepke ........................................ Comparative Study by Media Ordinarily Used for the Growth of Brucella Species Genesio Pacheco and Milton Thiago de Mello ............................ The Carbon Dioxide Requirements of Brucella Abortus A. G. Marr and J. B. Wilson .......................................... Use of the Embryonating Egg for Isolation of Brucella in a Public Health Diagnostic Laboratory S. R. Damon and Kathleen Gay ........................................ Diagnóstico Bacteriológico y Biológico de la Brucelosis Luis A. Philipps...................................................... What Should Be Done with the Brucella Skin Test Karl F. Meyer ......................................................... Modalidades Clínicas de la Brucelosis Humana T. de Villafañe Lastra ................................................ Preparation and Properties of Brucella Abortus Vaccine (Strain 19) Desiccated by Lyophilization W. F. Verwey, N. H. Harringtonand Clara Matt ........................ Brucelosis Caprina en la República Argentina R. A. Maubecin, B. L. Morán, F. R. Jurado y V. C. Cedro ................ Controlling Brucellosis in Colorado Goats George W. Stiles....................................................... El Programa de Erradicación de Brucelosis en Puerto Rico Enrique R. Toro, Jr................................................... Control of Brucellosis in the United States B. T. Simms ......................................................... Trend of Nation-wide Brucellosis Eradication in the United States W. D. Knox ........................................................... Chemotherapy of Experimental Brucella Abortus Infection in Guinea Pigs and Mice A. Pomales-Lebrón, Howard E. Hall and C. Fernández.................. Estudios sobre Terapéutica de la Brucelosis M. Ruiz Castañeda,G. Guerrero Ibarray C. Carrillo Cárdenas ............ La Antibrucelina en el Tratamiento de la Brucelosis Febril Enso Criscuolo, F. Ramacciotti, Esther R. W. de Paolasso, H. Vacchiani, F. Bergagna, N. PierangeliVera y A. Ceballos ................. 285, Terramycin, Chloramphenicol and Aureomycin in Acute Brucellosis; A Preliminary Report John H. Killough, Gordon B. Magill and Richard C. Smith ................

115

122

133

145 151

157 165 177 191

209 216 226 238 244 252

260 276

288

296

ALPHABETICAL INDEX OF AUTHORS Page

Page

Driver, Fred C. U. S. Bureau of Animal Indus133 try, St. Paul, Minn ........... Emik, L. Otis 28 Scientist, Atlanta, Ga .......... Errandonea, W. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Fernández, Carlos San Juan, Puerto Rico ........ 260 Fernández Ithurrat, E. M. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, 15 Argentina..................... Gay, Kathleen Bureau of Laboratories,Indiana State Board of Health, Indian157 apolis, Ind .................... Gorelick, Arthur N. Camp Detrick, Frederick, Md... 83 Guerrero Ibarra, G. Departamentode Investigaciones Médicas, Hospital General, Mé276 xico, D. F., México ........... Hall, Howard E. 260 Baltimore, Md ................. Hall, Wendell H. Laboratory Service, Veterans Administration Hospitaland the Department of Medicine, University of Minnesota Medical 87 School, Minneapolis, Minn..... Harrington, N. H. Sharp and Dohme, Inc., Glenol-

Abaracón, D. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Basso, G. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, F 15 Argentina ..................... Bergagna, Fabio H. Clínica de Enfermedades Infecciosas, Facultadde Ciencias Médicas, Universidad de Córdoba, 288 Argentina..................... Berman, David T. Wisconsin Agricultural Experiment Station, University of Wisconsin, Madison, Wis .......... 104 Blood, Benjamin D. Pan American Sanitary Bureau Washington, D. C .............. 122 Braun, Werner Camp Detrick, Frederick, Md.. . 83 Carrillo Cárdenas, C. Departamento de Investigaciones Médicas, Hospital General, Mé- al 276 xico, D. F., México ............ Ceballos, Alberto Clínica de Enfermedades Infecciosas, Facultadde Ciencias Médicas, Universidad de Córdoba, 288 Argentina..................... Cedro, V. C. Ministerio de Agricultura y Ganadería, Buenos Aires, Argen216 tina ......................... Curbelo, Arturo Sección Experimental, Cátedra de Bacteriología, Universidad de 37 La Habana, Cuba ............. Criscuolo, Enso Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Cór285, 288 doba, Argentina ........... Damon, S. R. Bureau of Laboratories,Indiana State Board of Health, In157 dianapolis, Ind................

den, Penn.....................

209

Hutchings, Leslie M. Agricultural Experiment Station, Purdue University, Lafa115 yette, Ind ...................... Infantozzi, J. M. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Ithurralde, D. Instituto Malbrán, Ministeriode Salud Pública, Buenos Aires, 15 Argentina..................... ix

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Page

Jurado, F. R. Ministerio de Agricultura y Ganaderia, Buenos Aires, Argentina ......................... 216 Kaplan, Martin M. Division of Epidemiology, World Health Organization, Geneva, Switzerland ................... 6 Killough, John H. U. S. Naval Medical Research Unit No. 3, Cairo, Egypt...... 296 Knox, W. D. Editor, Hoard's Dairyman, Fort Atkinson, Wisc................ 252 Kraft, Mary Camp Detrick, Frederick, Md.. . 83 Magill, Gordon B. U. S. Naval Medical Research Unit No. 3, Cairo, Egypt ...... 296 Magoffin, Robert L. Department of Medicine, University of Minnesota Hospitals and Medical School, Minneapolis ........................... 94 Márquez, Viola Sección Experimental, Cátedra de Bacteriología, Universidad de La Habana, Cuba ............. 37 Marr, A. G. Departmentof Agricultural Bacteriology, University of Wisconsin, Madison, Wisc ............ 151 Matt, Clara Sharp and Dohme, Inc., Glenolden, Penn................... 209 Maubecin, R. A. Ministerio de Agricultura y Ganadería, Buenos Aires, Argentina ...................... 216 Mead, Dorothy D. Camp Detrick, Frederick, Md.. . 83 Mello, Milton Thiago de Army Veterinary Service, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil............. 59, 145 Meyer, K. F. George Williams Hooper Foundation, University of California, San Francisco, Calif........... 177 Miyara, S. Instituto Malbrán, Ministerio de Salud Publica,Buenos Aires, Argentina ..................... 15

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Molinelli, Ernesto A. Instituto Malbrán, Ministeriode Salud Pública, Buenos Aires, Argentina..................... 15 Morán, Benjamín L. Ministerio de Agricultura y Ganadería, Buenos Aires, Argentina ......................... 216 Murdock, Fred M. U. S. Bureau of Animal Industry, St. Paul, Minn........... 122 McCullough, Norman B. Microbiological Institute, National Institutes of Health, Public Health Service, Bethesda, Md ........................... 76 Paolasso, Esther R. W. de Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 285 Pacheco, Genesio Instituto Oswaldo Cruz, Rio de Janeiro, Brasil................ 145 Pandolfo, G. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, Argentina..................... 15 Pelaiz, Ana J. Centro de Brucelosis, Instituto Nacional de Higiene, Habana, Cuba ......................... 48 Pierangeli Vera, Néstor Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 288 Piñón, J. C. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección 69 de Ganadería, Uruguay........ Pomales Lebrón, A. San Juan, Puerto Rico ........ 260 Philipps, Luis A. Instituto Nacional de Biología Animal, Ministerio de Agricultura, Lima, Perú.............. 165 Ramacciotti, Félix Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 285

ALPHABETICAL INDEX OF AUTHORS

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Page

Page

Roepke, Martin H. University of Minnesota, St. Paul, Minn............... 123, 133 Ruiz Castafeda, M. Departamento de Investigaciones Médicas, Hospital General, México, D. F., México ......... 276 Simms, Bennett T. Bureau of Animal Industry, Agricultural Research Administration, United States Department of Agriculture, Washington, D. C ...................... 244 Smith, Richard C. U. S. Naval Medical Research Unit No. 3, Cairo, Egypt ..... 296 Speroni, A. Instituto Malbrán, Ministerio de Salud Pública, Buenos Aires, Argentina..................... 15 Spink, Wesley W. Department of Medicine, University of Minnesota Hospitals and Medical School, Minneapolis ........................... 94 Steele, James H. Veterinary Director, Atlanta, Ga........................... 28 Stella, J. L. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino," Dirección de Ganadería, Uruguay........ 69

Stiles, George W. Laboratory Section, Colorado State Department of Public Health, Denver, Col ............ 226 Szyfres, B. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino," Dirección de Ganadería, Uruguay ........ 69 Toro, Enrique R., Jr. Negociado de Industria Animal, Bureau of Animal Industry, San Juan, Puerto Rico ....

238

Trenchi, H. Servicio de Brucelosis, Laboratorio de Biología Animal "Dr. Miguel C. Rubino," Dirección de Ganadería, Uruguay........ 69 Vacchiani, Hugo Clínica de Enfermedades Infecciosas, Facultad de Ciencias Médicas, Universidad de Córdoba, Argentina ............... 288 Villafañe Lastra, T. de Comisión Permanente para el Estudio de la Brucelosis, Circulo Médico de Córdoba, Córdoba, Argentina ............... 191 Verwey, W. F. Sharp and Dohme, Inc., Glenolden, Penn..................... 209 Wilson, J. B. Department of Agricultural Bacteriology, University of Wisconsin, Madison, Wisce ........... 151

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INTER-AMERICAN COMMITTEE ON BRUCELLOSIS President-Alice C. Evans, Washington, D. C. Secretary-M. Ruiz Castañfeda, Mexico City, Mexico UNITED STATES COMMITTEE ON BRUCELLOSIS (Committee on Brucellosis, National Research Council) W. W. Spink, University of Minnesota, Chairman W. L. Boyd, University of Minnesota C. F. Jordan, Tarrant County Health Dept., Fort Worth, Texas L. M. Hutchings, Purdue University C. K. Mingle, United States Department of Agriculture C. L. Larson, United States Public Health Service A. C. Evans, Washington, D. C. PAN AMERICAN SANITARY BUREAU Regional Ofgice, World Health Organization Fred L. Soper, Director J. R. Murdock, Assistant Director M. E. Bustamante, Secretary General B. D. Blood, Veterinary Public Health Section H. L. Hayward, Conference Section ALPHABETICAL LIST OF PARTICIPANTS Dr. E. M. Aldrich Inspector in charge of Tuberculosis and Brucellosis Eradication, Boston, Mass. Miss Virginia Allen State Laboratory of Hygiene, Madison, Wisc. Dr. Ramón Atalaya Cultural Attaché, Colombian Embassy, Washington, D. C. Dr. Juan Carlos Bacigalupi Chief, Municipal Bacteriological and Vaccine Service, Montevideo, Uruguay Dr. Henry Bauer Director, Medical Laboratories, Minnesota State Department of Health, Minneapolis, Minn. Dr. Alfonso Bautista Bustos Caracas, Venezuela Dr. Walter R. Baynes Assistant State Veterinarian, North Carolina Department of Agriculture, Raleigh, N. C. *Dr. Hans C. Bendixen Royal Veterinary College, Bülowsvej 13, Copenhagen, Denmark * Member of WHO/FAO Expert Panel on Brucellosis. xiii

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Dr. Ramón S. Berges Cultural Attaché, Dominican Republic Embassy, Washington, D. C. Dr. David T. Berman Assistant Professor Veterinary Science, University of Wisconsin, Madison, Wisc. Dr. Irving H. Borts Laboratory Director, State Hygienic Laboratory, Iowa State Department of Health, Iowa City, Ia. *Dr. Werner Braun Biological Department, Chemical Corps, Camp Detrick, Frederick, Md. Dr. C. E. Brooke Chief, Clinical Investigations Branch, Camp Detrick, Frederick, Md. Dr. Harold S. Bryan Assistant Professor of Veterinary Pathology and Hygiene, College of Veterinary Medicine, Urbana, 1ll. Dr. Bert J. Cady Veterinarian in charge, Bureau of Animal Industry, Augusta, Me. Dr. Hugh S. Cameron Professor of Veterinary Medicine, University of California, Davis, Calif. *Dr. Birdsall N. Carle Chief, Brucellosis Unit, Microbiological Institute, National Institutes of Health, Bethesda, Md. Dr. Charles M. Carpenter Professor of Infectious Diseases, School of Medicine, University of California, Los Angeles, Calif. Dr. Clemente Carrillo-Cárdenas Assistant Chief, Medical Research Laboratory, Hospital General, México D. F., Mézxico Dr. Alejandro Chediak Professor, School of Medicine, University of Habana, Habana, Cuba Dr. C. W. Christensen Associate Director of Laboratories, Difco Laboratories, Detroit, Mich Dr. Alfred Cohn Bacteriologist, Montefiori Hospital, New York, N. Y. Dr. Marion B. Coleman Senior Bacteriologist, Department of Laboratory and Research, New York State Health Department, Albany, N. Y. Dr. Elvin Coon Veterinarian in charge, U. S. Bureau of Animal Industry, Charleston, W. Va. Dr. Cornelia Cotton Research Bacteriologistin Brucellosis, Livestock Sanitary Service, University of Maryland, College Park, Md. Dr. Enso Criscuolo Professor of Infectious Diseases, School of Medicine, Córdoba, Argentina. *Sir Weldon Dalrymple-Champneys, Bt. Deputy Chief Medical Officer, Ministry of Health, Whitehall, London, U. K. Dr. K. S. R. Damon Director of Laboratories, State Board of Health, Indianapolis, Ind.

COMMITTEES AND PARTICIPANTS

xv

Dr. I. S. Danielson Assistant Director of Animal Industry, Lederle Laboratorios,Division of American Cynamid Co., PearlRiver, N. Y. *Dr. Rebbi Durusan Head, Brucellosis Laboratory, Institute of Veterinary Bacteriology, Etlik, Ankara, Turkey Dr. Sanford Elberg Associate Professor of Bacteriology, University of California, Berkeley, Calif. Dr. L. Otis Emik Scientist (R), Communicable Disease Center, U. S. Public Health Service, Atlanta, Ga. Dr. José Carlos Falçao de Melo Technical Assistant of the Army, Biology Institute, Sao Paulo, Brazil Dr. Alberto Eduardo Fors Habana, Cuba Dr. E. R. Gentry Chief, Medical Division, Department of Medicine and Surgery, Veterans Administration, Washington, D. C. Dr. Herbert L. Gilman Professor of Bacteriology, New York State Veterinary College, Cornell University, Ithaca, N. Y. Dr. Kenneth Goodner Professor of Bacteriology, Jefferson Medical College, Philadelphia, Penn. Dr. Arthur N. Gorelick Medical Bacteriologist, Camp Detrick, Frederick, Md. Dr. Paulo Gouvea Department of Health, Niteroi, Brazil. Dr. Charles G. Grey Office of Experiment Stations, U. S. Department of Agriculture, Washington, D. C. Dr. Joseph Franklin Griggs Managing Director, Claremont Medical Research Foundation, Claremont, Calif. Dr. Patrick D. L. Guilbride Veterinary Surgeon, Department of Agriculture, Hope Kingston, Jamaica, B. W. I. Dr. Harold T. B. Hall Veterinary Consultant of FAO, Washington, D. C. Dr. Wendell H. Hall Chief, Bacteriology Section, Veterans Administration Hospital, Minneapolis, Minn. *Dr. Harold J. Harris Reserve Consultant in InternalMedicine, U. S. Naval Hospital, St. Albans, N. Y. Dr. Raymond J. Helvig Veterinarian, U. S. Public Health Service, Washington, D. C. Dr. Stanley L. Hendricks Public Health Veterinarian, Iowa State Department of Health, Des Moines, la.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS Dr. Luis Alfredo Herrera Rodriguez Chief, Sanitary Inspection Section, Ministry of Public Health, Caracas, Venezuela Dr. David A. Hill State Supervisor, Brucellosis Control, Department of Agriculture, Columbus, O. Dr. Nell Hirschberg Bacteriologist,North CarolinaState Laboratoriesof Hygiene, Raleigh, N. C. Dr. Gladys L. Hobby Brooklyn, N. Y. Dr. Monroe A. Holmes Senior Assistant Veterinarian, U. S. Public Health Service, State Department of Health, Seattle, Wash. Dr. I. Forest Huddleson Professor of Bacteriology and Public Health, Michigan State College, East Lansing, Mich. Dr. Charles A. Hunter, Director of Laboratories, Kansas State Board of Health, Topeka, Kan. *Dr. Marcel M. J. Janbon Infectious Disease Clinic, School of Medicine, Montpellier, France Dr. Herbert John Jenne Chief, Bureau of Brucellosis Control, New Jersey Department of Agriculture, Trenton, N. J. Dr. Edwin P. Johnson Animal Pathologist, Virginia Agricultural Experimental Station, Blackburg, Va. Dr. Mary Barnette Johnson Senior Bacteriologist, Bacteriology Division, Alabama Public Health Department, Montgomery, Ala. *Dr. Christian L. Jorgensen Aarhus, Denmark Dr. James C. Kakavas Professorof Bacteriology, University of Delaware, Newark, Del. Dr. Martin M. Kaplan Veterinary Officer, World Health Organization, Geneva, Switzerland Dr. Emily H. Kelly Bacteriologist, Camp Detrick, Frederick, Md. Dr. J. Kiser Lederle Laboratories, Pearl River, N. Y. Dr. William D. Knox Secretary, National Brucellosis Committee, Ft. Atkinson, Wisc. Lt. Col. Ludwig R. Kuhn MSC Chief, Department of Bacteriology, Army Medical Department Washington, D. C. Mrs. Edith Kuhns Director, BacteriologicalLaboratory, State Board of Health, Helena, Mont. Dr. A. K. Kuttler Veterinarian in charge, Brucellosis and T. B. EradicationDivision, U. S. Bureau of Animal Industry, Department of Agriculture, Washington, D. C.

COMMITTEES AND PARTICIPANTS

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Dr. Félix Luciani Lairet Chief, Biological Productions, Institute of Hygiene, Caracas, Venezuela Dr. Martín A. Landa Habana, Cuba Dr. J. D. Leaming Lederle Laboratories,Pearl River, N. Y. Dr. Frank M. Lee State Public Health Veterinarian, Columbia, S. C. Dr. Ralph W. LeGrow Professor of Research, Ontario Veterinary College, Guelph, Ont., Canada *Dr. Israel Live Associate Professor of Bacteriology, University of Pennsylvania, Philadelphia Dr. Owen L. Lockwood Inspector in charge of Brucellosis and Tuberculosis Eradication in the States of Maryland and Delaware, Baltimore, Md. Dr. Leo Lowbeer Director of Laboratories, Hillcrest Memorial Hospital, Tulsa, Okla. Dr. C. D. Lowe Bureau of Animal Industry, U. S. Department of Agriculture, Washington, D. C. Dr. Frank H. Manley Professor of Infectious Diseases, Alabama Polytechnic Institute, Auburn, Ala. Dr. Charles H. Mann Medical Director of Heyden Chemical Corp., New York, N. Y. Dr. C. Manthei U. S. Bureau of Animal Industry, Animal Disease Station, Beltsville, Maryland *Dr. Giuseppe Mazzetti Professor of Hygiene, Institute of Hygiene, University of Florence, Florence, Italy Dr. José González Medina Department of Agriculture, Caracas, Venezuela Dr. Marco Antonio Mena Brito Chief, Laboratories, Children's Hospital, Mexico, D. F. Dr. Karl F. Meyer Director, Hooper Foundation, San Francisco, Calif. Dr. C. A. Mitchell Dominion Animal Pathologist, Department of Agriculture, Animal Disease Research Institute, Hull, Quebec, Canada Dr. P. Morales-Otero San Juan, Puerto Rico *Dr. Benjamin Lucas Morán Chief, Division of Brucellosis and T. B., Ministry of Agriculture, Buenos Aires, Argentina Dr. Charles E. Morris Assistant Inspector in charge, Veterinary Livestock, Albany, N. Y.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Dr. Fred M. Murdock In charge of Production and Research, Anchor Serum Co., South St. Joseph, Mo. *Dr. Norman B. McCullough Surgeon, U. S. Public Health Service, Chicago, Ill. Mrs. Nell J. McNulty Hospital Bacteriology Department, Veterans Administration, Little Rock, Ark. *Dr. Leo Olitzki Professor of Bacteriology, Hebrew University, Jerusalem, Israel Dr. Galo Oliva Director General of Livestock, Ministry of Economy, Quito, Ecuador Mrs. Grace O'Neill Executive Offcer, U. S. World Federationfor Mental Health, New York, N. Y. Dr. Carlos Ortiz Mariotte Chief, Department of Epidemiology, Ministry of Health and Welfare, Mexico D. F., Mexico *Dr. Genesio Pacheco Oswaldo Cruz Institute, Rio de Janeiro, Brazil Dr. Ana Peláiz de Arán Chief, Brucellosis Center, Ministry of Health, Habana, Cuba Comdr. Frank R. Philbrook, MC, USN Research and Development Board, Department of Defense, Washington, D. C. Dr. Américo Pomales-Lebrón Professor of Bacteriology, University of Puerto Rico, San Juan, Puerto Rico Dr. Oswaldo Portella Assistant of the National School of Medicine, Rio de Janeiro, Brazil Dr. Horace M. Powell Head of Bacteriology, Lilly Research Laboratories, Indianapolis,Ind. Lt. Col. William D. Preston Chief, Internal Medicine Department of the Air Force, Washington, D. C. Dr. Jorge Quirós Sáenz, (Representing the Government of Costa Rica), College Park, Md. *Dr. Gérard Renoux Microbiology Laboratory, School of Medicine, Montpellier, France Dr. Mila E. Rindge Epidemiologist, Connecticut State Department of Health, Hartford, Conn. Dr. Alfonso Rivera Chief Veterinarian, Department of Agriculture and Commerce, San Juan, Puerto Rico Comdr. Robert E. Rock Bureau of Medicine and Surgery, U. S. Navy, Washington, D. C. *Dr. Martin H. Roepke School of Veterinary Medicine, University Farm, St. Paul, Minn. *Dr. Maximiliano Ruiz-Castañeda Director, Institute of Medical Research, General Hospital, Mexico, D. F. Mexico Dr. Kogi Saito Chief, Animal Hygiene Section, Ministry of Agriculture and Forestry, Tokyo, Japan

COMMITTEES AND PARTICIPANTS

xix

Dr. John H. Scruggs Veterinary Officer, U. S. Public Health Service, Indiana State Board of Health, Indianapolis, Ind. Dr. Sidney Silverman Bacteriologist, Camp Detrick, Frederick, Md. *Dr. Bennett T. Simms Chief, Bureau of Animal Industry, Department of Agriculture, Washington, D. C. Dr. Lawrence W. Slanetz Professor of Bacteriology, University of New Hampshire, Durham, N. H. *Dr. Arthur Wallace Stableforth Director, Veterinary Laboratory, Ministry of Agriculture, New Haw, Weybridge, England *Dr. James H. Steele Chief, Veterinary Public Health Service, Communicable Disease Center, Atlanta, Ga. Dr. George W. Stiles Director, Laboratory Section, Colorado State Department of Public Health, Denver, Colo. Dr. Luis Francisco Thomen Ambassador of the Dominican Republic to the United States, Washington, D.C. *Dr. Axel Thomsen Chief, Department of Veterinary Medicine, State Veterinary Serum Laboratory, Copenhagen, Denmark Dr. W. T. S. Thorp Chief, Comparative Pathology and Hematology Section, National Institutes of Health, Bethesda, Md. Dr. Enrique R. Toro, Jr. Assistant Veterinarian in charge, U. S. Bureau of Animal Industry, San Juan, Puerto Rico Dr. W. F. Verwey Director of Bacteriology Research, Medical Research Division, Sharp and Dohme, Philadelphia, Penn. Dr. Kenneth F. Wells Associate Chief Veterinarian, Dominion Department of Agriculture, Ottawa, Ont., Canada Dr. Joe Bransford Wilson Associate Professor, Department of Agricultural Bacteriology, University of Wisconsin, Madison, Wisc. Dr. W. H. Wiswell Assistant to Veterinarian in charge, Brucellosis and T. B. Eradication, Baltimore, Md. Dr. Teresa T. Woo Pediatrician,Health Department, Washington, D. C. Dr. Samuel H. Work Experimental Station, Department of Agriculture, Washington, D. C. Dr. George G. Wright Chief, Immunology Brucellosis, Camp Detrick, Frederick, Md. Dr. Adalbert O. Zink Lederle Laboratories, New York, N. Y.

ADDRESS OF WELCOME By Dr. AICE C. EVANS President, Inter-American Committee on Brucellosis It is a high honor and a great pleasure for the President of the Inter-American Committee on Brucellosis to have this opportunity to welcome to her home city the Third Inter-American Congress on Brucellosis. The Congress was organized by the United States Committee on Brucellosis of the National Research Council, which acts also as our national branch of the Inter-American Committee. I shall speak for a few moments as a member of the Organizing Committee. You honor us by your presence here, and we thank you for coming. We want this to be a week of benefits to all in the sharing of knowledge and experience in brucellosis problems. And we want it to be a week when new ties of international friendship are formed, and old ties are strengthened. In addition to the scientific program, a social program has been arranged which will provide an opportunity for an informal exchange of ideas about brucellosis problems. At these social gatherings there will be opportunity to enhance the appreciation of cultural differences which always grace and enliven international meetings. We want you to enjoy our city. We hope that you will find the time to see something of our national shrines, our government buildings, our art galleries and our parks. One of the features of this city in which we scientists take especial pride is its technical libraries. You are welcome to use them during your visit here. The Library of the United States Department of Agriculture and the Army Medical Library, both located within a few blocks from this building, and also the Library of Congress may hold source material which for a long time you have wanted to find. The staffs of those libraries will be eager to help you in the search for any information which you may want. We have a Hospitality Committee with members who speak Spanish and Portuguese. This committee has a desk near the registration desk. We hope that you and the members of your family who accompany you will make full use of this service in obtaining any kind of information which you may want. Since many of you are attending an Inter-American Congress on Brucellosis for the first time, a brief résumé of its historical background may be appropriate. In May, 1939, the First National Congress for the study of Malta Fever was held in the city of Torreon, Mexico. Continuing the series, the Fifth Mexican Congress was held in Mexico City in October-November, 1946. Invitations were sent to many leading scientists of North and South America who were interested in brucellosis. Delegates attended from 8 American countries. The First Inter-American Congress on Brucellosis grew out of the Fifth Mexican Congress when an Inter-American Committee of three members was elected. A few days after the close of the First Congress Dr. Mazza, the Argentine member of the Inter-American Committee, passed away. Homage was paid to his memory and a plaque was placed on his tomb in exercises held during the 1

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Second Congress held in Buenos Aires in 1948. Your Committee felt keenly the loss of Dr. Mazza's counsel. His place should be filled by election at the final session of this Congress. The purposes of this Inter-American organization are to stimulate research into brucellosis problems and to facilitate the dissemination of information gained through research. To accomplish these purposes the Inter-American Committee was charged with two chief duties: the promotion of a series of congresses and the organization of. national committees on brucellosis in the various countries. Each of the national committees has the responsibility of stimulating the study of brucellosis problems and the dissemination of information on brucellosis in the respective country. The national committee is the medium through which the international organization reaches the scientists and other interested persons. National committees have been organized in 13 countries, as follows: Argentina, Bolivia, Brazil, Canada, Colombia, Cuba, Chile, United States, Mexico, Panama, Peru, Puerto Rico and Venezuela. (The listing is in the alphabetical order determined by the Spanish spelling, as adopted by the Pan American Sanitary Bureau.) It is hoped that those countries which have not yet organized a national committee will do so in order that they may take active part in the growing movement. The Second Inter-American Congress on Brucellosis was held in Argentina in 1948, with delegates present from eight American countries. Sessions were held in Mendoza, November 17 to 20, and in Buenos Aires, November 22-26. The Congress was held under the auspices of the Government of Argentina and the Pan American Sanitary Bureau, in collaboration with the Inter-American Committee. During the Second Congress plans were made for the present Congress, which is held under the joint auspices of the United States Committee on Brucellosis of the National Research Council, the Pan American Sanitary Bureau and the Inter-American Committee on Brucellosis. We are happy to welcome to this Third Congress the European delegates who represent the World Health Organization. From the beginning the Inter-American Committee has borne in mind that when the World Health Organization should develop, brucellosis problems would force themselves upon it. The intention of the Inter-American Committee was always to adjust itself to play a part in the world-wide organization. The relationship between the two organizations is a matter which should be under your consideration this week. The InterAmerican Committee will appoint a subcommittee to meet with representatives of the World Health Organization to work out a plan for the future status of the Inter-American Committee in relationship to WHO. The frequency of meeting is a subject which should be reconsidered. Some members of the U. S. Committee on Brucellosis think that the interval between meetings should be longer than two years. Discussion and action on these matters should take place at the final session of this Congress. In closing, may I say again that we heartily welcome you all into our midst.

WELCOME ADDRESS By Dr. MIGUEL E. BUSTAMANTE

Secretary General, Pan American Sanitary Bureau Dr. Evans, Delegates to the Third Inter-American Congress on Brucellosis, ladies and gentlemen: The Pan American Sanitary Bureau, Regional Office of the World Health Organization for the Americas, because of its continued interest in brucellosis and the important part it plays in its health program for the Americas, has sponsored and assisted in the preparation of this Third InterAmerican Congress on Brucellosis. It is gratifying to add the name of the capital of the United States to those of Argentina and Mexico where the two former Congresses on Brucellosis, the First and Second, were held in 1946 and 1948, respectively, and in the preparation of which the Bureau also assisted. The Director of the Pan American Sanitary Bureau, Dr. Fred L. Soper, who is absent, has requested me to convey to you his regrets at being unable to welcome you to this city of Washington where the headquarters of the Bureau are located. Here, the Veterinary Public Health Section of the Division of Public Health devotes much time and effort in an endeavor to solve the many problems related to the international aspects of zoonoses, mainly brucellosis, a disease which greatly concerns not only the Departments of Health and of Agriculture, but the medical and veterinary professions as well. International gatherings such as this afford excellent opportunities for the interchange of ideas and discussion of the scientific, social and economic implications of a disease which plays such havoc with human and animal lives alike. Scientists all over the world are enabled to benefit by such discussions, by the results obtained thereof, and from the papers presented by their colleagues; all of which serves to stimulate them to carry on their active work with added zest. Brucellosis is a menace to man and domestic animals, impairs health in many ways and its multiple and variable behavior is a source of concern to clinicians, veterinarians, microbiologists and pathologists. Answers to the ever increasing number of questions regarding both diagnosis and treatment are yet to be supplied by the laboratory and the hospital. The importance of public health to the development and welfare of society is evidenced in diseases like brucellosis which present an example of a pathological problem to man and animals. Aside from its seriousness as an individual disease, brucellosis hampers the advancement of nutritional standards and endangers likewise the development of animal industry and of agriculture. The principles of prevention, control and eventual eradication of disease in general apply to the measures taken to cope with the problem of brucellosis in goats, pigs and cattle, to the studies made to check its spread to other domestic or wild animals, and above all to prevent by every possible means the spread of this disease among men. Clinical and laboratory research studies are being undertaken throughout the world by you, the members of the medical, veterinary and allied professions, and progress demands the exchange of such knowledge through the media of dis3

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

cussions, explanatory talks or suggestions, as well as through the adoption of resolutions at congresses of this nature, in order that the results obtained therefrom may be brought to the consideration of the World Health Organization and of the Pan American Sanitary Bureau. It is fitting, therefor, to expect that this Congress shall motivate the results we are all seeking in terms of better health for each and every one of the countries throughout the Americas. In welcoming you to Washington, congratulating ourselves for having you in our midst, I have the pleasure of extending to you the greetings of the Director and of the professional staff of the Bureau, and also of inviting you to visit our headquarters in this city.

OPENING REMARKS By WESLEY W. SPINK, M.D.

Chairman, United States Committee on Brucellosis Dr. Evans, members of the Congress and guests: On behalf of the United States Committee on Brucellosis I am glad to welcome you to the Third InterAmerican Congress on Brucel]osis. Two years ago the Committee on Public Health Aspects of Brucellosis of the National Research Council was designated as the American Committee on Brucellosis and entrusted with the responsibiilty of organizing the scientific sessions of this Congress. The Committee has worked hard to present a representative program embracing the various aspects of the brucellosis problem. Many of you have come a long way to attend this meeting, and we hope that the effort will prove worthwhile. In organizing this Congress the Committee has had the aid of many individuals and groups, without whose assistance this meeting could not have been held. All the financial support for the Congress has come from private sources; in fact, a group of private industries in the United States provided the necessary money. With this fund we have been able to provide a simultaneous translation service such as is used at the meetings of the United Nations. Each paper and discussion will be presented in English and in Spanish. This fund has also provided for a reception to be held in the Pan American Union this evening. The Committee is desirous of publishing the papers presented at this Congress as soon as possible. Since there are not enough available funds for this purpose, a registration fee of $5.00 is being charged for each person who desires a copy. In order to keep down the expense of the publication no discussions will be included. The American Committee on Brucellosis is also deeply indebted to the personnel of the Pan American Sanitary Bureau, especially to Dr. Miguel E. Bustamante and to Dr. B. D. Blood. This Congress could not have been held without the aid of this group. In addition, we have received considerable assistance from the Division of Biology and Agriculture of the National Research Council, particularly Dr. LeRoy Voris, Executive Secretary of the Agricultural Board. Each paper will be limited to 20 minutes for presentation and each discusser will be allowed 5 minutes. Adherence to this time limit is essential if the program is to be completed within a reasonable length of time. Although differences of opinion will most certainly arise as the meeting progresses, let the discussion be frank but friendly.

5

BRUCELLOSIS-A WORLD PROBLEM By MARTIN M. KAPLAN, V.M.D., M.P.H. Division of Epidemiology, World Health Organization, Geneva, Switzerland The organization of three Inter-American Congresses on Brucellosis in the past several years attests to the increasing awareness of public health workers to the magnitude of brucellosis as an international problem. A cursory glance at the available statistics on this disease, particularly when they are compared with those of other communicable diseases like tuberculosis and malaria, might give the impression that we are unduly concerned about this problem. Unlike tuberculosis and malaria, however, which patently afflict large sections of the world's population, the ravages of brucellosis are in great part still not recognized as stemming from a specific entity, because of the insidious nature of the disease and the difficulties involved in its diagnosis. Thus, brucellosis might be compared to an iceberg, since only its peak is visible to the casual observer. However, even our limited knowledge of this infection reveals that the prolonged physical suffering and reduced work capacity caused by brucellosis affect large segments of agricultural workers and other exposed groups, and that huge economic losses are resulting from decreases in milk production and breeding efficiency in affected livestock. In France, for example, it is estimated that the economic losses from brucellosis infection based on work days missed, care of the sick, and decreases in meat and milk production, exceed 100 million dollars annually;' in the United States the same figure is estimated for annual losses to the agricultural economy alone, apart from the human element involved. 2 These facts urge on us the conclusion that, in restoring and improving the health and economic well-being of countries throughout the world, brucellosis must be given primary consideration. Realizing the importance of the problem, various member countries of the United Nations have requested the specialized agencies, WHO and FAO, concerned with disease problems in man and animals to co-ordinate an international attack on brucellosis. Let us first examine briefly the known prevalence of brucellosis at the present time. Since this Congress will hear other papers on the status of brucellosis in North and South America, and most of you are aware of the general scope of the problem in these countries, we shall be concerned here principally with areas outside the Western Hemisphere. Table I is the officially reported incidence of human brucellosis infection in 1949. 3 6

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BRUCELLOSIS-A WORLD PROBLEM

The number of undiagnosed and unreported cases was undoubtedly far greater than these official figures. Thus, despite the fact that only 4000 to 6000 cases per year for the past several years have been officially reported in the United States, it has been estimated that the number of cases of brucellosis occurring each year in this country is perhaps between 40,000 and 100,000.4 Comparable figures for France are 1400 reported, and a minimum of 9000 cases as having actually occurred last year.' When one considers the nature of this disease, which may last over a period of years, the implications of these figures become more apparent. TABLE I-Reported cases of brucellosis in man for 1949 Continent

Africa

America

Country

Algeria Eritrea French Morocco Tripolitania Canada Chile Costa Rica Cuba Mexico Peru Unites States

Asia

Indo-China Irak

Oceania

Australia Hawaii New Zealand

Cases

Continent

Country

46

Europe

Austria Belgium Denmark France Germany Greece Ireland Italy Malta Netherlands Norway Poland Spain Sweden Switzerland Trieste Turkey Yugoslavia

42

9 9 188 32* 3* 28 1369* 491 4124 6 1* 38 4 30

Cases

24 8 196 1400 207 75* 6* 9426

910* 14 0 32 5484 20 196 2* 26* 73

*Incomplete.

Information concerning the prevalence of brucellosis infection in animals is at the present time very incomplete. Published reports are usually based on local surveys of a very limited nature. For the purposes of this paper, therefore, it was decided to send personal inquiries to government officials and brucellosis authorities in various countries outside the Western Hemisphere. The outline given below summarizes the answers received to date, and also includes personal observations of the author. References are used where reports in literature have been consulted. AFRICA Belgian Congo.-Bovine and caprine brucellosis sporadic in different parts of colonies. Approximately 7% of pregnant cattle in more highly developed areas of colonies abort, probably due in large part to brucellosis. In the region of

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Stanleyville, Br. Abortus isolated from cow, and 20% of 200 tested animals were positive reactors. Egypt.-Br. abortus infection common in cows and buffaloes; extent unknown. French Somaliland.-Apparently no bovine brucellosis; suspected in sheep and goats, but never confirmed, and extent in these animals is unknown. Madagascar.-Apparently no indigenous infection; one case reported in cow recently imported from France. Union of South Africa.5 -Between 25 and 50% of bulk milk reaching urban communities shows positive ring tests. Very few brucellosis-free areas in the country. Br. abortus prime offender; Br. melitensis believed to be present although not confirmed as yet. Fairly numerous human infections have been reported from Eritrea, Algeria, Kenya, Morocco, Tunis and Tripolitania; extent of animal infection in these countries unknown. Animal infection known to exist in Rhodesia, Ethiopia and Mauritius. ASIA China.-Br. melitensis and Br. abortus known to exist in human beings and animals; extent unknown. India. 6-Disease found in 10 to 50% of village cattle in areas of heavy rainfall; Br. abortus infection usual, but approximately 40% of Indian field strains had typing characteristics intermediate between Br. abortus and Br. melitensis. Israel.-Only few herds are infected at present. Japan.-Br. abortus infection in cattle fairly common; extent unknown. Br. suis isolated in 1940. Siam.-No cases of brucellosis in man or animals reported. Turkey.7-Approximately 18% of bovine found infected out of survey comprising over 8000 animals. Both Br. abortus and Br. melitensis infection exist in cattle and buffalo. Br. melitensis infection common in sheep and goats. Animal infection known to exist in Irak, Israel, Lebanon, Ceylon, Pakistan, Indo-China, Indonesia and Philippines; extent in these countries unknown. EUROPE Austria.-Approximately 20% of cattle infected. Belgium.-Approximately 5 to 6% of cows abort each year primarily due, it is believed, to brucellosis. Bulgaria.-Br. melitensis infection very common in sheep and goats; Br. abortus infection troublesome in herds around urban centers; extent of infection in animals unknown. Cyprus.-Free of brucellosis. Czechoslovakia.-Between 30 and 40% of cattle herds infected. Denmark.-Approximately 11% of cattle herds infected. Eire.-Br. abortus infection common in cattle; extent unknown. Finland.8-As of January 1, 1949, 627 infected herds. France.-Fifteen to 20% of cattle herds infected with Br. abortus (one au-

BRUCELLOSIS-A WORLD PROBLEM

9

thority gives this figure as exceeding 33%9); 15 to 40% of sheep and goat herds infected with Br. melitensis. Germany.9-Twenty-five to 50% of cattle herds infected. Greece.-Br.melitensis infection very common in sheep and goats; Br. abortus infection troublesome in herds around urban centers; extent of infection in animals unknown. Hungary.-Br. abortus infection common in cattle; extent unknown. Italy.-Br. abortus infection common in cattle of northern plains and large milking herds in northern Italy; approximately 30 to 40% of these herds infected. Cattle in central and southern Italy frequently infected with Br. melitensis. Sheep and goats are heavily infected in the mountainous areas of northern Italy; the central and particularly the southern portions of the country are likewise infected. Malta.-Cattle and goats heavily infected; Br. melitensis predominant. Netherlands.-Thirty to 40% of herds infected; approximately 8% of total number of cattle infected. Norway.-Disease eradicated except for two infected herds of cattle. Poland.-Between 25 and 35% of cattle herds infected. Rumania.-Cattle brucellosis known to be present; extent unknown. Spain.-Br. melitensis infection predominant. Cattle, sheep and goats heavily infected; extent unknown. Sweden.-Approximately 1% of cattle infected out of a total of 370,000 animals. Switzerland.9-Twenty-five to 40% of cattle herds infected. United Kingdom.-No recent data on prevalence of infection. In 1939 a minimum of 20% of the cattle in England were infected. Present policy of vaccination is believed to have reduced this figure considerably. Br. abortus only variety found, apart from a few isolations of certain strains which are indistinguishable, biochemically and serologically, from Br. melitensis. USSR.-Brucellosis in cattle, pigs, sheep and goats known to be troublesome in European and southern part of USSR; extent unknown. Yugoslavia.-Br. melitensis infection in cattle, sheep and goats very heavy in north-western part of country; disease believed widespread throughout remainder of country but extent unknown. Approximately 5% of cattle herds believed infected with Br. abortus. OCEANIA Australia.-Br. abortus infection in cattle fairly common; extent unknown. Hawaii.-Human infections reported but extent of disease in animals unknown. New Zealand.'O-Average annual incidence of abortions in dairy cattle of all ages is approximately 5%, apparently attributable in large part to brucellosis infections. The incompleteness of the above information illustrates the woeful lack of knowledge on the prevalence of brucellosis in large areas of the world. It is hoped that some of these gaps will be filled in next year through local and regional surveys which are planned with the assist-

1

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

ance of the WHO/FAO brucellosis centers now being established in various countries. AN INTERNATIONAL APPROACH

Until recently, mass inoculation campaigns and quarantine regulations were almost the sole forms of international action against communicable diseases. WHO and FAO have gone much further in their efforts in the international control in man and animals. These agencies are concerned with the epidemiology and epizootiology of diseases in the widest sense, as embracing the entire disease complex and the social and ecological factors involved. Brucellosis provides a challenging problem with respect to this concept of international action, in that an effective approach to the disease requires adaptation to various conditions and economies. Fortunately, however, for the convenient prosecution' of a world-wide program, the major aspects of brucellosis are of immediate importance to both advanced and undeveloped countries. I have considered in another paper" the main needs and problems encountered in brucellosis at the present time, but a brief recapitulation of a few of them might be helpful in evaluating, from an international viewpoint, what has already been done and what steps are indicated for the future. The needs and problems may be summarized as follows: (a) Surveys on the prevalence of the disease in man and animals, and the improved reporting of statistics. (b) The relative importance of contact exposure versus ingestion of infected food products in the transmission of brucellosis in different localities. (c) The importance of brucellosis as a cause of abortion in women and as an infection of infants and children. (d) The international standardization of the sero-agglutination test; clarification of the limitations of this test and of the complement-fixation test, and their role in the diagnosis of brucellosis. (e) The improvement and standardization of intradermic diagnostic agents for man and animals. (f) Simplified bacteriological technique for cultural and typing purposes. (g) Vaccine and chemotherapy in human brucellosis. (h) The use and improvement of vaccines and other control techniques in brucellosis of livestock. The need for more accurate knowledge on the prevalence of brucellosis has been mentioned previously, and the advantage of periodic compilation and distribution of statistical reports by an international agency is evident. The problems indicated in (b) and (c) above are suitable for local investigations, perhaps through some of the brucellosis centers set up by WHO and FAO. In this connection, it would be desirable to carry out experiments on the persistence of Br. abortus and melitensis in meat and dairy products under the prevailing conditions of preparation and

BRUCELLOSIS-A WORLD PROBLEM

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consumption of these products in various localities. Reports on this subject are sufficiently meagre and conflicting to warrant further study. Perhaps the most acute need from the point of view of international co-ordination and agreement in the field of brucellosis is the standardization of biological products and laboratory techniques. A case in point is the sero-agglutination test, which will be discussed further in other papers at this Congress. Surveys have clearly demonstrated that there are wide variations in the results obtained from, and the techniques of performing, the sero-agglutination test in different medical and veterinary laboratories. These variations include antigens of unequal sensitivity, the use of different serum dilutions, and divergent interpretations as to what constitutes a positive titer. Some efforts have been made, particularly in the veterinary field, to standardize the sero-agglutination test. In 1937, for example, the International Office of Epizootics adopted a standard serum and recommended its use to member countries with the hope that a degree of uniformity in the test would be achieved in veterinary laboratories. This project was interrupted by the war and was resumed in 1948. At the last meeting of the Office in May, 1950, it was resolved to advance the standardization of this test by the use of standardized antigens. It is to be hoped that through this Congress, and through the WHO/ FAO brucellosis centers, an extension of the principle of standardization will be achieved so that results of sero-agglutination tests reported by different laboratories will have a similar basis of reference. The same principle should be applied, wherever possible, to the various intradermic diagnostic agents at present used in man, milk ring test antigens, and vaccines used in human therapy and for the prevention of brucellosis in animals. In any international approach to a disease problem, we must always keep before us the fact that we are dealing in large part with economically underdeveloped countries. If our efforts at disease control are to be successful in these countries, simplified procedures and techniques must be developed. Much remains to be done in this connection in the field of brucellosis. Thus, simplified bacteriological techniques in isolating and typing Brucella, and in the production of Strain 19 vaccine are very much needed if these measures are to be used on the widest possible scale. More effective vaccines and improved diagnostic techniques for sheep, goats and swine would be of great assistance in many countries. The new antibiotics, although far from being the ultimate in brucellosis therapy, are giving promising results in human beings, but they are beyond the economic reach of the majority of sufferers; for this reason alone a critical analysis of vaccine therapy and perhaps of other inexpensive therapeutic procedures would be worthwhile. The WVIO and FAO have attempted to meet some of the problems

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

discussed previously by organizing WHO/FAO brucellosis centers in a number of countries to serve the needs of various regions. The following centers have already been established: Veterinary Microbiological Institute Botanikos Athens, Greece Dr. Costas Melanidis, Director

State Veterinary Serum Institute Bülowsvej 27 Copenhagen, Denmark Dr. Axel Thomsen Chief of Brucellosis Department

University of Florence Institute of Hygiene Center for the Study of Brucellosis Viale G. B. Morgagni 48 Florence, Italy Prof. G. Mazzetti, Director

Centre de Recherches sur la Fievre Ondulante Institut Bouisson-Bertrand Rue de l'Ecole-de-Médecine Montpellier, France Prof. L. Carrére, Directeur

Onderstepoort Veterinary Laboratory Onderstepoort Union of South Africa

Brucellosis Centre State Laboratory of Hygiene Rijeka, Yugoslavia Dr. J. Srakocic, Chief, Brucellosis Department

Commonwealth Serum Laboratories Parkville N. 2 Victoria, Australia Dr. F. G. Morgan, Director Ministry of Agriculture and Fisheries Veterinary Laboratory New Haw, Weybridge Surrey, England Dr. A. W. Stableforth, Director

State Veterinary Institute Etilik, Ankara Turkey Dr. M. Z. Muslu, Director

Centers are planned for at least two South American countries; Mexico; United States, and Asia. The laboratories in these countries have not yet been selected. These centers are occupied in studies of the bacteriology, transmission, diagnosis and therapy of the disease. They also serve as teaching centers for clinical and laboratory workers of their own and nearby countries, and in their work, emphasis is placed on the aspects of brucellosis of most importance to their particular regions. Thus, the centers in France and Yugoslavia are investigating the use of intrapalpebral inoculations of a non-antigenic intradermal agent for the diagnosis of brucellosis in sheep and goats, and the possible application of the milk ring test for these animals; in Copenhagen, studies are in progress on the comparative efficacy and standardization of milk ring test antigens; and consideration is being given in other centers to the problem of agglutinin-blocking antibodies in the sero-agglutination test, and the simplification of Strain 19 production. Fellowships have been awarded to workers in various centers to study brucellosis techniques in countries other than

BRUCELLOSIS-A WORLD PROBLEM

13

their own, and financial assistance has been provided, where necessary, for the purchase of apparatus and materials. In addition, literature on the latest advance in brucellosis is circulated to these centers by WHO and FAO. A WHO/FAO Expert Panel on Brucellosis comprising medical and veterinary experts drawn from all parts of the world has been organized to formulate specific recommendations for the guidance of WHO and FAO in their present and future activity in brucellosis. Moreover, the WHO and FAO are collaborating with the International Office of Epizootics in this work. By these means the agencies of the United Nations Organization are endeavoring to bring about closer relationships between brucellosis workers everywhere, and thereby to achieve a concerted attack on this problem on a global basis. We welcome warmly the valuable contribution of the Inter-American Congresses in the fight against brucellosis. REFERENCES (1) Carrere, L., Directeur, Centre de Recherches sur la Fievre Ondulante, Montpellier, France: Personal communication to the author. (2) Mingle, C. K.: Correlation of research with brucellosis control. Vet. Med. S4: 255 (July) 1948. (3) WHO Epidemiological and Vital Statistics Report, 3: 110 (May) 1950. (4) Jour. Am. Med. Assoc. editorial, 134: 876 (July 5) 1947. Evans, Alice C.: Brucellosis in the United States. Am. Jour. Pub. Health 37: 139 (Feb.) 1947. (5) Van Drimmelen, G.: The brucellosis survey in South Africa. Jour. S. Afr. Vet. Med. Assoc. 30: 178 (Dec.) 1949. (6) Polding, J. B.: Brucellosis in India. Ind. Jour. Vet. Sc. 17: 147, 1947. Abst. Vet. Bull. 19: 651 (Dec.) 1949. (7) Said Bilal Golem: L'Etat actuel des brucelloses en Turquie. Rev. turque d'Hyg. et Biol. Exp. 9: 63, 1949. Abst. Bull. Off. Int. Epiz., 33: 213 (Mar.-Apr.) 1950. (8) Holmberg, J.: Experiences in the control of contagious abortion in Finland. Proc. XIVth Int. Vet. Cong., London, 1949. (9) Verge, J.: La prophylaxie des brucelloses-Etat actuel de la question en médicine humaine et en médicine vétérinaire. Rev. Path. Comp. 50: 157 (Mars) 1950. (10) Buddle, M. B.: Vaccination against bovine brucellosis in New Zealand. Proc. XIVth Int. Vet. Cong., London, 1949. (11) Kaplan, M. M.: A Summary of the present status of brucellosis. Bull. World Hlth. Org. In press. LA BRUCELOSIS-PROBLEMA MUNDIAL (Sumario) En muchos países empieza a reconocerse que la brucelosis es una enfermedad a la cual no se le da la importancia que merece. La gravedad del problema en el Hemisferio Occidental es bien conocida. Se estima que en el continente europeo, exceptuando a los paises escandinavos, del 15 al 30% del ganado lechero está infectado. En aquellas partes del mundo adonde se han introducido razas de ganado europeo, tales como partes de Africa, India, China, Japón y Oceanfa, se ha presentado la brucelosis. La Brucella abortuses la especie que predomina en

lo

14

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

dichas zonas. Es interesante observar que la Br. suis aparentemente tiene poca significación fuera del Hemisferio Occidental, pero sobre este particular queda mucho por hacer. En Turquía, Grecia, Yugoeslavia, España y partes de Italia y Francia, la Br. melitensis es la causa principal de esta enfermedad en el hombre. A principios de 1950, la Organización Mundial de la Salud, a solicitud de las Naciones Miembros, comenzó a prestar ayuda en la coordinación de la campaña contra la brucelosis en el mundo. El trabajo se emprendió en cooperación con la Organización de las Naciones Unidas para la Agricultura y la Alimentación. Debido a la confusión que resulta de la multiplicidad de procedimientos empleados para el control de la enfermedad en el hombre y en los animales, muchos de ellos de muy dudoso valor, fué indicado que el problema de brucelosis debería ser enfrentado en forma internacional. La OMS y la FAO han tratado de resolver este problema designando catorce centros OMS/FAO para brucelosis en distintos países, destinados a satisfacer las necesidades de varias regiones. Estos centros han emprendido estudios sobre la epidemiología, epizootiologia, diagnóstico y terapia de la enfermedad; y sirven como centros de enseñanza para personal de las clínicas y laboratorios, locales y de otros paises. Hay becas disponibles para estudios. Por medio de estos centros la OMS y la FAO están también fomentando la estandardización de los procedimientos de laboratorio para el diagnóstico de la brucelosis en el hombre y en los animales, con el objeto de que, en cuanto sea posible, los estudios publicados sobre esta enfermedad en cualquier país, tengan una base común de referencia en otras partes. El Grupo de Peritos de la OMS y la FAO para la brucelosis celebrará sesiones en Washington durante e inmediatamente después de las sesiones del Tercer Congreso Interamericano. Se pedirá al grupo que formule recomendaciones específicas para el tratamiento y control de la brucelosis en el hombre y en los animales, y que indique pautas convenientes que puedan servir de orientación a la OMS y a la FAO. La OMS y la FAO han solicitado la colaboración de la Oficina Internacional de Epizootía y la del Comité Permanente de Congresos Internacionales de Veterinaria en la realización del programa de brucelosis. Se espera que por medio de estas organizaciones se puedan tomar medidas para limitar el tráfico internacional de animales infectados.

EPIDEMIOLOGIA DE LA BRUCELOSIS HUMANA EN LA REPúBLICA ARGENTINA II. LA BRUCELOSIS DEL ESTE Y DEL OESTE

Por E. A. MOLINELLI, E. M. FERNÁNDEZ ITHURRAT, D. ITHURRALDE, G. BASSO, S. MIYARA, A. SPERONI Y G. PANDOLFO Instituto Malbrán del Ministerio de Salud Pública de la Nación En la República Argentina la brucelosis del hombre está directamente vinculada a las enzoosis provocada en tres ganados domésticos (caprino, porcino y bovino)' por el parasitismo de microorganismos del género Brucella. Se ha demostrado que la enfermedad humana incide en el territorio comprendido entre los paralelos 22 y 46 de latitud sur, pero se carece de datos respecto de los residentes de la Patagonia austral (Santa Cruz, Tierra del Fuego e Islas Malvinas). A casi 3,500 enfermos asciende el número de brucelosos estudiados por nosotros con la cooperación del cuerpo médico nacional. Su edad oscila entre 8 meses y 82 años. Predominan netamente los adultos jóvenes, ya que algo más de la mitad corresponde a individuos de 21 a 40 años. En cuanto al sexo, la brucelosis humana indígena priva entre los varones-a éstos corresponden las tres cuartas partes de la morbilidad total -en razón de que, por la índole de las profesiones que afecta, la mayoría de las personas expuestas al contagio son hombres. El sexo masculino prevalece significativamente en los diferentes grupos de edades, excepto en menores de 10 años y en mayores de 70, entre los cuales la distribución es prácticamente igual en ambos sexos. Tanto si se trata de enfermedad profesional como si se debe a otro modo de contagio, la morbilidad estacional es mayor en primavera y verano y menor en otoño e invierno. Entre nosotros la brucelosis humana suele contraerse por: (1) contacto directo profesional con animales brucelosos y subproductos de éstos; (2) ingestión de alimentos infectados; (3) concomitancia de contacto profesional e ingestión de alimentos infectados; (4) permanencia en ambientes infectados o en la proximidad de los mismos; (5) infección accidental en laboratorios de bacteriología, y (6) contagio interhumano. La importancia porcentual de cada uno de estos mecanismos de infec1 Hasta ahora ni en los ovinos ni en los equinos de la Argentina ha sido aislada ninguna especie de Brucella. Los estudios existentes o arrojan resultados negativos (Sautu Riestra, 1943; Manzullo, 1949, etc. en ovinos) o sólo relatan la comprobación de bajos títulos aglutinantes en pocas cabezas de estos ganados (Pardal, 1933; Quevedo, 1939; Harispe y Ruiz, 1945; Minoprio, 1947; etc.) 15

16

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

ción brucelosa indígena, es la siguiente:

Fruencrwa %

Contacto directo profesional con animales brucelosos ................ Ingestión de alimentos infectados ..................................... Contacto profesional e ingestión de alimentos infectados .............. Permanencia en ambientes infectados por brucelas o en su proximidad Infección de laboratorio .............................................. Interhumano (lactancia materna, coito) ..............................

42.0 22.0 31.0 4.0 0.6 0.2

Los casos de brucelosis por contagio directo profesional con animales brucelosos se registran entre: (a) individuos dedicados a la faena, industrialización y transporte de reses para el consumo (personal de mataderos y frigoríficos que sacrifican ganados o elaboran subproductos animales; técnicos de la inspección veterinaria; personal ocupado en el transporte y recepción de hacienda y visceras; carniceros, etc.); (b) individuos que crían y explotan ganados (cabreros, tamberos, mayordomos y peones de campo, veterinarios, hacendados, granjeros, criadores de cerdos, etc.) en el curso de diversas tareas pecuarias (cuidar, ordeñar, desollar, etc., animales brucelosos; asistir vacas en trance de parición; extraer a mano restos de placenta; manipular fetos, etc.), y (c) individuos que manipulan tripas, cebo, o elaboran chacinados; obreros de barracas o curtiembres en contacto con cueros frescos o salados, etc. En estos casos (a, b y c), aunque aparentemente la única puerta de entrada es la piel, intacta, fisurada o con soluciones accidentales de continuidad es imposible excluir la probable contaminación por brucelas a través de las mucosas ocular, nasal, labial y bucal. En cambio, por la mucosa de revestimiento del tubo digestivo2 han

contraído la brucelosis aquellas personas que entre nosotros registran como único antecedente epidemiológico la ingestión de alimentos infectados de origen animal, a saber: leche cruda de vaca y cabra, queso fresco de vaca, queso fresco-'quesillo" (requesón y naterón)-y queso estacionado de cabra; cremas; "ricota" y manteca sin pasteurizar; carne y vísceras de cabrito insuficientemente cocidas. Además, en el centrooeste argentino el agua acumulada en pequeñas represas rurales ("aguadas"), de los puestos de cabras especialmente, debe ser considerada en potencia como fuente de infección brucelosa humana, pues esta agua estancada satisface simultáneamente las necesidades de hombres y animales. En la nosología argentina, la brucelosis por ingestión de leche cruda 2 En este caso se habla genéricamente, por no saberse aún a ciencia cierta en cuál de sus tres sectores (ingestivo, digestivo y eyectivo) tiene lugar la penetración de las brucelas. Sin embargo, el documentado trabajo experimental de U. Pagnini (Gior. Batt. Imm. 1939, 22, (4): 595-617), nos inclina a sostener también para el hombre, la importancia predominante de la mucosa bucofaringea como puerta de entrada de la infección brucelosa, en condiciones naturales y en cuanto al aparato digestivo se refiere.

BRUCELLOSIS HUMANA EN ARGENTINA

17

de vaca no tiene la importancia epidemiológica que reviste en otros países, dada la feliz circunstancia de estar muy arraigada la costumbre de hervir este alimento antes de ingerirlo. La mayor parte de los casos en que se registra el antecedente de consumir leche cruda de vaca, acaece entre los miembros de la colectividad anglosajona o entre personas que por considerar de mejor calidad supuestas "leches pasteurizadas", las ingieren sin someterlas previamente a la ebullición. El consumo exclusivo de otros lacticinios de vaca, tampoco tiene importancia estadística en la determinación de enfermos de brucelosis. Dentro de su escasa incidencia, merece agregarse el de un catador de cremas destinadas a la elaboración industrial de manteca, el cual-por más que sea obvio decirlo-tan sólo gustaba las muestras sometidas a su examen. Contrariamente, alto valor epidemiológico tiene la ingestión de lacticinios de cabra, en particular el denominado "quesillo", alimento muy difundido en el centro, oeste y Patagonia argentinos. Como casos excepcionales citaremos el de dos lactantes que adquirieron brucelosis al nutrirse con leche materna. Ambas madres padecían la noxa y hallábanse en pleno periodo de estado. De una de las leches se aisló Br. melitensis. En cambio, cuando el contagio se establece por permanencia en ambientes contaminados (casi siempre motivada por razones profesionales) o en sus proximidades, entonces sí puede afirmarse categóricamente que son puertas de entrada las mucosas ocular, nasal y labial y que el mecanismo de infección ha sido respectivamente facilitado por la adhesión, inhalación y deglución de partículas infecciosas. A la circunstancia de permanecer en ambientes infectados deben imputarse los casos de brucelosis que hemos registrado entre el personal de mataderos y frigoríficos de carnes sin contacto directo con reses o derivados (v.g., jefe y obreros de la sección mecánica, jefes administrativos y oficinistas, servicio médico del establecimiento, policía interna, ordenanzas, choferes, albañiles, foguistas, ascensoristas, pintores, carpinteros, electricistas, tacheros, hojalateros, vendedor de diarios, etc.) A la contingencia de la simple estadía, breve o prolongada, en las proximidades de un ambiente contaminado, cabe atribuir el caso del bruceloso cuyo único antecedente epidemiológico fehaciente era habitar, calle por medio, frente a una plaza ferroviaria de estacionamiento de ganado; a aquellos ocurridos entre rematadores de hacienda, etc. Y lo mismo puede decirse de otros casos singulares. Nos referimos, por ejemplo, a quienes han contraído brucelosis sin más antecedente probado que el de: (1) haber concurrido a "puestos" de cabras; (2) estar dedicados al comercio de cabras o de guano; (3) ser jardinero en un mercado de hacienda en pie; (4) visitar circunstancialmente un laboratorio en momento en que se filtraban varios litros de cultivo de Br. melitensis (de

18

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

la señorita protagonista de este último caso, pocas semanas después-por hemocultivo-se aislaba Br. melitensis).3 Según lo establece nuestra encuesta epidemiológica, la brucelosis profesional de los técnicos que trabajan en laboratorios de bacteriología, contraída por infección accidental, es debida casi siempre a la manipulación de cavias inoculados con materiales brucelosos y la subsecuente adhesión y deglución de polvo atmosférico cargado de detritus de estos animales de experimentación. La infección accidental de laboratorio ocurrió en uno de los autores (E. M. F. I.), a pesar de manipular los cavias con guantes de goma que protegían sus brazos hasta los codos. En la brucelosis contraida por contacto sexual, el agente etiológico llega al organismo por la mucosa genital. Nuestra casuística, que comprende dos mujeres esposas de brucelosos en actividad, incide muy pobremente en el total de enfermos estudiados. En cuanto al posible mecanismo potencial de la infección brucelosa interhumana por efecto de la transfusión de sangre (dadores individuales o material de "bancos de sangre"), ni en nuestro historial clínico ni en la bibliografía nacional, se registra caso fehaciente alguno. Ya reseñados los distintos mecanismos de la infección brucelosa indigena, es oportuno decir que cada una de estas modalidades de infección gana o pierde importancia epidemiológica en las diferentes regiones geográficas donde incide la brucelosis, y dentro de estas últimas, de acuerdo a la condición de vida, urbana o rural, de sus habitantes. Así, en las ciudades y pueblos del litoral argentino, donde están instalados los grandes mataderos y frigoríficos de carnes, la brucelosis se contrae generalmente por contacto directo profesional con animales brucelosos o subproductos, y la permanencia en ambientes contaminados por brucelas; en tanto que, en las ciudades y villas de las regiones andina y central, la noxa se adquire por ingestión de lacticinios infectados (preferentemente por consumo del "quesillo" de cabra). A mayor abundamiento, cabe agregar que en los grandes centros urbanos del litoral (Capital Federal, Rosario, Avellaneda, Zárate, Berisso, Colón y Valentín Alsina) donde no se crían ni se sacrifican caprinos en cantidades significativas, el 90% de los casos de brucelosis con comprobación bacteriológica, ocurre entre el personal de los mataderos y frigoríficos de carne, donde se faena exclusivamente hacienda bovina, porcina y ovina. En los ambientes rurales argentinos la modalidad prevaleciente es el contacto directo profesional con animales brucelosos; sobre todo en el cuidado, cría, explotación y comercio de los ganados vacuno y porcino, en la región litoral, y del ganado caprino en el centro, región andina y Patagonia. En estas tres últimas zonas, se agrega la ingestión de leche ' Una contaminación similar ocurrió en dos técnicos del Instituto Malbrán, los cuales sólo presenciaron el filtrado de cultivos para elaborar melitina.

BRUCELLOSIS HUMANA EN ARGENTINA

19

y lacticinios de cabra y el consumo de carne y vísceras de cabrito, insuficientemente cocidas. Si a lo expuesto anteriormente se agrega que las tres cuartas partes de los enfermos estudiados por los autores han contraído la enfermedad TABLA I.-Confrontación de los datos de la encuesta epidemiológica con la especie

de Brucella aislada en 800 casos humanos de brucelosis de la Argentina Fuente de Infección

1

2

3

4

5

6 7 8 9 10 11

Caprinos y/o ingestión de leche cruda de cabra y/o ingestión de lacticinios de cabra.............. Idem + otros ganados (bovinos, procinos u ovinos) con/sin ingestión de leche cruda de cabra o vaca y/o ingestión de lacticinios de cabra y/o vaca .............. Ambientes donde hay ganado caprino con infección brucelosa (sin contacto directo con estos animales) ........................... Bovinos y/o ingestión de leche cruda de vaca y/o ingestión de lacticinios de vaca.......... (A-a) Idem + otros ganados (porcinos u ovinos) con/sin ingestión de lacticinios de vaca............ (B-b) Porcinos ..................... (C-c) Idem + ovinos ................ (D) Ovinos ......................... (E) Ambientes infectados de mataderos o frigoríficos de carne*........(F) Infecciones de laboratorio ......... Contagio interhumano .............

Br. melitensis

272

Br. abortus Bru Bzr.sis Br. cortus

-

86

5

Buel Brucella atípica

1

-

2

-

1

-

-

-

11

91

111

-

3 1

46 62 6 21

20 5 2 1

-

1 2 1

30 3 -

10 2 -

-

382

261

152

-

3

1

5

* Los enfermos incluidos en el renglón 9 son aquellos que por razones de trabajo (mecánicos, electricistas, albañiles, pintores, carpinteros, oficinistas, sección vigilancia, foguistas, etc.) frecuentan los distintos ambientes de recepción de hacienda, matanza y elaboración de carnes de estos establecimientos, sin realizar tareas que exigen contacto directo con animales o subproductos.

durante el ejercicio de su trabajo u oficio, emerge entonces, con toda claridad, la extraordinaria importancia que en la Argentina tiene la brucelosis profesional. A los efectos de establecer la relación entre fuente de infección y especie de Brucella infectante, se han confeccionado las Tablas I, II y III. En la Tabla I hállanse confrontados los datos de la encuesta epidemiológica con el resultado de la clasificación bacteriológica correspondiente a 800

20

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

casos humanos de la Argentina, en los cuales los autores aislaron la Brucella causal. En las Tablas II y III, tales datos han sido discriminados para facilitar la mejor comprensión de su signifcado, según correspondan o no a casos infectados en mataderos y frigoríficos de carnes. La lectura de la Tabla I demuestra que cuando la brucelosis humana depende exclusivamente de la: (1) enzoosis caprina, siempre se aisla Br. melitensis (Tabla I, renglón 1); (2) enzoosis bovina, se aisla en orden decreciente de importancia Br. abortus, Br. suis y Br. melitensis (Tabla I, renglón 4); (3) enzoosis porcina, se aisla casi siempre Br. suis, en pocos casos Br. abortus y excepcionalmente Br. melitensis (Tabla I, renglón 6). TABLA II.-Discriminaciónde 328 brucelosos de la tablaI, los cuales contrajeronsu enfermedad trabajandoen mataderos o frigoríficos de carnes y derivados* Fuente de infección

A B C D E F

Br

Bovinos ......................... Idem + porcinos y/u ovinos ..... Porcinos ................... Idem + ovinos .................. Ovinos ................... ....... Ambientes infectados de estos establecimientost................

B s m¢litensis

1 1 1...... 1

r.

is

aborBr. t atípica ca

86 44 58 6 21

45 16 2 2 1

-

1

30

10

1

4

245

76

3

2 -

* Una parte de los enfermos correspondientes a los renglones A-E tienen contanto con animales en pie, pero la gran mayoria trabaja con las carnes y subproductos de la especie animal indicada en cada renglón. t Véase llamada (*) de la Tabla I.

Por tanto, los datos precedentes ponen de manifiesto que también en nuestro territorio hay intercontaminación por brucelas, que afecta a los ganados bovino (con Br. suis y probablemente Br. melitensis) y porcino (con Br. abortus).4 La intercontaminación del ganado vacuno con Br. suis resulta muy elevada, de atenernos a los datos de la Tabla II, renglón A, o sea calculando la frecuencia (65%) de aislamiento de Br. suis entre los individuos que contraen brucelosis manipulando reses vacunas y subproductos en los mataderos y frigorificos de carne. En cambio, entre los individuos que se contagian de brucelosis por ingerir leche cruda o lacticinios de vaca, o trabajar con ganado vacuno fuera del ambiente de mataderos 4 Desde el punto de vista bacteriológico cabe consignar que: (1) En regiones argentinas carentes de cabras hemos aislado Br. melitensis de enfermos con antecedentes de contacto profesional con vacas o de ingerir lacticinios crudos de vacas; (2) Fernández Ithurrat y Molinelli (1934) y Cedro (1945) han aislado Br. suis de muestras de leche de vacas de tambos de la ciudad de Buenos Aires y partidos circunvecinos; (3) Aun no se ha aislado Br. melitensis de bovinos.

21

BRUCELLOSIS HUMANA EN ARGENTINA

y frigoríficos de carne, la frecuencia del aislamiento de Br. suis se reduce al 7% (Tabla III, renglón a). En cuanto a los 22 brucelosos cuyo antecedente de contacto profesional señala al ganado ovino como única fuente de infección (Tabla II, renglón E), cabe destacar que todos ellos eran obreros que trabajaban en plazas de matanza de frigoríficos de carne, situadas justamente en un sector contiguo y dentro del mismo ambiente destinado al sacrificio de hacienda porcina. Esto explica la aparente discrepancia entre este dato (Tabla TABLA III.-Discriminación de 472 brucelosos de la Tabla I, infectados en zonas rurales o urbanas sin conexión con mataderos of frigorificos de carne Fuente de Infección

Caprinos y/o ingestión de leche cruda y/o lacticinios de cabra ........... (1) Idem + otros ganados (bovinos, porcinos u ovinos) con/sin ingestión de leche cruda y/o lacticinios de cabra y/o lacticinios de cabra y/o vaca ......... (2) Permanencia en ambientes donde hay ganado caprino con infección brucelosa (sin contacto directo con estos animales) ............................ (3) a) Bovinos y/o ingestión de leche cruda y/o lacticinios de vaca ............. (4) b) Idem + otros ganados (porcinos u ovinos) con/sin ingestión de leche cruda y/o lacticinios de vaca ....... (5) c) Porcinos .......................... (6) Infecciones de laboratorio ........... (10) Contagio interhumano .............. (11)

Br meitensis

Br. suis

272

--

86

2

5

Br. abortus

1

-

1

-

-

10

5

66

2

2 4 3

4 3 2

2 1 378

Brucela atipica

-

-

16

76

1

-

2

II, renglón E) y la inexistencia de infección brucelosa en el ganado ovino en la Argentina.' Finalmente, la Tabla II permite establecer que los ambientes de mataderos y frigoríficos de carnes están altamente contaminados con Br. suis, como lo demuestra particularmente el renglón F, pues entre 42 enfermos, sin antecedentes de contacto con reses o subproductos (personal de la sección mecánica, policía interna, oficinistas, electricistas, albañiles, pintores, foguistas, carpinteros, etc.), 30 estaban infectados por esta especie de Brucella. Por otra parte, en ciertos brucelosos de nuestro país, la encuesta epidemiológica señala a más de una especie animal como fuente de origen de la noxa. En estos casos, cuando entre ellas figuran los caprinos (Tabla I, renglón 2), en el 97% de los enfermos se aisló Br. melitensis; y cuando

22

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

figuran los bovinos y porcinos (Tabla I, renglón 5), en el 71% de los casos se aisla de los enfermos Br. suis. Estos resultados sugieren la mayor importancia que en la Argentina tiene Br. melitensis y Br. suis como especies causales de la brucelosis del hombre. II. LA BRUCELOSIS DEL ESTE Y DEL OESTE

El estudio epidemiológico, bacteriológico y clínico de esta zoonosis, permite fundamentar el concepto de la existencia en la República Argentina de dos grandes grupos nosográficos: (1) La brucelosis del Este y (2) La brucelosis del Oeste, que, en t.érminos latos, encuentran en la enzoosis bovino-porcina y en la enzoosis caprina, su respectiva fuente de infección. La brucelosis del Este incide entre los habitantes que viven en todo el territorio de la Capital Federal, provincias de Buenos Aires, Santa Fe, Entre Ríos, Corrientes, gobernación de Misiones, parte oriental de La Pampa, planicie de Córdoba, departamento Rivadavia de Santiago del Estero y parte oriental de las gobernaciones de Chaco y Formosa. La brucelosis del Oeste afecta a pobladores de las provincias de Jujuy, Salta, Catamarca, La Rioja, San Juan, Mendoza, San Luis, Tucumán, la casi totalidad de Santiago del Estero, noroeste de Córdoba, centro y oeste de La Pampa, oeste del Chaco y Formosa y los territorios de Río Negro, Neuquén y Chubut. La autenticidad de ambos grupos está sólidamente corroborada por el resultado del estudio bacteriológico hecho en el Instituto Malbrán. Así, la Tabla IV demuestra la frecuencia preponderante de Br. suis y Br. abortus en los enfermos del Este y la presencia casi exclusiva de Br. melitensis en los del Oeste. Allí donde la enfermedad se origina por la infección de los ganados bovino y porcino, lo habitual es comprobar casos singulares de la noxa en cada hogar. En cambio, en las zonas de infección caprina, lo frecuente es observar varios miembros de una misma familia simultáneamente atacados de brucelosis en actividad. La enfermedad humana en la Argentina reviste, en general, una sintomatología y evolución diferente según se trate de casos originados en las regiones del este o del oeste del país. En el oeste, donde los casos humanos se deben a Br. melitensis, la enfermedad es más grave (antes del advenimiento de la aureomicina, cloromicetina y terramicina el promedio de duración de la enfermedad era de ocho a diez meses y la mortalidad arrojaba una cifra próxima al 8%); la fiebre adquiere casi uniformemente el tipo ondulatorio; la sintomatología nerviosa, digestiva (hepática, especialmente) y articular es más severa; el curso de la dolencia es poco influenciado por la vacunoterapia específica y son más frecuentes las recaídas después de administrar los antibióticos precitados.

23

BRUCELLOSIS HUMANA EN ARGENTINA

En el este, donde la enfermedad se debe principalmente a Br. abortus entre los individuos sin contacto con ganados y a Br. suis entre el personal de mataderos y frigoríficos de carnes, los casos son comparativamente menos graves; tanto por la escasa mortalidad que arrojan (alrededor del 2%), como por su más reducida duración (promedio cuatro a seis meses); la tendencia a la recuperación espontánea y los frecuentes éxitos terapéuticos, sin recaídas, obtenidos mediante la medicación por "shock" específico (utilizando la vacunoterapia por vía endovenosa) o la administración de los modernos antibióticos antes mencionados. TABLA IV.-Especie de Brucella aisladaen 800 enfermos de la Argentina y

distribución geográfica de los casos Región geográfica

Br. abortus

Br. suis

Br. melitensis

Brucella atípica

Total

Este .......... Oeste .........

148 4

260 1

14 368

4 1

426 374

152

261

382

5

800

Finalmente, desde el punto de vista de la temperatura corporal, la diferencia entre los brucelosos del este y del oeste se aprecia principalmente en el trazado de la curva termométrica. Los primeros muestran a menudo un brote febril único, en lugar de las clásicas ondas febriles, que caracterizan a los brucelosos de la zona oeste. BIBLIOGRAFÍA Amuchategui, S. R.; Villafafie Lastra, T. de; Goobar, J. K.; Elkeles, G.; Tey, J. A. y Schwartz, R.: Endemogeografia humana y animal de la brucelosis. Rev. Méd. de Córdoba, 35, (10): 523-528, 1947. Aribazalo, J. B. y Pomina, C. P.: La fiebre ondulante en los territorios de Rio Negro y Neuquén (República Argentina). Sem. Méd., 1, (17): 1442-1444, 1933. Atlas, J.: Epidemiología de la brucelosis en la provincia de Santiago del Estero. Dia MEdico, 19, (54): 1732-1736, 1947. Basso, G.; Miyara, S. y Molinelli, E. A.: La brucelosis en la provincia de Mendoza. Sem. Méd., 2, (28), 126-133. Addenda et Corrigenda, (29): 216, 1943. Basso, G. y Miyara, S.: Brucelosis en las provincias de Cuyo. Rev. Asoc. Méd. Arg., 62, (625-626): 119-125, 1948. Cedro, V. C. F.: Eliminación de Brucella por la leche de vacas reactoras. Rev. Méd. Cien. Af., 10, (12): 755-764, 1948 y 11, (1-3): 1-16, 1949. Cortelezzi, E. D.: Consideraciones epidemiológicas sobre brucelosis. Sem. Méd., 1, (8), 365-371, 1945; Dia Méd., 17, (38): 1062-1068, 1945; Anales de la Segunda Convención de los Médicos de la Industria, 1946, 105-119. Buenos Aires, Instituto Argentino de Seguridad, editor. Crescentino, H. H.: La fiebre ondulante en la provincia de San Juan. Semana Méd. 2, (36): 712-715, 1933. Rev. Méd. de Cuyo, 9, (84): 249-251. 1934. Criscuolo, E.: Epidemiología y tratamiento de la brucelosis. Rev. Méd. Córdoba, 33, (9): 657-659, 1945.

24

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Criscuolo, E.: Indice de infección brucelosa en la provincia de Córdoba. Rev. Méd. Córdoba, S4, (1): 3-12, 1946. Rev. San. Mil., 45, (3): 376-383, 1946. Fernández Ithurrat, E. M.: Mis investigaciones sobre las Brucellas. Rev. Méd. Vet., 23, (9-10): 366-377, 1941. Harispe, C. M. y Ruiz, G.: Indice de morbilidad de la brucelosis. La infección en la especia ovina. Anuario de la Facultad de Medicina Veterinaria, La Plata, 8, 67-80, 1945. Manzullo, A.: La infección brucelosa de los ganados ovinos de la República Argentina. Datos relativos a la Gobernación Maritima de Tierra del Fuego. Primera Reunión Panamericana de Enfermedad de Chagas, Primera Reunión conjunta de la Sociedad Argentina de Patología Infecciosa y Epidemiología y de la Sociedad Argentina de Patología y Epidemiología de Enfermedades Transmisibles. Tucumán, Salta y Jujuy, julio 11-16, 1949 (actas inéditas). Comunicación personal, octubre de 1950. Mazza, S.: Un foco antiguo del país y el más intenso de brucelosis de origen caprino en la provincia de Tucumán. VIII Reunión de la Sociedad Argentina de Patologia Regional del Norte, Santiago del Estero, 2 y 3 de octubre de 1933, 2: 825-829. Buenos Aires: Imprenta de la Universidad, 1934. Mazza, S. y Basualdo, C.: Foco de fiebre ondulante caprina en Angaco Sud (San Juan). IX Reunión de la Sociedad Argentina de Patologia Regional, Mendoza, octubre 1-4 de 1935, S, 1814-1815. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Canal Feijoo, E. J.: Foco de fiebre ondulante en el departamento de Choya y reacciones de Wright de animales de la provincia de Santiago del Estero. Publicación No. 12 de la Misión de Estudios de Patología Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S.; Caro, A. y Cores, L. B. de: Comprobación de focos de fiebre ondulante en los departamentos de Metán, Anta y Rosario de la Frontera, provincia de Salta. Publicación No. 10 de la Misión de Estudios de Patologia Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S. y Giménez, A. V.: Un afo de observación de fiebre ondulante en el departamento de Santa Maria, provincia de Catamarca. Publicación No. 11 de la Misión de Estudios de Patología Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S. y Herrera, J. Ch.: Observaciones de focos de fiebre ondulante en Andalgalá (Catamarca). IX Reunión de la Sociedad Argentina de Patologia Regional, Mendoza, octubre 1-4 de 1935, 3, 1815-1824. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Herrera, J. Ch: La brucelosis en el Departamento Andalgalá de la Provincia de Catamarca. Pren. Méd. Arg., 28, (41): 1963-1965, 1942. Mazza, S. y Lovaglio, J. A.: Brucelosis en el Dep. de Cafayate, provincia de Salta y regiones vecinas. IX Reunión de la Sociedad Argentina de Patalogia Regional, Mendoza, octubre 1-4 de 1935, 3, 1780-1802. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Rebosolan, J. B.: Observaciones de brucelosis en los departamentos de Choya, Guasayán y Moreno (Sgo. del Estero) IX Reunión de la Sociedad Argentina de Patologia Regional, Mendoza, octubre 1-4 de 1935, 3, 1758-1763. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S. y Ruchelli, A. P.: Un año de observación de la fiebre ondulante en los departamentos de Belén y Tinogasta, provincia de Catamarca. Publicación No. 11 de la Misión de Estudios de Patología Regional Argentina, Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S. y Ruchelli, A.: Observaciones de brucelosis en el departamento Tino-

BRUCELLOSIS HUMANA EN ARGENTINA

25

gasta, provincia de Catamarca. IX Reunión de la Sociedad Argentina de Patología Regional. Mendoza, 1 al 4 de octubre de 1935, 3, 1828-1843. Buenos Aires: Imprenta de la Universidad, 1939. Mazza, S., Ruchelli, A. y Arroyabe, V.: Comprobación de focos de fiebre ondulante en la provincia de La Rioja. Publicación No. 10. Misión de Estudios de Patologia Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1933. Mazza, S.: Santillán, P. y Gutdeutsch, H.: Sobre focos de fiebre ondulante en la provincia de Tucumán y regiones limítrofes. Publicación No. 6, de la Misión de Estudios de Patología Regional Argentina. Buenos Aires: Imprenta de la Universidad, 1932. Pren. Méd. Arg., 19, (21): 1306-1312, 1932. Minoprio, J. L.: Nota sobre infección brucelósica en ovinos. Haumania, 1, (1): 42-44, 1947. Molinelli, E. A.: Infección por Brucella en los frigoríficos y mataderos de la República Argentina. Sem. Méd. 1, (14): 1176-1179, 1933 a. Molinelli, E. A.: Estudio de la infección profesional por Brucella en ambientes urbanos de la República Argentina. Sem. Méd., 2, (50): 19191923, 1933 b. Molinelli, E. A.: La infección profesional por Brucella en algunos ambientes urbanos y rurales de la República Argentina. Rev. Asoc. Méd. Arg., 48, (337): 863-884, 1934. Sem. Méd., 2, (43), 1248-1258, 1934. Molinelli, E. A.: Misión sanitaria a Tinogasta (Prov. de Catamarca) con especial referencia a la brucelosis autóctona. Bol. San. Dep. Nac. Hig., 1, (4): 293-300, 1937. Molinelli, E. A.: Basso, G. y Miyara, S.: Epidemiología de la brucelosis humana en la República Argentina. Rev. Asoc. Méd. Arg., 61, (601-602): 182188. Primera Reunión Interamericana de la Brucelosis, México, 28 de octubre al 3 de noviembre de 1946: 43-61. Ediciones del Hospital General, Secretaría de la Salubridad y Asistencia, México, D. F., 1948, editor. Molinelli, E. A. y Fernández Ithurrat, E. M.: Estudio de la infección por Brucella en las vacas de los tambos de la ciudad de Buenos Aires. Sem. Méd., 2, (29): 176-186. Rev. Méd. Vet., 16, (2): 13-36, 1934. Molinelli, E. A.; Fernández Ithurrat, E. M.; Basso, G.; Miyara, S. y Fernández Ithurrat, M. C.: A survey of brucellosis in the Argentine Republic. Part I: Introduction. Part II: Clinical study. Part III: Biological diagnosis. Part IV: Confrontation between autochthonous cases of the west and littoral regions. Part V: Occupational brucellosis. Proceedings of the Sixth Pacific Science Congress (held at Berkeley, Stanford and San Francisco, July 24 to August 12, 1939), 5, 267-291. Berkeley, California: University of California Press, 1942. Molinelli, E. A.; Fernández Ithurrat, E. M. e Ithurralde, D.: Epidemiologia de la brucelosis humana en la República Argentina. Rev. Asoc. Méd. Arg., 62, (625-626): 111-119, 1948. Molinelli, E. A.: Fernández Ithurrat, E. M.; Basso, G.; Miyara, S.; Fernández Ithurrat, M. C.; Piorsky, I.; Zuccarini, J.; Ithurralde, D.; Gambino Rosa, V. de; Cornejo Saravia, E.; Burlando, A. J.; Iacapraro, G.; Insausti, T.; Malbrán, J.; Dambrosi, R. G.; Royer, M.; Thompson, V. E. R.; Pandolfo, G. P.; D'Alessandro, N. V. y Gambino, L. R.: Brucelosis humana en la República Argentina. Memoria del Primer Congreso Nacional de la Brucelosis, Montevideo, diciembre 15-17, 1947: 439-491. Montevideo: Imprenta Letras, editor, 1948. Panzeri, A.: Brucelosis en Serrezuela. Rev. Méd. Córdoba, 35, (11): 611-615, 1947.

26

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Pardal, E.: Fiebre ondulante. Investigaciones epidemiológicas realizadas en la provincia de San Luis. Sem. Méd. 1, (7), 590-594, 1933. Pardal, E.: La Brucelosis. Investigación, morbilidad, distribución y epidemiologfa en la provincia de San Luis. Sem. Méd., 2, (50): 941-948, 1945. Quevedo, J. M. (h.): Papel de las brucelas en los equinos y lesiones atribuibles a su acción. Presentación de algunos casos. Rev. Méd. Vet., 21, (7-8): 298-306, 1939. Rosas Costa, G. y Petric, J.: Investigación sobre divulgación brucelósica entre el personal de la industria de la carne en la provincia de Entre Rios. Primera contribución. Bol. Inform. del Ministerio de Salud Púb. de Entre Ríos, 1, (6): 8-15, 1949. Rosas Costa, G. A. y Puskovich, F.: Investigación sobre divulgación brucelósica entre el personal de la industria de la carne en la provincia de Entre Rios (segunda contribución). Bol. Inform. del Ministerio de Salud Púb. de Entre Rios, 1, (12): 11-23, 1949. Ruchelli, A.: La fiebre ondulante en el noroeste de la provincia de Catamarca. Tesis No. 4809 de la Facultad de Ciencias Médicas de Buenos Aires. Buenos Aires: Imprenta de la Universidad, 1935. Sautu Riestra, M. R. de: La brucelosis en la inspección de carnes. Rev. Méd. Cienc. Af., 5, (12): 901-910, 1943.

EPIDEMIOLOGY OF HUMAN BRUCELLOSIS IN THE ARGENTINE REPUBLIC (Summary) In the Argentine Republic human brucellosis is connected with the incidence in goats, swine and bovines. Brucella organisms have not been isolated from sheep. Human brucellosis is encountered between the 22 and 46 degrees latitude, South. Seasonal morbidity is greater in spring and summer and lesser in autumn and winter. Out of 3,000 patients, more than half range between the ages of 21 and 40, males being prevalent, except in cases under 10 and over 70 years of age, where the number of male and female cases is about the same. The source of infection is as follows: Occupational contact with Brucella infected animals .......... 42% 22% Infected food ............................................ 31% Occupational contact and infected food ..................... 4% Exposure in places infected with Brucella or their proximity ..... 0.6% Laboratory infection ...................................... Human contact (coitus, breast feeding) ..................... 0.2% In large towns of the Argentine littoral 90% of brucellosis cases are found among slaughter house and meat packing plant workers while in the towns and villages of the Andean region the chief source of human infection is goat milk products, cheese principally. In rural areas, infection from occupational contact with bovines and swine is prevalent in the eastern littoral region, while infection from goats is chiefly observed in the central, Andean and Patagonian regions. The drinking of milk is not an important epidemiological factor as the habit of boiling milk is widespread.

BRUCELLOSIS HUMANA EN ARGENTINA

27

Epidemiological data and bacteriological tests show intercontamination with Brucella organisms between bovines (with Br. melitensis and Br. suis) and swine (with Br. abortus); this is not observed in goats. In Argentina the danger to man of exposure to animals infected with Brucella is, in decreasing order of importance, goats, swine, bovines. Human brucellosis in Argentina may be classified into two nosographic groups: (1) Brucellosis of the East and (2) Brucellosis of the West, each having marked epidemiological, bacteriological, clinical and therapeutic characteristics.

BRUCELLOSIS INCIDENCE IN THE UNITED STATES By JAMES H.

STEELE,

D.V.M., and L. OTIS EMIK, Ph.D.

Veterinary Director and Scientist (R), respectively, Communicable Disease Center, PublicHealth Service, Federal Security Agency, Atlanta, Georgia The annual incidence of brucellosis in the United States is represented by the number of new cases of the disease occurring in the country during a year. This figure will never be known with great accuracy under conditions now existing. The reported figures represent attacks of brucellosis which may be either primary, relapses, or exacerbations of chronic cases. Some of these non-primary attacks have never been reported as primary cases, others have. The available figures are more closely akin to prevalence than incidence. The best approximation to true incidence could be calculated if the rates and methods of infection of man from source animals and the prevalence in animals could be determined. Surveys made up to the present are neither adequate nor appropriate for making any national extrapolations. Where appropriate population figures on the occupational occurrence of the disease are available, as in certain states, occupational rates may be calculated on the basis of these data, and national estimates calculated from them. It is necessary to assume that the occupational rates are fairly constant from state to state and that the variability of rates between states used to calculate the standard rates is a measure of the statistical error. The complete accuracy of this estimated figure will depend also upon the correctness of the primary assumptions, the selection of proper populations, and the reliability of sources of reported attacks. It is also possible to make some estimate of a national figure on the basis of laboratory results. The degree of error will be high due to the incompleteness and variability of the data at hand. Essentially, the method is to determine the ratio of acceptable cases to positive serum agglutinations at each different titer level and to estimate cases from laboratory results. The year 1949 has been chosen for this investigation. SELECTION OF METHODS AND SOURCE DATA

The accuracy of any data on brucellosis depends upon many factors. Particularly, diagnosis and reporting of cases must be relatively accurate in any area to be used as a standard. Epidemiologic investigations should be relatively complete to provide the necessary classification of the individual case, as well as to act as a check on diagnosis and reporting. Minnesota meets these requirements well. Iowa, Illinois, and Wisconsin 28

BRUCELLOSIS INCIDENCE IN THE UNITED STATES

29

provide good information but certain details are lacking. These four states were selected as the source of data for this phase of the study. The other states contributing information have not been solicited to as great an extent. Information from previous investigations in Iowa (Jordan-1942, 1950; Jordan and Borts-1947) and in Minnesota (Magoffin, Kabler, Spink and Fleming-1949) demonstrated the magnitudes of various attack rates for specific occupations. High rates were found for packinghouse and rendering-plant workers, veterinarians, and animal production farmers; and intermediate rates for butchers, processors, stock buyers, and stock handlers. The attack rates for children, housewives, and the remainder of the population were relatively low. TABLE 1.-Brucellosis cases by occupation in Minnesota, Iowa, Illinois, and

Wisconsin for 1949 State

Wisconsin. Iowa ...... Illinois.... Minnesota.

Veter- Packing house inarjano workers

Other animal animal contact

kes14

2 9

19 28

7 13

4

47

3

am Farmers

115 173 176 141

ChilHouseuse ghi"-Other wives

36 51 124 33

14 19 14 20

Other know known

Subtotal

Un kown

47 65 74 41

240 358 388 289

23 116 61

240 281 504 350

Rates for any occupational group can be calculated if the number of cases and persons in the group can be adequately determined. Selection of specific occupation groups which are limited to the same occupational exposure hazards as the cases to which they will be related should provide maximum accuracy in the derived rates. The available data for attacks of brucellosis by occupation in Minnesota, Iowa, Illinois, and Wisconsin during 1949 are presented in Table 1. Bureau of the Census figures were available for packing-house workers, farmers, children, housewives, and the remainder of the population. The number of veterinarians in the United States was obtained from their national directory. Farmers were defined as those engaged in animal husbandry, including dairy and livestock farms, plus subsistence farms and general farming. Undoubtedly, farms are not perfectly split this way. Many agronomists will have a few animals around, and some subsistence farms will have none. Children's rates were considerably more constant when only rural-farm children were considered. The same was true for rural housewives. No population figures could be specifically determined for those workers such as butchers, rendering-plant workers, stock buyers and handlers, dairy workers, and others having contact with animals or their products. Since this group is rather intimately related to slaughter, for the most part, it appeared most appropriate to add these cases to those of packing-house

30

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

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BRUCELLOSIS INCIDENCE IN THE UNITED STATES

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workers. By adding all packing-house workers, and those processing meat as well, into one population, the variability of the rate for this group of cases was reduced by 60%. The remaining cases of known occupation gave most stable rates in the rural population. The populations for these various occupation groups are listed in Table 2 for the four study states and for the entire country. The average rates for the four study states by occupation group and their standard deviation, calculated by the method given in Pearl (1941) are presented in Table 3. TABLE 3.-Brucellosis average attack rates by occupation for Minnesota, Iowa,

Illinois and Wisconsin for 1949 Occupation

group

.

Veterinarians ............................ Packing house plus other animal contact workers .............................. Animal farmers ......................... Rural housewives ...................... Rural farm children. Remainder (rural) .8.53

Average annual rates .. (100,000)

Standard deviation of rate (%)

950.6

28

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22 15 24 9 14

The adjustment of numbers of reported cases on the basis of laboratory results is subject to large sources of error. This method may be fairly good for one particular purpose, namely, to estimate the number of cases which would have been discovered if epidemiologic investigation were pursued diligently on all serum agglutination tests positive at dilutions of 1:80 or greater. To obtain the ratio of cases to titers, the distribution of titers in culturally proven cases determined by Magoffin et al. (1949), was compared to the distribution of titers for the ten states in Table 4 listing number of positive agglutination tests. The ratio of Minnesota titers to those in the ten states and the ratio of culturally proven cases to total cases reported in Minnesota are necessary to complete the estimates for the ten states. RESULTS AND DISCUSSION Estimated numbers of cases were calculated for each occupation group and for each state simply by multiplying the rate by the population and taking the sum of the calculated cases. There were 10,709 estimated cases for the United States in 1949 as compared to 4,143 cases officially reported by the National Office of Vital Statistics. The standard deviations of the rates for the average specific population units of the four states, used to calculate the rates, vary from 9 to 28% as shown in Table 3. Extrapolating and combining the occupational groups gives a standard

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Equine brucellosis was detected by serological tests in animals that suffered from fistulous whithers and periodic ophthalmia, but it was found also in horses that did not show any evidence of either disease. A strain of Brucella abortus was recovered from a dog in one instance. Two out of 90 sheep showed anti-Brucella agglutinins in significant titers. For the sake of conciseness the data ware tabulated in Tables I-VI, and we shall present some general remarks on bovine brucellosis, the only type that has merited more attention. The tables and the detailed sheets from which they were derived showed that there are highly infected herds in regions where other herds are immune or present a low incidence rate. We think that this difference must be credited to two principal items. Firstly, the lack of any sort of prophylaxis in contaminated herds, a fact that gradually increases the number of infected animals in a single herd. Secondly, the relatively natural segregation of some herds due to the great expanses of land where they are raised, often limited rather by the very frequent topographical irregularities than by fences or restrictive measures to the dispersion of cattle. A fact that must be stressed in connection with the low rates in some regions is the type of cattle raising in practice in a large part of the country; the animals live in immense pasturages or tracts of land and are assembled sometimes only once a year; thus animal to animal infections have little chance of occurring. Moreover, the eliminated microorganisms on the ground are more easily destroyed by the natural physico-chemical agents, before they have the opportunity of infecting other animals. To support this fact it suffices to mention the high rates in dairy cattle from the States of Rio Grande do Sul, Rio de Janeiro, Sao Paulo and Pará, compared with the relatively low rates in the range or common cattle of the same regions. Prophylactic measures against animal brucellosis are hardly used in the country. Of course the importation of infected animals from other countries is forbidden. All animals to be shown at Federal or State exhibitions must be free from the disease, and a certificate of negative serological test must be presented when registering animals in the herdbooks; this is partly the reason for the low rate in selected cattle. As has been observed in other countries, serological surveys have given rise in recent years to a gradual spread of the infection beyond most of the primitive foci because as soon as the breeder knows that some of his animals are reactors he sends them to the public market where they may be bought by farmers whose herds do not have the disease. In the States of Sao Paulo and Rio de Janeiro official measures concerning the control of brucellosis in dairy cattle were recently introduced; the animals must be submitted to periodic blood tests and reac-

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68

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

tors are not allowed to continue as dairy animals. These tests showed that the disease is still prevalent in many dairy herds. In a very few instances the reactors have been segregated. Slaughtering following a positive test is very rare because no indemnity is paid; the farmers not realizing the importance of this measure for their herds, prefer to sell the reactors. Strain 19 vaccine has been used in calves, in some States, without any particular planning except in the State of Sao Paulo. As its use is increasing at present, we think that all data based upon serological reactions henceforth must be regarded with caution if it is not stated whether the animals were vaccinated or not. This is of special importance in the State of Sao Paulo where a large scale trial mass vaccination of cattle, including adults, is in progress, mainly on dairy cattle. The results obtained until now are fairly good, but the persistence of agglutinins may restrict future control measures based on blood tests. The abortion ring test is being slowly introduced as an auxiliary tool for detecting infected dairy animals, but it is still in the experimental stage. Human infection parallels the animal disease, obviously bovine and porcine in origin, and is observed mostly in regions where there is a large number of animals. The rare cases in which Br. melitensis was recovered were doubtful or were observed in persons who possibly had been infected outside the country. Several resolutions have been approved in successive Congresses of Veterinary Medicine recommending that studies on brucellosis be intensified, but even a general knowledge of the disease is far from being achieved. The importance of the disease, both as an economic and public health problem, is not generally realized. It is necessary to encourage and to support the work of the few groups who, for many years, have been doing free-lance studies of the disease. We think that the need for information, standardization of methods for the diagnosis, plans for the control of the disease adapted to each particular region of the country, and many other items concerned with human and animal brucellosis in Brazil indicate that the organization of a Center for the study of the disease is worthy of consideration. We hope that in the near future this Center will be a reality. LA BRUCELOSIS ANIMAL EN EL BRASIL (Sumario) Los datos sobre brucelosis bovina muestran que la enfermedad está diseminada en el pais, tanto en el ganado para el consumo como en el ganado lechero, en una proporción del 10 al 20%. La incidencia de la infección en los cerdos es más alta llegando hasta 30 ó 40%. Como es de suponer, la enfermedad ha sido más estudiada donde se encuentran las concentraciones más grandes de ganado, como en los estados de Rio Grande do Sul, Sáo Paulo, Minas Gerais y Rio de Janeiro.

LA BRUCELOSIS ANIMAL EN EL URUGUAY

69

El diagnóstico ha sido establecido mediante el aislamiento de microorganismos de la placenta, fetos o leche, pruebas de aglutinación y manifestaciones clfnicas.

La brucelosis caprina es rara y solamente se han encontrado casos esporádicos por medio de pruebas de aglutinación en focos de brucelosis bovina. Hasta el presente, esto no tiene ninguna importancia epidemiológica. La brucelosis equina y canina es también esporádica y algunos casos han sido descubiertos mediante pruebas de aglutinación en focos de brucelosis bovina. En un caso se aisló la Brucella abortus de un perro. No es posible afirmar si la infección se propagó de algunos focos existentes o si habla existido por muchos años sin ser descubierta. La evidencia, sin embargo, parece indicar que ya existía en los lugares donde ha sido observada hasta ahora, y en muchos otros desconocidos aún, porque se han descubierto focos nuevos cada vez que se han efectuado estudios serológicos en rebaños ubicados en lugares donde no parecia existir la enfermedad. La infección humana, indudablemente de origen bovino o porcino, es comparable a la enfermedad animal. Los pocos casos en que se aisló la Br. melitensis fueron dudosos o fueron de personas que posiblemente hablan contraído la infección fuera del país.

LA BRUCELOSIS ANIMAL EN EL URUGUAY Por B. SZYFRES, J. L. STELLA, W. ERRANDONEA, H. TRENCHI, D. ABARACóN, J. C. PIÑON y J. M. INFANTOZZI

Servicio de Brucelosis. Laboratorio de Biología Animal "Dr. Miguel C. Rubino", Dirección de Ganadería, Uruguay El Uruguay es un país eminentemente ganadero. Los productos animales, lana y carne, constituyen su principal renglón de exportación. El número de ganado bovino, en su predominancia de producción de carne, se calcula actualmente entre 7 y 8 millones y el de ovinos entre 25 y 28 millones de cabezas. La brucelosis es uno de los principales problemas sanitarios de nuestra ganadería. Por la merma que ocasiona en el procreo y en la producción de leche, la brucelosis repercute directamente sobre la riqueza básica del país. Este informe tiene por objeto dar a conocer el estado actual de la cuestión en el Uruguay. Reunimos en él los datos existentes sobre la difusión de la enfermedad y las medidas que ha adoptado el Poder Ejecutivo para contrarrestar su extensión y reducir los estragos causados por ese mal. Los resultados obtenidos hasta ahora no son satisfactorios. El problema de la profilaxis tendrá que ser reconsiderado en su conjunto, con el fin de proveer al país de un programa nacional de control de la enfermedad, en consonancia con los últimos adelantos científicos y las características de nuestro medio.

70

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS DATOS SOBRE LA DIFUSIóN DE LA BRUCELOSIS ANIMAL

Brucelosis bovina.-La infección a Br. abortus fué comprobada en el Uruguay, en el año 1928, por A. Cassamagnaghi (1). En 1932 y 1933, el Laboratorio de Investigaciones (actual Laboratorio de Biología Animal de la Dirección de Ganadería) hace un estudio sistemático, con el fin de conocer el grado de difusión de la enfermedad en los establecimientos lecheros del Departamento de Montevideo (2). Se ha practicado el diagnóstico en 1,790 animales lecheros pertenecientes a 224 tambos. En 116 de esos establecimientos, es decir en un 51.7% de los examinados, se han encontrado animales con reacciones positivas a la prueba de la aglutinación. De los 1,790 animales examinados, el 20.3% dieron reacciones positivas y el 14.7% reacciones dudosas. El Laboratorio de Biología Animal de la Dirección de Ganadería (4) dió a conocer los datos correspondientes a las pruebas efectuadas entre 1932 y 1947. Esos datos se refieren a 113,645 muestras de sangre de bovinos, procedentes de 1,161 establecimientos repartidos por la República. Los exámenes efectuados respondieron a distintas razones, tales como: estudio epizootiológico, diagnóstico por haberse producido abortos en el establecimiento de origen, saneamiento de establecimientos infectados, exportación, y desde 1942, el dar cumplimiento a la Reglamentación de Exposiciones, Ferias y Remates. El 31.9% de los 1,161 establecimientos, de los cuales hemos examinado muestras de sangre, con un promedio de 97 muestras por establecimiento, contenían animales positivos a la seroaglutinación. Las siguientes cifras indican el índice de animales infectados: Reacciones positivas ................................... Reacciones dudosas .................................... Reacciones negativas .................................. Total .............................................

5,964 3,415 104,266 113,645

(5.2%) (3.0%)

El resultado de estos exámenes indicaría que del 5 al 8% de los bovinos del Uruguay estarían infectados. LA INFECCIóN BRUCÉLICA EN OTRAS ESPECIES ANIMALES

La difusión de la brucelosis suina es poco conocida. Por los pocos datos existentes parecería que no estuviera muy extendida. La infección a Br. suis fué reconocida recién en el año 1943 (5), cuando se aisló por primera vez el germen en causa, pero ya anteriormente se habían encontrado aisladamente algunas muestras con reacciones dudosas y en 2 ocasiones con reacciones positivas. El primer foco de brucelosis suina, reconocido en el país, fué originado por la importación de una hembra de la República Argentina con reacción negativa, que abortó a los 3 meses en el establecimiento de destino, propagándose luego los abortos a 12 hembras más. De 95 suinos examina-

LA BRUCELOSIS ANIMAL EN EL URUGUAY

71

dos de ese establecimiento 42 resultaron positivos y 13 dudosos. Otros focos que se constataron a continuación fueron casi siempre originados por la importación de animales infectados, no reaccionantes a la seroaglutinación. El examen practicado por el Laboratorio de Biología Animal, en los años 1933-1941, en 204 muestras de suinos faenados en el Frigorífico Nacional de Montevideo, y 146 muestras de otras procedencias, dió por resultado 8 reacciones dudosas y las restantes negativas (5). En 1941, Scaltritti (3) examina 588 muestras de sangre de cerdos faenados en el Frigorífico Nacional, encontrando una muestra positiva. Pradines (3) practica la seroaglutinación en 751 muestras de suinos sacrificados en el mismo Frigorífico, encontrando sólo 4 reaccionantes dudosos. Según los datos que anteceden, parecería que la brucelosis suina está poco difundida en el Uruguay. La población caprina en el Uruguay es muy reducida. Según el censo de 1937 sería de 28,129 cabezas en toda la República. Las cabras no son explotadas en forma industrial, y el intercambio de especímenes es casi nulo. Esta circunstancia explica por qué esta especie animal no fué objeto de estudio en cuanto a infección brucélica. En los ovinos del país no se han encontrado reaccionantes positivos o dudosos en los 2,834 animales que se han examinado, procedentes de 8 departamentos de la República. El mismo resultado negativo se ha obtenido en cerca de 2,000 reproductores ovinos importados. LA LUCHA CONTRA LA BRUCELOSIS

Desde que la infección brucélica fué comprobada en el país, la Dirección de Ganadería se ha esforzado constantemente' por proveer al país de una legislación y de un plan de lucha para contener la difusión de la enfermedad y para reducir los estragos que ésta ocasiona. Los esfuerzos de la Dirección de Ganadería se han encontrado, sin embargo, con dificultades insalvables para lograr sus propósitos. La política profiláctica se puede dividir en 2 etapas, la primera, de 1928 a 1933, consistió en medidas severas y compulsivas para intentar erradicar la infección y la segunda, de 1933 a la fecha, tiene como base la profilaxis libre, opcional. Los progresos hechos, tanto en la primera etapa como en la actual, son muy poco satisfactorios, quedando como único saldo positivo la experiencia adquirida y la posibilidad de estructurar en el futuro un programa que se ajuste más a nuestra realidad epizootiológica y características de la explotación ganadera. Pasaremos rápidamente revista sobre la legislación profiláctica en el Uruguay, tratando de establecer en lo posible las circunstancias que se han opuesto a su ejecución. En 1928, en seguida de reconocida la infección, la brucelosis es incluida entre las enfermedades que dan lugar a la aplicación de las medidas sani-

72

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

tarias establecidas para las demás enfermedades infectocontagiosas (ley del 13 de abril de 1910). Esta inclusión, como asimismo el decreto de 21 de mayo de 1930 y del 16 de junio de 1932 implicaban la denuncia obligatoria de la enfermedad, el aislamiento de los animales infectados y la puesta del establecimiento bajo el control directo de la Dirección de Policía Sanitaria (hoy Dirección de Ganadería), estando ésta facultada para efectuar "las pruebas diagnósticas periódicas que crea necesarias". En la época en que fueron concebidas estas medidas, en el medio rural se sabia muy poco sobre la verdadera entidad y el alcance que tenía la infección brucélica para el ganado. La profesión veterinaria, por otra parte, desconocía el grado de difusión de la infección, por carecer de estudios nacionales sobre el tema, ni tenía la experiencia ni los medios necesarios para encarar la lucha contra la enfermedad. Estas medidas tan rigurosas se encontraron desde el principio con la resistencia de los ganaderos que veían lesionados sus intereses, y tuvieron que ser revocadas por el decreto de octubre de 1933. Con esa fecha "se faculta a la Dirección de Policía Sanitaria de los Animales para tomar en cada caso concreto en que se constata la infección, las medidas que la prudencia aconseje para localizar el mal y evitar su difusión". La Dirección de Ganadería de actualmente facilidades a los ganaderos que quieran sanear sus haciendas. A todo establecimiento en saneamiento se le ha eximido del pago por las serorreacciones que se efectúan en el Laboratorio de Biología Animal, siendo asimismo gratuito el servicio del veterinario que se traslada al establecimiento para extraer las muestras de sangre. La única exigencia es que el propietario "se comprometa a aceptar y dar cumplimiento a las indicaciones que se le formulen respecto al destino a darse a los sujetos reaccionantes". El único método oficialmente reconocido para el control de la brucelosis es el de la prueba serológica y la eliminación o aislamiento de los reaccionantes. La pérdida ocasionada por el sacrificio de los reaccionantes corre por exclusiva cuenta del propietario, no pagando el Estado ninguna indemnización. La vacunación con gérmenes vivos, tal como el empleo de la cepa 19, no está reglamentada y su elaboración, venta y uso son considerados ilícitos. Los resultados alcanzados por ese plan son muy poco satisfactorios. Un escaso número de establecimientos ganaderos se ha acogido a los beneficios otorgados. Por una parte, los conocimientos sobre la brucelosis y la manera de combatirla no tuvieron la difusión necesaria; por otra parte, es muy difícil conquistar la cooperación del hacendado para un programa de profilaxis opcional, exigiéndole sacrificios económicos. Si en Estados Unidos de Norte América el método de prueba serológica y sacrificio obtuvo algunos resultados significativos, es porque el gobierno federal y los estatales disponían de fondos necesarios para indemnizar a los propie-

LA BRUCELOSIS ANIMAL EN EL URUGUAY

73

tarios por sus pérdidas y pudieron de esa manera conseguir su colaboración. En nuestras condiciones, sin indemnización y sin un programa concreto de lucha con la correspondiente financiación para los gastos de operación, el procedimiento de prueba serológica y de sacrificio estaba desde el principio destinado al fracaso. En primer término, han recurrido lógicamente a la Dirección de Ganadería los hacendados que más tangiblemente han sufrido los estragos de la brucelosis, es decir, los propietarios de establecimientos donde la infección era de reciente instalación y de rápida difusión. En estos casos, como es sabido, el método de prueba y sacrificio es sumamente oneroso y casi siempre inoperante. Los propietarios de rodeos, con un índice bajo de infección, han sido los que en menor número se acogieron a las facilidades otorgadas, pero siempre que se actuó en estos establecimientos se obtuvieron resultados satisfactorios, con la diferencia de que la erradicación de la enfermedad era mucho más lenta en haciendas con un número elevado de animales, que en aquellas con un número más reducido. En términos generales, podemos decir afirmándonos en la experiencia adquirida, que no se puede construir, en el Uruguay, un plan de control de la brucelosis exclusivamente a base de prueba serológica y sacrificio. En el mejor de los casos, aun contando con la cooperación de los hacendados, no disponemos del personal técnico necesario para esa empresa. En efecto, en muchos departamentos, con una extensión de 8 a 10 mil kilómetros cuadrados, con varios cientos de miles de cabezas de ganado vacuno y muchos cientos de establecimientos ganaderos, hay un solo veterinario y que lógicamente, sólo en forma parcial, puede dedicarse a la lucha contra la brucelosis. No menos importante es el hecho de que por la característica de la explotación extensiva es sumamente difícil tomar las medidas de higiene indispensables, quedando muchas veces el material infeccioso por un tiempo indeterminado en el campo. En vista de los factores arriba mencionados, abogamos para que se incorpore en la lucha contra la brucelosis, mientras no haya un agente inmunizante mejor, la vacuna a cepa 19, porque entendemos que es la única manera de conquistar la cooperación de los hacendados y de extender la profilaxis de la brucelosis sobre todo el país. Desde el año 1942, la Dirección de Ganadería, por intermedio del Laboratorio de Biología Animal, está elaborando y aplicando la vacuna a cepa 19 en establecimientos ubicados en varios departamentos de la República, a título experimental y en forma gratuita. El Primer Congreso Nacional de la Brucelosis, realizado en Montevideo en diciembre de 1947, ha aprobado una moción presentada por los técnicos del Servicio de Brucelosis del Laboratorio de Biología Animal de la Dirección de Ganadería, solicitando a los Poderes Públicos que se autorice y reglamente la elaboración y uso de la vacuna a cepa 19.

74

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Esta iniciativa ha encontrado una acogida favorable en el ambiente ganadero, como lo atestigua la solicitud de la Asociación Rural del Uruguay dirigida al Ministerio de Ganadería y Agricultura, pidiendo se autorice el uso de la cepa 19, bajo el control estatal (6). El empleo de la vacuna a cepa 19 se encuentra actualmente a consideración de la Dirección de Ganadería. Entre las otras medidas preventivas, adoptadas por el Uruguay, mencionaremos el Reglamento de Importación y Exportación de Animales, del 8 de junio de 1934, y el Reglamento de Exposiciones, Ferias y Remates de Ganado, del 7 de octubre de 1942. En el primero de estos reglamentos se establece que todos los reproductores de las especies bovina, equina, porcina, caprina, y ovina, serán sometidos, mientras cumplen la cuarentena, a las pruebas diagnósticas para el reconocimiento de la brucelosis. De la importancia de esta medida hablan las cifras que arroja el suero-diagnóstico de animales importados entre los años 1943 y 1946. De 5,622 reproductores bovinos importados, de los cuales 5,437 corresponden a la República Argentina y los restantes de ultramar, 476 ó sea el 8.4% dieron reacciones positivas y 417 ó sea el 7.4% reacciones dudosas. El Reglamento de Exposiciones, Ferias y Remates establece que todo reproductor bovino o suino macho o hembra, de pedigree o puro por cruce, que concurra a exposiciones, ferias o liquidaciones de estancias, tiene que estar provisto de un certificado de "libre de brucelosis". Esta reglamentación tenía ante todo por objeto poner coto al fraude que significaba el vender reproductores infectados y de poco rendimiento por animales que, a juzgar por sus cualidades zootécnicas y de pedigree, serían de alto valor. En cuanto a la brucelosis suina, no hay ningún programa estatal de control, excepto los 2 reglamentos ya mencionados, debiéndose mencionar que la Dirección de Ganadería ha procedido en varias ocasiones, al constatar la infección, al sacrificio de todos los animales del establecimiento infectado, indemnizando al propietario. REFERENCIAS (1) Cassamagnaghi, A.: Enfermedad de Bang. Bol. Pol. Sanit. Anim., año 12, No. 1, p. 21, 1928. (2) Rubino, M. C.: El aborto epizoótico o enfermedad de Bang. Bol. Pol. Sanit. Anim., año 19, No. 10, p. 501, 1935. (3) Purriel, P., Risso, R., y Espasandín, J.: Brucelosis. Estudio de esta enfermedad en el Uruguay. Editorial Independencia. Montevideo, 1944. (4) Szyfres, B.; Rodríguez García, J. A.; Giacometti, H.; e Infantozzi, J. M.: Primer Congreso Nacional de Brucelosis. Memorias. Montevideo. p. 82, 1947. (5) Rubino, M. C.; Szyfres, B.; y Tortorella, A.: Núm. Cient. Acc. Sind., año 7, No. 1, p. 13, 1945. (6) Rev. Asoc. Rural del Uruguay, p. 56, sbre. 1949.

LA BRUCELOSIS ANIMAL EN EL URUGUAY

75

ANIMAL BRUCELLOSIS IN URUGUAY (Sunzmary) Bovine brucellosis was recognized in Uruguay in 1928. Of 113,645 bovine blood samples examined, 5.2% were positive and 3% suspicious. These samples came from 1161 ranges and farms, distributed all over the country. Of these herds 31.95% contain one or more reactors. Of the 224 dairies located in the capital, Montevideo, which were examined in 1932-33, 51.7% were infected. Of a total of 1172 of these animals 20.3% were positive reactors and 14.7% suspicious. All purebred and crossbred animals shipped to expositions or auction sales must be provided with a brucellosis-free certificate. The control-program in Uruguay consists of free assistance on behalf of the Bureau of Livestock to any herd owner who desires to eradicate the infection from his herd. Test and slaughter or test and segregation is the procedure employed. Only a few herds are under this program and up to the present, only a few have been freed from infection, especially the smaller ones and with a low percentage of infection. No indemnity payment and scarcity of personnel are the principal difficulties in this work. Strain 19 vaccination is now under the consideration of the Ministry of Agriculture and Livestock. Immunization with Strain 19 is being employed in a limited number of herds, under experimental conditions. Brucella suis infection in Uruguayan swine herds was recognized in 1943. It seems that swine brucellosis is not very widespread. Brucella infection in sheep and goats has not been found yet and Br. melitensis has not been isolated from other sources.

BRUCELLOSIS-A PACKING PLANT PROBLEM By NORMAN B. MCCULLOUGH, PH.D., M.D. MicrobiologicalInstitute of the National Institutes of Health, Public Health Service, Bethesda, Maryland A few years after the discovery of the etiology of Malta fever, the importance of raw goat's milk in the epidemiology of the disease in man was revealed. With the demonstration of brucellosis in man due to Brucella abortus and Brucella suis, further evidence accumulated of the importance of milk in the transmission of this disease. For some years raw milk was considered as the prime source of brucellosis in man. In the last decade or two with pasteurization of milk being practiced more extensively, it has become increasingly apparent that contact with infected animals and their tissues is now equally, if not more, important than milk in the spread of this disease to man. In the United States it would appear that the number of cases occurring after exposure to infected animals or their tissues may exceed the number deriving their disease from milk. The incidence in workers coming directly in contact with infected animals far exceeds that in the general population. In Iowa, in an investigation of 2,405 case reports of human brucellosis occurring over a seven year period, 75% of the cases gave a history of direct contact with farm animals.1 In a study of the epidemiology of 268 culturally proved cases in Minnesota, at least 60% were classified as occupational in origin.2 In Argentina, similar findings have been reported.3 Brucellosis is now recognized as an occupational disease. With the growing recognition of brucellosis as an occupational hazard, there has been much speculation and confusion in regard to the epidemiology and incidence of this disease in packing plant personnel. Opinions range from (a) that the exposure incidence is extremely low and the hazard relatively unimportant to (b) that simply walking through the packing plant or stock yard area constitutes exposure to brucellosis. The true incidence of human brucellosis in the general population is difficult to determine. If we were to count only those cases which are culturally proved, the number would be small. However, when we consider the high incidence of dermal sensitivity to Brucella and also of positive agglutination titer, it would appear that a large segment of the population has at some time come in contact with the organism. How many of these individuals may have had clinical undulant fever is unknown. Dermal sensitivity occurs in from 10 to 25% of the population in different parts of the country. In certain occupational groups, such as veterinarians and packing house workers, it is much greater than this. In tests of 11,000 sera derived from people living mostly in Chicago, some degree of agglutinins (1/25 or higher) was found in 1.55 per cent. 4 76

BRUCELLOSIS-A PACKING PLANT PROBLEM

77

In other areas where exposure to raw milk and infected animals is greater, the incidence is increased. In limited surveys of packing house workers, positive agglutination tests have been reported in from 10 to 30% of the workers.s, s Our knowledge of the extent of the hazard due to brucellosis in packing plants is incomplete. It is based largely upon the results of surveys employing dermal sensitivity and the agglutination test and a few well conducted epidemiological studies. These studies have not been extensive. Recently we have determined the presence of agglutinins in the personnel of three small specialty plants-a glue plant, a fertilizer plant, and an extract plant-located in the Chicago Stock Yards area. The standard test tube agglutination test was employed with incubation at 37 ° C for forty-eight hours, using our standardized antigen. The data obtained appears in condensed form in Table I. TABLE I.-Brucella agglutination titers of personnel employed in a glue plant, extract plant, and fertilizer plant Agglutination Titers Plant

Glue ........ Extract...... Fertilizer....

No. Men

294 77 133

~

0

146 24 57 7 102 16

45 9 12

27 3 3

16 1 0

12 0 0

7 0 0

4 0 0

6 0 0

2 0 0

~

No. Pos.

5 0 0

148150.34 2025.97 31 23.31

% Pos.

These plants were selected as representing end-points of the slaughterhouse processing line. The incidence of 50.34% positive tests obtained in the glue plant personnel speaks strongly for the intensity of exposure at this level and contrasts with the findings of 25.97% and 23.31% in employees working in the extract and fertilizer plants. The incidence of high titers was confined almost wholly to glue plant personnel. Of 148 glue plant workers with demonstrable agglutinins, 52, or 35.1%, had titers of 1/160 or higher. In the extract plant, only one of 77 men tested had a titer of 1/160, and in the 133 men working in the fertilizer plant there were no titers higher than 1/80. The marked difference in the reflected degrees of exposure is presumably due to the different nature of the materials handled in the respective plants. Commencing two years ago, we examined the submaxillary lymph nodes of slaughtered hogs by weekly sampling over a period of six months. 8S 9 One lymph node was excised from each carcass and cultured for Brucella. In this unselected series of 5,000 marketed hogs, Brucella was isolated in thirty-five instances, or 0.7% of the hogs. In addition to Br. suis, Br. abortus and Brucella melitensis were repeatedly recovered.

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Br. melitensis infection in hogs had been reported previously in Iowa. t° Since our preliminary report, Damon et al. have isolated this species from one hog in Indiana." There is growing epidemiological evidence that cases of Br. melitensis infection in man in this country are derived from hogs.2 11 '12 13 With the isolation of all three species of Brucella from hogs slaughtered in a large Chicago packing plant, it is apparent that persons engaged in the slaughtering of hogs and the processing of their meat are exposed to all three species. We have observed cases of undulant fever due to Br. abortus occurring in individuals working exclusively on the hog kill and have also reported a case of brucellosis due to Br. melitensis in a meat inspector engaged in inspecting the head glands of hogs.9 Our knowledge of the incidence of brucellosis in the hog is incomplete, but it appears high. Various surveys indicate that this incidence may be from 1 to 20%. 14' 15, 16, 1 In 1,008 hog sera o]btained during the previously mentioned survey, 40% were found to have agglutination titers of 1/20 or higher, and 6.45% of 1/160 or higher.9 If one were to take only the 0.7% of culturally proved brucellosis as the true incidence of infected carcasses giving exposure to the workers, in large plants, each individual handling the carcasses would come in contact with Brucella several times a day. If the incidence of high agglutination titers found in this survey is taken as the real incidence of infection, then "several" must be changed to "many". Since the inception of the brucellosis control program in cattle, great numbers of Bang's reactor cattle have been slaughtered. Workers handling these carcasses take no extra precautions. It has been assumed by some and all too often stated that these animals offered little risk to the worker. This opinion was based on statements that brucellosis in the cow is a localized disease and that only the uterus and udder are infectious. This, of course, is not so. Although there is this tendency to localize, there is ample evidence to show that the disease may be generalized for months after its inception. In a number of studies, Br. abortus has been recovered from the blood stream of cattle.' 8' 19, 20 Recently, Manthei and Carter studied both artificially and naturally infected cows. 2' Of 270 cows examined, positive blood cultures were obtained from 172. In eighteen animals studied for a prolonged period, 80% yielded positive cultures for five months, with bacteremia persisting in one animal for nearly two years. The organism has been isolated from many portions of slaughtered animals. In an attempt to obtain more direct data bearing on this problem, we recently cultured the carcasses of 100 Bang's reactor cattle slaughtered in a Chicago plant.22 Cultures were obtained from over twenty locations, mostly lymph nodes, throughout the carcass. As was to be expected, in a number of these cattle, no isolations were made. Br. abortus

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was recovered from some tissue in 42% of the animals. In 29%, isolations were made from locations other than the uterus and supramammary lymph nodes. In 10%, there were numerous recoveries from many locations. With the culturally proved incidence of brucellosis in slaughter-house hogs, the high incidence of agglutination titers found in these animals, and the high incidence of generalized infection in slaughtered Bang's reactor cattle, one must conclude that packing plant workers suffer relatively heavy exposure to Brucella from these sources. There has been much speculation concerning the role of air and other environs in the dissemination of brucellosis among plant personnel. Although such factors may contribute to the epidemiology, one would hardly expect them to be as major a factor as repeated daily contact with heavily infected animal tissues. Aside from the exposure offered by the fresh carcass, the viability of Brucella during processing of meat products may be important. It has long been known that the viability of Brucella is great. Recently, Hutchings et al. have studied the viability of Br. suis in the carcasses of slaughtered hogs subjected to the usual degree of refrigeration. 2 3 They were able to isolate Brucella from many locations in the carcass for as long as three weeks after slaughter. We have confirmed this fact and have recovered Br. suis from refrigerated lymph nodes forty-nine days after slaughter. In collaboration with Dr. L. M. Hutchings, we conducted experiments on the fate of Brucella in naturally infected pork when subjected to curing and smoking.2 4 A herd of infected hogs was obtained. When isolated, the infecting agent proved to be Br. melitensis. The hogs were slaughtered, and the hams and shoulders were subjected to the usual curing process employed in one of the large packing plants. Trained packing plant personnel carried out the procedures. Plant conditions were simulated as closely as possible. Cultures were made from the pickled hams and shoulders at various intervals. Br. melitensis was isolated repeatedly for as long as three weeks. A number of cured hams regularly yielding positive cultures were then subjected to smoking. This procedure was carried out under packing plant conditions. During smoking the hams are subjected to relatively high temperatures. No isolations were made after smoking. It would appear that during the smoking process Brucella is destroyed, but that the organism survives during pickling. Extensive and comprehensive surveys of the personnel of packing plants to determine how many of the workers actually contract brucellosis have not been made. Such studies, employing not only the usual serological and dermal sensitivity techniques, but also blood cultures, history taking, physical examinations, and competent appraisal of the health status of each worker examined, would do much to clarify our

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knowledge of the incidence and extent of clinical brucellosis in this group of workers. The packing industry has been reluctant to accept brucellosis as an occupational disease. However, for a number of years our South American neighbors have recognized brucellosis as an occupational and compensable hazard. Three years ago the state of Iowa similarly recognized its importance and passed an occupational disease law to cover the situation in that state. This enlightened attitude is to be commended. As Borts has pointed out, "That brucellosis is an occupational hazard is not denied by those who are fully informed and are willing to admit facts and potentialities as they exist." 25 Until such time as brucellosis in domestic animals is eradicated or so reduced in incidence as to be negligible, the occupational hazard of brucellosis will continue. The question arises as to what precautions might be instituted to reduce the danger. At present in most plants none are taken. It is difficult to formulate a plan until all the facts are at hand. Here we are still badly in need of facts. Further investigations of the incidence and epidemiology of the disease are needed. The people working in these plants should be studied to delineate the degree and extent of disability involved. There are some protective measures that could be used even now. An educational program seems indicated. Individuals should be urged to report any illness or injury promptly. Even minute cuts and abrasions should be given prophylactic care. Personal hygiene should be stressed. It would be quite feasible to go even further than this, particularly in the case of Bang's reactor cattle. Such material might even be slaughtered separately by workers already immune to the disease. Education would appear to be the prime tool in this program. REFERENCES (1) Jordan, C. F.: Symposium on Brucellosis, A. A. A. S. The Epidemiology of Brucellosis, pp. 98-115, 1950. (2) Magoffin, R. L.; Kabler, P., Spink, W. W.; and Fleming, D.: An epidemiologic study of brucellosis in Minnesota. Pub. Hlth. Rep., 64: 1021-1043 Aug. 19, 1949. (3) Molinelli, E. A., Basso, G., and Miyara, S.: Epidemiologia de la brucelosis humana en la República Argentina. Rev. Asoc. Med. Arg., 61: 182-188, 1947. (4) Unpublished data. (5) Huddleson, I. F.: Brucellosis in man and animals. New York, The Commonwealth Fund, 1943. (6) Topley and Wilson's Principles of Bacteriology and Immunity, 3rd ed., Baltimore, The Williams &Wilkins Co., 1946. (7) McCullough, N. B.: Symposium on brucellosis, A. A. A. S., Laboratory Tests in Brucellosis, pp. 116-121, 1950. (8) McCullough, N. B.; Eisele, C. W.; and Pavelchek, E.: Isolation of Brucella abortus from hogs. Pub. Hlth. Rep., 64: 537-538 Apr. 29, 1949.

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(9) McCullough, N. B.; Eisele, C. W.; and Pavelchek, E.: A Survey of brucellosis in slaughtered hogs. Pub. Hlth. Rep. In press. (10) Borts, I. H.; McNutt, S. H.; and Jordan, C. F.: Brucella melitensis isolated from swine tissues in Iowa. Jour. Am. Med. Assoc. 130: 966, 1946. (11) Damon, S. R., and Scruggs, J. H.: Recovery of Brucella melitensis from the hog. Pub. Hlth. Rep., 65: 374 Mar. 17, 1950. (12) Jordan, C. F., and Borts, I. H.: Occurrence of Brucella melitensis in Iowa. Jour. Am. Med. Assoc. 1S0: 72-75 Jan. 12, 1946. (13) Kabler, P.; Bauer, H.; and Nelson, C. B.: Human Brucella melitensis infections in Minnesota with hogs as the probable source. Jour. Lab. & Clin. Med., 32: 854-856, 1947. (14) Boak, R. A., and Carpenter, C. M.: Brucella abortus agglutinins in porcine blood. Jour. Inf. Dis., 52: 425-429, 1930. (15) McNutt, S. H.: Incidence and importance of Brucella infection of swine in packing-houses. Jour. Am. Vet. Med. Assoc., 39: 183-191, 1935. (16) Boyd, W. L.; Kernkamp, H. C. H.; Roepke, M. H.; and Blye, C. E.: Incidence of brucellosis in swine. Proc. 46th Ann. Meeting U. S. Livestock San. Assoc., pp. 124-128, 1942. (17) Hutchings, L. M.: Brucellosis in swine. Proc. 47th Ann. Meeting U. S. Livestock San. Assoc., pp. 52-58, 1943. (18) Fitch, C. P.; Bishop, L. M.; and Kelly, M. D.: The isolation of Brucella abortus from the blood stream of cattle. Proc. Soc. Exp. Biol. & Med., 34: 696-698, 1936. (19) Lubke, A.: tUber das Vorkommen von Bang-bakterien im Blut von Kihen aus banginfizierten Bestanden, Ztschr. f. Infektionskr., 47: 240, 1935. (20) Soule, M. K.: Bacteriological and serological findings in Brucella abortus infections in animals and man. Premier Congrés International de Microbiologie, 1: 606, 1930. (21) Manthei, C. A., and Carter, R. W.: Persistence of Brucella abortus infection in cattle. Am. Jour. Vet. Res., 11: 173-180, 1950. (22) McCullough, N. B.; Eisele, C. W.; and Byrne, A. F.: Incidence and distribution of Brucella abortus in slaughtered Bang's reactor cattle. Pub. Health Rep. In press. (23) Hutchings, L. M. et al.: In press. (24) In preparation for publication. (25) Borts, I. H.: Some observations regarding the epidemiology, spread and diagnosis of brucellosis. Jour. Kansas Med. Soc., 41: 399, Dec. 1945. LA BRUCELOSIS COMO PROBLEMA DE LAS PLANTAS DE ENVASE (Sumario) En los Estados Unidos el número de casos de brucelosis humana observados después de la exposición a animales infectados o los tejidos de éstos, parece exceder el número de los que contraen la enfermedad por la leche. En una investigación de 2,405 casos de brucelosis humana observados en Iowa, 75% acusaron antecedentes de contacto directo con animales de granjas. En un estudio epidemiológico de 268 casos comprobados por medio de cultivos en Minnesota, por lo menos 60% se clasificaron como ocupacionales. Se han comunicado hallazgos semejantes en Argentina. La brucelosis se reconoce ya como enfermedad ocupacional. La frecuencia de la sensibilidad dérmica a la brucela y de las aglutininas del

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suero es mucho mayor en grupos ocupacionales específicos que entre la población general. Se examinó en busca de aglutininas en el suero al personal de tres pequeñas plantas de envase: una planta de cola, una planta de abonos, y una planta de extractos, que representan los puntos terminales de las operaciones de un matadero. En el personal de la planta de cola se encontró que la frecuencia de las reacciones positivas ascendía a 50.34%, llegando a 25.97 y 23.31%, respectivamente, entre los empleados de la planta de extractos y de abonos. Los títulos elevados se limitaron casi por completo al personal de la planta de cola. En una serie no seleccionada de 5,000 cerdos mercados, se aislaron brucelas de los nódulos linfáticos submaxilares de 35 (0.7%). Se descubrieron las tres especies de Brucellas, encontrándose por primera vez la Br. abortus en los cerdos infectados naturalmente. Se ofrecen pruebas epidemiológicas indicativas de que el cerdo constituye una fuente de las infecciones humanas por Br. abortus y Br. melitensis. Se examinó la carne de 100 cabezas de ganado que habían acusado reacciones positivas para la Br. abortus. Se prepararon cultivos con 23 tejidos, principalmente nódulos linfáticos. Se aisló la Br. abortus de una o más localizaciones en 42 animales. En 10 de los animales se aisló de numerosos sitios. Aunque el útero y los nódulos linfáticos supramamarios fueron los órganos invadidos con más frecuencia, se encontró la infección en zonas muy esparcidas. Los perniles y cuartos delanteros de los cerdos infectados naturalmente con Br. melitensis fueron sometidos al proceso comercial de salazón. La Br. melitensis se aisló repetidamente de los jamones en conserva durante tres semanas. No se aisló la brucela después del ahumado. Se discute el riesgo ocupacional de la brucelosis en la industria de envases.

VARIATION AS A TOOL IN BRUCELLOSIS RESEARCH

By WERNER BRAUN, ARTHUR N. GORELICK, MARY KRAFT AND DOROTHY D. MEAD Camp Detrick, Frederick, Maryland The striking differences in colonial types of Brucella are usually associated with differences in virulence. They, therefore, provide simple markers for determining the fate of variants in vivo after simultaneous or consecutive inoculation of two or more variant types. It is of considerable value for various phases of brucellosis research to utilize such marked cell types for in vivo studies. First, by adjusting infective doses of consecutively administered inocula in such a manner that ID 50 's are comparable, it becomes possible to study certain immunologic and pathogenetic problems. Since descendants of the first inoculum can be easily distinguished from the progeny of the second inoculum by observing colony-types on plates streaked with tissue samples from the infected animals, the proportion of cells recoverable from the primary or secondary infection can be ascertained rapidly in any tissue. Second, this technique permits the investigation of certain problems associated with a chronic disease such as brucellosis. Previous in vitro studies had shown that the less virulent nonsmooth types do not establish themselves in originally smooth cultures which are maintained in the presence of small amounts of normal serum from susceptible animals. In contrast, a rapid establishment of these less virulent nonsmooth variants was observed in vitro in the presence of sera from infected or immunized animals. These effects were shown to be independent of the presence of agglutinins. On the basis of these results it was believed that less virulent nonsmooth types may also establish themselves in vivo some time after an acute infection but may be replaced eventually by virulent types when selective conditions return to the status analogous to normal hosts. It was suggested that such population changes may play a role in the frequently observed relapses. The findings in one extensive experiment with guinea pigs, in which an intermediate variant of Br. suis gradually replaced a smooth type, seemed to substantiate this assumption. However, it was not possible to detect a similar replacement of smooth types by nonsmooth types in several subsequent guinea pig experiments. In order to obtain further information on this point, as well as information of the kind which we discussed earlier, guinea pigs were inoculated subcutaneously or intracardially with massive doses of a mucoid variant of Brucella suis one week or five weeks after primary infection with smooth organisms. The intervals of one week and five weeks between S and M infections were chosen because in vitro studies had shown that the selective activity of guinea pig sera was unaltered one week after S 83

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infection but had undergone changes five weeks following S infection. The smooth Brucella suis cells were injected subcutaneously in the right inguinal region; each animal was infected with 30 cells which represents approximately 10 ID60 's. One group of animals was then inoculated one week later with 2 X 109 mucoid cells per animal subcutaneously near the site of the primary infection; another group was inoculated intracardially one week after the smooth infection. Two additional groups of animals were similarly inoculated 5 weeks after the primary S infection. A total of 80 guinea pigs were thus infected; in addition 21 control animals were infected with S only, 24 other animals were infected subcutaneously or intracardially with M cells only. Animals were sacrificed 1, 2, 4, and 6 weeks following the inoculation of M cells and 17 different tissues of each animal were cultured after thorough grinding with mortar and pestle. The distribution of recoverable M cells differs significantly depending on the route of inoculation. One week after the subcutaneous M injection, M cells were recoverable only from the right inguinal lymph node, left axillary lymph node, spleen, liver, renal or lumbar lymph node, and to some extent from the bone marrow. In contrast, all tissues from intracardially challenged animals sacrificed at the same time permitted the recovery of a generally high percentage of M type cells. The relatively low percentage of M type cells recoverable from the right inguinal lymph node and the lumbar lymph node of these "intracardial" animals is noteworthy, especially since these two tissues yielded the highest recovery of M types in subcutaneously challenged animals. This may be attributable to some kind of blocking effect towards the intracardially introduced M cells in the tissues closest to the site of the primary infection. In all instances the lumbar or renal lymph node yielded recoveries identical to those of the regional right inguinal lymph node, just as if there were a direct connection between those two lymph nodes. In any event a comparison between the two groups of animals indicates that a blocking effect by the lymphatic system follows the subcutaneous administration, whereas intracardial infection permits a general distribution of the variant type throughout the body of the host. The next point of interest is the rapid disappearance of the M cells from most tissues within 4 weeks after infection. The different tissues seem to possess a differential ability to rid themselves of the M types, as shown by the greater persistence in the mesenteric and cervical lymph nodes. It was also observed that the M type disappears much faster from the tissues of animals previously infected with S type cells than from previously uninfected animals. Practically no M cells were isolated from animals sacrificed 2 weeks or more after the secondary M infection, whereas in M infected, previously unexposed, animals the

same M type was found to persist for more than 4 weeks.

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The disappearance of M types is even more rapid in animals which were challenged with M 5 weeks after S infection. No M types could be recovered at all from the tissues listed one week after subcutaneous M infection of previously S-infected animals. M types could actually be recovered from all these animals when abscesses near the site of infection were cultured. Whereas intracardial M challenge one week after S infection permitted good recovery of M types for at least two weeks, a similar challenge 5 weeks after the S infection yielded no M recoveries two weeks later. In addition to these recoveries from various tissues, localized abscesses found in a number of animals were cultured. Most of these abscesses yielded M cells even when they came from animals which did not permit recovery of M cells from any of the other tissues. It was possible to isolate M cells from an abscess of an animal (No. 30) four weeks after the subcutaneous M infection. At that time no M cells could be isolated from any other tissue. An animal (No. 60) which was sacrificed 6 weeks after intracardial M infection contained an abscess in the submaxillary lymph node which yielded 5% M and 95% S cells. The following results resembled the Koch phenomenon: When M cells were injected subcutaneously into animals 5 weeks after the S infection, no M cells could be recovered from any of the usual tissues cultured, but for 2 weeks following the M infection enormous numbers of M cells were found in localized abscesses at the site of injection. Two more examples showed the survival of less virulent nonsmooth types in localized lesions four and six weeks after intracardial M infection or 9 and 11 weeks after the S infection. One animal had a lung abscess containing 100% M cells, the other animal had a pericardial abscess containing 98% nonsmooth cells of a rough type plus 2% S cells. (Incidentally, cervical and bronchial lymph nodes, as well as the spleen, appear to be preferential sites for long persistence of S cells.) In reviewing these data we must conclude that, at least as far as the nonsmooth type used in these studies is concerned, there appears to be no opportunity for the less virulent type to establish itself for prolonged periods in what we might call the "open" tissue of the host, even though it was introduced at a time when the selective serum activity of the host had changed. However, less virulent nonsmooth types, either introduced by injection or appearing spontaneously in vivo, seem to be able to persist in localized abscesses. Further studies are required to confirm these isolated observations, but it should be pointed out that such behavior would be in agreement with recent results obtained in test tube experiments. Studies reported by Goodlow, Mika, and Braun revealed that population changes in vitro are controlled by the accumulation of a toxic metabolite in originally smooth cultures. It was demonstrated that Brucella cells produce alanine as an end-product of normal me-

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tabolism. When alanine accumulates to a certain level in the culture it inhibits the growth of smooth types but permits the establishment of spontaneously arising nonsmooth mutants with greater alanine-resistance. These nonsmooth types continue to produce alanine and will be replaced eventually by other, even more alanine-resistant, nonsmooth types, or in some strains, by a highly alanine-resistant smooth type. Preliminary evidence also indicated that the addition of serum from a normal, susceptible host may modify the metabolism so that no alanine is produced and population changes are avoided. While this information was of great value for the understanding and control of variational processes, it also suggested that selective phenomena may be entirely different in vitro and in vivo. The data cited had shown that the accumulation of a specific metabolite plays a major role in the creation of specific environmental conditions which will favor the nonsmooth variants, but this accumulation took place in test tubes, i.e. in a closed system. The animal body probably does not represent such a closed system but rather an open system from which accumulating metabolites are removed continuously. Therefore, it would not be expected that conditions which will affect population changes in vitro will be created to the same extent in most tissues of a host. However, a high metabolite concentration could be expected in a closed environment as represented by abscesses. Localized abscesses may, however, constitute the site at which the nonsmooth types find the environmental conditions that are necessary to endow them with a high selective value. The value and significance of the previously reported serum effect, i.e. the activity of the SS factor in vitro, does not appear to be diminished in view of these results. Time does not permit citation of unreported data which further illustrate how closely the in vitro activity of the SS factor correlates with stages of immunity and susceptibility, but we may add that it is entirely possible that the establishment of less virulent types in vivo may require both the availability of a closed system and an alteration of the SS factor activity of the host. The obvious question whether it might be possible to modify the course of the disease by raising the in vivo level of metabolites involved in favoring the establishment of less virulent types cannot be answered as yet, but it illustrates the new type of approach to economically important problems which can be developed by using variation as a tool in brucellosis research. LA VARIACIóN COMO UN INSTRUMENTO EN LA INVESTIGACION DE LA BRUCELOSIS (Sumario) Las diferencias en los tipos de colonias, que por lo común indican diferencias de la virulencia, facilitan puntos de referencia sencillos para comprobar el destino de las variantes in vivo después de la inoculación simultánea o consecutiva de dos o más tipos variantes. Para comprobar la posibilidad de que las variantes no

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lisas menos virulentas de la Brucella suis persistieran in vivo después de las infecciones prolongadas con microorganismos lisos sumamente virulentos (S), se inocularon cobayos con dosis masivas de una variante mucoidea (M) una semana o cinco semanas después de la infección primaria S, utilizando la via subcutánea o intracardiaca. Los animales fueron sacrificados a la semana, dos semanas, cuatro semanas y seis semanas después de la inoculación de las células M, cultivando una gran variedad de los tejidos. La distribución y persistencia de los tipos M en los distintos tejidos variaron en forma notable según la via de inoculación. Después de la administración subcutánea se observó bloqueo del sistema linfático. Los diversos tejidos revelaron al parecer distinta capacidad para desembarazarse del tipo M. Con excepción de algunos abscesos localizados, los tipos M no persistieron en ningún tejido durante períodos prolongados. Se presentan los resultados de estos experimentos, así como la relación que guardan con los resultados de los estudios in vitro, y se discuten los problemas de la patogenia.

THE BACTERICIDAL ACTION OF HUMAN BLOOD AGAINST BRUCELLA AND ITS SPECIFIC INHIBITION* By WENDELL H. HALL, M.D., PH.D. Laboratory Service of the Veterans Administration Hospital and the Department of Medicine, University of Minnesota Medical School As a part of a study of the pathogenesis of human brucellosis an investigation of various serologic changes was carried out. A review of the literature indicated that, whereas such antibodies as agglutinins and opsonins have been studied thoroughly, little attention has been paid to bactericidal antibodies in human brucellosis. Considerable work has been carried out with the blood of various animals (principally cows). Only the most pertinent observations will be reviewed here. In 1932 Rundberg' published a thesis concerning the bactericidal action of human and animal sera upon Br. abortus in which he concluded that the bactericidal antibody did not increase upon immunization. He

found the antibody was heat-stable, but also decided that it did not require the presence of complement in order to lyse Brucella. The latter observation was contrary to the findings of Mackie and Finkelstein 2 and many subsequent investigators. 3 4, 6 Irwin and Ferguson 6 found that upon recovery from Br. abortus infection the titer$ f bactericidal antibody in the serum of cows increased from 1:25 or less to 1:625 or more. More recently Huddleson 7 noted that the plasma bactericidal activity of Brucella infected cows was less than that of normal cows when tested against Br. abortus. He did not reconcile this observation * Sponsored by the Veterans Administration and published with the approval of the Chief Medical Director. The statements and conclusions published by the author are the result of his own study and do not necessarily reflect the opinion or policy of the Veterans Administration.

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with the work of Irwin and Ferguson. 6 In 1947 the present author 8 reported similar observations with the blood of normal humans and patients having brucellosis. It was pointed out at that time that the reduction in bactericidal activity of the serum of patients infected with Brucella was due to the prozone inhibition phenomenon. The actual titer of the bactericidal antibody had increased during the disease. This observation has subsequently been confirmed in animal experiments by Berman and Irwin 9 and by Huddleson.'° It is the purpose of the present communication to offer an explanation for the mechanism of this prozone of inhibition of bactericidal activity. The methods employed in the studies to be reported here have been given in detail in a thesis submitted to the faculty of the Graduate School of the University of Minnesota and will be published elsewhere. They will therefore be described only briefly here. (1) The "standard" bactericidal test. Serial tenfold dilutions of a suspension of Brucella were added to equal quantities of fresh defibrinated blood, serum or plasma. After 24 hours incubation at 37°C a loopful of each mixture was subcultured upon an agar plate in order to demonstrate the viability of the Brucella. The bacterial inoculum was controlled in each experiment by four plate colony counts. (2) The "dilution" bactericidal test. The "standard" test was altered through the use of serial tenfold dilutions of serum inactivated at 56°C for 30 minutes. After the addition of Brucella, complement was added. Since guinea pig complement was ineffective, either diluted fresh infant's serum or rabbit serum was used as the source of complement. (3) Absorption of Brucella antibodies was carried out with bacterial antigens prepared by the addition of diethyl ether to heavy suspensions of the organisms in physiologic saline solution. After evaporation of the ether, nine volumes of the antigen were added to one volume of the normal or immune serum. After incubation for 2 hours at 37°C and 18 hours at 5°C the mixtures were centrifuged and the supernatant fluid was tested for antibody content and re-absorbed with the same antigens. Serial absorptions were carried out 3 to 4 times in most cases. Observations in normal human subjects.-The blood of well over 100 children and adults who did not have brucellosis was examined for Brucella antibodies including bactericidins. The age of the subjects appeared to influence the presence or absence of bactericidins significantly. Although the seruff of infants 3 months or less in age was strongly bactericidal for Br. abortus, two thirds of the infants between 4 and 16 months of age had little or no bactericidal activity. The serum of all children 18 months of age and older, as well as that of all normal adults, was strongly bactericidal for Br. abortus. The antibody was undoubtedly transferred passively to young infants from their mothers. The pathogenesis of its appearance in older children remains a mystery comparable to that which surrounds the development of iso-hemagglutinins.

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The velocity of the lethal effect of human serum upon Br. abortus was moderately rapid; the majority of bacterial cells were killed within 2 to 4 hours at 370°. The remaining bacteria, however, were killed only at a much slower rate, suggesting that two factors were operating, a fact which was soon confirmed. While fresh normal adult serum was strongly bactericidal for Br. abortus, this property was almost completely lost after heating to 56°C for 30 minutes. However, the addition of diluted fresh serum from an infant or rabbit, which was not bactericidal per se, restored the adult serum to its full activity. Thus, it became apparent that the bactericidal effect of serum depended upon the presence of a heat-stable substance (antibody) and a heat-labile substance (complement). Another factor which governed the bactericidal phenomenon was the variety of Brucella. It was conclusively demonstrated that, whereas Br. abortus was easily killed by normal human serum, Br. suis and Br. melitensis were resistant to its action. This observation was of interest in view of the greater virulence of the latter varieties for man. It was also noted that the temperature of incubation had a marked influence upon the ability of human serum to kill Br. abortus. Both serum and citrated whole blood from normal adults and patients having acute brucellosis, though actively bactericidal for Br. abortus at 37°C, had little lethal effect upon these bacteria at 40C. In fact, we have isolated viable organisms after a small inoculum (5 cells per ml.) of Br. abortus was introduced into citrated blood and stored 125 days at 4°C. This fact is of obvious importance in the operation of blood banks. All donors should be carefully screened in order to rule out brucellosis if transmission by transfusion is to be avoided. The inhibitory effect of low temperatures upon this bactericidal system appeared to be due to the fact that complement failed to unite with the antibody-sensitized bacteria. The principal factors responsible for the bactericidal property of human blood for Br. abortus appeared to reside in the serum or plasma and not in the cells. The latter, by themselves, had little or no such activity. However, whole citrated or defibrinated blood from normal adults and from patients having brucellosis was often more strongly bactericidal for Brucella than plasma or serum. The difference was not well correlated with the opsonic index of the blood, and it may have been due to nonopsonic phagocytosis of the Brucella. Dilution of normal adult serum beyond a certain point removed its bactericidal activity even though the complement was restored. It was established that the maximum titer of the heat-stable bactericidal antibody for Br. abortus ranged from 1:42 to 1:420 in the serum of 6 normal adults. A fact of considerable significance was disclosed by absorbing normal human sera with various bacterial antigens. It was found that the heatstable bactericidal antibody for Br. abortus was absorbed equally well by

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Br. abortus, Br. melitensis and Salmonella oranienburg. Since the latter organism contained no antigen in common with Brucella, it must be concluded that the natural bactericidal antibody is not specific. Observations in patients having brucellosis.-No qualitative difference was disclosed between normal human bactericidal antibody and that found in the serum of patients suffering from brucellosis. It soon became apparent however that, although the serum of patients having acute brucellosis (symptoms for less than 3 months) was strongly bactericidal for Br. abortus, the undiluted serum of patients who had the disease for a long period of time was not bactericidal. These statements are based upon the study of the blood of 119 patients who were judged to have brucellosis. The diagnosis was proved by the isolation of Br. abortus in 38, Br. suis in 1 and Br. melitensis in 3 patients. The most unequivocal data on this point were gathered by analysis of 79 bactericidal tests using the serum of 22 patients having proved brucellosis. Br. abortus was isolated from 20 and Br. melitensis from 2 of the patients. None had prior Brucella vaccine therapy nor skin tests. In those patients whose symptoms had been present for one month or less, the mean bactericidal power of the serum for Br. abortus was high (10 - 6 dilution of Brucella sterilized). At the end of 2 months this figure had declined to 10- 7 , and by the end of 3 months it had reached 10-8. The mean serum bactericidal power did not rise above this figure thereafter though the symptoms of the disease had been present for 2 years or longer. The serum of none of the patients killed a significant number of Br. suis or Br. melitensis at any stage of their disease irrespective of the variety of infecting Brucella. The importance of obtaining serum before Brucella vaccine therapy or skin tests was emphasized by the observation that the ability of the undiluted serum of patients to kill Br. abortus sharply declined shortly after the intradermal injection of several Brucella antigens. Cholera vaccine also caused a similar decline in the bactericidal activity of human serum for Br. abortus, as well as an increase in serum Brucella agglutinins. This was attributed to the fact that Brucella contain an antigen similar to the H antigen of Cholera vibrio. Though the (undiluted) serum of patients having prolonged symptoms of brucellosis often failed to kill Br. abortus, whole defibrinated or citrated blood from the same patients was often strongly bactericidal. The difference in activity was not accounted for by specific opsonins; it was not well correlated with the opsonic index (Huddleson). The blood cells from such patients were not bactericidal per se. The presence of "nonspecific" opsonins might explain the difference. The low bactericidal activity of the (undiluted) serum from patients having chronic brucellosis was regularly increased during therapy with such antibiotics as streptomycin, aureomycin and chloramphenicol. Streptomycin therapy enabled the blood to kill large numbers of Brucella

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of all varieties. Aureomycin had somewhat less effect, and chloramphenicol had the least. The effect of dilution of the serum upon its bactericidal power for Br. abortus was studied by a method allowing restoration of complement activity. When tested by this method, the titer of heat-stable bactericidal antibody was 1:42,000 in 1 patient and 1:420,000 in 2 other patients having acute brucellosis. These patients had been ill for 2 months or less. Repetition of such experiments with serum from patients having chronic brucellosis was of greater interest. While such sera were usually unable to kill Br. abortus before dilution, after dilution to 1:420 or even 1:42,000 they became strongly bactericidal. One such serum manifested bactericidal activity even after dilution to 1:4,200,000. It was clearly evident that the lack of bactericidal activity on the part of such (undiluted) sera was due to the prozone inhibition phenomenon. The mean titer of maximum bactericidal activity against Br. abortus was 1:4,200 in the serum of 15 patients having brucellosis and manifesting such prozones. The average duration of symptoms in these patients was 13 months (range 2 months to 20 years). When non-bactericidal (undiluted) sera from patients having brucellosis were added to an equal quantity of normal human serum, the resulting mixture was not bactericidal for Br. abortus. In other words, prozonal sera contained an inhibitor preventing bactericidal action against Br. abortus by normal bactericidal antibody. There was no evidence that complement activity was interfered with. The titer of this inhibitor was determined in the serum of 15 patients having chronic brucellosis. It ranged from 1:7 to 1:42,000 (mean = 1:420). The titer of the inhibitor in all sera was always less than the titer of maximum bactericidal activity. The inhibitor was heat-stable and was not inactivated in the presence of crystalline human or bovine albumin. Brucella antibodies were absorbed from the serum of several patients having acute brucellosis due to Br. abortus. Bactericidins for Br. abortus were completely removed by one absorption with either Br. abortus, Br. melitensis or S. oranienburg. It was concluded that the bactericidal antibody found in the serum of patients having acute brucellosis was non-specific and did not differ from natural bactericidal antibody. In a similar fashion Brucella antibodies were absorbed from the serum of several patients having chronic brucellosis by repeated exposure to bacterial antigens. After 1, 2 and occasionally even after 3 absorptions with Br. abortus or Br. melitensis these non-bactericidal sera became increasingly bactericidal for Br. abortus though their Brucella agglutinins and opsonins had been completely absorbed. After 4 absorptions the sera lost their bactericidal antibody. The phenomenon was not due to serum dilution. Serial absorption of these sera with S. oranienburg

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did not remove Brucella agglutinins nor opsonins nor did it cause the sera to become bactericidal for Br. abortus. Addition of absorbed and unabsorbed sera from patients having chronic brucellosis to normal human serum indicated that absorption with Br. abortus and Br. melitensis antigens removed the bactericidal antibody inhibitor. Absorption with S. oranienburg did not do so. The bactericidal antibody inhibitor in such sera therefore appeared to be specific for Brucella. Confirmation of this was given by the fact that (undiluted) serum from patients having chronic brucellosis, though not bactericidal for Br. abortus, was strongly bactericidal for S. oranienburg. The facts appear to justify the conclusion that the inhibition prozone, which appeared in the serum of patients having brucellosis for several months and rendered their (undiluted) serum non-bactericidal for Br. abortus, was not due to the production of an excess of (non-specific) bactericidal antibody but was the consequence of the stimulation of a specific inhibitor which prevented the lethal activity of that antibody (plus complement) upon Br. abortus. Sera containing this inhibitor became bactericidal for Brucella only after absorption with Brucella or after the addition of antibiotics capable of killing Brucella. No blood product was found which was capable of overcoming the inhibitor. REFERENCES (1) Rundberg, G.: Eine Experimentelle Studie iber die Immunitatsverhaltnisse bei Bang's Abortus Bazillus. Thesis, Svenska lak-sallsk. forhandl. 58: 77-152, 1932. (2) Mackie, T. J., and Finkelstein, M. H.: Natural bactericidal antibodies; Observations on the bactericidal mechanisms of normal serum. Jour. Hyg. 31: 35, 1931. (3) Irwin, M. R., Beach, B. A.; and Bell, F. N.: Studies on the bactericidal action of bovine whole blood and serum towards Brucella abortus and Brucella suis. Jour. Inf. Dis. 58: 15-22, 1936. (4) Shrigley, E. W., and Irwin, M. R.: On the differences in activity of serum complement from various animal species. Jour. Immunol. 32: 281-290, 1937. (5) Irwin, M. R., and Beach, B.A.: Differential bactericidal activity of bovine serum toward strains of Brucella abortus of high and low virulence. Jour. Agr. Res. 72 (No. 2): 83-91 Jan. 15, 1946. (6) Irwin, M. R., and Ferguson, L. C.: Increase of bactericidins in the serum of cattle following recovery from infection with Brucella abortus. Proc. Soc. Exp. Biol. and Med. 38: 451-452, 1938. (7) Huddleson, I. F., Wood, E. E., Cressman, A. R. and Bennett, G. R.: The bactericidal action of bovine blood for Brucella and its possible significance. Jour. Bact. 50: 261-277, Sept. 1945. (8) Hall, W. H. and Spink, W. W.: Studies of immunity in brucellosis: The bactericidal action of human blood against Brucella, Proc. Am. Soc. Clin. Invest., Atlantic City, May 1947. Abstract, Jour. Clin. Invest. 26: 1183, Nov. 1947. (9) Berman, D. T., and Irwin, M. R.: Further studies of the bactericidal action of

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bovine serum for Brucella. Proc. 48th Meeting Soc. Am. Bact., Minneapolis, May, 1948, pp. 38-39. (10) Huddleson, I. F.: The potentiating action of sulfonamides on the Brucella antibody-complement system. Am. Jour. Vet. Res. 9: 277-285, 1948.

ACCIÓN BACTERICIDA DE LA SANGRE HUMANA CONTRA LA BRUCELA Y SU INHIBICIÓN ESPECIFfCA (Sumario) Desde hace cerca de veinte años se ha reconocido que la sangre del hombre y animales normales es capaz de destruir las brucelas, especialmente la Brucella abortus. Los experimentos realizados por el autor han demostrado que los anticuerpos bactericidas humanos son termoestables, pero que requieren la presencia de un complemento (de hombre o de conejo) para la máxima actividad. Asi-

mismo se demostró que eran activos en la ausencia de leucocitos, que eran activamente bactericidas a 37° C pero no a 4° C, y que eran absorbidos tan ávidamente

por la Salmonella oranienburg como por la brucela, y que por lo tanto eran noespecíficos. El titulo de anticuerpos bactericidas en sueros de personas normales varía entre 1:42 y 1:420. El suero de enfermos de brucelosis aguda era fuertemente bactericida para la Br. abortus. El titulo de los anticuerpos bactericidas de tales sueros variaba entre 1:42,000 y 1:420,000. Los anticuerpos bactericidas inmunes no se diferenciaban cualitativamente de los anticuerpos bactericidas normales, y eran también noespecíficos. El suero no diluido de la mayor parte de los enfermos, que han padecido de brucelosis durante dos o más meses, no pudo destruir la Br. abortus en número apreciable. Esta falta de actividad bactericida se debe al fenómeno de inhibición de prozona. Dichos sueros resultaron bactericidas para la Br. abortus al diluirse de 1:420 a 1:42,000 y activarse con complemento. Las mezclas de dichos sueros con suero normal no resultaron bactericidas a menos que se diluyeran mucho los primeros. El suero no bactericida, sin diluir, contenía un anticuerpo inhibidor bactericida. Este inhibidor fué la causa del fenómeno de prozona, y su titulo era menor que el de los anticuerpos bactericidas. No obstante, dicho inhibidor era especifico para la brucela y se combina a está con mayor avidez que los cuerpos bactericidas. La actividad bactericida de dicho inmunisuero podía aumentarse

con la adición de antibióticos como estreptomicina, aureomicina, y cloranfenicol.

CLINICAL COURSE OF HUMAN BRUCELLOSIS IN MINNESOTA By WESLEY W. SPINK, M.D. AND ROBERT L. MAGOFFIN, M.D. Department of Medicine, University of Minnesota Hospitals and Medical School Studies on global epidemiology in recent years have emphasized that the clinical manifestations of an infectious disease may vary from one geographical area to another. Brucellosis is a disease that reflects a variable clinical course, even when studied in one locality. A classification of the signs, symptoms and duration of brucellosis based upon observations in a limited geographical area may provide misleading information when applied to other regions. For example, the disease varies according to the species of Brucella that is prevalent in a given area. The over all clinical picture of the disease due to Brucella melitensis differs from that caused by Brucella abortus. There is also a variation in the animal reservoir of the disease throughout the world, embracing such factors as the species of animal harboring the infection, and the number of animals infected. Local habits and customs will greatly influence the opportunity of human exposure to the disease. The underlying state of health of a regional population will have an important bearing on the severity and duration of the illness. In view of these variables, it is desirable to have reliable data on the natural course of the human disease in a given area to serve as a sound basis for evaluating therapeutic measures. For these reasons, it is essential to carry out detailed studies of human brucellosis as it exists in different localities. As a contribution to this effort, this report is based upon personal observations made on a group of patients in Minnesota during the years of 1937 to 1950. ECOLOGY OF BRUCELLOSIS IN MINNESOTA

Minnesota possesses a large rural population engaged in dairy farming and the raising of hogs. There is an extensive reservoir of brucellosis in the dairy herds, approximately 20% of the herds showing one or more reactors for Bang's disease. There are several large meat packing plants in the state, and thousands of animals are brought in from neighboring states for processing. In general, the people are an economically independent, hardy pioneer stock of Nordic ancestry and in an excellent state of nutrition. Animal and human brucellosis in Minnesota is primarily a disease due to Br. abortus, though hogs in this area serve as a reservoir for both Brucella suis and Br. melitensis. Several factors have permitted a comprehensive study of the human 94

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disease as it exists in this state. The population is relatively stable. The referring physicians have been most cooperative, and the administrators in the packing plants have been very helpful in our investigations. It should be emphasized that the University Hospital is a state institution, receiving patients predominantly from small towns and rural areas. Since Minnesota embraces a recognized endemic area of brucellosis, the hospital staff has been alerted for many years to the presence of the disease. Adjacent to the University Hospitals are the Laboratories of the Minnesota State Department of Health with well trained personnel and available diagnostic techniques. Diagnostic services are not only available without charge to physicians by the Minnesota Health Department but the Department has also carried on a consistent campaign for years urging physicians to utilize these services, particularly cultural techniques. As a result, physicians throughout the state have been on the alert for the disease and have channelled both questionable and problem cases of brucellosis into the hospital. It is significant that although several hundred patients have been examined for brucellosis each year, the diagnosis of active disease has been established in only 186 patients between 1937 and 1950. This number is in contrast to the larger numbers of cases reported by other investigators in the United States. This discrepancy reflects the difference in criteria utilized for the diagnosis of active brucellosis, a feature which will be discussed shortly. COMPOSITION OF CLINICAL MATERIAL

Of the 186 cases, 101 were proved bacteriologically (Table 1). Only 18 were females and the remaining 168 were males. This preponderance of males over females is due in part to the inclusion of a large number of employees of packing plants among the cases. The over all sex distribution for all reported cases in the State of Minnesota is 78.5% males and 21.5% females. The age distribution of the University Hospital cases is detailed in Table 2. The preponderance of cases was in males in the age group of 20 to 45 years, which reflects the high proportion of cases having an occupational source of infection among farmers and packing plant employees. While the University Hospital serves primarily the rural population in the state, only 30% of the cases originated in rural areas. The large number of urban cases is due again to the preponderance of packing plant employees. The occupation and residence of all the cases are summarized in Table 3. It should be stressed that the proportion of cases with severe illness and complications was greater than usually encountered, because the severity of the disease prompted the physicians to seek hospitalization for the patients in this category.

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TABLE 1.-Cases of brucellosis observed. University of Minnesota Hospitals and

Clinic, 1937-1950 Total cases diagnosed ........................................... Brucella isolated ............... ........ .. .............. Br. abortus.............................................. 89 Br. suis .......... ........................... 7 Br. melitensis ........................................... 5 TABLIE

186 101

2.-Age and sex distribution. 186 cases of brucellosis Males

Females

0-4 5-9 10-19

2 2 9

0 O 2

20-29

58

3

30-39

61

4

40-49 50-59

21

2 5

23l

60-69

3

2

5

Totals

168

18

186

Age Group

12

Both Sexes

Per Cent

8.1

61}

67.7 21.5

2.7

TABLE 3.-Occupation and residence. 182 cases of brucellosis Rural

Farmers and farm laborers....

Urban

44

Farm wives ....................

8

Farm children .................

2

Packing plant and stockyard workers ....................... Laboratory personnel ........... Veterinarians .................. Housewives and children ........ Miscellaneous trades ............

Total ......................

54

Total .........................

DEFINITION OF "ACUTE"

AND "CHRONIC"

88 6 2 12 20 128

BRUCELLOSIS

Because of the variability in the manifestations of brucellosis, it is difficult to define the disease in terms of "acute" brucellosis and "chronic" brucellosis. Since these terms have been used with different and conflicting interpretations by various workers, it is not easy to employ them without some misunderstanding. Therefore, we should like to make it clear that we are using the terms "acute" and "chronic" primarily with reference to the duration of the illness. Thus "acute" brucellosis signifies an illness of short duration. In addition, according

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to common usage, it also implies a degree of severity. A "chronic" illness means one of long duration, and as a corollary, an illness usually not manifested by severe symptoms. But brucellosis may be manifested by a brief and mild illness, and a long, protracted illness may have severe and debilitating complications. In this report, however, the terms "acute" and "chronic" will apply principally to the duration of the illness, regardless of the severity. As in previous statements, we will regard brucellosis as being acute, only if the illness has endured for 3 months or less. We have previously considered a case chronic, if the illness extended beyond 3 months. However, in the light of an extended follow-up study of the disease, we now reserve the term "chronic" for those cases in which illness endured for at least 1 year or longer. This is more in accordance with the frequent use of the term "chronic brucellosis" in the literature to designate an illness of several years' duration. For an illness of intermediate duration of 3 to 12 months, we will employ the term "subacute." MANIFESTATIONS OF EARLY STAGES OF BRUCELLOSIS

In order to formulate as accurately as possible the clinical manifestations and course of the disease as it occurs naturally with little or no alteration by effective therapy, we have made a careful analysis of 68 patients observed for the most part before the advent of the antibiotics. None of these patients received aureomycin, chloramphenicol or the combination of streptomycin and sulfadiazine. The majority of these patients did receive one of the sulfonamides at some time during their illness and six patients received a short course of streptomycin. A few patients also received vaccine. About one-fourth of this group received no chemotherapy or vaccine. Though it is possible that the illness in some of these patients was favorably influenced by the therapy received, it is now generally recognized that the sulfonamides and streptomycin, when used alone, do not consistently alter the pattern of disease. These 68 patients have been included in the "untreated" group. Brucella were isolated from 44 of the 68 patients. The species of Brucella included 40 strains of Br. abortus, 2 of Br. suis, and 2 of Br. melitensis. Three of the patients died within the first year of the disease of subacute bacterial endocarditis due to Br. abortus, leaving 65 cases for a long follow-up study. All of the patients have been followed for a minimum of one year and the majority for over three years, while some have been seen intermittently for 7 to 13 years. The average follow-up period has been 4.5 years. It should be emphasized again that many of the patients included in this study were seriously ill, and there were several individuals with painful and disabling complications. The incubation period of brucellosis in these patients, when it could be determined, was found to vary from a few days to several months.

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The onset varied widely. Some patients experienced a gradual onset with a sense of general malaise and a mild indisposition to prolonged physical and mental activity. Fever was detected upon examination, but often the patients did not realize that they had an elevated temperature. On the other hand, in many of these patients after a prodromal period of a day or two, the onset was very abrupt and severe with the appearance of chills, fever, sweats and headache. The most constant symptoms elicited were weakness and a tendency to fatigue more readily than usual. Other common manifestations were headache, a feeling of chilliness or the presence of rigors, nocturnal sweating, generalized aches and nervousness. The most constant objective physical finding was the presence of fever. A tendency to "nervousness" associated with tremors of the hands was frequently observed. The palms and soles were usually moist and mottled in appearance. A majority of the patients demonstrated enlarged cervical lymph nodes, and less commonly, axillary and epitrochlear lymphadenopathy. Splenomegaly was demonstrable in about one-third of all the cases, but usually was associated with the more severely ill patients. The laboratory findings during this early phase of the illness were unequivocal. In 44 patients the organism was isolated. Agglutinins were demonstrable in all cases in a titer of 1:80 or higher, though in one or two instances the initial test was negative because the patients were seen so very early in their illness. The most reliable laboratory procedure for screening the suspected cases of brucellosis was the agglutination test, utilizing the tube-dilution method with an antigen of Br. abortus obtained from the Bureau of Animal Industry of the United States Department of Agriculture. Misleading information has found its way into the literature on the agglutination reaction in brucellosis which has been based on the use of unreliable antigens, and the employment of rapid slide techniques of doubtful accuracy. One unsubstantiated and highly misleading statement repeated over and over is that "organisms are frequently isolated from patients who have a negative agglutination reaction." All too frequently the studies cited to support this assertion make no mention of the type of agglutination test employed or the antigen used. Convincing evidence supporting such a statement in a significant number of cases in which reliable techniques were used is lacking. But there is considerable available evidence that if a reliable antigen is used with the tube-dilution technique, one rarely will isolate Brucella from patients with absent agglutinins, and only infrequently from patients having a low titer of agglutinins. The Laboratories of the Minnesota Department of Health routinely culture approximately 800 specimens of citrated whole blood submitted annually from suspected cases of brucellosis (1). During the four year period of 1945-1949, approximately 3200 samples were cultured, from which 350

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isolations of Brucella were made. Only two of the bloods from which Brucella were cultured failed to show the presence of agglutinins. All the other bloods from which the organisms were isolated revealed a titer of 1 to 80 or above. None had a maximum titer of only 1 to 40.. It is significant that the majority of the 3200 bloods studied had either absent agglutinins, or a low titer of agglutinins, so that ample opportunity was afforded for the discovery of organisms from patients of this type. Similar evidence has been accumulated by Damon (2) in Indiana where apparently a less sensitive antigen is employed for the agglutination test. Clots from all bloods submitted by physicians for Brucella agglutination test were cultured. In a study of 7906 clotted specimens, 23 isolations were made from the 513 specimens which revealed agglutinins in a titer of 1:40 or above. Of the remaining 7393 clots which showed no agglutinins present in a titer of 1 to 40 or above, only two isolations were made. In our experience, extensive cultural studies have been made of the blood, bone marrow, liver biopsies, lymph nodes and spleen, and no isolations of Brucella have been accomplished except in those patients having agglutinins. When a correlation is made between the titer of agglutinins and positive blood culture, over 90% of our patients have had a titer of 1 to 160 or above. Since many normal and healthy individuals in Minnesota have a low titer of Brucella agglutinins, a titer of less than 1:160 frequently has doubtful clinical significance (3). It has been our practice to obtain at least three specimens of blood for culture on three different days in suspected cases of brucellosis. Brucella have been cultured from approximately 50% of our cases of brucellosis. In three instances, organisms have been recovered from aspirated sternal bone marrow, when simultaneous cultures of venous blood have remained sterile. Other helpful aids in screening the early case of brucellosis have been the total leukocyte and differential blood counts. Usually the total count has been normal or reduced, and has rarely exceeded 10,000 cells per cu mm. A relative lymphocitosis was usually present. It is of interest that since individuals entering the University Hospitals come from an endemic area of brucellosis, approximately 20% of all adult patients were found to exhibit a positive reaction following the intradermal injection of Brucella antigen. Neither the intradermal test nor the opsonocytophagic reaction has been found to be a reliable diagnostic aid, and these tests are not used by our group. The clinical and laboratory data which have been described to this point constitute the characteristic features of the initial or early phase of illness as seen in our patients. In a number of cases the early phase had completely or almost completely subsided before the patient came under our care.

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This initial period of the illness which is referred to by some authors as the acute phase, represents the period of relatively sustained symptoms until a temporary remission or recovery ensues. In the 65 untreated cases previously referred to, the initial attack lasted from three to four weeks up to eight or nine months. In a substantial majority, however, the early phase of the illness lasted from one to three months. SUBSEQUENT COURSE OF BRUCELLOSIS

Little controversy exists over the clinical manifestations which have been described for the early phase of brucellosis, but general agreement is lacking as to the total duration of the illness and ultimate outcome of the patients. In attempting to determine when the patient had fully recovered from the disease, we have depended upon objective and subjective data. A patient was not considered as being restored to normal health unless he said so. He was still considered as being ill if he complained of symptoms, even though no objective findings were demonstrable. Acute brucellosis.-Of the 65 living patients, 10 or 16%, recovered completely within three months, and have remained well for one to seven years. Subacute brucellosis.-There were 25 cases in which the illness endured for three to twelve months. This group comprised 39% of the cases. The disease persisted in these cases because of first, the initial phase frequently associated with persistent bacteremia, endured beyond three months; second, the patients experienced one or more relapses after periods of relative well-being lasting for several weeks or months; and third, there was a protracted period of convalescence without objective evidence of active disease being present. A demonstrable complication was responsible for prolongation of the illness in only one of the patients, and this one patient had encephalo-meningitis. Thus, thirty-five of the total number of patients, or 54% having subacute or acute illness had recovered completely within one year (Table 4). Chronic brucellosis.-This group included 30 cases or 46% of the total, who were ill for one year or longer. These patients can be divide into one of three groups. First, were those individuals having evidence of continued active infection, which was manifested by recurring relapses, but without evidence of localization. Both laboratory and clinical evidence of active disease was present, such as was found in the initial phase of the illness. There were only five cases in this group. All of these five patients eventually recovered, and have remained well for one to three years. The second group of cases with an illness lasting for more than a year included 12 individuals who had localizing evidence of the disease associated in some cases with frequent recurrent relapses of systematic

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illness. The localized complications observed in these 12 patients were: five of spondylitis; two of meningo-encephalitis; one of meningo-encephalitis and spondylitis; one of cholecystitis; one of pericholecystic abscess; one of bone lesion of right hip; and one of radiculo-neuritis. Not included in this group of complications are the three patients who died of subacute bacterial endocarditis. The third group of chronic cases comprised 13 individuals in whom all objective evidence of active illness had subsided, but who still complained of ill health. No significant evidence of disability was present. These 13 cases represented one-half of the patients who had been ill for more than a year, or 24% of all the cases. It is this group of patients that represents the difficult problem of chronic brucellosis today. Fever TABLE 4.-Duration of symptoms in 65 cases untreated brucellosis. (1937-1950) 37 Br. abortus, 2 Br. suis, and 2 Br. melitensi No. Cases

Per cent

Acute ....................

10

15

Subacute ................ Chronic .................

25 30

39 46

was not demonstrated in any of these patients after 12 to 18 months of illness. Repeated cultures of blood remained sterile, and the agglutinin titer had gradually diminished. It should be emphasized that in all the cases of chronic brucellosis it is most unusual in our experience to isolate Brucella from cultures of blood after the illness has endured for one year. Because these 14 patients presented a difficult therapeutic problem they have been studied very thoroughly, and several features of their illness stand out. In the first place, several of these patients had underlying emotional difficulties. Two of the patients had been discharged from the armed services during World War II because of inability to adapt prior to the onset of brucellosis. Two others were alcoholics of long standing. Only three of the patients were females, and each of them had had difficulty in meeting their daily problems. One cannot deny that this group of patients were ill. It is well known that brucellosis does have a serious impact upon the nervous system, and that underlying personality disorders and conflicts may be intensified by the disease, but after fever and other objective evidence of active infection have subsided, it is our view that nothing is to be gained by continuing to direct therapy against presumptive infection that cannot be demonstrated. On the contrary, much harm may be done by encouraging the patient to feel that he had a chronic illness for which nothing can be done and, therefore, he need not make any further attempt to cope with the exigencies of earning a living. He is sick; he is blameless for his failures. Another

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factor that has entered into the prolonged illness of some of these patients, has been financial compensation for disability under the Workmen's Compensation Act. Brucellosis is a compensable disease in the meat packing plants of Minnesota. A careful study of some of these patients has convinced us and our associates that the convalescence has been prolonged because of compensation received or because of unsettled claims for compensation. And finally, there is a very small group of chronically ill patients who appear to have been emotionally stable individuals prior to illness, yet who have had protracted symptoms. In the absence of objective evidence of localization of the disease of continued active infection, it is our conclusion that this type of illness again represents a post-infectious syndrome, which is more directly attributable to a reaction pattern set up during the course of the active disease. TABLE 5.-Duration of disability in 65 cases untreated brucellosis Months

No. Cases

Per cent

0-3 3-6 6-9 9-12 12-24

23 14 13 7 8

35 22 20 11 12

Of the 30 chronically ill patients, 18 have been observed to recover completely, and they have remained well for one to seven years. Six of these 18 patients had an illness persisting for two to five years. The remainder of these 18 patients were recovered by the end of two years. In 12 of these 30 chronic cases, the patients still had complaints at the time of the last follow-up so that the ultimate duration of illness could not be determined. Thus, in our series of 65 patients, over 80% are known to have recovered completely and have remained well for at least a year when last seen, and 31 cases are known to have remained well for three or more years. In our experience then, the great majority of patients have had a self limiting illness with subsequent recovery. PERIOD OF DISABILITY CAUSED BY BRUCELLOSIS

In the foregoing discussion, we have been concerned with the total duration of time in which the patients had symptoms. An analysis of the total period of disability has also been made. A patient was considered disabled if he could not carry on with the routine daily work as he could before he became ill. This included those individuals who were only partially disabled. There is a marked contrast between the duration of illness when based on subjective complaints, as compared

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to disability caused by the disease (Table 5). While 30 of the 65 cases, or 46%, complained of symptoms beyond one year, only 8 cases, or 12%, were disabled beyond that period. In fact, 37 of the cases, or 57%, were able to return to their usual routine at the end of 6 months, and 77% at the end of nine months. Factors prolonging disability were a relapsing illness and the presence of complications. In addition, there were those individuals who could not endure the day's work, although no organic cause for their disability could be ascertained. REFERENCES (1) Bauer, H.: Personal communication. (2) Damon, S. R. and Albright, K.: The isolation of Brucella from blood clots. Brucellosis. Am. Assoc. Adv. Sc., p. 122, Washington, D. C., 1950. (3) Spink, W. W., and Anderson, D.: Brucella studies on bank blood in a general hospital. Jour. Lab. and Clin. Med., 35: 440, 1950. LA EVOLUCION CLÍNICA DE LA BRUCELOSIS HUMANA EN MINNESOTA (Sumario) El estudio clínico de 186 casos de brucelosis humana observados en Minnesota durante un periodo de 13 años, ha recalcado el hecho de que la enfermedad se limita a si misma. La clasificación de la brucelosis clínica propuesta se basa en la duración de la enfermedad desde la iniciación de los síntomas. Los casos en que la enfermedad ha durado menos de tres meses se clasifican como agudos, de tres meses hasta un año como subagudos, y cuando la enfermedad persiste durante más de un año se clasifica como brucelosis crónica. En Minnesota la inmensa mayoría de los casos de brucelosis se deben a la Br. abortus. En un grupo de 65 enfermos que no recibieron los medicamentos considerados eficaces en la brucelosis, se observó otro rasgo notable en la evolución clínica. El grupo comprendió 24 (39%), cuyos síntomas persistieron durante un año después de la iniciación de la enfermedad, pero sólo 8 (13%), no pudieron reanudar sus labores diarias al cabo de un año. El problema básico de la brucelosis crónica afecta hoy día como a 20% de todos los casos; este es un grupo de enfermos relativamente pequeño, pero importante. Estos son los sujetos con síntomas subjetivos, pero que revelan pocos signos clínicos convincentes o pruebas de laboratorio indicativas de enfermedad activa.

THE NATURAL COURSE OF BOVINE BRUCELLOSIS* By D. T. BERMAN Department of Veterinary Science, Wisconsin Agricultural Experiment Station, University of Wisconsin Bovine brucellosis (Bang's disease, contagious abortion) is an infectious disease, due in the majority of cases to Brucella abortus, and characterized by inflammatory changes of the endometrium and placenta, and often resulting in the premature expulsion of the fetus. The dramatic nature of the cardinal sign of abortion, and the lack of other manifest signs of disease has led to the development of a concept of bovine brucellosis as a placental disease, ignoring the often presented evidence for its generalized nature. All too often efforts at control are based upon this concept rather than upon the complete picture of the infection (see Elberg and Silverman).' In this paper an attempt will be made to describe the events in the natural course of bovine brucellosis, emphasizing the generalized nature of the infection, as demonstrated by many investigators. Because of time limitation the discussion of certain aspects of the disease, which do not bear directly upon this central theme and which have been reviewed adequately by others, will be curtailed. ROUTES OF INVASION

The earliest demonstrations of the infectious nature of bovine contagious abortion were accomplished by reproducing the typical syndromel2 3' 4 with intra-vaginal inoculations of pregnant cows with portions of fetal membranes, or uterine exudate discharged in abortions. Bang 4 with Stribolt in 1897 proved the etiological significance of the organism now known as Brucella abortus by producing typical abortions after intra-vaginal instillation of pure cultures isolated from the uterine exudate of an aborting cow. In this work epizootological evidence incriminating the bull as a source of infection for cows was also presented. Although it was shown' that bulls may develop genital infection, with shedding of organisms in the semen, after instillation of cultures into the prepuce or after intravenous inoculation, 6 the evidence for transmission to cows by natural service by such bulls has been inconclusive. 7 More recently Bendixen8 and Seit 9 have reported the spread of infection to previously clean herds by the use of artificial insemination of semen from infected bulls. Experimental confirmation has been obtained by Manthei et al.0 Agglutinins in seminal plasma have been described by Christensen."l * Published with the approval of the director of the station. 104

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While it is not improbable that contamination of the vagina with infected bedding, or by the use of infected semen, may be responsible for naturally acquired infection, it appears that ingestion is the most important means by which the organisms gain access to the body. Bang' 2 and others have shown that it is possible to reproduce the disease in pregnant heifers and cows by feeding infectious material or cultures, and it has been suggested that infection probably occurs through contaminated feed. The large numbers of organisms present in the uterine discharges of infected animals, and their resistance to most natural conditions with the exception of direct sunlight,' 3 14 offer sufficient opportunity for the contamination of pastures and feed. Experimentally it has been found possible to infect cattle through the abraded, or even intact skin.?5 Infections may also be produced by instillation of infective material into the conjunctival sac.l 6 The latter method has been employed to a great extent in research on bovine brucellosis because of its simulation of natural infection through a mucous membrane, and because it is possible to administer known numbers of organisms to all experimental animals. Of interest for further investigation are the reports of infection by arthropod vectors,' 7 by intrammary inoculation' 8 and by the respiratory route.l1 INFECTIVE NUMBER OF ORGANISMS FOR NORMAL CATTLE

The results of a number of trials with conjunctival exposure of normal cattle with varying numbers of either of two strains of virulent Br. abortus have been reviewed recently.2 o 21 In the series with one strain (528), exposure to approximately 15 million organisms produced results which were not significantly different from those obtained when the dose was 150 million or 1 billion organisms (55 abortions in 61 animalswith an infection rate somewhat higher). When the dosage was reduced to approximately 1 million organisms a significant reduction in the abortion rate was obtained (8 abortions in 19 animals) while the reduction in infection rate was not so great (13 infected of 19 animals). Less than a million organisms caused 3 abortions in 9 animals, with 5 animals infected, and somewhat more than a thousand organisms caused no abortions in 9 individuals, 2 of which became infected. The titration of the second strain (2308) showed no difference in abortion or infection rates in animals given 26 or 15 million organisms (29 abortions in 30 animals, all of which were infected). With 740 thousand organisms 7 of 9 animals aborted, and 8 were infected. A dose of 370 thousand produced abortions in 5 of 9 animals, and infections in 7 of these. From these results it would appear that there is a real difference in the virulence for cattle of these two strains of Br. abortus, and this

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impression is confirmed by the results of titrations in guinea pigs. 2 ' Further trials using a variety of strains, and larger numbers of animals will be necessary to arrive at numbers for general application. In investigational work the number of organisms used must be conditioned by the questions which the animals are being used to answer. SUSCEPTIBILITY OF NORMAL CATTLE TO INFECTION

A summary20 of the results of exposing normal, pregnant animals by the conjunctiva to at least a million organisms (of a variety of strains)usually many times this number-showed that in 36 trials there had been 303 abortions among a total of 353 animals. Analysis of the results indicated that the variations of the percentages of abortions around the average (85.8%) would occur infrequently by chance alone, meaning that the underlying probability of abortion could not be considered uniform from trial to trial. Among the variables encountered were differences in the stage of pregnancy at which animals were challenged, and the number of organisms of many different strains used. Sexually mature, unbred heifers have been considered relatively resistant to infection by Br. abortus. This opinion is supported in part by the small percentage of reactors in this classification observed in official testing programs, and in part by the study of Edgington and Donham, 22 in which 15 heifers exposed before breeding failed to abort during the subsequent pregnancy, and apparently failed to become infected. This apparent resistance, manifested particularly by failure to abort, has been the goal of prophylactic vaccination with living cultures. While the resistance engendered may protect against abortion, the vaccinated animals may become infected with the vaccine strain, and may shed the organisms in the milk, and/or uterus. Definitive evidence of the susceptibility of unbred animals has come from a number of workers, and here it is necessary to differentiate between the infectious disease, characterized by abortions, and infection per se. Bang et al.23 produced infection in 8 unbred heifers by feeding cultures. The infection was manifested by the dissemination of the organisms through the blood stream to various body lymph nodes of all, the liver of one, the spleens of 4, and the udders of 4. The nongravid uteri were not invaded. Unfortunately agglutination test results are not given in this paper. Manthei 21 has also reported an outbreak of brucellosis in a group of open heifers naturally exposed. Six of 24 animals developed agglutinins, Brucella were isolated from the udder secretions of 5, and from the uterine discharges of one of the 3 animals which had normal calves after the subsequent breeding. Approximately 3 years after exposure organisms were recovered from the tissues of 5 of the animals. Manthei also stated that a number of non-pregnant heifers have been experimentally exposed with resultant bacteremia and udder infection. Similar results4 have been obtained in our laboratory.

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The resistance of sexually immature animals is apparently of a different order. Calves infected in utero or from their surroundings after birth remain infected for a short time only unless they are kept in contact with infectious material, or fed infected milk. Within several weeks from the time calves are removed from the presence of the organisms they are usually free from infection, and develop into uninfected, susceptible cattle. While Carpenter 2 5 has shown that organisms fed young calves invade the lymph nodes of the head and digestive tract, and in a few cases may be found in the spleen, or liver, Brucella are eliminated within 4 to 5 weeks after the cessation of feeding. An attempt 2 6 to overcome the resistance of young animals by the administration of estrogenic hormones during exposure was unsuccessful. Further investigations of this type using more animals, and various hormone preparations would seem desirable. DISTRIBUTION OF BRUCELLA IN THE BODY OF THE HOST

A review of the studies of the distribution of the organism in the host emphasizes the significance of extra-genital sites of infection. Schroeder and Cotton2 7 tested by guinea pig inoculation the tissue of a group of naturally infected cows for the presence of Br. abortus. The tissues examined included the blood, spleen, liver, kidney, brain, ovary, uterus, milk, synovial fluid, and lymph nodes from all parts of the body. With the exception of the udders and supramammary lymph nodes, the organism was isolated in only one instance-from a pelvic lymph node. Birch and Gilman28 recovered Br. abortus from the supramammary lymph nodes of 7 of 17 cows, the lymph nodes of the head of 2 of 19, the bronchial and mediastinal glands of one animal, and the spleen of one. The mammary gland and inguinal lymph nodes were found to be infected in each of 3 cows examined by Roots and Ridala.29 The spleens of 2 animals yielded Br. abortus, as did the fluid of a carpal hygroma. Krüger30 isolated Br. abortus from the muscular pillars of the diaphragm of 2 animals which failed to react to the agglutination test. One of the most definitive studies was that of Bang, Bendixen and Oerskov2 3 who studied the distribution of Br. abortus in the bodies of 8 non-pregnant heifers fed large numbers of organisms, and killed at intervals from sixth to the ninetieth day after exposure. They found that in early stages of infection the organism was located principally in the lymph nodes of the head and digestive tract. After a month the bacteria were distributed throughout the reticulo-endothelial system. In animals killed later the organisms were found primarily in the mammary glands and supramammary lymph nodes. Doyle3 ' made a detailed examination for the presence of Brucella in the tissues of 32 naturally infected cows, and made recoveries from one or more sites in 26 animnals. Of the 13 tissues from which isolations

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were made 8 were lymph nodes, and Doyle concluded that the principal site of Br. abortus in the carrier cow, in addition to the udder, appears to be the lymphoid tissue. In an unpublished study3 2 Br. abortus was isolated from the supramammary lymph nodes of 13 of 33 reactor cows eliminated from herds in the test and slaughter control program. Isolations from various lymph nodes including those of the head and thorax, pelvis, and digestive tract have been made from 5 animals examined in this laboratory. The livers of 3 of these animals and the spleens of 2 were also infected. Manthei and Carter33 have tabulated the results of a study of the sites of localization among 15 experimentally infected cows, on which complete hemocultures were also performed. Isolations were made only from the reproductive tract, mammary gland and various lymph nodes. Infection of the supramammary lymph nodes was always associated with mammary infection. Brucella infection of the blood stream of infected cattle was demonstrated by Soule 34 and Fitch et al., 36 and a comprehensive study has been made recently by Manthei and Carter.3 3 The latter authors concluded from their results that the incidence and persistence of bacteremia may be used as criteria for the estimation of degrees of resistance and susceptibility of cows exposed to virulent Brucella. Bacteremia was not demonstrable in vaccinated animals which resisted the induced infection, while in unvaccinated susceptible animals there was a high incidence and persistence of blood stream infection. Strain 19 vaccinated animals not fully protected but with varying degrees of resistance showed a transient bacteremia. Persistent bacteremia was more frequently associated with abortions and weak calves at parturition than with normal calvings. Bacteremia persisted for 5 months in a high proportion of experimentally infected unvaccinated cattle, after which the incidence declined. Approximately 5 to 10% of the animals continued to exhibit intermittent bacteremia for 2 years. Manthei and Carter 33 have reviewed the studies of uterine infection in cattle. Of particular interest is the work of Fitch et al.34 in which Br. abortus was isolated from 24 of 66 uteri showing no evidence of recent parturition, and from 23 of 27 where there were signs of recent parturition. The organism was found in one non-gravid uterus 195 days after parturition. Similarly Birch and Gilman 28 have reported finding Br. abortus in the genital mucosa of 4 artificially infected animals 15, 13, 8 and 7 months respectively following parturition. These authors considered that true localization had occurred. In the study of Fitch et al.36 41 gravid uteri were examined, and Br. abortus was not isolated from any in which pregnancy of less than

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four and a half months existed. The organism was isolated from 45% of those more than four and a half months in gestation. Gwatkin 3 7 and Fitch et al.38 isolated Brucella from the uterine material of approximately 20% of reactors at the time of full term parturitions. From these studies it is possible to reconstruct a more complete picture of the course of natural infection in cattle. Following invasion through mucous membranes the organisms set up infection in the regional lymph nodes, multiplying therein for a variable period of time. They are then carried by the lymph to the blood, from which they may be isolated regularly or intermittently depending upon the numbers and frequency with which they are showered from the foci of infection. Dissemination to other lymph nodes, liver and spleen, the mammary gland and uterus is probably hematogenous. Invasion of the gravid uterus rarely occurs before the second trimester of pregnancy, and persistent localization following an infected parturition is probably relatively infrequent. In most of the 20% of animals which become persistent uterine shedders the reproductive tract is probably reinvaded during subsequent pregnancies, while in some animals true localization in the uterus apparently occurs. Among the gaps in this picture is the part played by the bone marrow as a site for localization. PATHOLOGY OF BOVINE BRUCELLOSIS

Considering the emphasis which has been placed on uterine infection, it is not surprising that most studies of the pathology of bovine brucellosis have been confined to the changes found in the uterus and mammary gland. Hallman's 3 9 study of the necrotic and inflammatory changes in the uterus and those of Ridala4 0 and Runnels and Huddleson 4l on udder inflammation have been reviewed adequately by Huddleson. 4 2 Histopathological examination 4 3 of infected lymph nodes reveals granulomatous changes similar to those described in the lymph nodes of infected swine by Brown et al.44 and in human bone marrow by Sundberg and Spink.45 The lesions observed in infected lymph nodes consisted primarily of a reticular hypertrophy and hyperplasia, with almost complete replacement of the medullary cords and parts of the cortex by swollen reticulum cells. There was also a lymphoid hyperplasia. Early focal changes were infiltration with polymorphonuclear leucocytes, monocytes and many plasma cells and proliferation of the capillary endothelial cells. Occasional giant cells could be seen, but necrosis was not apparent. In older lesions the lymphoid tissue was replaced by stellate collections of reticulum cells with pyknotic nuclei, which seemed to represent an effort at healing in the absence of fibrosis. These were analogous to the "reticulum cell scars" described by Brown et al.44

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The major route of elimination of the organism from the body of the infected cow is with the discharges from the uterus at parturition. Fitch et al.4 6 have shown that the abortion bacillus may be isolated from the vaginas of cows for 25 days following abortion or infected full term parturitions. Manthei and Carter3 3 studied the genital infection of 18 experimentally infected cows through 2 pregnancies after exposure, and demonstrated persistent genital elimination of organisms in more than 80% of the animals for at least a month after parturition, and found one cow which shed Brucella for as long as 101 weeks. Brucella abortus was not isolated from the genital tract of 12 of these cows between conception and the second parturition, at which time 3 of them shed the organism. Experiments previously cited indicate that approximately 20% of reactors to the agglutination test are uterine shedders at full term parturitions. Since the first discovery of udder infection in 1911, by Schroeder and Cotton 47 and Smith and Fabyan 4 8 it has been demonstrated by many workers that infected cows may shed Br. abortus from the mammary gland for years. The most recent study of this type is that of Manthei and Carter.33 Fitch et al.49 have demonstrated the organism in urine and feces from infected animals, but concluded that this is not a common occurrence. It would be difficult to rule out contamination by discharges from the reproductive tract. Reference has been made already to elimination of Br. abortus from the male reproductive organs. SEROLOGICAL CHANGES

In animals infected during pregnancy the agglutinin titer generally rises before abortion to 1:200-1:1000 or higher. The incubation period is variable and may last from 1 to more than 30 weeks, depending in part upon the number of organisms.? 0 Some animals may fail to react to the agglutination test until after the termination of the pregnancy, while a few others may fail to react to the agglutination test at all, although definitely infected.30o 31 Even after initial infection a considerable group of animals, which react to the agglutination test in diagnostic titers, are not shedders of infection.? 2 Many of these can be shown to harbor organisms in lymph nodes, and may be considered as "arrested" cases. Some reactors may fail to shed Brucella at a number of parturitions, and then at some later date become persistent shedders. 33 With the passage of time some infected animals cease to react to the agglutination test in diagnostic titers. Beach et al.62 found 14 such ceased reactors in 37 animals within 2 years after experimental infection.

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Winner 5 3 observed an incidence of about 17% in several thousand animals tested in an official eradication program. It has been concludeds 3 that the probability of a ceased reactor being a carrier of Brucella is extremely small. Further Beach et al.64 have shown that recovery from infection, as evidenced by the loss of agglutinins, leaves the animal with a high degree of resistance to reinfection, which it has not been possible to duplicate by vaccination with Strain 19. Other serological changes which have been described during infection are an increase in specific bactericidins66 and a loss of a serum selective factor for smooth organisms. CONCLUSION Examination of the natural course of bovine brucellosis makes untenable the original concept that infection manifests itself as a placental disease, with little or no disturbance of health other than abortion. The research reviewed here shows that infection of cattle with Brucella results in a generalized disease analogous to the disease in guinea pigs, swine and human beings. These facts must be considered in the interpretation of the results of vaccination, and in the establishment of control programs. Real progress in defining the mechanisms of resistance requires that infection per se not be confused with the placental disease. REFERENCES (1) Elberg, S. S. and Silverberg, S. J.: Immunology of brucellosis, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 62, 1950. (2) Franck, 1876. Cited by Huddleson.4 2 (3) Lehnert, 1878. Cited by Wilson, G. S. and Miles, A. A.: Topley and Wilson's Principles of Bacteriology and Immunity. Third Edition, Williams & Wilkins Co., Baltimore, 1708, 1946. (4) Bang, B.: Die Aetiologie des Seuchenhaften (Infectiosen) Verwerfens. Zeitschrift fur Tiermedizin 1: 241, 1897. (5) McFadyean, J., Sheather, A. L., and Minett, F. C.: Researches regarding epizootic abortion of cattle. Jour. Comp. Path. 26: 142, 1913. (6) Seddon, H. R.: Studies in abortion disease. Jour. Comp. Path. 32: 1, 1919. (7) King, R. O. C.: Brucella infection in the bull: A progress report of mating experiments with naturally infected bulls. Australian Vet. Jour. 16: 117, 1940. (8) Bendixen, H. C.: Et Filfaelde af akut Brucellainfecktion i Pars glandularis ductus deferentis hos Tyr, Maanedsskr. Dyrl. 66: 1, 1944. (9) Seit, B.: Kastningssmitte ved kunstig Inseminering, Maandsskr. Dyrl. 56: 12, 1944. (10) Manthei, C. A., DeTray, D. E., and Goode, E. R.: Brucella infection in bulls and the spread of brucellosis in cattle by artificial insemination. I. Intrauterine injection, cited in Manthei.21 (11) Christensen, N. O.: Studies on the agglutinin formation in brucellar infection of the genitals of the bull. Acta Path. et Microbiol. Scand. 25: 202, 1948. (12) Bang, B.: Infectious abortion in cattle. Jour. Comp. Path. 19: 191, 1906. (13) Cameron, H. S.: The vitality of Br. abortus. Cornell Vet. 22: 212, 1932.

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(14) Bosworth, T. J.: Fourth Rep. Director Inst. Anim. Path., Cambridge 65: 1934-35. (15) Bang, O. and Bendixen, H. C.: Does the Br. abortus go through the normal hide and is this an important way of infection in cattle? Saertryck af Medlemsblad for Den danske Dyrlaeg. 15: 1, 1932. (16) Thomsen, A.: Antibody production in infectious abortion, especially in cattle. Skand. Veterinaertidsk. 8: 233, 1918. (17) Tovar, R. M.: Infection and transmission of Brucella by ectoparasites. Amer. Jour. Vet. Res. 8: 138, 1947. (18) Washko, F. V., Hutchings, L. M. and Donham, C. R.: Studies on the pathogenicity of Brucella suis for cattle. Amer. Jour. Vet. Res. 9: 342, 1948. (19) Elberg, S. S. and Henderson, D. W.: Respiratory pathogenicity of Brucella. Jour. Inf. Dis. 82: 302, 1948. (20) Berman, D. T., Irwin, M. R. and Beach, B. A.: Statistical considerations of controlled experiments in brucellosis. Amer. Jour. Vet. Res. 10: 130, 1949. (21) Manthei, C. A.: Brucellosis in cattle, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 172, 1950. (22) Edgington, B. H. and Donham, C. R.: Infection and reinfection experiments with Bang's disease. Jour. Agric. Res. 59: 609, 1939. (23) Bang, O., Bendixen, H. C. and Perskov, J.: Ruft Brucella abortus Bang eine universelle Infektion beim Rinde hervor? Acta Path. et Microbiol. Scand., Suppl. 16, 7, 1933. (24) Berman, D. T.: Unpublished data, 1950. (25) Carpenter, C. M.: Bacterium abortus invasion of the tissues of calves from the ingestion of infected milk. Cornell Vet. 14: 16, 1924. (26) Mitchell, C. A. and Gwatkin, R.: The relationship of oestrin to colonization of Brucella abortus in calves. Canad. Jour. Comp. Med. 9: 16, 1945. (27) Schroeder, E. C. and Cotton, W. E.: Some facts about abortion disease. Jour. Am. Vet. Med. Assoc. 50: 321, 1916. (28) Birch, R. R. and Gilman, H. L.: The agglutination test in relation to the persistence of Bacterium abortus in the body of the cow. Ann. Rept. N. Y. State Vet. Coll., Cornell Univ., Ithaca, N. Y., 56, 1931. (29) Roots, E. and Ridala, W.: Untersuchungen iber das Wesen der Brucellose u. uber die Ansiedlungsorte der Br. abortus im Korper des Rindes, in besondere in der Thyreoidea, Eesti Loomaarst 8: 165, 1932. (30) Krtger, H.: Ueber das Vorkommen von Bang bacterien im Fleisch geschlacteter Rinder, Deuts. Tierárztl. Wschr. 40: 481, 1932. (31) Doyle, T. M.: The distribution of Br. abortus in the system of "carrier" cows. Jour. Comp. Path. &Therap. 48: 192, 1935. (32) Berman, D. T.: Unpublished data, 1946. (33) Manthei, C. A. and Carter, R. W.: Persistence of Brucella abortus infection in cattle. Am. Jour. Vet. Res. 11: 173, 1950. (34) Soule, M. K.: Bacteriological and serological findings in Brucella abortus infections in animals and man. Premier Congrés International de Mi crobiol. 1: 606, 1930. (35) Fitch, C. P., Bishop, L. M. and Kelly, M. D.: The isolation of Brucella abortus from the blood stream of cattle. Proc. Soc. Exp. Biol. & Med. 34: 696, 1936. (36) Fitch, C. P., Boyd, W. L., Bishop, L. M. and Kelly, M. D.: Localization of Brucella abortus in the bovine uterus. Cornell Vet. 22: 62, 1939. (37) Gwatkin, R.: Incidence of Brucella abortus in the fetal membranes of fulltime, reacting cows. Cornell Vet. 22: 62, 1932.

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38) Fitch, C. P., Boyd, W. L. and Bishop, L. M.: A study of the vaginal content of pregnant Bang infected cows for the presence of Brucella abortus. Jour. Amer. Vet. Med. Assoc. 92: 171, 1938. (39) Hallman, E. T.: Further studies in the reproductive organs of cattle. Cornell Vet. 14: 254, 1924. (40) Ridala, V.: Inquiries into the pathogenic changes produced by Brucella abortus in the udder and certain organs of the cow. Vet. & Milk Hyg. Inst., Univ. Tartu, Estonia, 1936. (41) Runnells, R. A. and Huddleson, I. F.: The nature of Bacterium abortus infection in the udder of the bovine. Cornell Vet. 16: 376, 1925. (42) Huddleson, I. F.: Brucellosis in man and animals. The Commonwealth Fund, New York, 1943. (43) Berman, D. T. and McNutt, S. H.: The reaction of the reticulo-endothelial system in naturally acquired bovine brucellosis. Unpublished data, 1947. (44) Brown, I. W., Forbus, W. D. and Kerby, G. P.: The reaction of the reticulo-endothelial system in experimental and naturally acquired brucellosis of swine. Amer. J. Path. 21: 205, 1945. (45) Sundberg, R. D. and Spink, W. W.: The histopathology of lesions in the bone marrow of patients having active brucellosis. Blood, Suppl. 1, 1947. (46) Fitch, C. P., Delez, A. L. and Boyd, W. L.: Duration of the elimination of Bacterium abortus in the vaginal and uterine discharges of infected cattle. Jour. Am. Vet. Med. Assoc. 76: 680, 1930. (47) Schroeder, E. C. and Cotton, W. E.: The bacillus of infectious abortion found in milk. 28th Ann. Rept. Bur. Ani. Ind., U. S. D. A., 139, 1911. (48) Smith, T. and Fabyan, M.: The pathogenicity of Bacillus abortus Bang. Centralblatt fur Bact. (Abt 1) 61: 549, 1911. (49) Fitch, C. P., Bishop, L. M. and Boyd, W. L.: A study of bovine blood urine and feces for the presence of Bacterium abortus Bang. Proc. Soc. Exp. Biol. & Med. 29: 555, 1932. (50) McEwen, A. D., Priestly, F. W. and Patterson, J. D.: An estimate of a suitable infective dose of Br. abortus for immunization test on cattle. Jour. Comp. Path. & Therap. 62: 116, 1939. (51) Birch, R. R., Gilman, H. L. and Stone, W. S.: The immunity created by vaccination of calves with Brucella abortus Strain 19. Cornell Vet. 35: 119, 1945. (52) Beach, B. A., Irwin, M. R. and Ferguson, L. C.: The significance of the "ceased" reactor to Bang's disease. Jour. Agric. Res. 61: 75, 1940. (53) Winner, W.: Personal communication, 1948. (54) Beach, B. A., Irwin, M. R. and Ferguson, L. C.: The response of "ceased" reactors in Bang's disease to reexposure. Jour. Agric. Res. 66: 523, 1942. (55) Irwin, M. R. and Berman, D. T.: Bactericidal tests in brucellosis, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 85, 1950. (56) Braun, W.: Variation in the genus Brucella, Symposium on Brucellosis. Am. Assoc. Adv. Sci., Washington, D. C., 26, 1950. LA EVOLUCIóN NATURAL DE LA BRUCELOSIS BOVINA (Sumario) Se discute la infección del ganado con Brucella abortus tomando en cuenta las vías de invasión, y el análisis estadístico de los coeficientes de infección en el ganado infectado experimentalmente. Se analiza también la influencia de distintas cantidades de microorganismos sobre el coeficiente de infección en las

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pruebas experimentales. Un repaso de los estudios de la distribución del microorganismo en el huésped recalca la importancia de las infecciones extragenitales. Los tejidos extragenitales invadidos con más frecuencia son los ganglios linfáticos, el bazo y el hígado, asi como la glándula mamaria. La enfermedad se caracteriza también por bacteriemia intermitente, en particular durante las fases agudas. El examen histopatológico de los nódulos linfáticos infectados revela alteraciones granulomatosas que se caracterizan por proliferación de los reticulocitos e infiltración con plasmocitos y monocitos, y en ocasiones formación de células gigantes, pero con poca necrosis. Se conoce más generalmente la necrosis y exudado descubiertos en la placenta y en las carúnculas uterinas. El microorganismo se elimina principalmente en las excreciones del aparato de la reproducción durante el parto, y durante períodos variables después. En la transmisión de la infección al hombre, la introducción del microorganismo en la leche reviste mayor importancia que su diseminación entre el ganado. Los toros con infección del aparato de la reproducción pueden eliminar brucelas en el semen. La infección bovina se caracteriza por la elaboración de aglutininas específicas, anticuerpos que fijan el complemento y aumento de la actividad bactericida específica del suero. También se ha descrito la pérdida de un factor del suero que fomenta el establecimiento de una brucelal lisa. Se discuten la frecuencia y el significado inmunológico de la reposición espontánea.

THE NATURAL COURSE OF SWINE BRUCELLOSIS* By L. M. HUTCHINGS, B.S., D.V.M., M.S., Pn.D. Although swine brucellosis has been discussed many times in the past thirty-five years, it seems appropriate to again review the more important aspects of the natural course of the disease at the Third InterAmerican Congress on Brucellosis. Since 1941, when Dr. Jacob Traum first reported the recovery of Brucella organisms from aborted swine fetuses there have been numerous investigations dealing with many phases of swine brucellosis. Unfortunately, however, we are unable to draw upon the vast volume of investigational work which has been reported on bovine brucellosis. It is now recognized that swine brucellosis, although not of paramount economic significance to the pork producer, is often a serious economic problem to the professional swine breeder. In addition to the economic loss to the swine industry, brucellosis of swine is a menace to human health and to the health of other species of animals. Knowledge has recently accumulated concerning naturally occurring cases of Brucella abortus and Brucella melitensis infection in hogs (McCullough et al., 1949; Jordan and Borts, 1946). Thus further emphasis may be placed on swine as a reservoir for human brucellosis, since any species of animal which has been shown to harbor all three species of Brucella must be regarded as a potential source of spread. Our understanding of the precise means by which Brucella infection spreads from swine to cattle and man is not complete, but reports of the recovery of Br. suis from naturally infected cow's milk have been made by Hasseltine (1930), Beattie and Rice (1934) and Borts et al. (1943). Washko et al. have demonstrated experimental infection of cattle by intramammary exposure to Br. suis. Several reports have shown that Brucella may be found in freshly dressed pork and that these organisms may survive for relatively long periods of time (up to at least 20 days) under refrigeration. Thus man could acquire brucellosis by contact with this infected meat as well as by contact with the live infected hog. From this brief introduction, it can be readily understood that an infection cycle between swine, cattle and man is of considerable importance, not only to the industries involved, but also to the epidemiology of brucellosis in human beings. Since brucellosis generally is not transmitted from man to man, domestic animals are regarded as the chief source of human infection. Therefore the control of the disease in man is largely dependent upon the control of brucellosis in animals. * Published as Journal Series Paper No. 475 of the Purdue University Agricultural Experiment Station, Lafayette, Indiana.

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This makes the problem of brucellosis control of vital importance to the veterinary profession. In the available time, an attempt will be made to discuss briefly the natural course of swine brucellosis from the standpoints of susceptibility, transmission, symptoms, resistance and therapy, diagnosis, prevention and control. SUSCEPTIBILITY

Swine of both sexes and all ages appear to be susceptible to brucellosis. Symptoms are more pronounced in adult swine than in pigs. Suckling pigs seem to be less susceptible than weanling pigs, but the suckling pig may be infected either artificially or under field conditions. These observations are similar to those made on cattle and human beings as far as age susceptibility is concerned. It is a common belief that pregnancy or sexual maturity are essential for infection of swine with Brucella. Observation and experience have shown this belief to be erroneous. There is ample evidence that males are as susceptible as females. Castrates may acquire the disease also. In considering susceptibility, it should be pointed out that swine of all ages and both sexes appear to have some natural resistance to brucellosis. This is indicated by the size of exposure doses usually employed in infection of swine experimentally and by the difficulties commonly encountered in experimental production of abortions. TRANSMISSION

There are some features of the spread of brucellosis which are not well understood in swine as in other species. However, from the available information, the author considers swine brucellosis to be spread primarily by animal to animal contact and secondarily by indirect means, such as contaminated transportation facilities, carrier animals and birds, contaminated feed sacks and the innumerable other methods of indirect contact. Transmission may occur by ingestion of infected material, contact with infected discharges, by breeding, transportation, shows, sales, fairs and probably many other ways. An infected boar may eliminate Brucella in the semen in tremendous numbers. Thus the infected boar is often observed to be the cause of spread, especially when introduced into a herd which is free of brucellosis. Although adult or breeding swine are most commonly considered in the spread of brucellosis, one must not overlook the possibility of spread from infected pigs. It is known that Brucella may localize in many if not all parts of the body and hence elimination of the organisms may occur in any of the natural body discharges such as milk, urine, and

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feces in addition to semen. Infected sows which abort or farrow normally may eliminate Brucella in the urogenital discharges for variable intervals after parturition. Br. suis is prone to produce abscesses; these abscesses may rupture from time to time and spread Brucella about the premises. The livability of Brucella suis outside the animal body has not been extensively studied, but must be considered in transmission of brucellosis. Such studies as have been made indicate that Br. suis may live for three or four months in such materials as tap water and sterile soil, but for shorter periods of a month or so in distilled water, urine, feces, and unsterile soil. SYMPTOMS

The variation in symptomatology which is observed in swine brucellosis is somewhat dependent upon the localization of the organisms in the body. Following exposure to Br. suis, swine often develop a bacteremia which persists for variable periods of time, usually not more than 60-90 days. During and subsequent to the bacteremia, the organisms tend to localize. The points of localization are unpredictable and Br. suis has been found in most if not all of the tissues and organs of infected swine. The lymphatic tissue and sexual organs are common sites for localization; but isolation has been made from liver, brain, spinal cord, joints, kidney, bladder, mammary tissue and other portions of the body. The gross symptoms most commonly observed are abortion, sterility, orchitis, lameness, posterior paralysis, and occasionally metritis and abscessation of the extremities or other parts of the body. The symptom of abortion is rather variable, hence it is extremely difficult to predict the number of abortions in any Brucella infected herd. The abortion rate may be 50 to 80% of the females, or, in other herds which are equally infected, as measured by the serum agglutination test, there may be no observed abortions. The stage of gestation at which infection occurs, the virulence of the organism, and the susceptibility of the swine probably all play a part in the outcome of pregnancy. Abortions may occur at any time during pregnancy; those which occur very early in gestation are often unobserved. The symptoms of lameness, posterior paralysis and abscessation are commonly associated with the localization of Brucella in the areas involved. Sterility in gilts, sows and boars is common and sometimes is the only outward sign of brucellosis in the herd. Sterility in the female is often temporary. Orchitis in the boar is frequently observed where brucellosis exists. The orchitis is usually unilateral. Sterility may or may not occur as a result of orchitis. Fertility appears to be lowered, but complete sterility may not be present.

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Brucellosis of swine appears to be a somewhat self limiting type of disease at least in so far as abortion and serum agglutination are concerned. These features of the disease have contributed to the reports claiming the development of a serviceable immunity through the use of live or killed vaccines.This has been especially true when the results of vaccination have been measured by breeding efficiency only rather than by the more rigid criterion of freedom from infection. There is ample evidence that a tolerance to Brucella is developed by swine through natural acquisition of brucellosis or through vaccination. It has been demonstrated many times that most female swine abort only once, a few may abort twice and very few become habitual aborters as a result of Brucella infection. This in itself is an indication of tolerance to the infectious agent. As yet investigators have not satisfactorily exploited such natural phenomena by means of vaccination to a point where the artificial production of immunity is considered practical. Chemotherapeutic and antibiotic agents and combinations of the two have been studied to a limited extent, but the results to date have not been sufficiently encouraging to justify field usage. From the brief summary of resistance and therapy for swine brucellosis, it seems obvious that control of the disease rests largely in our ability to diagnose the infection and then make suitable disposition of the infected herds and animals. DIAGNOSIS

The principal method of diagnosis of swine brucellosis in current use is the serum agglutination test, the same as for the diagnosis of bovine brucellosis. The complement fixation test and various intradermal allergic tests have been tried but have been discontinued as too cumbersome or less accurate than the standard agglutination test. It is generally accepted that the serum agglutination test in swine is effective in determining the presence or absence of brucellosis in the herd, but has its limitations in detecting brucellosis in individual animals. Therefore it is necessary to use the agglutination test for diagnosing the disease in a herd and to base attempts at control on entire herds or units rather than on individual swine. It is now considered necessary to conduct the test in serum dilutions of 1:25, 1:50, 1:100 and above. The interpretation of the test requires some judgement since in nearly any sizeable herd of swine low-titered reactions occur in the absence of demonstrable infection. As a practical rule serum agglutination reactions below the 1:100 dilution are not considered indicative of brucellosis unless there are definite reactors at the 1:100 dilution or higher in the herd. Purchase of individual swine which exhibit a low-titered agglutination response should be avoided unless the status of the entire herd of origin is known.

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It is beyond the scope or time allotted to this paper to discuss the details of control other than to point out that two methods have been successful. 1. Test and prompt sale for slaughter of the entire herd if infection is detected. 2. Test, segregation, and delayed slaughter of the infected herd. The essential steps in this plan are: (a) Blood test the entire breeding herd. (b) If infection is present, consider the entire herd as infected, rather than remove the positively reacting animals. Manage the herd as a unit. (c) Raise the pigs from this infected unit. Wean and test the pigs at 8 weeks of age. Isolate the negative pigs on clean premises as far removed as possible from the infected parent herd. Maintain this isolation until it is practical to dispose of the infected parent herd. (d) Blood test the pigs up to, and during, the first pregnancy. Remove all reactors as they occur. Breed only those gilts which are negative to the blood test to non-infected boars. (e) Dispose of the original infected herd as soon as suitable negative replacements are available, or as soon as it is obvious that the plan is giving satisfactory results. (f) Premises where the infected herd was kept should be cleaned and disinfected throughly prior to admission of the clean replacement herd. Citations to the literature have been kept to a minimum for purposes of clarity and conservation of space; however, a partial bibliography appears at the end of this discussion. BIBLIOGRAPHY Beattie, C. P., and Rice, R. M. (1934): Undulant fever due to Brucella of the porcine type Brucella suis. Jour. Am. Med. Assoc., 102: 1670. Borts, I. H., Harris, D. M., Joynt, M. F., Jennings, J. R., and Jordan, C. F. (1943): A milk-borne epidemic of brucellosis. Jour. Am. Med. Assoc., 121: 319. Borts, I H. (1945): Some observations regarding the epidemiology, spread and diagnosis of brucellosis. Jour. Kansas Med. Soc. Dec., 1945, pp. 1-7. Cameron, H. S. (1943): Brucellosis in swine. I. The interpretation of low titer reactions in experimental and field infections. Am. Jour. Vet. Res., 4: 169-172. Cameron, H. S., Gregory, P. W., and Hughes, E. H. (1943): Inherited resistance to brucellosis in inbred Berkshire swine. Am. Jour. Vet. Res. 4: 387-389. Cameron, H. S. (1947): Brucellosis eradication and its effect on production in a large swine herd. Cornell Vet., 37: 55-58. Connaway, J. W., Durant, A. J., and Newman, H. G. (1921): Infectious abortion in swine. Missouri Agr. Expt. Sta. Bull., 187. Cotton, W. E., Buck, J. M., and Smith, H. E. (1938): Communicability of infectious abortion between swine and cattle. U. S. D. A. Tech. Bull., 629. Graham, Robert, Boughton, I. B. and Tunnicliff, E. A. (1930): Studies on porcine infectious abortion. Univ. Illinois Agr. Expt. Sta. Bull., 343. Hadley, F. B., and Beach, B. A. (1922): An experimental study of infectious abortion in swine. Univ. Wisconsin Agr. Expt. Sta. Res. Bull., 55.

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Harms, Amanda H. (1932): Cattle as a possible source of infection from Brucella abortus var. suis. Jour. Am. Vet. Med. Assoc., 34: 246-249. Hashizumo, K., Kamoto, K., and Kurizawa, T. (1940): Swine brucellosis. Res. Bull. Imp. Zooteck., Expt. Sta. Tibasi, 42: 1-113. Hasseltine, H. E. (1930): The relationship of human and animal brucellosis. Jour. Am. Vet. Med. Assoc., 29: 330-338. Hutchings, L. M. (1943): Brucellosis in swine. Proc. 47th Annual Meeting U. S. Livestock Sanitary Assoc., pp. 52-58. Hutchings, L. M., Delez, A. L., and Donham, C. R. (1944): Studies on brucellosis of swine. I. Infection experiments with weanling pigs. Am. Jour. Vet. Res., 5: 195-208. Hutchings, L. M. (1944): Report of further studies of brucellosis in swine. Proc. 48th Annual Meeting U. S. Livestock Sanitary Assoc., pp. 105-109. Hutchings, L. M., and Andrews, F. N. (1946): Studies on brucellosis in swine. III. Brucella infection in the boar. Am. Jour. Vet. Res., 7: 379-384. Hutchings, L. M. (1947): Field control experiments with brucellosis in swine. Proc. 51st Annual Meeting of U. S. Livestock Sanitary Assoc., pp. 124-136. Johnson, H. W., and Huddleson, I. F. (1931): Natural Brucella infection in swine. Jour. Am. Vet. Med. Assoc., 78: 849-862. Jordan, Carl F. (1942): Undulant fever in Iowa. Proc. 46th Annual Meeting of the U. S. Livestock Sanitary Assoc., Chicago. Jordan, C. F., and Borts, I. H. (1946): Occurrence of Brucella melitensis in Iowa. Jour. Am. Med. Assoc., 130: 966. Kernkamp, H. C. H. (1942): Forum on infectious and transmissible diseases of swine. Proc. 46th Annual Meeting U. S. Livestock Sanitary Assoc., p. 63. Kernkamp, H. C. H., and Roepke, M. H. (1948): The interpretation of low agglutination titers in the control of swine brucellosis. Am. Jour. Vet. Res., 9: 46-49. Manthei, C. A. (1948): Research on swine brucellosis by the Bureau of Animal Industry (1941-1947). Am. Jour. Vet. Res., 9: 40-45. McCullough, N. B., Eisele, C. W., and Pavelchek, Emma (1949): Isolation of Brucella abortus from hogs. Pub. Hlth Repts., 64: 537-538. McNutt, S. H. (1935): Incidence and importance of Brucella infection of swine in packing houses. Jour. Am. Vet. Med. Assoc., 86: 183-191. McNutt, S. H. (1938): Brucella infection in swine. Proc. 42nd Annual Meeting of the U. S. State Livestock Sanitary Assoc., Chicago. Moore, V. A. (1929): Relation of undulant fever in man to livestock sanitation. J. Am. Vet. Med. Assoc., 27: 605-617. Stone, W. S. (1943): Brucellosis in swine. Cornell Vet., 83: 115-119. Thomsen, Axel. (1934): Brucella infection in swine. Acta Path. Microbiol. Scand. Suppl. 21. Translation by Hans Anderson, M.D. Washko, F. V., Hutchings, L. M., and Donham, C. R. (1948): Studies on the pathogenicity of Brucella suis for cattle. I. Am. Jour. Vet. Res., 9: 342-350. EL CURSO NATURAL DE LA BRUCELOSIS PORCINA (Sumario) En la actualidad, la brucelosis porcina está reconocida como una enfermedad impotante, tanto bajo aspectos económicos como de salud pública. Se ha demostrado que el cerdo es susceptible a las tres espeies de Brucella, esto es, Br. abortus, suis, y melitensis.

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El hombre puede contraer la brucelosis por contacto, ya con cerdos vivos infectados o, potencialmente, por contacto con carnes de cerdos infectados. Se ha demostrado que la Brucella permanece activa por lo menos 20 dfas en los tejidos del cerdo infectado en forma natural. Los síntomas de brucelosis observados con más frecuencia en los cerdos son: aborto, esterilidad, lisiamiento, orquitis, parálisis posterior y abscesos. Los sfntomas dependen en parte de la localización de los organismos. Los cerdos de toda edad y de ambos sexos son susceptibles, pero los efectos son más graves en animales adultos. La transmisión ocurre principalmente por contacto directo de animal con animal. La propagación puede ocurrir indirectamente a través de medios de transporte, alimentos contaminados, pocilgas y, quizás, hasta por agentes mecánicos. La Brucella puede ser eliminada en la leche, orina, heces, semen y exudaciones urogenitales de los cerdos infectados. El diagnóstico está basado en la interpretación de los resultados de la prueba normal de aglutinación del suero, tal como sucede en la brucelosis bovina. Esta prueba es exacta cuando se toma procedimiento de diagnóstico en un rebaño, pero tiene ciertas limitaciones para el diagnóstico individual de la enfermedad en el cerdo. El control de la brucelosis porcina depende del diagnóstico, así como de ciertas formas de segregación o eliminación de cerdos infectados o de rebaños enteros. La vacunación y el tratamiento no han demostrado aún ser satisfactorios para el control de la brucelosis porcina.

STUDIES OF THE PHYSICAL PROPERTIES AND AGGLUTINABILITY OF BRUCELLA ANTIGENS USED IN THE AMERICAS* By FRED M. MURDOCKf, MARTIN H. ROEPKES AND BENJAMIN D. BLOOD§ The blood serum agglutination reaction is a relatively simple test widely used for the diagnosis of brucellosis in both animals and man. Unfortunately the antigens and techniques for this test have not been standardized on an international basis. The results have thus been subject to controversy and misinterpretation. This situation was recognized in the recommendation of the XII Pan American Sanitary Conference (Caracas-1947), "that the methods and means of brucellosis diagnosis be made uniform throughout the American Republics". The Second Inter-American Congress on Brucellosis (Buenos Aires-1948) also emphasized the need for standardization of diagnostic methods for human and animal brucellosis and recommended that the agglutination reaction with standard antigen be employed. The purpose of the present studies was to determine the similarities or variances that existed between Brucella antigens used in the Americas. The Pan American Sanitary Bureau obtained the antigens for this study by arrangements with various governmental laboratories and agencies concerned with serum agglutination tests for the diagnosis of brucellosis. The antigens were received from countries throughout the Americas; in addition, several antigens produced in Europe were included. Thirty-six antigens, including seventeen for the tube test and nineteen for the plate test, from twenty-five governmental and private laboratories in fifteen different countries and territories, were studied and are included in this report. Governments which cooperated in the project were those of Argentina, Brazil, Canada, Chile, Cuba, Denmark, Dominican Republic, Ecuador, Great Britain, Jamaica, Mexico, The Netherlands, Peru, United States and Venezuela. Brucella Species.-The species of Brucella used in the preparation of those antigens for which information was supplied, were as follows: * Paper No. 2608, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota. This study was supported by a grant from the Pan American Sanitary Bureau. t United States Bureau of Animal Industry, St. Paul, Minnesota. At present

employed by the Anchor Serum Co., South St. Joseph, Missouri. : University of Minnesota, St. Paul, Minnesota. § Pan American Sanitary Bureau, Washington, D. C. 122

PHYSICAL PROPERTIES OF BRUCELLA ANTIGENS Brucella Brucella Brucella Brucella Brucella

abortus ......................................... suis ............................................. melitensis ....................................... abortus and Brucella suis ........................ abortus and Brucella melitensis ..................

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19 antigens 2 antigens 2 antigens 1 antigen 2 antigens

Culture Media.-For the preparation of the several antigens, the Brucella cultures were grown as follows: Medium

No. Antigens

Potato agar ..................................................... 14 Liver infusion agar .............................................. 4 Peptone agar .................................................... 6 Tryptone agar .................................................. 3 Glucose agar .................................................... 2 Dextrose agar ................................................... 2 Hartly digest agar .............................................. 1 Medium prepared from beef broth and peptone with dextrose & glycerine ..................................................... 1 Medium prepared from meat infusion and peptone ............... 1

Growth Period.-The period of growth of the cultures was 48 hours for 15 antigens, 72 hours for 18 antigens and 96 hours for 1 antigen. Harvesting Solutions.-The growth was harvested with: 0.5% phenol in physiological salt solution ................. 13 antigens 0.5% formalized physiological salt solution ............... 6 antigens Physiological salt solution ................................ 5 antigens 12% salt solution ......................................... 4 antigens Physiological salt solution containing 1% formalin ........ 2 antigens 2 antigens 1.2% salt solution, containing 0.5% phenol ............... 12% salt solution plus 1% phenol ........................ 1 antigen Physiological salt solution plus 1% glycerine and 0.25% phenol .............................................. 1 antigen

Killing and preservation.-In the majority of instances the bacteria were killed by heating the suspensions to temperatures ranging from 60 to 100°C for periods ranging from 10 minutes to 60 minutes, or by boiling for 10 to 30 minutes. In 6 instances, the organisms were killed with 0.5% formalin, 2 by undiluted formalin and 1 by 95% alcohol. For a high percentage of the antigens, the killed cells were suspended in a sodium chloride solution of concentrations ranging from 0.85% to 1.2% and containing as a preservative 0.5% phenol. In some instances, small amounts of dyes, such as crystal violet and/or brilliant green, were added. In 2 instances, 1% formalin plus merthiolate were added as preservatives; in 1 instance, 1% phenol, and in another, 0.25% formaldehyde, for the same purpose. Cell concentration.-Standardization of the density or concentration of cells in 8 cases depended upon the McFarland nephelometer, in 7

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cases, upon the per cent by volume of centrifuged cells as determined by the Hopkins vaccine tube or the Fitch-modified-Hopkins vaccine tube; in 4 instances upon testing with serum of known titer; in 2 upon the Fitch-modified-Hopkins vaccine tube and the testing with sera of known titers; in 4 upon comparison with former lots of antigen prepared in the laboratory; in 1, upon comparison with antigen from another country and testing with sera of known titers; in 1 upon an absorptiometer; and in 1, upon comparisons with another antigen. Intended use.-The species for which the antigens were to be used were designated as follows: Human ............................................................ Bovine ............................................................. Bovine, porcine and human ........................................ Porcine. ........... ......................................... Bovine and porcine ................................................ Porcine, caprine and human ........................................ Bovine, porcine and caprine ........................................ Melitensis infections ...............................................

5 4 2 2 2 1 1 2

TUBE TEST METHODS A summary of the instructions given for conducting the tube agglutination test follows: Dilutions.-The volume of the serum-antigen mixture was 1 cc for 11 of the antigens and 2 cc for the remaining 7. The diluent to be used for the tube test was 0.5% phenol in physiological salt solution for 9 of the antigens, and plain sterile physiological salt solution for 8 of the antigens. The tests were to be incubated. Incubation Period.At 37°C for 72 hours, 6 antigens; for 48 hours, 2 antigens; for 24 hours plus 8 hours at room temperature, 1 antigen; for 24 hours plus 24 hours at room temperature, 1 antigen; for 24 hours plus over night at room temperature, 1 antigen; for 18 hours plus 20 to 24 hours at room temperature, 1 antigen; for 20 hours, 2 antigens; for 2 hours plus 22 hours in the refrigerator plus 2 to 3 hours at room temperature, 1 antigen; for 2 hours plus over night in the refrigerator, 1 antigen; At 56°C for 20 to 25 hours in a water bath, 1 antigen. Reading Instructions.-In almost all instances, the instructions with respect to the lighting arrangement involved a shaded light for good illumination of the specimens and a dark background, for visual reading of the degree of agglutination. In one instance a low power magnifying lens was recommended for better observation of the agglutination re-

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action. The instructions for the reading of the tests, in a high percentage of the cases, recommended designating the degree of agglutination for each dilution as negative, partial or complete agglutinations. In a few instances, it was recommended that the degree of reaction be graded from negative to 3 or 4+, for each dilution in the test. PLATE TESTS

With respect to the plate test, recommendations for the time of reading were: 15 minutes for 4 antigens 8 minutes for 3 antigens 5 minutes for 3 antigens 3 minutes for 2 antigens 8 to 10 minutes for 1 antigen 5 to 8 minutes for 1 antigen 2 minutes for 1 antigen and one antigen, after rocking the plate back and forth 15 to 20 times. LABORATORY COMPARISON OF ANTIGENS

Laboratory studies made on the antigen included the following procedures: microscopic and cultural examinations for purity; concentration of the antigens, in terms of the volume of centrifuged cells per 100 cc, as determined by centrifugation at 2800 r.p.m. for 75 minutes with the Fitch-modified-Hopkins vaccine tube, and agglutination tests on 55 bovine and caprine serums of various titers to determine agglutinability and sensitivity of the antigens. Purity.-The per cent of extraneous cells in the antigen, as determined by microscopic examination of stained specimens, varied from 0 to 13%. The bacterial count of viable cells per cc of plate antigen, or concentrated tube antigen as received, was in 18 cases 0; in 12 antigens, the counts ranged from 1000 to 8500; in 5 antigens, from 51,000 to 84,000; in 1 antigen, 360,000, and in 3 antigens, 3 to 10 million. The viable cells found were not Brucella. As near as could be determined from observations on agglutinability with the 55 sera used, the extraneous cells found did not materially interfere with the agglutination reaction. Agglutinability.-The bovine sera with low to high titers used for the agglutination studies were obtained either from known infected herds, or from animals located in the stockyards at packing plants. The sera with negative titers were obtained from a negative herd. The titers of the serum ranged from negative at the 1:25 dilution to complete agglutination at 1:1600 as determined by the tube method of the United States Bureau of Animal Industry. The titers of the 24 sera used to

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calculate the relative sensitivity of the antigens, ranged from incomplete or partial agglutination at the 1:50 dilution, up to partial agglutination at 1:200. The instructions supplied with each of the antigens were followed as closely as possible with respect to the preparation of the test, period of incubation and the reading of the test. Tests were read in such a way that the operator had no knowledge of the number of the serum concerned, in order to insure unbiased readings. Table I summarizes observations on the antigens studied. Under the columns with headings, "Country", and "Laboratory", respectively, Roman numerals designate countries; capital letters designate the laboratories or private concerns which submitted the respective antigens; the Arabic numerals indicate the different antigens from each laboratory. Antigen concentration is in terms of per cent by volume of centrifuged cells. Concentrations are listed for the final dilution of the antigen as present in the test. In the case of plate test antigens, concentrations are those found in the antigens as received ready for use. The relative sensitivity of the antigen, as given in the sixth column, is numerically the average titer obtained on 24 sera in the low and intermediate titer range. The average titer obtained for an antigen with the 24 sera was calculated as follows: each dilution of a single test was given a value of 1 for complete agglutination, 0.5 for partial or incomplete agglutination, and 0 for no agglutination. Thus, with a test starting at the 1:25 dilution, a titer on a serum showing incomplete agglutination at 1:100 was given a titer value of 2.5. A serum showing complete agglutination at 1:50 was given a titer value of 2. Likewise, a serum showing incomplete or partial agglutination at 1: 50 was given a value of 1.5, and so on. By adding the titer values of each of the 24 sera and dividing by 24 the average titer value was obtained. This average titer value was then reconverted to an average titer. For example: an average titer value of 2.68 is equivalent to complete agglutination at a dilution of 1 to quantity 50, plus 68% of the difference between 50 and 100, or complete agglutination at 1:84. The relative sensitivity would then be 84. The method of determining the relative sensitivity of antigens for comparative purposes, by conducting tests on 20 to 30 sera of low to intermediate titers, with unbiased readings of the test, has been found to be very satisfactory. The relative sensitivity values obtained as noted may be duplicated within plus or minus 10%, in a very high percentage of cases. If small differences in antigen sensitivity are to be accurately evaluated, an appreciable equal number of sera must be used, and readings must be entirely independent. The next column lists the minimal significant titer where instructions included the interpretation of the agglutination reactions. In those instances in which the minimal significant titer was given as partial agglutination at a given dilution, the significant titer was designated as

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midway to the next lower dilution. For example, a significant titer or partial agglutination at 1:100 is designated as equivalent to complete agglutination at 1:75, i.e., midway to the next lower dilution. Likewise, partial agglutination at 1:80 is considered equivalent to complete agglutination at 1:60, and partial agglutination at 1:40 equivalent to complete agglutination at 1:30. This was'done to facilitate comparisons of the various antigens and calculation of "relative applied sensitivities". The eighth column contains calculated values of "the relative applied sensitivity". These values combine the factors of (a) relative sensitivity and (b) significant titer. They were computed by dividing the relative sensitivity value by the significant titer value and multiplying the quotient by 100. Since these values combine the relative sensitivity and the interpretations of the agglutination test, they indicate the sensitivity of the test as applied under field conditions. In Table I, under "Interpretation" are interpretations of the 24 sera tested, in terms of significant, doubtful and negative titers. Under "Readability of Test," subjective evaluations are given with respect to the ease of reading the test. These evaluations were made from the standpoints of character of agglutination and sharpness of end-point. The agglutination reactions obtained with antigens XI A-1 and XII A-2 were so erratic that relative sensitivity calculations could not be made. With these two antigens, the tests were negative on a number of the high titered sera. Antigen XII A-1 is a special type of antigen to be used with a special technic of conducting the test, using one dilution. The results of agglutination reactions obtained with this special test appeared to be quite satisfactory from the standpoint of separating sera giving titers above 1:100 with the majority of the antigens from those below 1:100. In several instances, information was not given with respect to significant titers. The relative applied sensitivity and interpretation values could thus not be determined in these cases. In two instances, insufficient amounts of antigen were supplied to conduct the test on all of the 24 sera used for sensitivity determinations. For these, values could not be given in the "Interpretation" column. Also, for these two antigens, the values given for relative sensitivity and relative applied sensitivity are not strictly comparable to those given for the other antigens. As shown in Table I the relative sensitivity values for the plate antigens are less variable than is true for the tube antigens. The ratio of maximum to minimum relative sensitivity for the plate antigens is 140 to 51 or a ratio of 2.74:1 which indicates that the most sensitive plate antigen was 2.74 times that for the least sensitive antigen. For the antigens used in the tube test, the range was from 538 to 36, or a ratio of maximum to minimum of 14.9:1. The relative applied sensitivities for the plate test varied from 187 to 51. In this case the most sensitive

128

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

TABLE I.-Summary of siudies on Brucella antigens used in the Americas and

several European countries I

-

Labo· Coun- ratory try &Antigen

II

III IV V VI VII VIII IX X XI XII

A-1 A-2 B-1 C-1 D-1 E-1 A-1 A-2 A-3 A-4 A-5 A-6 B-1 C-1 C-2 A-1 A-2 B-1 A-1 A-1

A-1 A-1 A-1 A-1 A-1 A-1 A-1 A-2 B-1 C-1 XIII A-1 B-1 B-2 XIV A-1 A-2 XV A-1

Species of Brucella

Type of Antigen

abortus abortus abortus abortus

Plate Tube Plate Plate Plate abortus Plate suis Tube suis Tube abortus Tube abortus Tube meliten. Tube meliten. Tube abortus Plate abortus Plate abortus Tube abortus Plate abortus Tube abortus Tube abortus Plate suis and Plate abortus abortus Tube abortus Tube abortus Plate abortus Tube abortus Plate Tube abortus abortus Special abortus Tube abortus Plate abortus Plate abortus Plate abortus Plate abortus Tube abortus Plate abortus Tubo Plate abortus of Ratio Maximum Minimum

RelaSignifi- tive Acant pied Titer tivity

Cell Conc.

Relative Sensitivity

13.7 0.041 3.1 2.2 12.5 10.3 0.017 0.027 0.017 0.028 0.009 0.024 11.0 14.0 0.140 12.0 0.035 0.028 10.2 4.4

47 86 140 125 65 55 458 467 499 400 538 313 57 55 39 63 86 111 62 136

75 75 75 100

63 115 187 125

6-14-4 14-9-1 22-2-2 18-3-3

50 60 60 60 60 60 60 100 100 100 100 150 100

110 763 778 831 667 897 522 57 55 39 63 86 74 62

24-0-0 24-0-0 24-0-0 24-0-0 24-0-0 24-0-0 4-17-3 5-16-3 5-13-6 2-20-2 9-14-1 10-14-0 6-16-2

0.100 0.037 0.7 0.134 5.2 0.024 20.0 0.080 12.0 7.2 7.0 11.0 ] 0.038 11.0 0.043 7.0

52 130 62 36 126

20

260

24-0-0

75 30 100

82 117 126

12-7-5 14-7-3 17-6-1

100 100

52 51

100 100 100 100

62 79 60 78

52 51 63 62 79 60 78 106

14.9 1

100

Interpre- Readability tation of Test (S*-D*-N*)

3-20-1 11-12-1 5-17-2 9-15-0

Normal Normal Normal Normal Poor Normal Fair Fair Fair Fair Fair Fair Normal Normal Normal Normal Normal Normal Poor Normal Normal Normal Poor Normal Normal Poor Poor Normal Normal Normal Normal Normal Normal Normal Normal

23 1

* S = significant titer, D = doubtful reaction, N = negative reaction. antigen, as far as application is concerned, was 3.67 times the sensitivity of the least sensitive antigen. In comparing the relative applied sensi-

PHYSICAL PROPERTIES OF BRUCELLA ANTIGENS

129

tivity of the tube antigens, the ratio of the most sensitive to the least sensitive was 897 to 39 or a ratio of maximum to minimum of 23:1. TABLE II.-Effect of concentration of Brucella cells and techniques of conducting

the tube agglutination on average titer or relative sensitivity S ecies

Labora-

%Cells CAs by by Vol.

Antigen

A-1 A-3 A-5 C-2 A-2 B-1 A-1

II

III IX

Ratio of

suis abortus melitensis abortus abortus abortus abortus

U. S. B. A. I. Conc. and Technique

Recommended Conc. and Technique

%Cells Cel by Vol.

Sensit i . SRelati*ívie ¡vi

538

Maximum

129

-129 74

14.9

36

Minimum

100 129 74 92 85 77 77

0.045 0.045 0.045 0.045 0.045 0.045 0.045

458 499 538 39 86 111 36

0.017 0.017 0.009 0.140 0.035 0.028 0.134

Relative Sensitivity

1.74

TABLE III.-Agglutination titers on the dried standard Brucella abortus agglutinating serum (International Office of Epizootics) Supplied by the Veterinary Laboratory, Weybridge, England (Tube Test Antigens) Titer on Standard Serum Country

II II

VI IX

XI XII XIII XIV

Laboratory and Antigen

A-2 A-1 A-3 A-5 C-2 A-2 B-1 A-1 A-1 A-1 A-2 B-2 A-2

tivity Values from Table I

Dilution 1:320

1:640

1:1280

1:2560

+ +

+

+ + + +

1 + + +

+ + 1

1 1

+ +3 + + + 3 + + + +

+ + + + + + + 1 + 1 + +

1:5120 1:10240 1:20480

+ + +

1 + +

1 1

86 458 499 538 39 86 111 52 36

1 + 1

79 78

Cell Concentration.-The observations made while conducting the agglutination tests suggested that possibly some of the marked variation in sensitivity found might be due to the particular strain of Brucella used to prepare the antigen and the method of growing the cultures as

130

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

well as harvesting and killing the cells. However, it appeared that the major factors responsible for the great variation in sensitivity were due to variations in the concentration of Brucella cells in the antigens and variations in the techniques of conducting the agglutination test. In order to investigate the latter possibility, seven of the tube antigens which showed a marked range in relative sensitivity were retested using a uniform concentration of Brucella cells and the technic of conducting and reading the test as recommended by the United States Bureau of Animal Industry. The relative sensitivity values obtained by the two methods are shown in Table II. For the recommended concentration and technic of conducting the test given for each of the antigens the relative sensitivities varied from 538 to 36 which is a ratio of maximum to minimum of 14.9 to 1. With a uniform technic of conducting the test and a uniform concentration of cells for all of the antigens the relative sensitivities varied from 129 to 74 or a ratio of maximum to minimum of 1.74:1. These results indicate that the major factors responsible for the marked variation in the results obtained with the tube tests were due, to a very great extent, to variations in the concentration of the Brucella cells in the antigens and to variations in the technic of conducting the test. Results with Standard Dried Serum.-The standard dried seruml of the International Office of Epizootics, kindly supplied by Dr. A. W. Stableforth of the Weybridge Laboratory in England, was used to test 13 of the tube test antigens. The titers obtained on this standard serum are given in Table III. The titers obtained on this one serum were, within experimental error, in general agreement with the relative sensitivity values as given in Table I. DISCUSSION In making the various tests on the antigens involved in this study, particularly the agglutination studies, it is to be expected that the results obtained would not be the same as those which might be obtained in the laboratory which submitted the antigen. Therefore, the values shown should be considered as only rough approximations. With several exceptions, the purity of the antigen from the standpoint of extraneous viable and non-viable bacteria, appeared to be satisfactory for use in the agglutination test. From the standpoint of the character of the agglutination reaction, the sharpness of the end-point of the agglutination titers and the absence of serious discrepancies from the normal with respect to the agglutination titers obtained with the different sera tested, the agglutinability of a high percentage of the antigens appeared to be normal. The studies indicate that uniformity of concentration of the antigen and technic of conducting the agglutination test as well as uniformity in the interpretation of the test would

PHYSICAL PROPERTIES OF BRUCELLA ANTIGENS

131

very materially reduce the wide range of relative applied sensitivities found for the antigens tested. As results of these studies indicate, it is difficult to compare the reports of brucellosis studies which involve serum agglutination tests from different laboratories and countries without satisfactory knowledge of the antigens used, the technics of conducting and interpreting the test. The quite extensive use of the serum agglutination test as a diagnostic test in the bovine brucellosis control program in the United States has demonstrated the importance of a uniform test throughout the country. In the early stages of the bovine brucellosis control program each state had its own particular antigen and technic of conducting the test. Other studies 2 . 3 on a number of such antigens indicated approximately the same marked degree of variation in sensitivity as was found in this study. It was difficult to compare the results obtained in a control program in one state with those of another. Appreciable difficulties also arose with respect to the interstate shipment of cattle which were subject to the serum agglutination test. Many of these difficulties were appreciably alleviated when nearly ten years ago, a uniform antigen and technic of conducting the test was adopted in the United States. ACKNOWLEDGMENT

It is desired to give credit to Dr. Peter J. Ostapchuk for very valuable technical assistance in conducting this study. REFERENCES (1) Stableforth, A. W.: Standardisation du diagnostic de la brucellose bovine par l'épreuve de séro-agglutination. Dix-huitiéme Session du Comité de l'Office International des Epizooties, XXIV: 229-247, (1950). (2) Fitch, C. P. and Thompson, C. M.: Studies of physical properties and agglutinability of Brucella abortus plate antigens from several sources. Cornell Vet., 26: 222-230 (1936). (3) Fitch, C. P., Roepke, M. H. and Thompson, C. M.: Studies of physical properties and agglutinability of Brucella abortus plate antigens from several sources. II. Cornell Vet., 27: 366-373 (1937). ESTUDIOS DE LAS PROPIEDADES FÍSICAS Y LA AGLUTINABILIDAD DE LOS ANTfGENOS DE BRUCELLA USADOS EN LAS AMÉERICAS (Sumario) Para realizar este estudio se utilizó un total de 36 pruebas de aglutinación de antígenos de Brucella, que fueron enviados por 25 laboratorios gubernamentales o particulares de 15 paises distintos. Se hicieron los siguientes estudios: examen microscópico y de cultivo para determinar la pureza de los antígenos; concentración de los antígenos mediante centrifugación para determinar el porcentaje por volumen de células de Brucella; y además, pruebas de aglutinación en 55 sueros de bovinos y caprinos de diversos titulos para determinar la capacidad de aglutinación y de sensibilidad de los antigenos.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

La pureza de los antígenos, con algunas posibles excepciones, pareció ser satisfactoria desde el punto de vista de su empleo. La concentración de las células de Brucella por volumen en los antígenos empleados para la prueba común de placa, o prueba rápida, varió de 0.70% a 14.0%, con un promedio de 8.6%. La concentración final de los antígenos, según las pruebas de tubo o prueba lenta, varió de 0.009% a 0.14%, con un porcentaje medio de 0.048% de células por volumen. A juzgar por los resultados obtenidos en las pruebas de

los 55 sueros mencionados, un alto porcentaje de los antígenos pareció indicar una capacidad normal de aglutinación. La sensibilidad relativa de los antígenos varió considerablemente entre 36 y 538 según pudo determinarse por los títulos promedios de 24 sueros de título bajo o intermedio. Los estudios efectuados indican que la uniformidad de concentración de los antígenos y la técnica que se siga para las pruebas de aglutinación, podrían reducir muy sustancialmente la variabilidad de sensibilidad relativa encontrada en los antígenos.

FIELD STUDIES ON THE DIAGNOSIS OF ANIMAL BRUCELLOSIS WITH SPECIAL EMPHASIS ON THE RING TEST* By FRED C. DRIVERt AND MARTIN H. ROEPKEt As other papers in this symposium no doubt will deal with the diagnosis of brucellosis in animals other than the bovine, this paper will be confined to the diagnosis of bovine brucellosis and particularly to the use of the ring test for the detection of Brucella infected dairy herds. The extensive use of the blood serum agglutination test for the diagnosis of brucellosis in this country as well as many other countries has, rather satisfactorily, demonstrated the accuracy and practical value of the test under field conditions. The blood test has, however, a handicap which is not related to the accuracy of the test but to the nature of the test, in that it requires a large number of personnel and appreciable funds to use it on an extensive scale in an eradication or control program. It is not practical to conduct county-wide blood tests in counties certified as modified brucellosis-free, at intervals shorter than three years. In such certified counties which adjoin districts not on the area plan, reinfection rates may be quite high, and, in many instances, an interval of three years between blood tests in a county is too long. Many new centers of infection have several years time to spread to neighboring herds. Also, because of the lack of veterinary personnel for the brucellosis control program in our state, we have been able to do very little more than to conduct county-wide blood tests for certification or recertification of the 23 counties which were on the area control plan by 1942. From 1942 to 1949, area control work was initiated in only 5 new counties, which is relatively slow progress considering that there are 87 counties in our state. It was with the thought in mind that the ring test, as an adjunct to the blood test, might provide a means of extending more rapidly or speeding up the control program in our state that a study of the ring test was initiated in the fall of 1947. The ring or ABR test for brucellosis was first described by Fleischhauerl and Fleischhauer and Hermann 2 in 1937 and 1938. The ring test which is applied to whole milk is an agglutination test and depends on the presence of Brucella agglutinins in the milk from an infected animal. The antigen used, a 3 to 4% suspension of Brucella abortus cells, is stained a deep blue color with hematoxylin so that the agglutination reaction may be observed in the presence of whole milk. * Paper No. 2605, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota. t Inspector in Charge, U. S. Bureau of Animal Industry, St. Paul, Minnesota.

$ University of Minnesota, St. Paul, Minnesota. 133

134

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

In positive samples, the agglutinated cells adhere to the cream droplets and are carried to the top with the cream to form a deep blue cream ring or line, whereas in milk from non-infected animals the stained antigen remains in the skim milk fraction to give it a light blue color, and the cream ring which forms is white. The nature of the reaction observed gives the test its name. The ring test is very easily performed in that one drop of antigen is added to 1 cc of milk and the mixture allowed to stand at room temperature from 60 to 90 minutes or incubated at 37°C for 45 to 50 minutes which is sufficient time for the cream to rise to the top, at which time the test may be read. The ring test was found to be so sensitive that in a high percentage of cases a positive test was obtained even though the milk from one infected animal was mixed with that from 5 to 15 negative or non-infected cows. This high sensitivity permitted the test to be used on pooled or mixed milk samples from a herd collected at the receiving platform in a dairy plant and thereby serve as a very rapid, and inexpensive screening test to locate infected herds. Since the original description of the test by Fleischhauer, a number of studies have been reported, particularly from Sweden 3 and Denmark.4- 9 These reports described improved methods of preparing the antigen, techniques of conducting the test and rather extensive studies showing the efficiency of the test in locating infected herds. Some idea of the efficiency of the test was presented in reports from Denmark by Seit and Jorgensen 5 and by Christiansen. 9 These investigators found that the ring test was positive on the milk-can samples of 82% of 2,031 herds classified as infected on the basis of the serum agglutination test results. Of the failures of the ring test, investigations of such herds disclosed that about two-thirds of the failures were due to the fact that the infected animals present were not in production. That is, heifers, dry cows or, in some instances, a bull or steer were the infected animals. It was considered likely that the majority of such herds would be detected on a second ring test in three to six months. Such a procedure would overcome, to an appreciable extent, this natural limitation of a single ring test on a herd. STUDIES IN MINNESOTA

The studies in Minnesota are part of a cooperative project between the State Livestock Sanitary Board, the Bureau of Animal Industry of the United States Department of Agriculture and the University of Minnesota. In the early part of the studies the investigations were limited to those counties in which county-wide blood tests were to be made for recertification of the county. The ring tests were made just preceding the blood tests in the county in order that a more accurate evaluation of the agreement between the two tests might be obtained.

DIAGNOSIS OF ANIMAL BRUCELLOSIS

135

The results of the studies in 9 such counties were so encouraging that the ring test work was extended to all of the counties which were on the area plan. In the latter counties, blood tests were made only in those herds which were positive to the ring test. The ring test at six month intervals as an adjunct to the blood test was adopted as a regular part of the control program in the counties under the area plan. At this writing, 25 of the counties have had at least four county-wide ring tests and the fifth test is under way. Methods.-As time does not permit the presentation of the details of the preparation of the antigen, the collection of the milk and cream specimens and the techniques of conducting and reading the test, reference is made for these detailed procedures to previous publications.'° - ' 2 The antigen was prepared from either the official U. S. Bureau of Animal Industry stock Brucella abortus tube or the plate antigen. The antigen was stained with hematoxylin by a slight modification of the method supplied by Dr. Aage Jepsen of the Royal Veterinary College, Copenhagen, Denmark on his visit to our laboratories in 1947. Composite milk samples of approximately 9 or 10 cc were obtained from each herd by collecting about 3 cc quantities of milk from each one of two or three milk cans received from a herd at the receiving platform of the dairy plant or creamery. Cream specimens from each herd were obtained in a similar manner. The ring tests were conducted in a portable laboratory, arranged in a county court house or a public building, or in a trailer laboratory centrally located from the standpoint of the creameries and dairy plants in the area under study. As a fairly high percentage of Minnesota dairymen market cream only, it was necessary to modify the test, to render it applicable to cream. The test on cream was performed by adding 12 drops of cream and one drop of antigen to a small tube containing 1 cc of physiological saline and two drops of a saturated sodium carbonate solution. The sample was then mixed thoroughly, left at room temperature and the results noted as for the test on milk. Arbitrary designations were used to indicate the degree of reaction to the ring test and were given designations ranging from negative to 4+ depending on depth of color of the cream ring as compared with the skim milk fraction. The tests were read as negative if the cream line or ring were lighter in color than the skim milk fraction; 1 + if the blue color of the cream ring were equal to that of the skim milk fraction; 2+ if the color of the cream ring were slightly darker blue than the skim milk fraction; 3+ if the color of cream ring were markedly bluer than the skim milk fraction and 4+ if the color of the cream ring were very dark blue and the skim milk fraction essentially white. Results of Field Studies: Table I is a summary of the study in the nine counties in which the herd ring tests preceded, by a short interval, the

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

136

county-wide blood test. As may be noted from this summary, the overall agreement between the two tests was slightly over 96%. This value by itself is not particularly significant in that the average percentage of infected herds in these counties was approximately 4.5%. If all of the ring tests had been negative there would have been approximately 96.5% agreement. Of major interest are the values shown in the right hand column which indicate the per cent efficiency of the ring test in locating the infected herds or the per cent of infected herds which were positive to the ring test. An average of 68% of the infected herds were positive to the test. Of the 120 herds which were negative to the test, 107 were investigated to determine the possible cause or causes for the failures. Of the 107 failures to detect the infected herds, 64% were due to non-producing reactors present. Usually only one such animal was TABLE I.-Comparison of results of blood tests with herd ring tests in counties with county-wide blood tests County

Koochiching Itasca St. Louis

No. Herds Ring Tested

228 584 2,704

Agreement of Ring and Blood Tests BloodRing -

210 573 2,502

Blood + Ring +

Per cent Agreem't

11 4 61

96.0 98.8 94.7

Disagreement Blood + Ring -

Blood Ring +

Per cent Efficiency in Locating Inf. Herds

5 2 20

2 5 121

69 67 75

49

48

1

100.0

0

0

100

Watonwan Wilkin Hubbard Kitson Roseau

828 669 1,054 990 1,363

748 558 1,014 906 1,326

56 69 15 33 14

97.8 94.8 98.5 94.9 98.1

15 15 15 39 10

9 26 10 12 13

79 82 50 46 58

Total

8,469

7,885

264

96.2

121

198

Lake

1

68

present per herd. The ring test was positive on approximately 90% of the infected herds in which one or more infected animals were in production at the time of the test. In the last three counties listed in Table I, the ring tests were conducted during the late summer or early fall months during which time an appreciable percentage of the dairy cows were dry. This suggests that to obtain best results the ring tests should be made during the season of highest milk production in an area. The number of herds positive to the ring test but negative to the blood test (false positive ring test) was unusually high in St. Louis County. This was due to a deliberate disregard of the warning of the Danish workers with respect to the collection of milk samples from the weighing tank on the receiving line in the dairy plants. The ring test is so sensitive that the small amount of milk from an infected herd which remains

DIAGNOSIS OF ANIMAL BRUCELLOSIS

137

in the weighing tank is sufficient, in many instances, to cause a positive reaction on the sample from a negative herd which follows. The results of blood tests in 650 herds positive to the ring test on cream and 650 herds positive to the ring test on milk are shown in Table II and Figure 1. An over-all agreement of the two tests of 70% for the positive ring test on cream specimens and 62% for the positive ring test on milk was found. It was considered that there was agreement between the two tests even though some herds were classified as suspicious on the basis of the blood test results. As might be expected, the percentage of herds classified as reactor herds was highest for those on which 3 or 4+ ring tests reactions were obtained. TABLE II.-Results of blood tests on herds positive to ring tests Positive Herd Cream Tests Comparisons

120 + Tests R* SI NI

197 ++ R

S

226 +++ N

R

S

98 ++++ N

R

Number of herds... 38 13 78 115 25 57 159 20 47 77 Per cent of herds... 29 10 60 58 13 29 70 9 21 79

S

N

Agreement

R

S

Tota No. Herds

N

7 14 385 65 96 650 7 14 60 10 30 's, 70 &

Positive Herd Milk Tests 88 + Tests

145 ++

150 +++ --

267 ++++

Number of herds... 10 9 69 42 18 85 79 23 48 193 28 46 324 78248 650 Per cent of herds... 11 10 78 29 12 59 53 15 32 72 11 17 12 38 ~, 62

R = Herds with one or more animals positive to blood test. S = Herds with one or more animals giving "suspect" titers but no reactors. N = Herds negative to blood test.

As may be noted from Figure 1, it would appear that the test as used on the cream specimens was less sensitive than that for milk. Another point of interest is that the percentage of herds classified as suspicious on the basis of the blood test results was relatively constant irrespective of the degree of reaction to the ring test either on milk or cream. Table III is a summary of the results obtained in those counties in which county-wide blood tests were made either between the second and third or between the third and fourth ring tests. The first two counties are predominately dairy counties whereas the other three counties contain a fairly high percentage of beef herds. Carlton County is on the border of a district which is not on the area control plan and has had a history of a high reinfection rate between county-wide blood test. The third ring test was made shortly after the county-wide blood test was started. The county-wide blood test was conducted by the

138

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

local veterinarians and a period of approximately 9 months was required to complete the work. It is reasonable to assume that some of the 17 infected herds disclosed first by the blood test became infected in the interval between the two tests. This is suggested by the fact that the fourth ring test which was made within two weeks after the completion of the county-wide blood test disclosed 12 new infected herds contained 18 reactor animals. Of the 17 infected herds which were first disclosed by the county-wide blood test, 3 were positive to the fourth ring test in the county. It is of considerable interest to note that the three ring tests plus the county-wide blood test disclosed a total of 97 Numbers on top of. blocts - Total numberherds in eochgroup

l[-

Herds Cassed os Negotive on Blood Test D* 'iuect» ' ..

_

88

. 145 197

129

...

t.nfected" .

150 226

"

267 98

6 4

2

·--

+-I+t'+ Rino Test Reodinos on Herds

+++'++

FIGURE 1.-Results of blood tests on herds showing various degrees of reactions to the ring tests

new infected or reactor herds, whereas the four ring tests alone would have disclosed at least 95 new reactor herds. The results obtained in Clearwater County, also a predominately dairy county, were very similar to those obtained in Carlton County. Of the four infected herds which were first disclosed by the county-wide blood test, 3 were positive to the third ring test in the county along with one additional new infected herd. Although there was a time interval of approximately four months between the completion of the county-wide blood test and the third ring test, the three county-wide ring tests disclosed as many new centers of infection as were found by two countywide ring tests plus the county-wide blood test.

DIAGNOSIS OF ANIMAL BRUCELLOSIS

139

If the relatively high percentage of beef herds in the other three counties listed in Table III are taken into consideration, the results obtained with the ring test in these counties would appear to be reasonably satisfactory. It is evident, however, that if a ring test program were to be adopted as an adjunct to the blood test for brucellosis control in TABLE III.-Results of county-wide blood tests following two or three county-wide ring tests for brucellosis Ring Tests New Infection County

No.s Herds

No. Test

New Inf. Herds

Carlton Clearwater Clay Marshall Norman

1601

1302

1506

2084

1588

New Inf. Cattle

New Inf. Herds

3 4th

80 12

365 18

(13, + ring

2 3rd

12 1

52 2

3, + + ring ring

3 4th

54

222 1

3 4th

65

2 3rd

39

1

9

10

%of Total New Infection Disclosed by Ring Test

County Blood Test Additional Infection

288 23

(20

233 24

48

New

Inf.

Herds

Cattle

19

82%

95%

5

75%

91%

41

75%

85%

45

76%

85%

92

45%

72%

Cattle

1, + ring

TABLE IV.-Partial county blood tests for recertification following three county wide ring tests in Pennington and Red Lake counties Pennington (Est. 1000 hds., 17,000 cat.) New inf. disclosed No. Herds

Three ring tests ........................ Blood tests (491 herds, 6926 cattle) ...... (243 herds, 4262 cattle) .....

18 3

No. Cattle

59 3

Red Lake (775 hds., 15,000 cat.) New inf. disclosed No. Herds

No. Cattle

36

156

2

2

Blood tests were conducted on only old reactor herds plus those herds which had not had one or more ring test.

areas of this nature, provisions should be made for blood tests at suitable intervals on all herds which are not covered by the regular ring test program. Table IV is a summary of the results obtained in two counties, also containing an appreciable percentage of beef herds, in which tests were made only in those herds which had not had one or more ring tests

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plus those herds which were on the infected list; The results in these two counties suggest that the major portion of the infection was confined to the herds producing market milk or cream. Relatively little infection was found in the large number of herds in these counties from which cream or milk samples had not been obtained for the ring test. CONCLUSIONS The fact that the ring test was negative on approximately one-third of the infected herds and from the fact that the blood tests were negative on approximately one-third of the herds which were positive to the ring test, the ring test cannot be considered a highly reliable test on a pooled milk or cream sample from a herd. It would seem more logical to consider the test as a highly sensitive presumptive or screening type of test which, to be effective, should be used in conjunction with the blood test in an official disease control program where there are provisions for follow-up blood tests in all herds which are positive to the ring test and where there are provisions to give assistance and counsel to the owners of the infected herd disclosed. It is a test that may be used to direct the blood testing work to those herds most likely to be infected, which provides a rapid and inexpensive means in conjunction with the blood test of locating infected herds or new centers of infection. Used in this manner, it may offer a means of appreciably speeding up the brucellosis control program in many areas where dairying is the predominate cattle industry without an appreciable increase in cost and in personnel, both of which are major factors at this time in a brucellosis control program. The inaccuracies and natural limitations of the test may be overcome to a great extent by repeating the test at six month intervals. The effectiveness when used in a program of this nature is indicated by the results in the two predominately dairy counties, namely Carlton and Clearwater. The number of new centers of infection which were disclosed by the county-wide blood test alone was very low. The results obtained in these two counties along with the evidence obtained from preliminary summaries of the results obtained in several other similar counties indicate to us that there is a rather limited justification for conducting county-wide blood tests for recertification of counties of this nature when the limited personnel we have and the funds involved could be used much more advantageously and effectively to extend the control and eradication program to new counties and districts. We are convinced that a control program of county-wide ring tests alone at six month intervals will keep the number of unknown centers of infection in a predominately dairy county at an appreciably lower level than would be obtained with county-wide blood tests alone at intervals of 3 years. The over-all requirements in terms of personnel and funds would also be appreciably lower.

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The relatively high percentage of false positive ring tests found in this study, although objectionable, does not seriously interfere with the use of the test as an adjunct to the blood test in counties of the type involved in this study. The average per cent of infected herds in the counties was below 2.5%. The problem of the false positive ring test is somewhat similar to that of the "suspect herd" found with the blood test, that is, the herd with one or two animals with suspicious or doubtful titers and all other animals negative to the blood test. We have frequently experienced with county-wide blood tests nearly as many, and in some instances more, suspect herds than herds classified as infected on the basis of the blood test results. Our data indicate an increased percentage of false positive ring tests with the successive tests in the counties. There would appear to be a low base line of false positive tests in counties in which there has been relatively little infection for a number of years. Another factor which contributes to the number of false positive ring tests is the tendency for a positive test on a herd to persist for a period of time after the animals designated as infected by the blood test are removed from the herd. The response of the herd owners to the ring test program has been excellent. In fact, the response might even be termed "enthusiastic". In the past the herds have gone from three to six years without a blood test. The dairymen become concerned regarding the status of their herds during these long intervals. The frequent checking of the herds provided by the ring test, is one of the chief reasons for the enthusiasm of the dairymen to the test. From the disease control standpoint, it is very desirable to locate new centers of infection as early as possible in order to reduce the spread of the disease both in the herd as well as to neighboring herds. In this respect, it has been of considerable interest to us to note on the successive ring tests in a county that an increased percentage of the new infected herds contained but one or two reactors. Early disclosure of an infection is of major importance to the herd owner. Very limited studies have been made on the effect of vaccination, with Brucella abortus Strain 19, on the test. A study of several adult animals which were followed closely after vaccination, along with the experiences obtained with the test in the field, suggest that vaccination with Strain 19 may interfere to some extent with the ring test as a diagnostic aid. Three adult animals vaccinated with Strain 19, although strongly positive to the ring test shortly after vaccination, became negative to the test in 4 or 5 months, whereas the blood titer remained appreciably above 1 to 100. These results along with the experiences of the test under field conditions suggest that the majority of animals with a persistent blood titer due to vaccination, are negative to the ring test. These limited observations are in agreement with the report of Traum and Maderious1 3 on milk-whey agglutination titers of animals with

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vaccinal titers as compared with those from which virulent Brucella had been isolated from the milk. Of 50 adult animals vaccinated with Strain 19, 48 became negative to the whey agglutination test at the 1:12.5 dilution within 3 months after vaccination although the animals remained positive blood reactors. Of 89 animals from which virulent Brucella had been isolated from the milk in 85 cases, the highest whey titers for the milk from any one of the 4 quarters were above 1:12.5. Similar observations were reported by Holm, Eveleth and Rheault with the ring test on milk from animals with persistent vaccinal titers when such milk was diluted to varying degrees with negative milk. They found in a high percentage of cases that the ring test on such milk at dilutions above 1:10 with negative milk, was negative whereas the opposite was true with milk from animals with positive blood reactions and a history of long-standing infection. The estimated cost of a county-wide ring test is about 10% of that of a county-wide blood test in areas where the infection rate is low, as was true in this study. However, the cost would increase appreciably as the infection rate increased due to the cost of conducting the necessary follow-up blood test on the herds which were positive to the ring test. The reports by Bruhn and by Christiansen on the results of simultaneous blood and ring tests on individual animals suggests that the ring test is of limited value on an individual animal basis. They found a significant percentage of positive ring tests on animals which were negative to the blood test. ACKNOWLEDGEMENTS

It is desired to give credit for the large amount of field work involved in this cooperative study to members of staff of the U. S. Department of Agriculture, Bureau of Animal Industry, namely, Doctors L. B. Calusen, J. E. Wentworth and Louis Olson. REFERENCES (1) Fleischhauer, G.: Die Abortus-Band-Ringprobe (ABR) zue Festellung von bangverdachtigen Vollmilchproben. Berl. tierarztl. Wchnschr., 53: (1937): 527-528. (2) Fleischhauer, G., and Hermann, G.: Ueber weiters Erfahrungen mit der Abortus-Bang-Ringprobe (ABR) bei der Untersuchung Von Milchproben auf Abortus Bang. Berl. tierarztl. Wchnschr., 54: (1938): 333334. (3) Norell, N., and Olsen, A.: Om várdet av serologiska mjolkundersoknigar medlst "ABR" (Abortus-Bang-Ringprobe). Skand. Vet.-tidskr., 38: (1943): 321-341. (4) Winther, O., and Hansen, A. C.: Undersgelser over Abortus-Bang-Ringproven. Maanedsskr. Dyrl., 55: (1943): 401-416. (5) Seit, B., and Leth Jergensen, K.: Abortus-Bang Ringprovens Vaerdi som diagnostisk Hjaelpemiddel. Maanedsskr. Dyrl., 56: (1944): 227-292. (6) Bruhn, P. A.: Abortus-Bang-Ringproven Valleog Serum-titer. Medlemsbl. Danske Dyrl., 27: (1944): 477-502.

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(7) Bruhn, P. A.: Problemer der Knytter sig til Abortus Bang-Ringproven. Skand. Vet.-tidskr., 38: (1948): 71-93. (8) Bruhn, P. A.: The Brucella Abortus Ring Test. Am. Jour. Vet. Res., 9, (1948): 360-369. (9) Christiansen, M. J.: Om Abortus-Bang-Ringproven og dens Anvendelse i den praktiske Kastningsbekaempelse. Maanedsskr. Dyrl., 69: (1948): 193-230. (10) Roepke, Martin H.; Clausen, Lawrence B.; and Walsh, Andrew L.: The milk and cream ring test for brucellosis. Proc. 52nd Ann. Meeting, U. S. Livestock Sanitary A., (1949): 147-159. (11) Roepke, Martin H.; Paterson, Katherine G.; Driver, Fred C.; Clausen, L. B.; Olson, Louis; and Wentworth, J. E.: The Brucella abortus ring test. Am. Jour. Vet. Res., XI (1950): 199-205. (12) Roepke, Martin H.: The Brucella abortus ring test. Brucellosis (A Symposium) by the Am. Assoc. Adv. Sci., Washington, D. C., 1950, pp. 126135. (13) Traum, J., and Maderious, W. E.: The interpretation of whey agglutination test results in cows vaccinated with Brucella abortus strain 19. Am. Jour. Vet. Res., 8 (1947): 244-246. DIAGNOSTICO DE LA BRUCELOSIS EN LOS ANIMALES, CON REFERENCIA ESPECIAL A LA PRUEBA DEL ANILLO (Sumario) De conformidad con el plan para el control de la brucelosis en toda una zona, en 28 condados se ejecutaron de tres a cuatro pruebas del anillo en muestras de leche y de crema. Al iniciarse los estudios menos de 5% de los rebaños estaban infectados. En nueve condados se hicieron pruebas sanguíneas de todo el ganado después de las pruebas del anillo. En otros seis condados las pruebas sanguineas se hicieron después de dos o tres pruebas del anillo. En estas condiciones (menos de 5% de rebaños infectados) se obtuvieron los siguientes datos del estudio comparado: cuando s61o se practicó una prueba del anillo, esta resultó positiva en mezclas de leche y crema del 68% de los rebaños infectados. Aproximadamente dos terceras partes de los fracasos de la prueba del anillo se debieron a que los animales infectados (por lo común sólo uno) no se encontraban en producción al hacer la prueba del anillo. Esos rebaños infectados se descrubren por lo general al realizar una prueba del anillo en todo el condado seis meses después, cuando los animales infectados están en producción. La prueba resultó positiva aproximadamente en 9 de 10 rebaños con uno o más animales infectados en producción. En los condados en que predomina la producción de leche, 3 6 4 pruebas del anillo en todo el ganado, realizadas con intervalos de 6 meses, revelaron aproximadamente 85% de los rebaños infectados y 95% del ganado infectado. En varios de los condados en que se hicieron pruebas de sangre entre la tercera y cuarta pruebas del anillo, se descubrió que las cuatro pruebas del anillo revelaron prácticamente el mismo número total de nuevos centros de infección que las tres pruebas del anillo más la prueba sanguínea en todo el condado. De los rebaños positivos a la prueba del anillo, aproximadamente la tercera parte resultaron negativos con la prueba sanguinea. Las discrepancias o errores de esta naturaleza son algo parecidos a las discrepancias observadas con la prueba sanguínea, en que muchos rebaños se clasifican como sospechosos debido

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a la presencia de uno o dos animales con un título sanguíneo sospechoso. Tomando por base los resultados de las pruebas sanguíneas posteriores, se clasifica como negativo un elevado por ciento de estos rebaños. Aunque ofrece reparos, el problema de las pruebas del anillo aparentements seudopositivas no afecta en forma marcada el empleo de la prueba como presuntiva para los rebaños. Como prueba presuntiva permite limitar las pruebas sanguíneas a los rebaños que es más probable estén infectados, permitiendo así evitarla en numerosos rebaños negativos o no infectados. Los estudios limitados en la leche de animales adultos vacunados con la Cepa 19 de Brucella abortus indican que en un por ciento relativamente elevado de casos a los 4 a 6 meses de la vacunación la leche de dichos animales es negativa a la prueba del anillo, o negativa a diluciones relativamente bajas con leche de animales no infectados.

COMPARATIVE STUDY OF MEDIA ORDINARILY USED FOR THE GROWTH OF BRUCELLA SPECIES By GENESIO PACHECO AND MILTON THIAGO DE MELLO The need for a liquid medium yielding good and early growth of Brucella species, stimulated a large amount of research in various laboratories. Tryptose broth and trypticase broth are at present the most widely used as were some time ago liver broth and placenta broth. Chemically defined media, although of great advantage for chemical studies of Brucellae are not well suited for rapid and simple production of large quantities of microorganisms. In order to reexamine this question, experiments were carried out with some of the media ordinarily used for the growth of Brucellae. Strains of the three Brucella species were grown in meat extract broth, veal infusion broth, dextrose veal infusion broth, liver citrate broth, bovine placenta broth and placenta glycerol broth. The intensity of growth was estimated with the aid of "Lumetron" (Mod. 400-G) interposing filter No. 530. The media were dispensed in test tubes in amounts of 10 ml and were inoculated with 0.1 ml of a 48 hour culture of each of 10 strains of Brucellae from our culture collection. After 48 hours of incubation at 37°C, the cultures were transferred to matched test tubes and the optical density was measured. We observed the following decreasing graduation in the ability of the media to support the growth of Brucellae: placenta broth, meat extract, dextrose veal infusion broth, meat infusion broth, glycerol placenta broth and citrate liver infusion broth. Similar experiments were conducted using veal infusion broth, dextrose veal infusion broth, liver infusion broth, placenta infusion broth and tryptose broth, but nine strains of Brucellae were used and the intensity of growth was measured by the same method after 20 hours. The best results were observed with veal infusion broth followed in decreasing order by placenta infusion broth, tryptose broth, dextrose veal infusion broth, and liver infusion broth. In both experiments liver infusion broth yielded the poorest growth. The preliminary experiments summarized above showed that Brucellae could be grown in relatively simple media and that veal infusion broth seemed better than tryptose broth for the early development and this fact could be seen even by visual comparison. The reading of the optical density of cultures in the "Lumetron" in low concentrations of microorganisms was not very sharp and this fact led us to more detailed work using greater number of media and measuring the intensity of growth with the Pulfrich nephelometer. All the results presented in this paper represent the differences between the initial 145

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relative turbidity of the media and the relative turbidity of the same media inoculated and after a variable time for each experiment. The relative turbidity was the mean of 5 readings in the apparatus using filter No. 1 (red) and standard turbidity disc No. 2. In all the experiments we used the strain 955 of Brucella suis received from "Instituto Biológico de Sao Paulo" and originally from Dr. Huddleson. The media were dispensed in standard 16 x 160 mm test tubes (Pyrex) in amounts of 10 ml unless otherwise stated and were inoculated with 0.1 ml of a 20-24 hour culture of microorganisms generally in tryptose broth. 1. In preliminary experiments we used media prepared by two types of pancreatic digestion (A and B) of bovine placenta, beef heart and bovine spleen and liver, dextrose broths prepared with the same organs, veal infusion and dextrose tryptose broth. Digested medium A was prepared according to the technique of Brown,* with 1% dextrose; in some batches a turbidity was observed after the sterilization and the medium was again filtered and sterilized the same way (115°C-20 minutes); the pH changed from 6.8-7.2 to 6.7-7.0; both were used in the tests. Digested medium B was being employed in this laboratory for production of gas gangrene toxins and was prepared as follows: to each 500 gm of ground fresh organs, 1000 ml of distilled water were added and the mixture was heated at flowing steam during 40 minutes; the reaction was adjusted to about pH 8.4 and 6 gmr of pancreatin were added. The mixture was digested at 500C overnight; in order to avoid contaminations some batches were digested under toluene; the following day the medium was filtered through cotton gauze and the pH adjusted 4.2; the medium was kept in the refrigerator at 5°C overnight; the clear supernatant was filtered through paper and the pH adjusted to 7.6; 1% peptone (Bacto), 1% dextrose (Pfanstiehl) and 0.5 sodium chloride were added. The medium was heated during 10 minutes at 115°C, filtered through paper while hot, distributed and sterilized (115°C for 20 minutes). Dextrose broth (1% peptone, 1% dextrose, 0.5% sodium chloride) was prepared by the conventional methods. Spleen infusion broth presented a high final pH (9.2) and was deep brown; it was adjusted to about pH 7.4 and cleared by mixing it with equal parts of a phosphate buffer solution. Liver infusion broth was cleared with equal parts of distilled water. The pH of all media before the final sterilization ranged from 7.4 to 7.9 but almost always dropped to very low figures after that, as seen in Table 1. Dextrose tryptose broth (1% dextrose) had a final pH of 7.2. Veal infusion broth was obtained from the stock of general media room of the Institute. * Brown, J. H.: Media prepared by the pancreatic digestion of meat. Jour. Bact., 1948, 55 (6): 817-872.

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In that first experiment turbidities were read in the nephelometer with different color filters and standard turbidity discs and as the results were almost the same, we will present only the data obtained with filter No. 1 (red) and disc No. 2, which were the same used in subsequent experiments. The results after 48 hours of incubation at 37°C are shown in Table 1. The results of this preliminary experiment indicated that the best media for the growth of Brucella suis strain 955 in 48 hours were the following, in decreasing gradation: (1) beef heart digested medium A; TABLE

1.-Difference between relative turbidity before and after 48 hour incubation of different media inoculated with Br. suis Medium

pH

Difference

6.8 6.8 6.5 6.5 6.1 6.5 6.1

161.8 106.1 82.3 71.4 52.6 42.2 17.0

6.7 6.5 6.4 6.6 6.5

53.8 7.1 71.7 0.5 22.4

6.3 6.5 6.5 7.8

81.6 147.7 8.3 0.7

7.0 6.5 6....

72.0 44.0

Digested medium A

Beef heart ............................... Beef heart (sterilized twice) .............. Bovine placenta (ster. twice) ............. Bovine liver ............................. Bovine liver (sterilized twice) ............ Bovine spleen ............................ Bovine spleen (sterilized twice) ........... Digested mediumz B

Beef heart (digested without toluene).... Beef heart (digested under toluene) ...... Bovine placenta (dig. under toluene)..... Bovine liver (dig. under toluene) ......... Bovine spleen (dig. under toluene) ....... Dextrose broth

Beef heart .............................. Bovine placenta .......................... Bovine liver ............................. Bovine spleen ............................ Controls

Veal infusion broth ...................... Dextrose tryptose broth .............

(2) dextrose placenta broth; (3) beef heart digested medium A sterilized twice; (4) bovine placenta digested medium A sterilized twice; (5) dextrose beef heart; (6) veal infusion broth; (7) placenta digested medium B under toluene, and (8) liver digested medium A. Some media yielded a fair growth and other a poor growth as some spleen and liver media. This fact was possibly due to repeated filtrations and sterilizations as well as variations in their final pH. 2. In another experiment the media were buffered during their processing and to part of them 1% peptone were added in order to see whether the growth could be improved. The media were prepared by pancreatic digestion following Brown technique and after filtration the

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volumes were completed to 1,000 with distilled water and the pH adjusted to 7.2. To each liter, 5.76 gm Na2 HPO 4 and 0.48 gm KH 2P04 were added; the media were heated in flowing steam for 10 minutes, filtered while hot and kept in the refrigerator overnight. To one part of the media 1% dextrose and 0.5 sodium chloride were added; to the other part the same substance and 1% peptone were added. They were heated again in flowing steam for 10 minutes, filtered, dispensed in flasks and sterilized at 115°C for 30 minutes. The slight precipitate formed was avoided by pippeting 10 ml of the supernatant to the tubes for the tests. Dextrose tryptose broth contained 2% tryptose, 1% dextrose and 0.5 sodium chloride. After inoculation the tubes were incubated and the turbidity read after 24 hours. TABLE 2.-Difference between relative turbidity before and after 24 hour incubation of different digested media buffered at pH 7.2 with and without peptone, inoculated with Br. suis Medium

Digested medium A Beef heart ........................................ Beef heart, with peptone ......................... Bovine liver ..................................... Bovine liver, with peptone ........................ Bovine placenta .................................. Bovine placenta, with peptone ................... Bovine spleen .................................... Bovine spleen, with peptone ...................... Controls Dextrose tryptose broth .......................... Veal infusion broth ...............................

Difference

5.5 0.4 1.1 0.2 4.8 0.6 0.5 0.2 17.5 21.1

The results are shown in Table 2. The results indicate that the buffering of the media does not improve the growth of the strain tested and was even unfavorable for that purpose, while in dextrose tryptose broth and veal infusion broth the growth was fairly good. Peptone did not improve the growth and some media, as beef heart, gave better results without peptone. 3. A new series of tests was conducted preparing media only with beef heart and following all the methods referred above. Placenta media were not prepared because they gave rise to difficulties during filtration. The results are shown in Table 3. Again in that experiment the controls supported better growth than the digested media; only in digested medium A good early growth was observed, which became better than that observed in dextrose tryptose broth in 48 hours.

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4. In order to verify whether beef heart digested medium A contained growth promoting substances which could be extracted from it and possibly improve the growth of Brucellae when added to other media, two parallel experiments were conducted. Beef heart digested medium A was mixed with ether (1:1) and kept undisturbed in a separatory funnel overnight; the medium in the botTABLE 3.-Difference between relative turbidity before and after 24 and 48 hour incubation of media preparedwith beef heart, inoculated with Br. suis Differences Medium 24 hrs.

48 hrs.

medium A, buffered ............. medium A ...................... medium A (sterilized twice) ... medium B, buffered ............. medium B ...................... broth ...........................

8.9 17.6 9.0 0.7 1.7 9.3

27.9 35.3 24.3 1.0 3.4 22.3

Controls Dextrose tryptose broth .............. Veal infusion broth ...................

19.6 19.8

30.4 45.1

Digested Digested Digested Digested Digested Dextrose

TABLE 4.-Difference between relative turbidity before and after 20 and 42 hour incubation of media with extracts of beef heart digested medium A, inoculated with Br. suis Differences Medium

Dextrose tryptose broth + ether - extract........... Veal infusion broth + ether - extract .............. Dextr. tryp. broth + acetone - extract ............. Veal infusion broth + acetone - extract ............ Digested medium A ether-extracted ................. Digested medium A acetone-extracted ............... Controls Digested medium A ............................. Dextrose tryptose broth ......................... Veal infusion broth .............................

20 hrs.

42 hrs.

16.4 17.8 10.8 14.8 4.8 2.9

24.3 36.8 16.4 28.5 11.7 9.6

4.4 13.6 14.1

10.6 24.4 28.5

tom was dispensed in the test tubes in amounts of 10.5 ml and the supernatant ether solution was added to dextrose tryptose broth and veal infusion broth (5 ml of ether solution and 10 ml of media). Another part of the digested medium was mixed with acetone (1:1), kept in the refrigerator overnight, and centrifuged. The acetone in the supernatant was evaporated in front of a fan and dispensed in the tests tubes in amounts of 12 ml. The precipitate was washed twice in acetone,

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centrifuged and partially dried; the residue was diluted in distilled water and 0.5 ml of the dilution was added to tryptose broth or to veal infusion broth. The media were sterilized at 115°C for 20 minutes, as well as the controls (medium without extraction, tryptose broth and veal infusion broth). The readings of the turbidity were made after 20 and 42 hours and the differences of turbidity are shown in Table 4. The experiment showed that veal infusion broth plus ether-extract of digested medium A was better than the other in 20 and 42 hours. Dextrose tryptose broth plus the same extract was better than the control in 20 hours and almost equal in 42 hours. CONCLUSIONS The experiments showed that very elaborated media are not necessary for the growth of Brucella and that the common media dextrose tryptose broth and veal infusion broth are adequate for that purpose, at least when stock cultures are being cultivated. For isolation we used any of the two referred media plus 5% horse serum. Some batches of tryptose were markedly bacteriostatic when the medium did not contain dextrose or horse serum. ESTUDIO COMPARATIVO DE LOS MEDIOS USADOS COMtNMENTE EN EL CULTIVO DE ESPECIES DE BRUCELLA (Sumario) Con el objeto de seleccionar los mejores medios comunes para el cultivo de especies de Brucella, realizamos experimentos preliminares de diez cepas de las tres especies de Brucella cultivadas en los siguientes caldos: infusión de res, extracto de carne, infusion de higado citrado, infusion de carne glucosa (dextrosa), infusion de placenta bovina y triptosa. Se midió la intensidad de crecimiento después de 24 horas de incubación a 37°C, con un colorímetro fotoeléctrico "Lumetron". En estos experimentos se comprobó que el mejor medio es la simple infusión de caldo de res, y después, la infusión de caldo de placenta bovina. Después de 48 horas de incubación, el crecimiento fué ligeramente más abundante en caldo de placenta bovina que en la simple infusión de caldo de res. Luego se realizó otra serie de experimentos con una cepa de Brucella suis cultivada en varios medios liquidos. La intensidad de crecimiento fué medida con un nelfelómetro (Photometre de Pulfrich pur la mesure des troubles). Los caldos usados fueron los mismos empleados en los experimentos preliminares, y además se utilizaron varios tipos de digestos trípticos de carne de ternera, músculo de corazón de res, hígado, bazo y placenta. Algunos de los medios fueron también tamponados al pH 7.2. La simple infusión de caldo de res produjo un crecimiento más abundante que los otros medios ensayados. En unos pocos casos, algunos medios mostraron notable resistencia a la Brucella suis, incluyendo algunos lotes de caldo de triptosa.

THE CARBON DIOXIDE REQUIREMENTS OF BRUCELLA ABORTUS

By A. G. MARR AND J. B. WILSON Research Assistant and Associate Professor, respectively, Department of Agricultural Bacteriology, the University of Wisconsin, Madison, Wisconsin A rational approach to chemotherapy of brucellosis requires that we have among other things an understanding of the physiology of the Brucellae. The establishment during the past fifteen years of the theory of competitive inhibition of enzyme systems in microorganisms of chemical agents has opened a new approach to the problem of chemotherapy. The literature on this subject and its application to treatment of disease has been clearly presented in the monograph, "The Basis of Chemotherapy" by Thomas and Elizabeth Work. This means, then, that we are in need of information on the mechanism of synthetic and of energy yielding reactions as carried out by the Brucellae. When we have this information we may be in a much better position to predict what agents might be used in chemotherapy of brucellosis. We must keep in mind, however, that the enzymatic reaction we wish to block in the bacterium should be unique to it and not be a reaction that would be blocked equally as well in the host. A study of the carbon dioxide requirements of Brucella abortus is indicated here for it is well known that cases yield cultures that require added carbon dioxide for primary isolation. Specific blocking of the mechanism for assimilation of carbon dioxide is a likely step in chemotherapy of Brucella abortus infections, since the metabolism of carbon dioxide in the parasite may differ from that of the host. It was recognized very early that Br. abortus was difficult to cultivate in air, but that growth could be obtained by using sealed culture tubes. Bang, in 1897, using different atmospheres of O2 and CO2 thought that growth of the organism was influenced by the lowered pO2. Novak, in 1908, cultivated Brucella abortus on the surface of solid media by enclosing the tubes in a jar with actively growing cultures of the highly aerobic Bacillus subtilis. Novak, as Bang had done previously, interpreted his data in terms of the lowered pO2 brought about by the growth of Bacillus subtilis. Numerous other workers reported growth of other pathogens such as the gonococcus and the meningococcus using Novak's technique, and their data were interpreted to mean these organisms required a lowered PO2. There were, however, some dissenters from this view and notable among them was Gates who, in 1919, thought that the change in pH brought about by the increased pCO2, might be a determining factor in the growth of the meningococcus. In 1921, Huddleson 151

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showed that chemically generated C02 introduced into the culture environment in a concentration greater than that of air would stimulate the growth of Brucella abortus and that the growth was not due to a reduced pO2. Huddleson concluded that a concentration of 10% C02 in the atmosphere was optimum for the growth of the abortion organism and that this pCO2 made the pH of the medium more optimal for growth. Next, Theobald Smith demonstrated that the size of the inoculum influenced the pCO2 required for growth; the larger the inoculum the less CO2 required. Smith showed also that C02 could not be replaced with hydrogen or nitrogen and he expressed doubts that the presence of C02 simply adjust the pH of the medium to that favorable for Brucella abortus to grow. Finally, in 1931, G. S. Wilson proved that C02 per se is required and that bicarbonate would not substitute for it. Wilson concluded that C02 acts not by altering the acidity of the medium but by virtue of its power of diffusing rapidly through the intact cell wall, and giving rise to an increase in the intracellular hydrogen-ion concentration. Let us look for a moment at the C02 requirements of other heterotrophic microorganisms. Valley and Rettger as well as Gladstone, Fildes, and Richardson have pointed out that the requirements for carbon dioxide among heterotrophic bacteria is extremely widespread. Apparently the requirement of Brucella abortus for a pCO2 greater than that of air is unique only in a quantitative sense. C02 is known to play an important role in heterotrophic metabolism for Wood and Werkman demonstrated ten years ago the fixation of CO2 by a heterotrophic bacterium and, more recently, Ajl and Werkman have shown C02 to be necessary in the cellular synthesis of dicarboxylic acids. Pappenheimer and Hattle and, also, Buchanan, Sonne, and Deluva have shown C02 to be necessary in the synthesis of nucleic acid constituents. Lwoff and Monod have indicated that C02 is probably involved in the synthesis of other, as yet unknown, intracellular catalysts. As a part of our investigations into the metabolism of the Brucellae, and in particular the C02 metabolism, the stability of Br. abortus for added carbon dioxide was determined. It is well known that strains of Br. abortus which require added C02 for growth can be trained to dispense with their added CO2 requirement. When slants are heavily inoculated with carbon dioxide requiring strains and incubated in air, usually one or more colonies develop on the slant. Theobald Smith interpreted this to mean that the single bacterium had been kept alive by the C02 production of many perishing bacteria. However, if these few colonies are tested it is found that they have lost the requirement for added carbon dioxide. This suggested to us a mutational loss of the added C02 requirement. For the determination of mutation rate, flasks containing 100 ml of Albini brucella broth were inoculated with 104 to 105 cells from a 24 hour

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culture grown under 10% carbon dioxide. One ml of the inoculated broth was then transferred aseptically to each of 15 sterile (1.5 x 9 cm) tubes or 10 ml to each of 10 sterile Pyrex milk dilution bottles and incubated in a 10% carbon dioxide-90% air mixture for 48 hours. Samples were taken from 2 or 3 cultures of each series for total counts which were determined by dilution and plating. These plates were incubated for 4 days in 10% carbon dioxide. Mutant counts were determined by direct plating of whole 1 ml cultures or aliquots of the larger 10 ml cultures and incubation of these plates in air. From the data obtained in these experiments mutation rates could be calculated from the total number of mutants or from the fraction of cultures containing no mutants using the formulas of Luria and Delbrúck as modified by Lederberg. The data given in this slide show very similar mutation rates for the nine strains tested. One of the strains, 6232C, was received by us with the history that it was CO2 stable; note that it has the lowest mutation rate of all the strains tested. The mean rate for this mutation is approximately 3 X 10- 1° per cell division. A much greater variation in mutant counts was found in a series of individual cultures than in replicate samples from the same culture, which indicate that the mutation is spontaneous and is not induced by the lower pCO2 of the air. Calculation of mutation rate from the total number of mutants depends on similar growth rates of mutant and parent in mixed culture where the ratio of mutant to parent is very small. To test growth rates under these conditions a flask of broth was inoculated with the parent Krause stock and a mutant strain derived from it. The initial ratio of mutant to parent was 1.0:107. After 48 hours incubation in 10% carbon dioxide the ratio was still 1.0:107 although total counts had increased 10-fold. Growth rates of the two strains in pure culture when incubated in 10% carbon dioxide were identical during the logarithmic phase of growth. It appeared possible that not all of the mutants were capable of forming colonies in the presence of large numbers of nonproliferating parent cells. To test this possibility a mutant strain from the parent Krause stock was plated in agar containing a background of 108 cells per ml of the Krause strain and in agar medium alone. The plates were then incubated in air. There was no significant difference in the counts of the two series, but the colonies of the mutant were much smaller when plated in the presence of large numbers of parent cells. In each of the mutation rate experiments, several colonies from the plates incubated in air were transferred to agar slants. These mutants which no longer required added CO2 for growth did not revert on serial subculture in either air or 10 per cent CO2. Cultures were tested for purity by Gram staining and high titer agglutination is anti-serum

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against Brucella abortus. Cultures were tested for colony type by the methods described by Henry and, more recently by Braun. Mr. White working in our laboratories has developed a method for detection of colonial variation that we find most useful. Cultures are streaked onto Albini brucella agar made to contain 1% glucose, 5% glycerol, and 2.5% agar. After incubation the plates are flooded with a dilute solution of crystal violet, the excess dye is removed, and the plates are observed with a low power dissecting microscope with the use of obliquely transmitted light. All of the cultures we used were smooth and the mutants derived from them were smooth. However, in order to show this technique for detection of colonial variants we took some kodachrome slides of other cultures of Br. abortus showing different colonial types. One of these slides presented the appearance of a normal smooth colony-the blue-green one-and a rough colony showing as red to purple. Another slide showed again a normal smooth colony as blue green and a mucoid colony which appears as dark purple with a cracked surface. Thus it is quite easy using this method that Mr. White has developed for a person with little experience to tell if he is working with a mixture of smooth and non-smooth organisms in a culture. In addition to the mutation studies, we have compared the metabolism of the parent and mutant for possible differences in metabolic pathways. The respiration rates of resting cell suspensions of the parent Krause strain and the mutant KC strain derived from it have been determined for a variety of substrates. There are no appreciable differences between parent and mutant strains in either rate of oxygen uptake determined as C0 2(N) or total oxygen consumed. These data indicate a fairly high degree of oxidative assimilation since the oxygen consumed per mole of substrate is considerably lower than that required for complete oxidation. The use of inhibitors revealed no differences in metabolic pathways in the parent and mutant strains. Sodium azide and 2,4-dinitrophenol in concentrations of M/600 and M/6000 respectively nearly doubled the rate of acetate oxidation and increased the total oxygen uptake to a value which approximated the amount required for complete oxidation. Acetate oxidation was inhibited by sodium arsenite and sodium bisulfide which indicates that alpha keto acids are intermediates in acetate oxidation in both parent and mutant. Conversely, the same concentrations of azide and dinitrophenol which stimulated the rate of acetate oxidation almost completely inhibited the oxidation of L-asparagine and L-glutamic acid. Asparagine oxidation was inhibited by an equimolar concentration of its enantiomorph while L-glutamic acid oxidation is not. No nutritional differences have been observed between parent and mutant strains other than the requirement for added carbon dioxide by the parent. The mutant strains examined thus far have been neither

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more nor less "exacting" than the parent strains from which they were derived. Earlier studies in our laboratories had shown that the CO2 requiring strains which we tested failed to grow in an Asparagine-Synthetic medium of Gerhardt and Wilson, while strains that did not require added C02 grew well in this medium. This indicated a correlation between the added C02 requirement and some other nutritional deficiency. We have found, however, that this nutritional deficiency is not linked with the CO2 requirement. Strains that require added C02 and that do not grow in the asparagine medium produce mutants that while they will grow in air on complex media fail to grow on the simplified asparagine medium. In so far as these experiments show mutant strains have the same nutritional pattern as their added C02 requiring parent. Also, we now have an added C02 requiring strain that will grow on the asparagine medium. Attempts to by-pass the added C02 requirement by the addition of some of the known C02 fixation products have not been successful. We have tried some of the Krebs intermediates and related amino acids, as well as adenine, quanine, and uracil. Culture filtrates and cell autolysates also failed to replace the added C02 requirements. CONCLUSIONS The results indicate that Brucella abortus loses its requirement for added carbon dioxide by spontaneous mutation at a rate of approximately 3 X 10 - 1° per cell division. Training of such cultures to dispense with their carbon dioxide requirements probably consists of a selection of spontaneously occurring mutants rather than gradual adaptation of the whole culture. The low mutation rate is indicative of a high degree of stability for carbon dioxide requirement in cultures of Br. abortus. The data on oxidative metabolism of various substrates associated with the Krebs cycle indicate a basic similarity of dissimilatory reactions in both parent and mutant types. This is confirmed by the similar behavior in the presence of various metabolic inhibitors. Thus far we have no information concerning the biochemical basis of the added carbon dioxide requirement of Br. abortus. Gerhardt and Wilson have demonstrated that some Krebs cycle intermediates do not by-pass the added carbon dioxide requirement. This does not necessarily preclude a basic biochemical similarity between the added carbon dioxide requirement and the "normal" heterotrophic requirement since several workers have reported that Krebs intermediates do not completely replace carbon dioxide. Currently we are using radioactive carbon in C02 to study this problem and we are comparing C02 fixation by the parent and by the mutant. We hope to report data from these experiments in the near future.

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DIOXIDO DE CARBONO QUE NECESITA LA BRUCELLA ABORTUS (Sumario) Se encontró que grupos pequeños de células en cultivos de cepas de Brucella abortus que requieren la adición de CO2, podían proliferar sin dicha adición. El análisis de la distribución de estas células es una variación mucho mayor en cultivos individuales que en muestra de control del mismo cultivo. Esto sugiere enfáticamente una mutación espontánea debida a que desaparece la necesidad de agregar C02. La proporción de esta mutación-de la necesidad de adición de CO 2 a la habilidad de poder crecer en el aire-ha sido calculada de la fracción de cultivos que no contenían mutantes (en una serie de cultivos similares) y del recuento total de mutantes de toda la serie. Para las cepas probadas, la proporción es aproximadamente de 5 X 10-1 ° por división de célula. Se realizó una comparación del metabolismo de célula en descanso de cepas mutantes y originarias, usando el microrespirómetro convencional de Warburg. En la proporción de oxidación por mutante y originaria de una variedad de substratos, no se ha observado diferencia alguna. El uso de inhibidores ha indicado modelos metabólicos muy similares. Las células en descanso, ya sean mutante u original, asimilan acetato y maletato por oxidación. La asimilación de acetato por ambas cepas es inhibida casi completamente por "azide" de sodio o dinitrofenol al 2.4. En cuanto a las necesidades de nutrición no se han observado otras diferencias aparte de la necesidad de agregar CO2. Se ha tratado de eliminar la necesidad de agregar CO 2, pero no se ha tenido éxito en este sentido.

USE OF THE EMBRYONATING EGG FOR ISOLATION OF BRUCELLA IN A PUBLIC HEALTH DIAGNOSTIC LABORATORY: A YOLK SAC TECHNIQUE By

KATHLEEN GAY,

B.S.,

AND S. R. DAMON, PH.D.

Senior Bacteriologist, and Director, Bureau of Laboratories, Indiana State Board of Health, Indianapolis, Indiana INTRODUCTION

Examinations for Brucella in the public health laboratory are generally made by culture or animal inoculation of blood specimens. The limitations of these technics are obvious to all who have had occasion to apply them and it is evident that a procedure which will permit more rapid reporting of results to the physician and also yield a higher percentage of recoveries is badly needed. Goodpasture and Anderson,' Buddingh and Womack,2 Ruiz-Castaneda, 3 and others have shown the causative organism of brucellosis to be preferentially an intracellular parasite. It seemed to the authors, therefore, that the embryonating egg might be useful as a medium for primary isolation. A survey of the literature revealed only a few specific references to the cultivation of Brucella in developing chick embryos; and of these reports, the majority dealt with the use of the fertilized egg as a means of evaluating therapeutic agents or as a technic for studying the pathology and invasive ability of Brucella. No reports of its use as a laboratory diagnostic aid were found. In undertaking a study of the value of the embryonating egg, the yolk sac inoculation technic was selected as the logical one to employ, since this area can receive a sizeable amount of blood without significant harm to the embryo, and the yolk itself is highly nutritious. The first step was to determine that age embryo for receipt of the inoculum which would provide optimum conditions for multiplication of small numbers of organisms. A preliminary experiment 4 indicated that 1-2 day embryonating eggs were of very doubtful value and that the three to six-day embryos provided optimum conditions. Three, four, and five-day embryos were finally selected for an evaluation series because they not only worked into a practical scheme for inoculation but also provided 9-11 days of further embryo development for multiplication of Brucella, before the yolk was so reduced that it was difficult to manipulate. The purpose of this paper is to describe a method which is proving successful, and to present experimental evidence for the selection of certain technics to be used in an evaluation of the three methods of isolating 157

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Brucella from the blood stream, namely, guinea pig inoculation, culture, and growth in the egg embryo. MATERIALS AND METHODS

Specimens.-Clots from blood specimens received routinely at the Indiana State Board of Health for diagnostic agglutination tests were employed. The sera were first examined for Brucella agglutinins with a commercial Brucella abortus rapid slide test antigen. If there was complete agglutination at the 1:20 dilution level, the clot was divided for culturing, guinea pig inoculation and egg injection. Part of the clot was injected directly into a tube of modified Huddleson crystal violet tryptose broth. The portion to be received by eggs and guinea pigs was forced through a syringe into bottles containing 5-7 ml tryptose broth and several small glass beads. These bottles were stoppered and shaken three minutes for further disintegration of the clot. Eggs.-White leghorn "hatching quality" eggs (avg. wt. 59 gm.) were obtained weekly from a local poultry supply house and held at 20°C until the day of incubation. That is, a week's supply was received on Wednesday and a sufficient number incubated each Monday and Friday, so that each day of the week 3-, 4-, or 5-day old live embryos were available. Before and after injection, all eggs were kept at 98-99°F (37°C-±), relative humidity 50-560, in an electric hatchery incubator. They were not turned at any time since doing so was found nonessential to the technic and also complicated the procedure for locating the yolk sacs. Candling of Eggs.-A small egg candler with a 100 watt bulb was installed in a plywood box 20" square. The interior was painted black and one side left entirely open to receive the egg racks. The opening was covered with a photographer's heavy black cloth. With this device it was possible to accurately candle embryos as young as three days. All eggs were candled immediately before use and the position of the embryo marked. Inoculation of Eggs.-Eggs which had incubated 3-5 days were first candled and those containing live embryos selected for inoculation. A small hole was drilled into the large end of each egg with an electric table model dental drill and § 22 S.S. White abrasive point. With a 20-gauge 12" hypodermic needle 0.5 ml of the blood-broth specimen was injected into each yolk sac. In making the inoculation the egg, with the marked embryo at the top, was placed horizontally on the table and the needle was inserted toward the center for 0.5-0.75". If the embryo was 3 days old, the direction of the needle was slightly upward; if 4 or 5 days, directly inward toward the center. Inoculations were made the day of the agglutination tests, although an occasional clot was held over the weekend. When this was done, the clot structure was not disturbed until the day of the inoculation.

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The area around each hole was swabbed before and after inoculation with tincture of iodine and the hole finally sealed with a swab of collodion. Examination of Eggs.-The examinations consisted of three subcultures from the yolk at about five day intervals and a final harvesting of all live embryos on the 18th or 19th day of total incubation, the embryo ages at each subculture being 9-10, 14 and 18-19 days, respectively. Several drops of yolk material from each egg were withdrawn and placed on dry tryptose agar (pH 6.6-6.7) plates, and streaked over the surface with a wire loop or sterile swab. All plates were held two weeks in an incubator with a C02 concentration of approximately ten per cent maintained at all time. If any embryos remained viable on the 18th or l9th day of incubation they were harvested and portions of the blood, yolk sac wall, internal organs, and mixed fluids streaked onto tryptose agar plates. Withdrawal of Yolk.-The yolk was withdrawn in a manner similar to that of the injections. However, since the position of the yolk sac gradually changes with further development of the embryo, allowances must be made for this by inserting the hypodermic needle at different angles and depths. When the egg is placed horizontally on the table, as a rule, the older the embryo the more the yolk position shifts from a high anterior position to a low posterior one. For the first withdrawal, when the embryo was 9-10 days old, the needle was inserted as for inoculation of a five-day embryo. However, for the second withdrawal at 14 days, the needle was inserted deeper and angled downwvard about 30 degrees. Occasionally, further probing was necessary to find a misplaced yolk. Identification of Brucella.-Suspicious colonies were fished to tryptose agar slants and incubated in the CO 2 incubator for 24-48 hours. A spot agglutination test was then made from the growth on the slant, using a commercial Br. abortus antiserum. If clumping occurred, duplicate tryptose agar slants were inoculated, one incubated in air and one in CO2 to check CO2 growth requirements. A saturated lead acetate paper was placed in the mouth of the latter to determine H 2S production. Growth from the original slant was also streaked onto basic fuchsin and thionin dye plates (dye conc. 1:100,000) for species typing. The remaining growth was then suspended in 0.5% formalinized saline, diluted to the proper density, and tube agglutination tests set up, with both the commercial antiserum and the homologous serum from the patient. EXPERIMENTAL RESULTS The first specimens of blood injected into eggs came from autopsied guinea pigs which had been inoculated subcutaneously with human blood from suspected cases of brucellosis, and from swine experimentally infected with Brucella. Fifteen specimens were injected into 3- and 5-day

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old eggs and three yolk subcultures made as previously described. Penicillin G was used in half the eggs in an attempt to control contamination. Four recoveries were obtained, all Br. suis strains-one from the guinea pig and three from the swine bloods, though only from eggs not receiving the antibiotic. In addition, a Br. melitensis strain was recovered from the blood of a treated ambulatory case when the clot was inoculated into 3-day old eggs. At this point it seemed desirous to have selected specimens in a number sufficient to make an evaluation test of the chick embryo method. Therefore, it was decided to select blood clots from those specimens being received routinely in the laboratory for agglutination tests. Experiment 1.-The specimens were treated as described in the foregoing section, except that any clots from sera showing even a partial agglutination were inoculated, three eggs receiving the blood clot in a low concentration of penicillin broth and three receiving the blood without the antibiotic. Seventeen specimens were injected. There were three yolk subcultures. Embryo tissues were not examined, otherwise. Thirteen specimens were negative, two unsatisfactory due to heavy contamination, and two yielded isolations of Br. abortus. Multiplication of Brucella was not detected in those eggs receiving penicillin except in the instance of a single embryo in which growth was quite delayed. By this time it was apparent that the low concentration of penicillin used did not control contamination and it also appeared that the antibiotic was inhibitory for Brucella. Consequently, any further attempt to control contamination was abandoned. Experiment 2.-In this group no penicillin was used. Each blood clot was injected into six eggs. As in Experiment 1 three yolk subcultures were made and the embryos were not harvested for a final examination. Sixteen clots were injected, with no recoveries and one unsatisfactory contaminated specimen. At this point, it was decided to employ more highly selected specimens, reduce the number of eggs per specimen and begin an evaluation series comprising inoculations into a broth for culture, into the guinea pig and into the eggs, using altogether the materials and methods described in the preceding section. The number of eggs per specimen was reduced from six to four since observations already made indicated this was probably a safe minimum. Further, as an additional examination of the eggs before discarding them, embryo tissues as well as the yolk were to be cultured. Experiment 3.-Each clot of this series was examined by inoculation subcutaneously into guinea pigs, inoculation into a tube of modified Huddleson's crystal violet tryptose broth (final dye concentration 1:1,000,000) from which periodic subcultures were made, and into embryonating eggs 3-5 days of age. Seventy-five specimens have been inoculated and examined as described in Materials and Methods. Seventeen were discarded as unsatisfactory due to con-

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tamination of the blood clot. From the 58 unsatisfactory specimens, there were 5 isolations, all Br. abortus. Three of the five recoveries were not obtained by either of the other methods. All five isolations were obtained by the seventh day following inoculation of the eggs, although an occasional egg of a group from which an isolation was made on the first yolk subculture did not show growth until the second withdrawal. Colonies of Brucella came up on the second day of the agar plate incubation. All five strains were typically Br. abortus. Table 1 summarizes all the recoveries made from blood clots inoculated into the yolk sacs of 3-5 day embryonating eggs. It is to be noted that TABLE 1.-The recovery of Brucella from blood clots Age of Embryo (days) Type

Lab. No.

Specimen Source

Titer*

At Injection

At Isolation

swine

5

13

12 19 5973

swine swine guinea pig

5 5 3

13 10 8

Melitensis

6356

human

1:320

3

12

Abortus

3338 3953 4046 4138 2172

human human human human human

1:320 1:160 1:80 1:320 1:160

4 4 3 4 4

9 10 10 9 9

Suis

2

* The highest serum dilution in which a 2+ agglutination reading was obtained.

only 5-9 days of incubation were required for isolating these Brucella strains. DISCUSSION

Although previous experiments have shown Brucella to multiply rapidly in 3-10 day old embryos, the optimum age range has been shown to be 3-6. The younger ages, 3-5 days, were selected for injection because in addition to providing optimum conditions for the growth of Brucella, they permit inoculation at that time when the yolk begins to swell. It is too early in the course of the experimental series to state that any one age embryo at the time of injection is superior to another; as recoveries have been made from all three ages employed. Attention has already been called to the fact that the final harvesting of the embryos and the third subculture of yolk material has not yielded additional isolations. Although these data have not been included in the experi-

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mental results, with the exception of half a dozen eggs, all live embryos at the pre-hatching stage were examined by staining tissue smears with Macchiavello's rickettsial stain.5 No additional infections were found. The fact should be mentioned that not all the specimens injected came from frank clinical cases. Although they usually came from a patient showing a fairly high titer of Brucella agglutinins, a significant percentage came from treatment checks and so-called chronic cases. Based on observations and results of the experimental data presented in this paper, the following procedure, which is applicable to use in any public health diagnostic laboratory and is now proving of considerable value in the hands of the authors, is suggested as one yielding a more rapid and efficient recovery of Brucella from the blood stream. It utilizes a simple type of specimen, which any physician prefers submitting. It provides for a preferentially-intracellular parasite the living tissue cells as in the guinea pig, without the concomitant danger to laboratory personnel and the expense and upkeep of animals; while at the same time it is a unit of culture medium which can be nearly as easily handled as the tube of broth. It can reduce the time interval between taking the specimen and receipt of the result by the physician to ten days in contrast to three or four weeks. The following scheme is suggested for laboratory use as a routine diagnostic aid: Eggs may be purchased once a week from any supply house handling "hatching" quality eggs and, if necessary, kept at least a week at approximately 20°C before incubating. If the eggs are set twice a week, that is, on Monday and Friday, either a 3-, 4-, or 5-day old embryo will be available each day except Sunday for inoculations. If the specimen is in the form of a clot, break up as previously described before injecting. Four eggs, as determined by the authors, is probably the minimum number per specimen to use. On the fourth or fifth day after inoculation withdraw a small portion of the yolk and streak onto a non-inhibitory plating medium, incubating the plates in an increased C02 atmosphere. On the fourteenth day, make a second and final yolk subculture. CONCLUSIONS 1. A method for using the embryonating chick egg for primary isolation of Brucella from the blood stream has been presented and supporting experimental data given. 2. Essentially, this technic consists of injecting specimens directly into the yolk sacs of 3-, 4-, and 5-day old embryos and subculturing periodically. 3. All three types of Brucella have been isolated and in many instances this technic has proved superior to guinea pig inoculation or culture.

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4. The methods described are being used in an evaluation of guinea pigs, eggs, and culture media for the isolation of Brucella from clotted blood. 5. The advantages of the egg embryo yolk sac technic are: (a) it provides living tissue cells in a compact medium; (b) it permits recovery of all three types of Brucella; (c) it gives quick results; (d) it is relatively inexpensive; and (e) it entails a minimum amount of danger for laboratory personnel. REFERENCES (1) Goodpasture, E. W., and Anderson, K.: 1937. The problem of infection as presented by bacterial invasion of the chorio-allantoic membrane in chick embryos. Am. Jour. Path., 13S 149. (2) Buddingh, G. J., and Womack, F. C.: 1941. Observations on the Infection of Chick Embryos with Bacterium tularense, Brucella, and Pasteurella Pestis. Jour. Exp. Med., 74: 213. (3) Ruiz-Castañeda, M.: 1947. Studies on the pathogenesis of brucellosis. Proc. Soc. Exptl. Biol. and Med.,64: 298. (4) Gay, Kathleen, and Damon, S. R.: Use of the chick embryo for isolation of Brucella: Multiplication of the organism in the yolk sac and selection of the embryo age optimal for isolation from blood. In press. (5) Smith, D. T., and Martin, S. M.: Zinsser's Textbook of Bacteriology, 9th ed., 1948. Appleton-Century-Crofts, Inc. AISLAMIENTO DE LA BRUCELLA MEDIANTE EL USO DEL HUEVO EN ESTADO DE EMBRIóN EN UN LABORATORIO DE DIAGNóSTICO (Sumario) Aunque desde hace algún tiempo algunos laboratorios de salud pública han estado interesados en el problema del aislamiento de la Brucella de muestras de sangre, muchos de ellos no han introducido todavía en sus prácticas de diagnóstico los procedimientos de cultivo de inoculación de cobayos. La vacilación en cuanto a la introducción de estos procedimientos se debe posiblemente al bajo porcentaje de los aislamientos de los tipos de especímenes generalmente sometidos, así como al riesgo que representan para el personal cuando se utilizan animales en el laboratorio. Durante algunos años, en el laboratorio de diagnóstico de la Junta de Salubridad del Estado de Indiana, se han empleado métodos de cultivo para el aislamiento de la Brucella. Sin embargo, desde la iniciación del Proyecto de Estudio de la Brucelosis en el Estado de Indiana, un aspecto que pareció merecer investigación fué si habría o no otros medios de aislamiento de estos organismos, medios que habrían de ser adaptables a la rutina de diagnóstico de laboratorio, y al mismo tiempo de valor igual o mayor que los métodos empleados en la actualidad. Por lo tanto, se consideró la posibilidad de usar huevos de gallina en embrión, como medio regular de cultivo. Los trabajos de Goodpasture y sus colaboradores han demostrado desde hace mucho tiempo que la Brucella puede sobrevivir y multiplicarse en huevos, en tanto que Spink y otros han utilizado embriones de huevos inoculados para probar diversos agentes terapéuticos, a pesar de no

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existir en forma impresa ninguna información relativa al uso del embrión de huevo como medio de cultivo para diagnósticos. A través de investigaciones preliminares, pronto se demostró el valor de los huevos en embrión como medio de cultivo; en consecuencia se seleccionaron embriones incipientes-de 3, 4 y 5 dias-que presentaban condiciones óptimas para la multiplicación de los tres tipos de Brucella. Además, pareció más ventajoso la inoculación de la muestra de sangre a través de la membrana vitelina. En sintesis, el procedimiento ha consistido en utilizar jeringuillas de 5 ml sin agujas, para introducir los coágulos de sangre en frascos con 7 ml de caldo de triptosa y bolitas de vidrio esterilizadas. La agitación de los frascos por 2 6 3 minutos sirve para desintegrar más los coágulos, después de lo cual se inyecta 0.5 ml en la membrana vitelina de cada embrión de huevo; se utilizan cuatro de éstos. La incubación de los huevos inoculados se hizo a 37°C, y después de 4

y 8 días se extrajeron porciones de yema para su cultivo en láminas con agar triptosa simple. El subcultivo final de la yema de los huevos se hizo después de 15 días de incubación, en esta oportunidad se recogieron los embriones con vida, y también se extrajeron y cultivaron el hígado, el bazo, la membrana vitelina, la sangre, el corazón y la vesícula biliar. Las láminas inoculadas se incubaron a 36°C, en una atmósfera de CO 2, y los organismos que surgieron fueron identificados mediante cultivo y aglutinación. Se han aislado las tres variedades de Bruecella de los coágulos de sangre mediante el método de inoculación de huevos de gallina en embrión. La técnica seguida es relativamente sencilla, y pueden obtenerse resultados con mayor rapidez que mediante cultivos o inoculaciones en cobayos.

DIAGNÓSTICO BACTERIOLÓGICO Y BIOLÓGICO DE LA BRUCELOSIS Por Luis A. PHILIPPS Jefe, Instituto Nacional de Biologia Animal, Ministerio de Agricultura, Lima, Perú El diagnóstico bacteriológico y biológico de la brucelosis es un problema de vital importancia; tanto en la medicina humana como en la medicina veterinaria. En el campo de la salud pública el problema reviste caracteres de vital importancia, si se tiene en cuenta que el hombre puede ser infectado por cualquiera de los tres tipos de Brucella conocidos y un diagnóstico rápido puede asegurar el tratamiento adecuado. En el campo veterinario, cuando se trata de aplicar un programa de erradicación de la brucelosis con miras a evitar la difusión de la brucelosis humana y aumentar la producción animal evitando los casos de aborto y de esterilidad. Realizada la siembra de un material sospechoso siguiendo las técnicas ya establecidas y sospechosa una colonia de Brucella es repicada en dos medios denominados "IM" y "SMG". Estos dos medios que fueron ideados por el bacteriólogo peruano, Dr. Hector Colichón, para el despistaje de colonias de tipo intestinal deben su nombre, el de IM a las dos primeras letras del Instituto "IMEX" (Instituto de Microbiología Experimental) del cual es Director el citado Doctor; el segundo, a su composición primordial: subacetato glucosa, manita (SMG). En nuestra práctica de laboratorio nosotros los hemos utilizado en una forma más amplia sirviéndonos para el despistaje de colonias pertenecientes a la familia Parvobacteriaceae; siendo las reacciones bioquímicas características que se traducen en cambio de color de los medios por la alcalinización o acidificación de ellos o por la fragmentación del medio sólido (SMG) y recolección de gas en la campanita del medio de "IM", cuando hay ataque de los azúcares hasta la descomposición de ellos con la consiguiente producción de gas. La composición de estos dos medios es la siguiente: "IM" Agua de peptona (ph 7.4) .................................... 100 cc

Mezcla triple carbohidrato (1)...............................

8 cc

1 cc Indicador de "IM" (2) ....................................... 5 cc Urea al 20% ................................................ (1) Mezcla triple carbohidrato: Lactosa al 20% .......................................... 75 cc 3.5 ce Sacarosa al 20% ......................................... 3.5 ce Manita al 20% .......................................... (2) Indicador de "IM" Es una mezcla de indicador de Andrade y bacto timol azul. Afiadiendo a 100 cce de este indicador 0.4 gm de bacto timol azul. 165

166

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

El agua de peptona se prepara con proteosa-peptona No. 2 Difco; cualquier otra peptona puede no ser adecuada; el uso de bacto peptona Difco no es recomendable para esta preparación. Disolver 10 gm de proteosa-peptona en 1000 cc de agua destilada (preferir bi-destilada) y luego agregar 5 gm de cloruro de sodio (NaC1) químicamente puro. Neutralizar y precipitar en caliente, filtrar por papel y ajustar a pH 7.4; repartir y luego esterilizar a 1 atmósfera por 15 minutos. Se emplea carbohidrato marca Difco o Merck y la esterilización se hace por filtración. El indicador de "IM" sirve para demostrar las variaciones alcalinas como las ácidas en los cultivos. El indicador de Andrade se prepara con 0.2 gm de bacto fuchsina ácida (National Aniline Div. Dye content 5.8%) disuelto en 92 cc de agua destilada, a la que luego disuelta se agrega 8 cc de NaOH normal para decolorarla. La urea igualmente debe ser esterilizada por filtración y se debe emplear urea químicamente pura. Preparación para el uso.-De acuerdo con las cantidades especificadas se agrega al agua de peptona la correspondiente cantidad de carbohidrato, luego el indicador y por último la urea. Agitar y repartir en tubos esterilizados como los usados para observar la producción de gas (Durham). El llenado de las campanitas se hace mediante una bomba de vacío, teniendo la precaución de calentarlos antes de someterlos a la cámara de vacío a fin de obtener en los tubitos interiores el lleno completo (todo indicio de burbuja obligará a desechar el tubo o a repetir el vacío en caliente). MEDIO "SMG" (Manita, glucosa-subacetato de plomo) Bacto-Tryptona Difco ................................ 15 gm Bacto-Proteosa Peptona Difco ........................ 5 gm 0.5 gm Subacetato de plomo (reactivo de Merck) ............. Tiosufalto de sodio (reactivo de Merck) ............... 0.2 gm Bacto-Manitol Difco .................................. 10 gm (50 cc sol. al 20%) Bacto-Dextrosa Difco .................................. 1 gm (5 cc sol. al 20%) Solución de rojo de fenol al 0.2% ..................... 15 ce Bacto-Agar Difco ................... 10 0................. gm Agua destilada ....................................... 1000 cc

Preparación.-Disolver las sales en el agua destilada, en seguida agregar las peptonas. Ajustar el pH a 7.1 y luego agregar la solución de rojo de fenol. Este medio no se filtra. La mezcla se esteriliza a 1 atmósfera durante 15 minutos. Al sacarlo del autoclave aun caliente se agrega asépticamente los carbohidratos, usando soluciones al 20% esterilizadas por filtración. Repartir con esterilidad en balones de 100 a 200 cc para uso cotidiano.

DIAGNÓSTICO DE LA BRUCELOSIS

167

La solución de rojo de fenol se prepara disolviendo un gramo de rojo de fenol en 40 cc de NaOH N/10 completando luego a 500 cc con agua destilada. Preparación para el uso.--Fundir el medio en baño de María a ebullición, dejarlo enfriar a 600C y luego repartir en tubos de 10 x 100 mm estérilmente y dejarlo endurecer en posición semi-inclinada; verificar un control de esterilidad antes de su uso. En estos dos medios la reacción de la Brucella es inconfundible. El medio de "IM" originalmente es de color amarillo, la producción de acidez se traduce en viraje de color desde el rosado al rojo y la alcalinidad por el viraje al azul. El medio "SMG" es de color rosado y la producción de acidez se traduce en variación al amarillo y la alcalinidad hacia el rojo. La producción de H 2S se revela por el ennegrecimiento del medio. CUADRO 1.-Distintas reacciones en los medios de "IM" y "SMG" de gérmenes cuyas colonias son similares a las de Brucellas 1"IMI"

"SMG"

Tipo bioqufmico

Pasteurella .........

Alc.

Ac.

-

Lig. Ac .

Streptococcus ....... Hemophilus ......... Alc. Brucella ............. Alc. Alcaligenes ..........

Ac. -

-

Gas

A

Alc.

Ac.

-

_ _ _Alc. Alc.

Gas

H2S

Lig. Ac.

-

-

Ac. Lig. Ac. -

-_ -

-

_ -

Alc. = Alcalino; Ac. = Acido; Lig. Ac. = Ligeramente ácido. DIFERENCIACIóN DE LAS BRUCELLAS ABORTUS, MELITENSIS Y SUIS POR EL USO DE DISTINTOS COLORANTES

Por la observación del siguiente cuadro que corresponde a un estudio comparativo de distintas cepas en lo referente al límite de tolerancia para su desarrollo en distintas concentraciones de colorantes, se puede concluir que en el tipado s61o son necesarios las siguientes concentraciones de colorantes: tionina al 1/30,000, fuchsina al 1/25,000 y cristal violeta al 1/50,000. Las demás concentraciones de tionina, fuchsina y cristal violeta no son necesarias. En la pironina las distintas cepas desarrollan en forma similar. En el cuadro siguiente las cepas numeradas del 1 al 9 corresponden a Br. suis; del 10 al 15 a Br. abortus y del 16 al 18 a Br. melitensis. DIAGNóSTICO BIOLÓGICO

Entre los distintos procedimientos de diagnóstico biológico la intradermo reacción es deficiente, ya que he podido observar que animales con reacción francamente positiva a la aglutinación tenían una intradermorreacción negativa; igualmente animales con aglutinación nega-

168

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS CUADRO 2 Tionina

Cepa

Fuchsina

No. 1/30,000

1 2

-++ ++ ++ ++ -++ -++ ++ ++ +-q +-q +-q

3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18

1/50,000

++ ++ ++ ++ ++ ++ ++ ++ ++

1/25,000

1/75,000

Cristal violeta

1/50,000

1/100,000

1/50,000

I

++ ++ ++ ++ ++ ++ ++ ++ q-q ++ q-q +-q

I I

1

++ ++ ++ q-q + +-q +

+

++ ++ +-q +-q +++ +q-

+

+

+

+++++++q+-

+

CUADRO 3 Piromina

H.S

Cepa No. 1/100,000

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15

16 17 18

++ ++ q-q ++ q-q ++ q-q ++ q-q ++ q-q ++ q-q ++ ++ + +f + + + + + + +

1/200,000

++ ++ ++ ++ ++ ++ ++ q-q ++ q-q +++ +-q ++ q-q + q-q +

+ q++q+ q+ ++ +++++-

+

tiva mostraban la intradermo reacción, unos positiva y otros negativa. Estas aseveraciones se ponen de manifiesto en el cuadro siguiente. La opsonocitofagia no es aconsejable para el diagnóstico de la bruce-

169

DIAGNÓSTICO DE LA BRUCELOSIS CUADRO 4.-Aglutinación introdermorreaccióny opsonocitofagia No. del Animal

ATlítuln o die Aglutinaci6n

Marcada

1921 2775 0034 2668 2568 2519 1926 2312 2423 1998 1450 1449 4320 6282 2156 2549 2261 2211 2580 1781 2437 2619

1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25 1/25

0501 1608 2356 0171 2302 2240 2511 1620 2498 8049 2771 2508 6304 1562 2212 2774 5658

1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50 1/50

o 1 o 1 112

5506 1627 0112 6379 1966 1495 2358

1/100 1/100 1/100 1/100 1/100 1/100 1/100

O 5 13 2 o 10 o

Moderada

Pequeña

Mo ,d

12 5 5

3 2 3 1 10

7

1 o 2 o 9 6 2 o 8 3 3 o 1 6

8 12 11 15 6 11 10 3 3 8 4 2 2

12 7 12 8 O 9 9 5 6 15 10 20 16

+

+ +++ ++ + ++ +++

7

9

+

3 9 9

20 9 12

+ +

8 1 2 3 15

16 14 3 16 2 8 1 21

++ ++ ++ ++ ++ ++ +

1 7

4 6 2 4 1 7

7

o 8 o 5 5 o 4

2

4

3

j

4

1 4 4

7

6

Intradermorreacción

++

3 10 5 6 3 o

o

No. índice Opsónico

11 9 8 3 6 17 3 14 25 11 24 6 13 13 22 6 19 14 24 17 6

7

4

Negativa

1

+ + + ++ ++ ++ + ++ + ++ + +

+

+++ +

+++ ++

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

170

CUADRO 4.-Agglutinación introdermorreaccióny opsonocitofagia (Cont.) No. del

Animal

Título de

Aglutinacideón

Marcada

Moderada

Pequefia

Negativa

No. índice

Intradermo-

Opsónico

Intradermoeacción

2481 2129

1/100 1/100

0 0

1 1

6 3

18 21

19 10

+ +

8052

1/400

4

8

3

10

43

+++

2532

0/0

4

7

10

4

47

-

CUADRO 5 Títulos de Aglutinación Antígeno*

Suero 1/25

2870

1/50

1/100

1/200

1/400

+ 1

2516

+

+ 2965

2540

+

+ + + + + + + + + + + + + + + +

I I I I I

+ + + +

q++ +++ + +-

171

DIAGNOSTICO DE LA BRUCELOSIS CUADRO 5.-(Cont.) Títulos de Aglutinación Antigeno*

Suero 1/25

2999

2351

1/50

1/0l

1/200

1/400

+

1 1 1

1 1 1 1 3195

3200

3198

* Las fechas de preparación de los antigenos fueron: A-enero 2 de 1941; B-junio 26 de 1942; C-julio 30 de 1943; D-abril 1 de 1944; E-abril 26 de 1945; F-marzo 12 de 1946; G-abril 16 de 1947; H-noviembre 15 de 1948. En las lecturas de las aglutinaciones de estos distintos sueros se observa que los antígenos no han sufrido cambio alguno en sus propiedades de aglutinabilidad.

172

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

losis ya que no hay ninguna relación entre el índice opsónico y el título de aglutinación, pues en el cuadro que antecede se puede observar que animales que tienen un título de aglutinación elevado poseen un índice opsónico bajo y a la inversa. Las aglutininas y las opsoninas son dos entidades completamente diferentes. En la opsonocitofagia se han seguido las técnicas de Keller, Pharris, Crit y Gaub consignadas en el libro de Métodos de Laboratorio Clínico de Kolmer y Boerner 1948. El CUADRO 6.-Estudio comparativo de la pruebas del anillo y las aglutinaciones del suero hemático y suero lácteo

·

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Pruebas del anillo (ring test)

++++ ++++ +++ ++ +++ ++++ +++ ++++

++ ++++ ++ ++++ ++ +++ ++ ++ +++ ++

I

Aglutinaciones No. S. Hemático

Suero Lácteo

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

1/800 1/50 1/100

1/200 1/25

1/50 1/100 1/50 1/100 1/200

1/200 1/200 1/50 1/50 1/50 1/400 1/50 1/100

42

43 44 45 46

1/100 +

1/50

1/200

<

Pruebas del anillo (ring test)

S. Hemático

Suero Látceo

1/50

-

1/25 1/25 1/400

-

1/50

-

1/100 1/100 1/100

-

++ ++++ ++++

++++ +++ +++ +++ ++

47

48 49 50

Aglutinaciones

1/50 1/50

-

-

++

·<

número de indice opsónico corresponde al de Foshay y Le Blanc, aunque la cuenta de leucocitos se ha hecho sobre 50 y no de 100. En la intradermorreacción se ha utilizado la brucelina. El "ring test" es de resultado práctico y una prueba bastante sensible pero no hay relación entre el "ring test" y la aglutinación del suero lácteo, pero sí en la mayor parte de los casos hay relación entre esta prueba y la aglutinación del suero hemático como se puede observar en el Cuadro 6 que bajo el título de estudio comparativo entre el "ring test" y las aglutinaciones del suero lácteo y hemático corresponde a un trabajo que

173

DIAGNóSTICO DE LA BRUCELOSIS

actualmente está realizando el Dr. Guillermo Montoya en el Laboratorio de Brucelosis de nuestro Instituto Nacional de Biología Animal. En la aglutinación del suero sanguíneo es necesario la estandarización de criterios de aglutinación y creo oportuno insertar un importante trabajo realizado por la Oficina de la Industria Animal de los Estados CUADRO 7.-Distribuciónde los títulos de aglutinación obtenidos por el método del tubo y de la placa Ejemplo

1 2 3 4 5 6 7* st 9 10 11 12 13 14 15

Método

Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo Placa Tubo

1/25 -

1/25 I

1/25

+

2 67 41 1 1 2 1 67 39 3

1/50

1/50

+

1/100 1

1/100

1/200 I

1/200

Total

68 41

68 41 68 41 68 41 68 41 68 41 68 41 67 40 67 40 68 41 68 41 68 41 68 41 68 41 68 41 68 41

16 23

31 16

16 1

2

1 1

4

1

25 18

13 11

1 3

39 28 6

20 18

8 10

1 1

17 6

6 3 5 2 5 2

24 9 1 1 28 10

34 6 22 22 2

26 26 10 12 4

7 8 3 1 11

13 3

20 9

18 29

39 27 39 19 29 24

16 10 20 17 25 11 1

3 1 1 3 9 3 14 3

29 23

18 12

6 3

1

10

19 10

11 8 24 20

1 1 15 1 8 1 3

15 5 2 2 5 2 5 3

1

1

* Uno de los operadores manifestó que habla recibido uno de los tubos roto. t Uno de los operadores no comunicó la aglutinación al título de 1/25.

Unidos, en el Animal Disease Station de Beltsville Research Center. Este trabajo consistió en el envío de 15 muestras de sueros a cada uno de los laboratorios de los Estados Unidos. Hubieron 68 operadores trabajando en la prueba en placa y 41 en la prueba en tubo. Se observará que hay una tendencia a la uniformidad, aunque los resultados obtenidos por algunos laboratorios fueron diferentes a los verdaderos resultados.

174

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

El antígeno utilizado fué preparado de acuerdo a las técnicas de la Oficina de la Industria Animal (V. Cuadros 7, 8 y 9). En lo referente al tiempo de duración de los antígenos podemos afirmar que mientras se conserven baj o refrigeración se pueden mantener inalterables por muchos años, como lo demuestra el trabajo que realizamos el año de 1949 con el Dr. Dale Suplee, en el Animal Disease Station de CUADRO 8 Sueros Positivos

Número de operadores en la prueba en tubo

Número de operadores en la prueba en placa

1-4-6-11-15 1-4--11-15 1 6-11-15 1--6--4-15 1---4 11 1 6-11 1- 4 -- -15 1 11-15 1 11 1- 15 1 1-2-4-6-11-15 1-2-4-11-15 1-2-4-6--15 1-4-6-9-11-15

21* 6 4 0 1 1 0 3 1 0 0 1 1 1 1

21* 12 3 1 0 1 4 9 2 1 11 0 1 1 0

Total ...............

41

68

* De acuerdo con los resultados del Animal Disease Station. CUADRO 9 Sueros Negativos

3-7-8 3-*-8 3-8

30 1 10

45 1 22

Total

41

68

* Muestra rota en tránsito. Beltsville. Se eligieron antígenos preparados desde el año de 1941 al 1948 y se utilizaron 9 muestras de sueros habiéndose obtenido aglutinaciones de título igual para cada suero, trabajando con los distintos antígenos. Esta inalterabilidad de los antígenos es de gran importancia, ya que se comprende que es difícil obtener antígenos que sean completamente iguales en su aglutinabilidad, y como su duración es larga, se pueden preparar lotes grandes que se puedan repartir en los distintos laboratorios. Los sueros números 4 y 15 son duplicados, y sin embargo se puede ver

DIAGNOSTICO DE LA BRUCELOSIS

175

cómo ha variado la distribución de los operadores según las aglutinaciones comunicadas. Los números colocados en columna debajo de cada título de aglutinación representan el número de operadores que manifestaron haber obtenido el título dado. En los cuadros 8, 9 y 10 se puede observar la distribución de los operadores según sus aciertos al determinar el título de aglutinación. Para aclarar el cuadro 8 tomaremos algunos ejemplos. Veintiún operadores trabajando con la prueba en tubo y 21 en la placa manifestaron que los sueros números 1-4-6-11 y 15 eran positivos y estuvieron acordes CUADRO 10 Sueros sospechosos

2- 5--9-10-12-13-14 2- 5-7-9-10-12-13-14 2 9-10-12-13-14 2-5 10-12-13-14 2-5-7-9-- 12-13-14 2-5-9-- 12-13-14 29-10-12-13 2-4- 9-10-12-13-14 - 5-7-9-12-13-14 2 9-12-13-14 2-5-9--12- 14 5-9-12-13-14 2-5--9-10 13-14 Total ....................... Operadores que obtuvieron otras combinaciones ...... Total .......................

Número de operadores en la prueba en tubo

Número de operadores en la prueba en placa

15* 2 4 3 O 1 2 2 2 1 O O 1

11* 7 4 0 4 5 1 O 0 2 2 2 2

33

40

8

28

41

68

* De acuerdo con los resultados obtenidos por el "Animal Disease Station."

con los resultados obtenidos por el Animal Disease Station. Seis operadores trabajando con la prueba en tubo y 12 con la de placa manifestaron que los únicos sueros positivos eran los números 1-4-11 y 15. En este caso se puede ver que fallaron en señalar al número 6 como positivo. En igual forma en los mismos cuadros se consigna la distribución de los operadores según su acierto en los sueros negativos y sospechosos. THE BACTERIOLOGICAL AND BIOLOGICAL DIAGNOSIS OF BRUCELLOSIS (Summary) Suspicious colonies of Bruecella are picked up and sown into two media called "IM" and "SMG". These two media are the formulas developed by Dr. Hector

176

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Colichon, a Peruvian bacteriologist, for the study and detection of intestinal types of bacteria. We have used these media for the detection of colonies of Parvobacteriaceaefamily. The biochemistry reaction of Brucella in these media is different from other members of the group, which is revealed by the change of color in the media. The original color of "IM" media is yellow; an acid reaction is noted by a pink or red color depending on the amount of acid produced. An alkaline reaction is noted by a blue color. The original color of "SMG" media is pink, and acid reaction is noted by a yellow color and an alkaline reaction by a red color. The different reactions of bacteria with colonies similar to Brucella are the following: Pasteurella, slightly acid in "IM" and "SMG" Brucella, alkaline in "IM" and "SMG" Streptococcus, acid in "IM" and "SMG" Hemophilus, alkaline in "IM" and acid in "SMG" For Brucella differentiation we used thionin 1/30,000; fuchsin 1/25,000; violet crystal 1/50,000; other dilutions of the dyes were not necessary. In diagnosis the intradermal reaction is inefficient. The opsonocytophagic test is negative because animals with high opsonic index have low agglutination titer in the blood serum and the inverse. The agglutinins and opsonins are two different things. The ring test is practical and has a high sensitivity, but there is no relation with milk serum agglutinations. With blood serum agglutinations there is a relation with the majority of examples. It is necessary to standardize the reading criteria in the blood serum agglutination. In reference to the expiration date of the antigen we can say that under refrigeration conditions they remain unaltered for many years.

WHAT SHOULD BE DONE WITH THE BRUCELLA SKIN TEST By K. F. MEYER, M.D. George Williams Hooper Foundation, University of California, San Francisco It has been suggested that the answer to the pertinent question "What should be done with the Brucella skin test?" be rendered by one who is at least historically responsible for introducing into the realm of brucellosis a diagnostic procedure of questionable value. This assignment is approached with a feeling of humility, since the appraisal and verdict will doubtless contribute little to ameliorate the temper of a heated controversy. I am keenly disappointed that other more urgent problems prevented the completion of the study of allergy in brucellosis instituted thirty years ago. Perhaps without these numerous interruptions the necessity to answer the question today might never have arisen. BACKGROUND

It is necessary now to look back into the history of the skin test and its interpretation as a diagnostic tool in Brucella infections. It begins as early as 1912 and 1913 when experience with "abortin", a diagnostic biologic product, was in part responsible for its use in the solution of an intriguing problem. In an effort to determine the frequency with which certified milk might be infected with tubercle bacilli, in 1916 and 1917, a large number of guinea pigs were inoculated with cream and sediment. This invariably produced in the animals a chronic disease simulating tuberculosis. In order to distinguish between Brucella abortus and Mycobacterium tuberculosis infections, intracutaneous tests with tuberculin and abortin were successfully employed. If the guinea pigs had lesions resulting from Brucella infection and positive spleen cultures and agglutination tests, the skin reactions were invariably strongly positive, but "typhoidin" and analogous preparations made from a variety of other micro-organisms invariably brought about no reactions. Detailed experiments furthermore proved that only an acute or chronic infection can produce specific hypersensitiveness in the guinea pigs. It was reasoned at that time that if man is infected with Br. abortus, in all probability lesions similar to those seen in guinea pigs would develop and, in consequence, similar cutaneous hypersensitivity should be present. To sound out the validity of this reasoning, 75 infants (2 to 4 years old) were tested with a standardized abortin preparation (not a broth filtrate as it has repeatedly been called in the literature). Prior to the test their source of milk had been found to be heavily infected with Brucella abortus. The injection of 1/500 mg of the alcohol-ether precipitated and dried 177

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bacterial suspension of Br. abortus, dissolved in 0.1 ml of slightly alkalinized phenolized saline produced significant reactions in only 2 of the 75 children. Since typhoidin and tuberculin induced similar cutaneous edema and erythema in the 2 patients suffering from chronic bone tuberculosis, the reactions were considered not specific, but rather in some way connected with the long-standing suppurative process. The agglutination test for Brucella conducted with the sera of the 2 patients was negative. At that time, epidemiologic inquiries among a great many children who consumed the Brucella-laden milk failed to produce clinical evidence that this organism was pathogenic for infants. Investigations then shifted to another important phase of allergy under discussion at that time. The resulting elaborate studies proved that the rabbit is an unsuitable model for the study of cutaneous hypersensitiveness to Brucella (it is still being used), and that a positive reaction does not reflect a state of immunity (Fleischner et al., 1919). Many a student of the experimental aspect of the intradermal test and the problem of cutaneous allergy has overlooked the important biologic differences between rabbits and guinea pigs (León and Sosa, 1947). A report by Burnet (1922a and b) that a filtrate from a Brucella melitensis culture, preferably 20 days old, so-called "melitin", elicited in persons ill (for not less than 5 days) or recovered (for over 3 years) from undulant fever a tuberculin-like reaction at the site of the intradermal injection initiated an effusion of papers. In countries bordering on the Mediterranean, this intradermal Burnet reagent was used for the most part as a confirmatory test. Results varied. Enthusiastically some reported specific reactions (Olmer and Massot, 1924; Debré et al., 1927; Lemierre et al., 1927; Cazalas, 1928; Liege and Castéran, 1929; Olmer, 1929; Dubois and Sollier, 1931; Rainsford, 1935), while others using control groups more freely and seriously questioned the specificity of the melitin test (Brugi, 1924; Montagnani, 1923; Sorge, 1925; Tapia and del Valle, 1928; Duffau, 1928). The evaluation studies by Trenti (1923) proved that the reaction is specific, but that active broth filtrate which would cause a reaction in diseased persons was not obtainable with every strain of Br. melitensis. Using dried suspensions of Br. melitensis (as originally recommended), Canale (1925) at the clinic in Florence demonstrated that intradermal injection produces in all patients suffering from undulant fever a characteristic reaction. Other bacterial antigens produced no skin reactions. Significant is the report that patients with other infections-typhoid, paratyphoid fever, tuberculosisoccasionally reacted quite intensely to the local administrationof the melitensis antigen. The superiority of a bacterial suspension as a test antigen was amply proved by several Italian (Fornaca and Bua-Fazio, 1924; Mitra, 1924; de Fermo Cesare, 1927) and American workers (Giordano, 1929; Simp-

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son and Frazier, 1929; Levin, 1930; Leavell and Amoss, 1931; Yeckel and Chapman, 1933). Even casual perusal of the papers dealing with this period stresses the simplicity of the test and the ease with which it can be applied by physicians in isolated districts devoid of other laboratory facilities. However, it was pointed out then that the interpretation is not uniformly simple and that the skin reaction must be read by an experienced observer. In order to enhance the specificity of the reaction, particularly to eliminate the supposedly false positive reactions, attempts to improve the reagent were undertaken. An active, acid, precipitable fraction prepared by Schoenholz and Meyer (1927) from a filtered Brucella abortus cell solution prepared by freezing and thawing proved highly active in tests on guinea pigs. Injection of the purified allergen caused hemorrhagic purpura, occasionally fatal, sometimes anaphylaxis without any cutaneous response. When diluted, it gave distinct, sometimes faint reactions in patients who had recovered from undulant fever, but the reactions were in no way superior to those obtained with the standardized bacterial proteins employed for many years in epidemiologic surveys by the staff of the Hooper Foundation. After experimentation with 22 different Brucella preparations, Leavell and Amoss (1931) concluded that none of the reagents is uniformly satisfactory. They also had an unduly high percentage of falsely positive reactions. In order to increase specificity and sensitivity without also increasing the frequency of severe local and systemic reactions, investigators have devoted a great deal of time, thought and energy to developing Brucella fractions with allergenic activity (Fugazza, 1933; O'Neil, 1933; Dacey and Korovin, 1934; Goldstein, 1934; Huston et al., 1934; Huddleson et al., 1937; Morales Otero and González, 1938, 1939; Krasov, 1941; Delez et al., 1947; Castañeda, 1949; Lebrón, 1949; Mosimann, 1949). Of the approximately two dozen different fractions described and lauded by the discoverers, the protein nucleate "brucellergin" of Huddleson ruled the realm of the intradermal diagnostic tests until Angle and his group (1938), Harris and Stevenson (1950) and others questioned the sensitivity of the reagents. Now it is known that even the purified nucleoproteins are capable of causing severe local and systemic reactions. Reading of the numerous reports with a charitable degree of critical appraisal, one is vividly reminded of the period between 1910 and 1930 when every clinician or tuberculosis specialist had his own tuberculin preparation and his own interpretation of the test. This state of affairs is not surprising since skin reactions are crudely evaluated in terms of symbols representing lesions of general size and intensity. Most investigators point out the essential features of the delayed reactionerythema, central blanching, increasing edema and necrosis-but pay no attention to what constitutes the degree of hypersensitivity.

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Basic studies by Miles and Pirie (1939a and b) both of antigens suitable for intradermal tests and of quantitative evaluation of the reaction have not received the attention they deserve. These investigators observed that the highly aggregated antigenic complex of Brucella melitensis, the disaggregated antigen, and a fraction found to be a formyl derivative of an amino polyhydroxyl (AP) compound were all able to produce skin reactions. The size of the lesions was calculated by measuring their height and the diameters of the edematous area and, by using a special formula, the volume of the lesions was expressed. A similar procedure has been used by Paterson, Pirie and Stableforth (1947) who isolated the "native" antigen from Br. abortusby phenol extraction and differential centrifugation. On plotting a comparison of the average volumes of the intradermal lesions caused by 0.2 mg of a protein-like material with those of the AP compound, the volumes of the AP compound rose rapidly to sharp single peak and declined steadily. By contrast, the protein-like substance, like whole bacteria, produced a slow rise and a characteristic prolonged response. In the AP compound the allergen is readily available, but it is evidently liberated slowly from the protein-like material. This protraction may lead to local antibody production and may in some way be connected with the phenomenon of delayed reactions. The clinical possibilities of the native antigens of Miles and Pirie (1939a) in the diagnosis of brucellosis have not been investigated, but the value of these studies lies in their revelation (a) of the relationship between the purity of the antigen and the response and (b) of the influence of antigenic mass on the response. For the sake of completeness, it is considered proper to mention the various other extracts and nucleoproteins tested on infected animals or on a limited number of Brucella-sensitive human patients. In the group of proteins may be placed the allergens of Morales Otero and González (1938), Castañeda and Carrilo (1941), Delez et al., (1947) and Lébron (1949). The chemical position of the specific soluble substance and those extracted with ether-alcohol and chloroform as described by Castañeda (1949), the Duponal WA (sodium lauryl sulfate or DEP) extract of Benedict (1950) and the nonantigenic acid hydrolysate of Plum and Russeff (1939), Krasov (1941), Live and Stubbs (1947) and Ottosen and Plum (1949) are not established. Several are excellent allergens. The gluco-lipid-amino acid complex, or endotoxins, although toxic for mice, are highly antigenic. They have given specific intradermal reactions and stimulated antibody production (Pop et al., 1938). Though polysaccharides had been repeatedly isolated and tested for antigenic properties (Favilli and Biancalani, 1934; Huston et al., 1934; Topping, 1934; Higginbotham and Heathman, 1936; Reiter, 1936), their suitability in the intradermal test has, until recently, received little attention. The new allergen of Mosimann (1949) is nontoxic for mice in

WHAT SHOULD BE DONE WITH THE BRUCELLA SKIN TEST 181 a dose of 670 mg per kilogram. As a hapten, it produces no antibodies. It is readily precipitable by the serum of Brucella-infected rabbits and by human antisera. In persons with no history of brucellosis, intracutaneous injections of 100 micrograms in phenolized saline produces, as a rule, no or very fleeting erythema. On the other hand, the injection of as little as 20 micrograms released specific cutaneous reaction in 28 patients suffering or recovered from brucellosis. The erythema with and without edema appears within 5 to 8 hours and reaches its peak within 10 to 12 hours. Intensively positive deep red reactions may persist for days, sometimes for 2 weeks or longer. Aside from moderate itching, necrosis has not been observed. A veterinarian who had recovered 26 years preceding the test had a strongly positive reaction. In a series of 8 persons with an indefinite history of Brucella infection, 5 reacted positively; the serum of these five reacted positively in the complementfixation and/or in the agglutination test. These preliminary studies prompted Fust and his associates (1949) to the conclusion that the intradermal test with Brucella polysaccharides may be useful in the execution of epidemiological surveys and the solution of certain diagnostic problems. One naturally awaits with interest a quantitative evaluation of this allergen in comparison with a carefully chosen protein preparation. The skin test for brucellosis has all the shortcomings of every intradermal test and many more. The diversity of allergens in use makes comparison and correlation of the results most difficult. However, certain facts concerning specificity and sensitivity of the intradermal Brucella test have been clarified. Tests conducted by experienced workers on infected laboratory animals leave no doubt that the allergens are strictly specific, and if properly standardized induce no reaction in healthy animals not infected with Brucella organisms. On the other hand, the numerous extensive surveys conducted with the aid of the skin test on supposedly normal populations and occupational groups in the United States, Sweden, Germany, Great Britain, Mexico and other countries have shown a variable percentage of positive reactions. When it was first recognized that the incidence of positive skin tests to various Brucella antigens is surprisingly high as, for example, 9.5 in school children (Angle et al., 1938; Angle and Algie, 1939) to 15% in healthy medical students (Levin, 1930; Favorite and Culp, 1935; Menefee and Poston, 1939; Goodman, 1946), to 19.5% in a rural population (Spink, 1947) and to 24.3% among Mexican army recruits (Glusker et al., 1950), the specificity has been questioned. It is the anthropocentrically adjusted interpreters not acquainted with the well-known biological combination -host-parasite-and the existence of invisible latent infections who consider these reactions as falsely positive. The concept and the recognition of the variable severity of a Brucella infection in man is a basic requirement for a proper understanding of the pathogenesis of undulant fever.

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Every publication wrestles with such terms as "natural resistance", "reaction of the host" and "subclinical infection". Many clinicians continue to consider Brucella infection to be synonymous with Brucella disease. They must realize that invasion of the body by this organism elicits a wide spectrum of reactions and that the reactions depend on the type of Brucella and on many highly complex host-determined factors-the individual's immunobiologic constitution, his past experience with Brucella, the degree and duration of contact. On exposure, immunobiologically efficient individuals acquire immunity with relative ease and as a rule exhibit hypersensitivity to bacterial allergens. This biologic behavior is responsible for the "atypical" missed mild or entirely asymptomatic latent infections and for what have erroneously been termed "false positive" reactions. Reactions in such instances are specific and denote the occurrence of past or present unrecognized infections. Increasing percentages of positive skin reactions have been observed in successive age groups. Surveys conducted on occupational groups have repeatedly demonstrated that the percentage of positive reactions is correlated with the opportunity and frequency of contact with the infective agent. The percentage of positive reactions varied from 2.6 to 11.1 in persons in sanatoriums who consumed infected milk (Levin, 1930; Straube, 1932; Goldstein, 1934) to 40 to 50% in packing-house employees (Dubois and Sollier, 1931; Heathman, 1934; Meyer et al., 1934; Molinelli, 1942) and to 20 to 90% in veterinarians, dairy workers and persons handling livestock (Meyer et al., 1934). Danish and Swiss epidemiologists have ascribed the high percentage of latent sensitizing infections to the necessarily intimate exposure of the veterinarians while treating cattle for sterility according to the method of Albrechtsen. As might be expected, the percentage of positive intradermal reactions elicited in persons drinking raw, presumably infected milk may be as low as 2% or as high as 54.9%. Specific sensitization to Brucella allergens is common among those who have had an opportunity to come in contact with the bacteria. There is at present no agreement concerning the immunophysiological foundation of the hypersensitivity. An impressive group of observers has concluded that intimate contact with the infective material, rather than past or present infection, is responsible for it. In fact, it has been claimed that latent infection which may be responsible for allergy cannot be proved. In other words, it is the opinion of some that hypersensitivity is acquired without infection. This interpretation is contrary to the generally accepted view that a positive tuberculin-like intradermal reaction elicited with a bacterial allergen when specific reaction denotes the occurrence of infection, but gives no reliable indication of current activity. Furthermore, those engaged in the experimental study of delayed skin reactions in diseases other than tuberculosis constantly realize that the tuberculin-like allergy in some way follows a focal tissue reaction resulting from injury by the microbian parasite.

WHAT SHOULD BE DONE WITH THE BRUCELLA SKIN TEST 183 Experimental studies indicate that the mere absorption of antigens by the cutaneous or intestinal route cannot incite cutaneous allergy in human beings. None of thirty medical students repeatedly injected Yith Brucella antigens had a positive skin reaction 4 weeks after the last inoculation of the immunogenic preparations (Meyer and Eddie, 1941). Ingestion of large quantities of dead Brucella organisms has also failed to produce dermal sensitivity (Braude et al., 1949; McCullough et al., 1949). The interpretation that only invasion and multiplication produces hypersensitivity in no way implies that Brucella sensitization is synonymous with active brucellosis. It is well known that hypersensitivity may last for 17 to 30 years after recovery from clinical disease (Olmer and Massot, 1924; Dubois and Sollier, 1931; Taylor et al., 1935; Olin, 1935; Fust et al., 1949), a stage when blood serum antibodies have completely disappeared. Observations on a few individuals over the past 40 years have shown that the state of allergy is constant while agglutinins and complement-fixing antibodies fluctuate frequently under the influence of intercurrent nonspecific infections, or because of renewed contact with Brucella-infected material. Claims based on experimental observations both on animals and man directed towards species-specificity of skin testing have not been confirmed (León and Sosa, 1947; Harris and Stevenson, 1950). This lack of species-specificity is not surprising since two antigenic substances which characterize smooth Brucella strains in the agglutination test have not been isolated (Miles and Pirie, 1939b). The sensitivity of the skin test has also been questioned. Anyone familiar with allergy recognizes how extensively hypersensitivity may vary. It is markedly influenced by hormonal and possibly by emotional factors (Harris, 1950). Reactivation may follow the accidental ingestion of antigenic material with milk or dairy products. Early students of the intradermal test were disconcerted to encounter a few cases of active disease in which the skin reaction developed late or not at all (Burnet 1922; Simpson and Frazier, 1929; Carpenter et al., 1929; Huddleson et al., 1937). Even when infection was conclusively proved by culture, the skin test remained negative (Dubois and Sollier, 1931; Taylor et al., 1935; Poston and Thomason, 1936; Vedel et al., 1925-1926; Menefe and Poston, 1939; Robinson and Evans, 1939). Even a casual reading of the case histories leaves little doubt that the state of anergia reflects the low level of the immunobiologic power of the infected individual. Just as in tuberculosis, the patients may manifest no cutaneous allergy on account of desensitization by circulating Brucella antigen. Another explanation is equally reasonable. The patients represent constitutionally defective individuals at the one extreme end of the infection spectrum who have an innate predisposition to accept the parasite and inability to dispose of it by autosterilization of the tissue.

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Any survey of chronically ill persons may disclose such anergic individuals. Anergia is not incompatible with experiences with other bacterial allergic skin tests. The conclusion that a negative skin test is strong evidence of absence of recent Brucella infection (Gould and Huddleson, 1937; Dalrymple-Champneys, 1950) requires more convincing evidence before it is generally acceptable. Considering the discrepancy between the results of the agglutination test and the intracutaneous test in cattle, Live and Stubbs (1947) concluded that "the more severe the infection, as judged by the agglutination test and the milk cultures, the less likely is there to be a demonstrable skin sensitivity, perhaps due to sensitization." FOREGROUND

In order to appraise the extent of Brucella infection in families, occupational groups and population aggregates in which the infective agent is freely prevalent in the animal kingdom, the skin test is a fully accredited tool. Epidemiologists all over the world have used it and continue to use it and make valuable observations (Jordan, 1950 and others). Numerous attempts have been made to determine the practicability of allergic skin tests for the detection of brucellosis in cattle, goats, sheep and hogs. The evidence thus far collected definitely demonstrates that the tests conducted with a variety of allergens are specific. Animals with histories of no exposure and negative serologic tests never yield positive skin tests. However, discrepancies between the results of the serologic, culture and intradermal tests conclusively prove that the latter cannot be relied on to make a diagnosis in individual cases of brucellosis. Equally important and significant from a comparative standpoint are the observations that animals negative to the agglutination test may yield positive skin reactions. In a herd of cows with brucellosis of long-standing, of 177 negative to the agglutination test, 24.8% had positive or suspicious skin reactions (Live and Stubbs, 1947). Taylor and his associates (1935) considered the intracutaneous far more sensitive than the agglutination test for detecting Brucella infection in goats. At least 31% more infections were detected in a flock of 142 goats with the skin test than with the blood tests. Similar findings have been made by others with respect to goats and sheep (Dubois and Sollier, 1931; Zdrodowski, 1930; Meyer and Eddie, 1935). Similarly, when swine are exposed to natural infection, sometimes more will have skin reactions than positive agglutination titers (Delez et al., 1947). The allergic skin test is of great value in surveys to detect the animals that have been in contact with infected sources. A logical conclusion from these findings led to the repeated recommendation that any flock which contains goats allergic to Brucella is infected, and skin reactors should be removed whether they are seropositive or not. Some are carriers and shedders while others

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are immune and fail to yield Brucella cultures by the usual somewhat limited procedures in which only a few organs are cultured (Meyer and Eddie, 1935). Comparative pathology thus supports and in many ways amplifies the available records in skin test surveys made on man. These achievements have become marred by unfortunate entanglements for which the test itself cannot be justly blamed. In spite of numerous findings to the contrary, sensitization, according to many clinicians, consistently accompanied the low-grade chronic form of undulant fever. Granted that the temptation is great if one does not realize the purpose and limitations of this test or if laboratory facilities are not available for more refined testing, yielding nevertheless is not clinically sound. For years responsible investigators and clinicians have emphasized and explained the pitfalls. They have pointed out that the responsibility for diagnosis in a given case cannot be met by the skin test alone, which was not intended for this purpose. They have shown that positive reactions to intradermal or agglutination tests in the absence of other findings rarely establish the diagnosis of chronic brucellosis (Spink, 1947; McCullough, 1950). Despite these warnings, even in recent years two groups of investigators (Spicknall et al., 1950; Eisele et al., 1950) have had to spend valuable time and energy to correct the wrong interpretation of the skin test. Finding positive skin reactions to Brucella antigen in patients with the diagnosis of multiple selerosis led to the suspicion of a possible relationship between brucellosis and structural disease of the central nervous system (Kyger and Haden, 1948). It is unnecessary to dwell on these errors. It is a continuous source of acute disappointment to realize that patients with other infections, with disease of the central nervous system or even with no disease whatsoever are stigmatized as victims of brucellosis through the misuse and then faulty interpretation of this diagnostic test. Suitable Brucella antigen, a recognized method of performing and interpreting the skin test in brucellosis must be systematically worked out by reliable immunobiologists. Until this is accomplished, it is imperative to join the National Research Council Sub-Committee on the Public Health Aspects of Brucellosis in condemning the routine use of Brucella antigens for skin tests in the diagnosis of Brucella infection. BIBLIOGRAPHY Angle, Fred E. and William Algie (1939): Chronic brucellosis (undulant fever); an analytical study of the positive reactors among school children.

Ann. Int. Med. 12: 1189-1193. Angle, Fred E., William H. Algie, Leona Baumgartner and W. F. Lunsford (1938): Skin testing for brucellosis (undulant fever) in school children. Ann.

Int. Med. 12: 495-502. Benedict, Albert A. (1950): Cutaneous hypersensitivity in experimental brucellosis. Thesis, University of California.

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Braude, Abraham I., David Gold and Dorothy Anderson (1949): Formation of antibodies in human subjects after the ingestion of heat-killed Brucella abortus. Jour. Lab. & Clin. Med. 34: 744-750. Brugi, A. (1924): L'intradermoreazione di Burnet nell'infezione melitense. Pensiero Med. 13: 361-363. Burnet, Et. (1922a): Recherches sur la fievre méditerranéenne. Arch. Inst. Pasteur de l'Afrique du Nord 2: 165-211; (1922b) Sur un nouveau procédé de diagnostic de la fievre méditerranéenne. Compt. rend. Acad. d. Sc. 174: 421-423. Canale, Piero (1925): Sulle intradermoreazioni a scopo diagnostico nelle febbri infettive (tifo e melitense). Riv. Clin. Med. 26: 527-536. Carpenter, C. M., Ruth Boak and O. D. Chapman (1929): The significance of Brucella abortus agglutinins in human serum. Jour. Immunol. 12: 65-83. Castañeda, M. Ruiz (1949): A short note on Brucella fractionation. Puerto Rico Jour. Pub. Hlth. 265: 72-74. Castefieda, M. Ruiz and Clemente Carrillo Cárdenas (1941): Treatment of brucellosis with Brucella antigens. Am. Jour. Trop. Med. 21: 185-190. Cazalas, Xavier (1928): Un cas de fievre ondulante. Valeur thérapeutique de la Mélitine de Burnet. Paris Méd. 69: 161-166. Dacey, H. Gladys and Nathan Korovin (1934): Intracutaneous reactions induced in guinea pigs inoculated with Br. abortus. Jour. Lab. & Clin. Med. 19: 589-592. Dalrymple-Champneys, Weldon (1950): Undulant fever. A neglected problem. Lancet 1: 429-435; 477-485. Debré, Robert, Julien Marie and Paul Giroud (1927): Fi¿vre ondulante autochtone. Intérét de l'épreuve a la mélitine de Burnet. Bull. Mém. Soc. Méd. d. Hóp. Paris 51: 1654-1663. Delez, A. L., L. M. Hutchings and C. R. Donham (1947): Studies on brucellosis in swine. VI. Clinical and histologic features of intracutaneous reactions to fractions of Brucella suis. Am. Jour. Vet. Res. 8: 225-234. Dubois, Charles and Noel Sollier (1931): Valeur de l'intradermo-réaction a la mélitine comme procédé de diagnostic de la fi¿vre ondulante de recherche des états d'allergie et d'immunité6 l'égard du Br. melitensis. Ann. Inst. Pasteur 47: 311-331. Duffau (1928): De l'utilisation du laboratoire dans le diagnostic de la filvre ondulante. Arch. Inst. Pasteur d'Algérie 6: 486-491. Eisele, C. Wesley, Norman B. McCullough and Grace A. Beal (1950): Brucellosis and multiple selerosis. Jour. Am. Med. Assoc. 143: 1473-1474. Favilli, G. and G. Biancalani (1934): Ricerche sulle sostanze idrocarbonate specifiche dei batteri del gruppo Brucella. Isolamento di un polisaccaride dalla varieta Br. abortus (bovina). Sperimentale, Arch. Biol. 88: 337343. Favorite, Grant O. and Curtis F. Culp (1935): The intradermal test in undulant fever. Reactions in healthy and infected individuals. Jour. Lab. & Clin. Med. 20: 522-526. Fermo, Cesare de (1927): Sul metodo ed il valore della intradermoreazione nella melitense. Gazz. d. Osp. 48: 869-871. Fleischner, E. C., K. F. Meyer and E. B. Shaw (1919): A résumé of some experimental studies on cutaneous hypersensitiveness. Am. Jour. Dis. Child. 18: 577-590. Fornaca, L. and F. Bua-Fazio (1924): Intradermal seroreaction in diagnosis of Malta fever. Riforma Med. 40: 393-394. Fugazza, Edvige (1933): Sulla allergia nella infezione sperimentale da Brucella Bang. Boll. d. Ist. Sieroterap. milanese 12: 142-147.

WHAT SHOULD BE DONE WITH THE BRUCELLA SKIN TEST 187 Fust, B., H. Loffler, W. Mosimann and M. A. Schoch (1949): Kutanreaktionen mit einem Polysaccharid-Allergen aus Brucella abortus Bang. Schweiz. Ztschr. f. Path. u. Bakt. 12: 484-488. Giordano, Alfred S. (1929): Brucella abortus infection in man. The intradermal reaction as an aid in diagnosis. Jour. Am. Med. Assoc. 93: 1957-1958. Glusker, David, Patricio Fuentes Villalobos and Carlos Gómez del Campo (1950): Ocurrencia de intradermorreacciones a la coccidioidina, brucelina, histoplasmina, haplosporangina y tuberculina, con relación a los rayos X, en conscriptos del Ejército Mexicano. Bol. Ofic. San. Panam. 29: 715-722. Goldstein, Jacob D. (1934): Cutaneous reactions in the diagnosis of undulant fever. Jour. Clin. Invest. 13: 209-218. Goodman, Morton J. (1946): Chronic brucellosis. Jour. Am. Med. Assoc. 131: 60. Gould, S. E. and I. F. Huddleson (1937): Diagnostic methods in undulant fever (brucellosis). With results of a survey of 8,124 persons. Jour. Am. Med. Assoc. 109: 1971-1974. Harris, Harold J. (1950): The possible influence of psychological factors in Brucella allergy. Internat. Arch. Allergy & Applied Immunol. 1: 109-115. Harris, Harold J. and Blanche J. Stevenson (1950): Brucellosis (undulant fever). Ed. 2, New York: Paul B. Hoeber, 617 pp. Heathman, Lucy S. (1934): A survey of workers in packing plants for evidence of Brucella infections. Jour. Infect. Dis. 55: 243-265. Higginbotham, Margaret and Lucy S. Heathman (1936): Precipitin and complement-fixation reactions of polysaccharide extracts of Brucella. Jour. Infect. Dis. 69: 30-34. Huddleson, I. Forest, Myrtle Munger, S. E. Gould and Doris Paulson (1937): A study of Brucella infection and immunity in humans. Am. Jour. Trop. Med. 17: 863-880. Huston, R. C., I. F. Huddleson and A. D. Hershey (1934): The chemical separation of some cellular constituents of the Brucella group of micro-organisms. Michigan Agric. Exper. Station Tech. Bull. No. 137, 25 pp. Jordan, Carl F. (1950): The epidemiology of brucellosis. In: Brucellosis. A symposium under the joint auspices of National Institutes of Health of the Public Health Service, Federal Security Agency, United States Department of Agriculture, National Research Council, September 22-23, 1949, Bethesda, Maryland. Washington: American Association for the Advancement of Science, pp. 98-115. Krasov, V. M. (1941): Biokhimicheskie i immunologicheskie svoistva fraktsii, vydelyaemylkh pri hidrolize bakterii brutsell. Veterinaria, Moscow No. 1, pp. 56-68. (Abstract: Vet. Bull. 12: 576, 1942) Kyger, E. R., Jr. and Russell L. Haden (1948): Brucellosis and multiple selerosis. Cutaneous reactions to Brucella antigens. Am. Jour. Med. Sc. 216: 689-693. Leavell, Hugh R. and Harold L. Amoss (1931): The endermic reaction in Brucella infections. Arch. Int. Med. 48: 1192-1197. Lebrón, A. P. (1949): A heat-stable, water-soluble Brucella allergen. Preliminary report. Puerto Rico J. Pub. Health 24: 337-342. Lemierre, A., G. Marchal and A. Jaubert (1927): Un cas de fievre ondulante autochtone. Valeur diagnostique et thérapeutique de l'intradermo-réaction de Burnet. Bull. Mém. Soc. Méd. d Hóp. de Paris 51: 1702-1709. León, Alberto P. and José Sosa (1947): Allergy in brucellosis. Am. Jour. Pub. Hlth. 37: 1033-1040. Levin, William (1930): The intradermal test as an aid in the diagnosis of undulant fever. Jour. Lab. & Clin. Med. 16: 275-281.

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Liege, R. and R. Castéran (1929): Un nouveau cas de mélitococcie guéri a la suite d'un injection unique d'une endo-protéine préparée avec le Bacillus abortus de Bang. Gaz. d. Hop. 102: 41-43. Live, I. and E. L. Stubbs (1947): Intracutaneous brucellosis tests in cattle. Am. Jour. Vet. Res. 8: 380-385. McCullough, Norman B. (1950): Laboratory tests in brucellosis. In: Brucellosis. Washington: Am. Assoc. Adv. Sci., pp. 116-121. McCullough, Norman B., C. Wesley Eisele and Grace A. Beal (1949): Oral administration of killed Brucella to man. Pub. Hlth. Rep. 64: 1613-1616. Menefee, E. E., Jr. and Mary A. Poston (1939): Significance of standard laboratory procedures in the diagnosis of brucellosis. Am. Jour. Med. Sc. 197: 646-653. Meyer, K. F. and B. Eddie (1935): The problem of caprine brucella infections in the United States. Jour. Am. Vet. Med. Assoc. 39: 286-303. (1941): Laboratory infections due to Brucella. Jour. Infect. Dis. 68: 24-32. Meyer, K. F., B. Eddie, L. Veazie, I. M. Stevens, B. Stewart and J. C. Geiger (1934): The heterogenous infection chains as occupational diseases. Arch. f. Gewerbepath. u. Gewerbehyg. 5: 514-559. Miles, A. A. and N. W. Pirie (1939a): The properties of antigenic preparations from Brucella melitensis: I. Chemical and physical properties of bacterial fractions. Brit. Jour. Exper. Path. 20: 83-98. (1939b): The properties of antigenie preparations from Brucella melitensis: III. The biological properties of the antigen and the products of gentle hydrolysis. Ibid. 20: 278-296. Mitra, Mariano (1924): La intradermoreazione nella febbre mediterranea dell'infanzia. Pediatrica 32: 721-724. Molinelli, Ernesto A. (1942): A survey of brucellosis in the Argentine Republic. Part I: Introduction. Proc. Sixth Pacific Sc. Cong. 5: 267-309. Montagnani, M. (1923): L'intradermoreazione nel tifo, paratifo A e B e nella febbre melitense con i filtrati di cultura. (Contributo clinico e sperimentale.) Riv. Crit. di Clin. Med. 24: 149-156; 161-163. Morales Otero, Pablo and Luis M. González (1938): Purified protein antigen from Brucella. Proc. Soc. Exper. Biol. &Med. 38: 703-705. (1939): Allergy in Brucella infections. Ibid. 40: 100-102. Mosimann, Walter (1949): Allergene aus Brucella abortus Bang. Schweiz. Ztschr. f. Path. u. Bakt. 12: 362-379. Olin, Gunnar (1935): Studien uber das Undulantfieber in Schweden. Acta Soc. Med. Suec. 61: 1. Reprint published by Buchdruckerei-Aktien-Ges. Isaac Marcus, Stockholm, 112 pps. Olmer, D. (1929): La mélitine de Burnet: son intérét pour le diagnostic, le traitement et la prophylaxie. Jour. méd. Franc. 18: 182-186. Olmer, M. D. and M. Massot (1924): Sur le diagnostic de la mélitococcie par l'intradermo-réaction. Marseille Méd. 61: 1206-1211. O'Neil, Alfred E. (1933): Preliminary note on the treatment of undulant fever in man with detoxified vaccine and with antiserum. Ohio State Med. Jour. 29: 438-439. Ottosen, H. E. and N. Plum (1949): A nonantigenic allergic agent for intradermal brucellosis tests. Am. Journ. Vet. Res. 10: 5-11. Paterson, J. S., N. W. Pirie and A. W. Stableforth (1947): Protective antigens isolated from Br. abortus. Brit. Jour. Exper. Path. 28: 223-236. Plum, N. and C. Russeff (1939): Immunobiological studies on Brucella abortus Bang for establishment of a serviceable allergic diagnostic means. Skandinav. vet. tidskr. 29: 31-53.

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Pop, Al., A. Damboviceanu, Cella Barber and I. Marinov (1938): Antigéne complet des Brucella. Propriétés biologiques. Compt. Rend. Soc. de Biol. 127: 733-736. Poston, Mary A. and Robert H. Thomason (1936): Meningitis due to Brucella in a child. Am. Journ. Dis. Child. 52: 904-906. Rainsford, S. G. (1935): Undulant fever, with special reference to cases seen in Malta. Jour. Roy. Nav. M. Serv. 21: 81-105; 182-216. Reiter, Dorothy 0. (1936): Studies on the extraction of a precipitable substance from the genus Brucella. Journ. Infect. Dis. 58: 45-58. Robinson, Frank H. and Alice C. Evans (1939): Chronic brucellosis in Charlotte, N. C. Report of cases; Jour. Am. Med. Assoc. 113: 201-206. Schoenholz, P. and K. F. Meyer (1927): The purification of abortin. Jour. Infect. Dis. 40: 453-468. Simpson, Walter M. and Eunice Frazier (1929): Undulant fever. Report of sixtythree cases occurring in and about Dayton, Ohio. Jour. Am. Med. Assoc. 93: 1958-1965. Sorge, G. (1925): Sul valore dell'intradermoreazione nell'infezione melitense dei bambini. Riv. de Clin. Pediat. 23: 471-481. Spicknall, Charles G., Leonard T. Kurland, B. N. Carle and Luther L. Terry (1950): Relation of brucellosis and multiple selerosis. Jour. Am. Med. Assoc. 143: 1470-1473. Spink, Wesley W. (1947): The problem of chronic brucellosis. Chicago Med. Soc. Bull. 50: 137-140. Straube, Ginther (1932): Untersuchungen über Bang-Allergie und Serumphanomene am Menschen. Med. Klin. 28: 1501-1502. Tapia, M. and A. del Valle (1928): Valor diagnóstico de las reacciones cutáneas con antígeno específico en la fiebre de Malta. Med. ibera 2: 317-321. Taylor, R. M., M. Lisbonne and L. F. Vidal (1935): La fievre ondulante en France d'apres les investigations du "Centre de recherches sur la fievre ondulante" de Montpellier. Mouvement Sanitaire, No. 130, 40 pp. Topping, Lucy E. (1934): Carbohydrate and nucleoprotein fractions isolated from the Brucella group. Jour. Path. & Bact. 39: 665-668. Trenti, Enrico (1923): Reazioni cutanee nell'infezione da micrococco melitense. Policlinico (sez. prat.) 30: 1249-1254. Vedel, A. Puech and M. Janbon (1925-1926): Deux cas de polynévrite mélitococcique. Bull. Soc. d. Sc. Méd. et Biol. de Montpellier 7: 406-410. Yeckel, H. C. and O. D. Chapman (1933): Brucella (Alcaligenes) infections in man. The intradermal reaction as an aid in diagnosis. Jour. Am. Med. Assoc. 100: 1855-1856. Zdrodowski, P., H. Brenn and B. Voskressenski (1930): Etude sur la fievre ondulante en Azerbaidjan. Recherches spéciales sur le groupe Brucella melitensis-abortus. Ann. Inst. Pasteur 45: 768-805.

¿QUE DEBE HACERSE CON LA CUTIRREACCION PARA LA BRUCELOSIS? (Sumario) La cutirreacción para la brucelosis con "abortin" se utilizó al principio con éxito para diferenciar la brucelosis experimental de la tuberculosis. Resulta difícil evaluar el empleo clinico subsecuente de la reacción en 75 jóvenes, pues aunque el abasto de leche se hallaba sumamente infectado, no había pruebas de que el microorganismo fuera patógeno para los niños. Las primeras investigaciones experimentales demostraron que el conejo no se presta para el estudio de la

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hipersensibilidad a la brucela, y que las reacciones positivas no indican forzosamente inmunidad. Más tarde se comenzó a utilizar en la clínica el "melitin" de Burnet, recomendando su empleo algunos investigadores y poniéndolo en duda otros. Aunque

la reacción es específica, no fué posible obtener un filtrado activo de caldo con todas las cepas de Brucella melitensis, y los enfermos con otras infecciones en ocasiones reaccionaban con mucha intensidad a la administración local del antígeno melitensis.

Desde un principio la cutirreacción contó con muchos defensores debido a que se ejecuta con suma sencillez, pero también desde el principio se ha recalcado que la interpretación no es sencilla y s61o deben probarla los observadores avezados. Por lo menos durante 20 años se ha tratado de mejorar el reactivo para aumentar la especificidad y eliminar las seudopositivas sin provocar reacciones muy graves. El éxito obtenido no ha sido sensacional ni siquiera adecuado para justificar el empleo de la prueba para la diferenciación diagnóstica delicada.

Miles y Pirie observaron que tres antígenos distintos de la Br. melitensis pueden producir experimentalmente cutirreacciones y midieron cuidadosamente las lesiones cutáneas, lo mismo que Paterson y colaboradores. Estos estudios, que ofrecen bases sólidas para más investigaciones y quizás para la evaluación clínica, pusieron de manifiesto la relación que existe entre la pureza del antígeno y la

reacción, y la influencia de la masa antigénica sobre la reacción alérgica. Tratando de descubrir la alergina ideal, se han utilizado extractos, nucleoproteinas, endotoxinas, polisacáridos y haptenos. El polisacárido de Mosimann parece prometedor.

La reacción ha sido criticada injustamente por los que se olvidan de que brucelosis no es sinónimo de invasión por microorganismos brucelosos. Si el enfermo no tiene manifestaciones clínicas de brucelosis, pero acusa una cutirreacción positiva, la prueba no es forzosamente seudopositiva; quizás refleje una

infección anterior que jamás se puso de manifiesto. Además de la constitución inmunológica del sujeto, la duración de la exposición quizás afecte la reacción. Con toda probabilidad, la reacción indica que en una ocasión u otra la brucela invadió los tejidos, pero no indica nada en cuanto a la actividad actual de la infección. La hipersensibilidad puede persistir hasta 30 años, y es más constante que la aglutinación o la reacción de fijación del complemento. También pueden afectarla factores hormónicos y emocionales. La ingestión de antígenos de brucelas no provoca por si sola ninguna reacción dérmica. La anergia constituye otro obstáculo al análisis exacto de la cutirreacción. En resumen, la cutirreacción es un arma epidemiológica bien acreditada que se ha utilizado para realizar observaciones científicas sólidas, pero en la actualidad no puede confiarse en ella para hacer el diagnóstico de un caso dado. La sensibilidad cutánea no indica forzosamente brucelosis crónica de poca intensidad. Antes de utilizar la prueba para el diagnóstico de un solo caso, los inmunobiólogos avezados deben preparar un antígeno adecuado de Brucella, y elaborar un método sólido para la ejecución e interpretación de la misma. Mientras no se logre esto, no puede menos que condenarse el empleo sistemático de los antígenos de Brucella para las cutirreacciones utilizadas para el diagnóstico de la brucelosis.

MODALIDADES CLINICAS DE LA BRUCELOSIS HUMANA Por el Dr. T. DE VILLAFAÑE LASTRA Miembro de la Comisión Permanente para el Estudio de la Brucelosis del Clrculo Médico de Córdoba, Argentina En una de las sesiones del Segundo Congreso Panamericano de la Brucelosis, reunido en Buenos Aires en noviembre de 1948, se resolvió que en esta tercera reunión se dedicara preferente atención al estudio del tema "Clínica de la brucelosis crónica". Como una aportación al anhelo expresado en esa asamblea es que he decidido hacer un análisis de las características clínicas que presentaron 550 de mis pacientes en quienes el diagnóstico de brucelosis pudo establecerse, de manera inobjetable, de acuerdo a nuestro criterio. Desde 1938 dedico especial atención al estudio de esta enfermedad y, en particular, al conocimiento de sus características clínicas, que valoramos indispensable para poder apreciar la verdadera trascendencia de la brucelosis en la especie humana. Este estudio lo hemos realizado en base a la observación minuciosa de nuestros enfermos, algunos por un período mayor de 20 años; utilizando todos los medios complementarios del diagnóstico clínico integral, bacteriológicos, biológicos, físicos, de especializados, etc. Las investigaciones anátomo-patológicas han merecido nuestro más vivo interés, especialmente el estudio macroscópico e histopatológico, tratando de valorar las características anatómicas e histopatológicas que esta enfermedad puede producir en los distintos parénquimas o sistemas del organismo. Son los estudios anatómicos realizados en los últimos 15 años sobre materiales de biopsias o de necropsias humanas y los realizados sobre piezas obtenidas de animales los que, al enseñarnos a conocer las características histológicas y anatómicas y su interpretación clínicoradiológica, nos van permitiendo comprender muchas de las modalidades de esta enfermedad que antes de estos estudios pudieron ser discutidos o aún desconocidos en su relación patogenética con la brucelosis. Dos tipos de padecimientos fueron observados en estos 550 enfermos y que denominamos "modalidad aguda" y "modalidad crónica". MODALIDAD AGUDA

Su clínica está en función del carácter septicómico con que evoluciona durante el período de iniciación y estado en general similar al de todas las enfermedades septicémicas. Patogenéticamente, esta modalidad aguda la creemos, en general, en relación de dependencia con una primoinfección brucelósica reciente, masiva, a gérmenes de virulencia exaltada y con condiciones inmunobiológicas especiales del paciente. Sin embargo, 191

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no creemos imposible de acuerdo al estudio de nuestra casuística que pueda ser debida a exacerbación endógena o reinfección exógena en sujetos que, habiendo superado una infección anterior, sean nuevamente victimas de reinfecciones masivas de gérmenes de marcada virulencia. Dos formas diferentes de iniciación hemos observado en la modalidad aguda: (1) de comienzo brusco y (2) de comienzo insidioso o subagudo; siendo la evolución y pronóstico en ambos, diferentes. La primera "de comienzo brusco" como su nombre lo indica, está caracterizada porque el paciente ha pasado súbitamente de la salud a la enfermedad; pudiendo precisar exactamente el día y la forma cómo se inició el padecimiento. Los sintomas iniciales suelen ser escalofríos, cefaleas, temperaturas continuas elevadas, 40°C y más, crisis sudorales, mialgias, estado nauseoso y marcada participación del estado general. En esta faz inicial suele haber reacción hepato-esplénica franca; como asimismo alteraciones del cuadro hematológico caracterizadas por anemia con leucopenia y neutropenia. Posteriormente las más variadas y graves localizaciones viscerales o tisurales suelen producirse como resultante de la severa septicemia brucelar que el enfermo soporta, debiendo destacarse por su pronóstico generalmente fatal, las localizaciones encefalíticas, las pancarditis donde la lesión más evidente suele ser de endocarditis vegetante y la forma hemorrágica o panmielotisis por agresión a órganos hematopoyéticos y sistema retículoendotelial. En estos pacientes las aglutininas suelen estar ausentes o sólo presentes a títulos bajos en los primeros 15 días de enfermedad. Posteriormente aparecen, alcanzando rápidamente cifras elevadas, de 1:500, 1:1000 ó más. La intradermorreacción suele también ser negativa. En cambio el hemocultivo es positivo a condición de que la siembra se efectúe en medios nutritivos adecuados y suele ser positivo reiteradamente en este periodo, demostrando esto, que el enfermo lleva una septicemia verdadera. Esta forma aguda, de iniciación brusca, corresponde a las descritas con los nombres de tificas, sudoroálgidas o ataxoadinámicas por las distintas escuelas que se han ocupado del estudio de esta enfermedad. Creemos necesario describir tres ejemplos de nuestra casuística. E1 primero se refiere a un hombre joven que vivió en zona apartada de contaminaciones masivas. Observacion 1.-O. D. A., argentino, 28 años. Era sano y vivió en la ciudad hasta poco antes de enfermar, que se establece como administrador de una estancia. Quince días antes de iniciarse su afección atiende el aborto de una vaquillona, efectuando extracción manual de placenta. El 28 de diciembre de 1942 muestra temperatura de 38°C, que persiste dos días, quedando posteriormente bien, según los familiares. El médico que lo

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examinó comprobó en esa oportunidad bazo grande, doloroso. El 7 de enero de 1943, temperatura entre 39 y 40°C; escalofríos intensos, malestar acentuado. Hay gorgoteo intestinal; bazo e hígado grandes. El examen de sangre demuestra: glóbulos rojos 5,100,000; blancos 1950; fórmula leucocitaria: neutrófilos 0%; basófilos 0%; eosinófilos 0%; linfocitos 98.75%; monocitos 0.25%; células de Turk 1%. El 10 de enero, presenta muy mal estado general. Temperatura 41 0C; escalofríos intensos, gran taquicardia, disnea. Gran excitación y a ratos obnubilación acentuada. Hay tos con expectoración purulenta. Soplo en base derecha y la radiografía de tórax demuestra infiltración de esa zona. Bazo grande, hígado grande y gran meteorismo abdominal. Huddleson positiva al 1:2,000. El hemocultivo da desarrollo de una Brucella clasificada como suis. Examen de sangre: glóbulos rojos 4,990,000; blancos 800. Fórmula leucocitaria: basófilos 0%; eosinófilos 0%; neutrófilos 1%; linfocitos 96%; monocitos 3%. No se observan formas prematuras ni alteraciones globulares en ambas series. Falleció en forma sincopal, a los quince días de su primer episodio febril. En resumen, el enfermo que consideramos libre de infección anterior se contaminó efectuando extracción manual de placenta, adquiriendo una septicemia brucelar sobreaguda. Los otros dos ejemplos corresponden a enfermos residentes en zona de alta infestación. El estudio anatómico de las lesiones óseas y neurológicas (médula y encéfalo) como asimismo el cuadro de panmielotisis de una de ellas, hacen que tenga especial interés. Observacion 2.-L. O., argentino, 50 años. Agua de Ramón, Depto. Minas, Prov. de Córdoba. Inicia su enfermedad en diciembre de 1940 con fiebre, astenia, cefaleas, zumbido de oídos, sordera bilateral progresiva. Al examinarlo por primera vez (2-VI-1941) nos llama la atención su psiquismo, enfermo apático, indiferente, contesta con marcado retardo. Inapetencia, rehusando alimentarse. Hígado grande y doloroso; bazo grande. Sistema nervioso: No hay Kroenig. Hay paraplejía total con atrofia flácida dolorosa. Lasegue bilateral. Reflejos abdominales, mediopubiano, patelares y aquilianos abolidos. Hormigueos y calambres en pantorrillas y pies. En el liquido céfalorraquideo: albúmina 0.85 gm por mil; cloruros 6.30 por mil; Nonne +++; Pandy + +; 39 elementos por milímetro cúbico: neutrófilos 31.50%; linfocitos 43%; células endoteliales 16.50%; células destruidas 9.%; Wassermann negativa; reacción de Mastic: curva meningitica. Reacción de aglutinación, técnica de Huddleson, en liquido céfalorraquideo positiva 1:200; en sangre positiva 1:500. Wassermann y Kahn en sangre negativas. El estudio radiográfico muestra alteración del segmento lumbar, único radiografiado. En la vista frontal se observa! lesión espondilitica tipo pico de loro en extremo lateral de tercera y cuarta vértebras lumbares; disminución del espacio que separa la segunda de la tercera vértebra lumbar. En el perfil obsérvase lesión espondilitica tipo pico de loro en extremo anterior de tercera y cuarta lumbares.

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En la necropsia se comprobó alteración evidente en bazo, higado, corazón y riñones. En cuanto al estudio anatómico del sistema nervioso, damos una síntesis del informe de los Dres. Peirotti y Pucheta Morcillo: "Descripción macroscópica: edema meníngeo, esencialmente notable en la bóveda cerebral, donde se comprueban francos exudados meningiticos." Cerebro: pálido (anemia). Vasos y nervios de la base normales. Configuración cerebral externa normal. Es muy evidente la condensación de la aracnoides en la región optoquiasmática y cuadrigeminal (leptomeningitis plástica), hecho que dificulta la separación del romboencéfalo. También se observan adherencias meníngeas en la región interhemisférica de lóbulos frontales. El corte revela ventrículos laterales aumentados de tamaño. No hay otra particularidad. Romboencéfalo: Nada de particular. Médula: Poliomielitis bilateral que toma tanto las astas anteriores como las posteriores, en el engrosamiento cervicodorsal. Pequeños puntos hemorrágicos a ese nivel que llegan casi a ser una mielomalacia en algunos sitios, especialmente a la izquierda. El estado congestivo persiste en toda la longitud de la médula dorsal, intesificándose en la mitad inferior de esta región. En la médula lumbar y sacra no se ven lesiones macroscópicas de mielitis, pero en la región lumbosacra y la cola de caballo, la leptomeninge ha sido asiento de un proceso plástico, con pérdida de su trasparencia, lo que ha producido el aglutinamiento de las raíces. Estudio microscópico: En el encéfalo se observa discreta reacción linfocitaria, meninges con vasos ligeramente aumentados de volumen en la zona subcortical y aun en plena corteza; algunos de estos vasos llevan un discreto acompañamiento de linfocitos a su alrededor. En un corte de la columna, comprendiendo desde tercera vértebra dorsal a cuarta lumbar inclusive, se puede observar lesión espondilftica de los cuerpos de segunda y tercera lumbares, con destrucción del disco intervertebral, el que hace hernia franca dentro del conducto raquídeo. El núcleo pulposo de la lla. dorsal está haciendo providencia dentro del cuerpo vertebral a través de una caries de esta vértebra (hernia intraesponjosa o nódulo de Schmorl). Se observa también la destrucción del disco que separa la 5a. de la 6a. vértebra dorsal. Interesa destacar que a pesar de existir tan s61o 14 días entre la muerte del enfermo y el momento de obtener las radiografías de columna descritas, la lesión espondilitica tan severa de las 2a y 3a vértebras lumbares y la hernia del disco que muestran los documentos anatómicos no eran presumibles en esas radiografías. S61o se velan lesiones del extremo anterior de los cuerpos vertebrales. Observaci6n 3.-R. de K. ingresó al servicio con epistaxis, gingivorragias y hemoptisis. Presentaba manchas esquimóticas y purpúricas en todo el cuerpo. Había iniciado su afección tres meses antes en los alrededores de la ciudad de La Rioja, con las características generales ya descritas para la forma aguda de iniciación brusca. A su ingreso, el examen de sangre era el siguiente: glóbulos rojos 1,450,000; glóbulos blancos 1,600; hemoglobina 24%; valor globular 0.80; plaquetas 78,000. Fórmula leucocitaria: neutrófilos 1%; linfocitos 93%; monocitos 6%; formas

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prematuras: un eritroblasto basófilo; alteraciones hemáticas: en series roja aniso y poiquilocitosis; en blanca ausente. La prueba hemogénica efectuada ese día, dió el siguiente resultado: (1) tiempo de sangría, 40 minutos; (2) tiempo de coagulación 9 minutos; (3) coágulo a 370 C, irretráctil hasta las 24 horas; (4) plaquetas no se aglutinan; hay anisoplaquetosis y anisocromia. Al cuarto día de su ingreso la fórmula era la siguiente: glóbulos rojos 800,000; glóbulos blancos 1,200; hemoglobina 16%; valor globular 0.62. Fórmula: neutrófilos 1%; linfocitos 93%; monocitos 5%. Formas prematuras ausentes en ambas series; alteraciones hemáticas globulares; en la fórmula roja: anisocitosis, poiquilocitosis, esquizocitosis, hipocromia, anisocromia. Serorreacción aglutinación positiva 1:200. Hemocultivo positivo Brucella melitensis. Falleció al séptimo día de su ingreso. Radiológicamente s61o se investigó columna lumbar, comprobándose lesión espondilitica en extremo anterior de 4a. lumbar e imagen de ósteartritis condensante en las articulaciones intervertebrales y partes óseas vecinas. La autopsia mostró alteraciones francas de bazo e hfgado. También lesión de sistema nervioso central y espondilitis grave. La pieza anatómica de la columna muestra lesión espondilitica a la altura de 7a, 8a y 9a dorsales. No hay absceso osifluente. Entre 10a y l1a vértebras dorsales hernia intraesponjosa y en las pequeñas articulaciones que unen l0a, 1la y 12a vértebras dorsales y sus arcos, ósteoartritis condensante. El corte mediano longitudinal de la columna dorsal muestra la lesión espondilítica de 8a y 9a dorsales, con desaparición del disco intervertebral y borramiento del canal raquídeo a su nivel. En el corte mediano longitudinal de columna lumbosacra en la parte correspondiente a arcos posteriores, articulaciones intervertebrales y apófisis espinosas, puede observarse marcada infiltración de las partes óseas. Estas lesiones de articulaciones y arcos posteriores las observamos con mucha frecuencia en brucelosis crónica, configurando tipos de reumatismo rizomiélico y por ello he estudiado estas piezas con detenimiento. La forma aguda de "comienzo insidioso o subagudo" corresponde a los cuadros ya clásicos conocidos con los nombres de fiebre ondulante o fiebre gástrica del Mediterráneo o fiebre loca. Las descripciones de Marston, 1861, de Hughes y la propia de Ramón, la definen admirablemente. De iniciación no bien precisada, el período de invasión es lento y de sintomatología vaga; el enfermo no puede indicar con exactitud cuando comenzó su enfermedad y en esto se diferencia fundamentalmente de la forma anterior. Paulatinamente se pasa del estado de salud al de enfermedad. Los síntomas iniciales suelen ser algias generalizadas, astenia, cefaleas, trastornos nauseosos, epistaxis, estados subfebriles, crisis sudorales, etc. Las características más salientes del período de estado suelen ser la fiebre que puede adoptar los más variados tipos, siendo entre nosotros frecuente el clásico tipo ondulante, los sudores, las algias y los dolores articulares. Manifestaciones gastrointestinales, hepato-esplenomegalia

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y modificación importante del cuadro hematológico (anemia y leucopenia con neutropenia). Una descripción de todas las manifestaciones clínicas de esta forma de la enfermedad es imposible dar en un trabajo de esta índole. Diremos aquí solamente que durante su evolución pueden aparecer las más variadas localizaciones; neurológicas, óseas, pulmonares, pleurales, cardiovasculares, genitales (orquitis u orcoepididimitis en el hombre; ovaritis, salpingo-ovaritis, metritis y para-metritis en la mujer), renales, hepáticas, vesiculares, intestinales, etc. La siguiente historia clínica con localización pulmonar no descrita, bronquiectasia a forma cavitaria es un buen ejemplo. Observación. 4-A. B., varón argentino, 15 años. Sebastián EI Cano, Prov. de Córdoba, cuidador de cabras. Siempre sano, hasta febrero o marzo de 1946 en que aparece pérdida de apetito, marcado decaimiento y sudores abundantes. En abril y mayo 1946 tos importante, seca al principio y después acompañada de esputos purulentos. El 25 de mayo 1946 hemoptisis abundante, ingresando por ella al Hospital Santa Rita donde permanece 4 meses. La hemoptisis duró 10 días, pero debió permanecer internado por temperatura del tipo ondulante. Hemocultivo positivo para brucelas. En diciembre 1946 nuevo empuje febril y hemoptisis que repite en marzo 1947, cuando le examinamos. Enfermo desnutrido, muy pálido, febril 38.50C. Nariz, boca y amígdalas normales. Tórax: Hay tos con esputos purulentos. El examen clínico no descubre nada especial. El estudio radioscópico permite observar una zona infiltro-ulcerosa en base derecha. Bazo muy grande. Hígado rebasa dos travesos de dedo la arcada costal. Examen de esputo reiterado: negativos para el Koch. Examen de sangre: glóbulos rojos 4,000,000; blancos 8,200; hemoglobina 62%; valor globular 0.77. Fórmula leucocitaria: polinucleares neutrófilos 45%; linfocitos 47%; eosinófilos 3%; monocitos 5%. Serorreacción de aglutinación, técnica de Huddleson, positiva hasta la dilución 1:5,000. Hemocultivo positivo; se aisla un germen que por sus caracteres morfológicos y requerimientos para la vegetación corresponde a una Brucella melitensis. La radiografía del tórax muestra en la parte inferior derecha una imagen anular. La broncografía corrobora la impresión de que esa imagen anular corresponda a una cavidad, penetrando el lipiodol muy fácilmente en ella. El enfermo falleció durante una de sus hemoptisis el 7 de junio de 1947. El Dr. Julio A. Herrero efectuó autopsia parcial. El informe es como sigue: "Pulmón derecho: se encuentra hepatizado. Al corte, el lóbulo inferior en su parte central presenta una cavidad del tamaño de una nuez de forma oval y de paredes lisas con depresiones en forma de criptas. El resto del parénquima pulmonar se presenta congestivo. Corazón: moderadamente aumentado de tamaño, pálido, con tendencia a

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aplastarse sobre la mesa. Abierto, según técnica, el miocardio aparece pálido. Aparatos valvulares de aspecto normal. Dilatación de las válvulas tricúspidas. Bazo: enormemente aumentado. Pesa 1,140 gm. Al corte sumamente congestivo. Riñones: muy pálidos. Al corte se aprecian bien ambas sustancias, existiendo congestión de la sustancia medular. Se descapsulan con facilidad. Estudio histológico: Pulmón: caverna ulcerosa complicada, revestida en gran extensión por epitelio bronquial. En los sitios donde éste falta, se encuentra tejido de granulación, muy rico en vasos inyectados de sangre. Los fenómenos de infiltración inflamatoria y hemorragia son profusos, observándose masas purulentas infiltradas por la sangre adheridas a la pared de la caverna. En la periferia se observa muy viva proliferación del epitelio bronquial que toma aspectos edematosos. El parénquima circundante presenta lesiones de alveolitis descamativa, alveolitis hemorrágica y edema. Bazo: congestivo, de éstasis con aumento de la pulpa roja por repleción venosa. Hígado: del tipo del hígado cardíaco en sus primeras fases. Hay además zonas de degeneración grasa y focos de infiltración inflamatoria crónica en los espacios de Kieman. Corazón: Miocarditis intersticial. Esclerosis del tejido reticular con fragmentación de las fibras musculares y discreta infiltración inflamatoria crónica. Riñones: tumefacción turbia de los túbulos y congestión. Diagnóstico anátomopatológico: caverna bronquiectásica ulcerosa supurada y hemorrágica. Miocarditis e insuficiencia hepatorrenal." En resumen, enfermo con brucelosis aguda a iniciación insidiosa, haciendo en el curso de ella manifestaciones pulmonares, por caverna bronquiectásica. También miocarditis e insuficiencia hepatorrenal. Nos interesa destacar sobre manera la caverna bronquiectásica que patogenéticamente la creemos debida a una localización visceral de la septicemia brucelósica que venía soportando este paciente. Fueron portadores de esta forma aguda o sea septicémica de brucelosis, 80 de nuestros 550 pacientes. De estos 80 enfermos agudos, 32 (40%) pertenecieron al tipo agudo de iniciación brusca, falleciendo de entre ellos 11 (34.3%). Al grupo agudo de iniciación insidiosa o subaguda pertenecieron 48 enfermos (60%), falleciendo 3 (5.2%). Del conjunto de estos 80 enfermos agudos, fallecieron 14 (17.5%); 18 curaron (22.5%) después de proceso septicémico prolongado; 48 (60%) fueron a la cronicidad, alcanzando secundariamente muchos de ellos la curación. MODALIDAD CRÓNICA Desde que Miss Alice Evans' publicara su articulo "Brucelosis Crónica" se han multiplicado los trabajos sobre este tema, pero aun no es totalmente aceptada ni existe acuerdo sobre sus características clínicas, frecuencia y trascendencia en patología humana.

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Si el término "agudo" o "crónico" se refiriera al tiempo de duración del padecimiento, quizás las discusiones serian inoficiosas. Con anterioridad a la era antibiótica (aureomicina y cloromicetina) sólo por excepción se velan curaciones radicales antes de los 6 meses de padecimientos. Pero, en una enfermedad como ésta, tan múltiple en sus aspectos clínicos y de evolución, importan más para hablar de modalidad aguda o crónica las características clínicas. Para nosotros la brucelosis a modalidad aguda lleva implícito, que el paciente está bajo la acción de un proceso septicémico, con sus características, cuadro toxi-infeccioso de altura variable, pero evidente; reacción febril, etc. Cuando los autores clásicos hablaban de enfermedad aguda en brucelósicos con evolución de 1, 2, ó 3 años, evidentemente se referían a las características clínicas de estos cuadros. Muchos de nuestros 80 enfermos agudos han llevado padecimiento de más de 18 meses, y en uno de ellos la evolución fué de 3 años y medio, falleciendo. Por presentar durante todo este período ondas febriles, con cuadro septicémico sostenido que dió reiterados hemocultivos positivos, lo consideramos "forma aguda de brucelosis". Para el concepto de cronocidad también, en nuestro sentir, importa más la característica clínica que el propio tiempo de evolución, que por supuesto debe ser prolongado. Llamamos, pues, "modalidad crónica" a todos aquellos cuadros de enfermedad cuyos padecimientos están caracterizados, como los describiera inicialmente Miss Evans, por una disminución fisica y psíquica del paciente comparable a la neurastenia, pero debiendo tenerse presente que durante el curso de su evolución pueden realizarse las más variadas localizaciones tisulares (óseas, ósteoarticulares, musculares, etc.) o viscerales (cardiovasculares, bronco-pleuro-pulmonares, neurológicas, genitales, etc.) que son las que darán las características clínicas de cada caso y las que regirán su evolución y pronóstico. Pero, hecho fundamental, el período de septicemia ya ha pasado o esta forma clínica fué sistémica, sin brotes septicémicos francos y por consiguiente, el hemocultivo es generalmente negativo. La fiebre, en general, es de bajo grado o está ausente. Claro está que hay ejemplos, como los de Miss Poston,2 en que el germen se pudo aislar de la sangre. En determinadas circunstancias podrán obtenerse cultivos positivos por siembras directas de sangre, expresando esto momentos de bacteriemias, previa inoculación al cobayo. ORIGEN DE LA BRUCELOSIS CRÓNICA

Primero: el padecimiento crónico puede ser resultante de una forma aguda anterior; segundo: es inicialmente crónico y la sintomatología de la enfermedad va apareciendo con carácter lento, insidioso, pero progresivo y prolongado en el tiempo.

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De nuestros 550 casos, 80 presentaron una forma aguda de comienzo y 470 (85.4%) presentaron una forma crónica durante toda su evolución. Cuando la modalidad crónica se origina en una forma aguda anterior, superada ésta y trascurriendo un tiempo'variable de 2, 3, 6 meses, un año o aun más, reaparecen algunos síntomas como dolores lumbares o articulares, o aparecen manifestaciones nuevas, rompiéndose así el aparente estado de salud que el paciente había alcanzado. CLÍNICA DE LA BRUCELOSIS CRóNICA

La modalidad crónica la describió Miss Alice Evans por primera vez,, definiéndola como "una enfermedad de característica indefinida que en su aspecto más dominante simula la neurastenia." Esta definición describe una de las formas clínicas más importantes, pero no engloba todos los cuadros clínicos y patológicos de la enfermedad crónica. Pocas afecciones como la brucelosis son tan proteiformes y capaces de atacar a los más variados parénquimas del organismo. Cada vez que un clínico especializado, pedíatra, neurólogo, psiquiatra, cardiólogo, gastroenterólogo, ginecólogo, traumatólogo, oculista, otorrinolaringólogo, alergista, etc. aborda el estudio de la importancia de esta enfermedad, sobre la etiopatogenia de las diferentes afecciones que dentro de su especialidad aun no tienen una causa etiológica claramente identificada, se encuentra con la sorpresa de que un número no despreciable de esos padecimientos pueden imputarse a una sepsis crónica por Brucella. Por ello su clínica estará durante un tiempo aun, en continua revisión y renovación. Las características clínicas están en concordancia o relación con el sistema del organismo preponderantemente atacado. De acuerdo a nuestra casuistica estudiamos las siguientes formas clínicas: (a) Afecciones de característica indefinida, que en su aspecto más dominante, simulan la neurastenia; (b) formas a manifestaciones neurológicas dominantes; (c) formas a localizaciones espondilíticas; (d) afecciones del tipo del reumatismo crónico y lesiones óseas; (e) formas a localización cardiovascular preponderante; (f) formas a localización bronco-pleuro-pulmonar; (g) procesos de manifestaciones alérgicas; (h) los más variados procesos viscerales, cuyo análisis realizaremos en forma sucinta. (a) Afecciones de característica indefinida que en su aspecto más dominante simulan la neurastenia.-La enfermedad se inicia con debilidad, astenia psíquica y física acentuada, cefaleas, irritabilidad, dolores de los más variados. Miss Alice Evans escribe al respecto: "El cuadro esquemático de neurastenia describe a la brucelosis crónica, a saber: agotamiento, insomnio, irritabilidad y quejas de algias y dolores sin signo objetivo que los justifique". Son enfermos en quienes se efectúan los más variados diagnósticos:

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debilidad, tuberculosis, neurastenia, etc., siendo frecuentemente este último el dominante. Frecuentemente hemos encontrado en estos enfermos lesiones parenquimatosas o viscerales y alteración del líquido céfalorraquídeo, con moderado aumento de albúmina. (b) Formas a manifestaciones neurológicas dominantes.-Comparten con las lesiones óseas y espondilíticas la preponderancia dentro de las manifestaciones clínicas de la enfermedad. Roger y Poursines 3 consideran a las brucelas como gérmenes de marcado neurotropismo. Esta capítulo por su importancia, significado y trascendencia escapa al dominio de un clínico especializado en enfermedades infecciosas, no neurólogo, pero a pesar de esto no puedo excusarme a describir lo que he observado en este campo de la patología, en mi contacto diario con una masa importante de brucelósicos. Hemos registrado afecciones del sistema nervioso central, de los nervios periféricos, de las meninges raquídeas (leptomeningitis), de la médula y de las raíces. Las lesiones de meninge raquídea, de médula y raíces en algunos casos han sido primitivas y en otros concomitantes o secundarias a lesiones óseas de vecindad. La manifestación del sistema nervioso central más frecuente entre los brucelósicos crónicos fué el fenómeno de hiperpresión endocraneal, con crisis comisiales y edema de papila. Este síndrome motivó en dos enfermos un diagnóstico erróneo de tumor de cerebro. En los nervios periféricos procesos de tipo neurítico, preponderantemente del ciático, ciatalgias a veces primitivas, frecuentemente están condicionadas por lesión de las partes óseas o membranosas vecinas. Los cuadros mielorradiculares o meníngeos rara vez son puros, condicionando generalmente meningo-mielo-radículo-neuritis, sobre todo de tipo dorso-lumbar o lumbar, constituyendo éstas las manifestaciones neurológicas más frecuentes en esta casuística. Observación 5.-S. M., argentino, 44 años. Cuidador de cerdos y vacas (Provincia de Santa Fé). Ningún proceso febril en los últimos 15 años. Desde hace varios años dolor de rodilla. Inicia su enfermedad actual dos años antes, en 1944. Flojedad de las piernas y rodillas. También desde esa época calambres, sensaciones de picotazos y hormigueos en ambas piernas. Sensación de piernas pesadas. En 1945 lo examina un neurólogo diagnosticando meningo-mielo-radículoneuritis que da paraplejía flácida. Sospecha que este proceso sea debido a una aracnoiditis, realizando investigaciones en busca del diagnóstico etiológico y para confirmar la presunción clínica de aracnoiditis. Las reacciones de Wassermann y Kahn fueron reiteradamente negativas en sangre. La reacción de aglutinación para brucelas, técnica de Huddleson, positiva en dos oportunidades hasta la dilución 1:200. El examen del líquido céfalorraquideo arroja el siguiente resultado: aspecto límpido, incoloro, no contiene pus ni sangre. Reacción de

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Pandy + ++; albúmina contiene 0.60 gm por mm. Citologia: 12 elementos por mm3. Wassermann en liquido, negativa. Las radiografías de la columna cervical, dorsal y lumbar no muestran lesión ósea significativa. El estudio lipiodolado del canal raquídeo muestra acúmulo importante de lipiodol en toda su extensión, confirmándose el diagnóstico de aracnoiditis. Con estos documentos lo vimos el 9 de enero de 1946. Camina con marcada dificultad. Músculos de cintura, pelvis, glúteos y muslos muy disminuidos; piernas musculatura conservada. Reflejos cremasterianos muy disminuidos, rotulianos y aquilianos en ambos lados abolidos; plantares ausentes. La flexión de tronco sobre muslo y de piernas sobre muslo demuestran una hipotonia muy acentuada. Repetimos reacción de Wassermann y de Kahn que resultan negativas. Reacción de aglutinación, técnica de Huddleson, positiva 1:200. Intradermorreacción a la melitina positiva franca hasta el 4° día. Con el diagnóstico de meningo-mielo-radiculitis y aracnoiditis se ordenó tratamiento. Hemos tenido otros pacientes brucelósicos que hicieron leptomeningitis raquideana intensa. En las piezas anatómicas de varios de los pacientes fallecidos por neurobrucelosis la leptomeningitis plástica existió. En la observación 2 de este trabajo se la describe en meninges craneales, región optoquiasmática y cuadrigeminal y en canal raquídeo, a nivel de médula lumbosacra. Consideramos a las brucelas uno de los gérmenes que produce con mayor frecuencia leptomeningitis plástica. Observación 6.-C. S., argentino, 40 años, soltero, trabaja en medio rural. Guttemberg, Prov. de Córdoba. En mayo de 1946, dolor, primero en columna lumbar que se propaga al estómago; este dolor calma pero aparece dolor interescápulo-vertebral; cree no haber tenido fiebre. Ambos dolores se acentúan y exacerban caprichosamente, durante unos meses, luego dolor y amortiguamiento por el territorio ciático de ambos lados, más a derecha, sobre todo con los esfuerzos de tos o estornudo. Estas parestesias en realidad inician la paraparesia y disminución sensitiva que se instala paulatinamente, según el neurólogo, Dr. M. Peirotti. Las piernas se ponen (mediados de 1946) duras como palos y amortiguadas; no hay dolor espontáneo ni provocado. Progresivamente sin otros síntomas llega la paraplejía; no hubo cefaleas, náuseas, vómitos, fiebre, ataque a los pares craneanos, ni manifestaciones en miembros superiores o trastornos esfinterianos. Reflejos cutáneos abdominales sólidos; medio pubiano, vivo en ambas respuestas. Atrofia discreta de nalgas y pantorrillas. Los movimientos pasivos (sobre todo de piernas sobre muslo) revelan al principio intensa espasticidad, que una vez vencida, se trasforma en hipotomia que permite desplazamiento anormal de piernas, muslos, etc. (estado flaxoespasmódico); la fuerza global muy escasa. Reflejo patelar, en ambos lados hipervivo, desencadenando poliquenecia inagotable; aquíleos abolidos; plantar, Babinsky intenso; todas las maniobras equivalentes positivas; clonus inagotables en ambas rótulas y pies. Es imposible investigar la coordinación por la parálisis.

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Sensación de quemaduras en ambos pies y de calor en las pantorrillas. Sensación al dolor y temperatura, abolida hasta la altura del apéndice xifoides (por detrás sube algo más la zona de anestesia); el tacto en esa zona muy embotado y en algunos puntos del pie y pantorrilla nulo. Sensibilidad profunda (diapasón) abolida hasta el apéndice xifoides. Posición de pie imposible. Psiquismo y lenguaje normales. El examen radiográfico de toda la columna muestra lesiones mínimas de cuerpos vertebrales, tipo pico de loro, especialmente en columna lumbar. Proceso condensante de arcos posteriores en región dorsal y lumbar; no muy evidentes. Análisis de sangre: Wassermann y Kahn negativas; aglutinación para Brucella, técnica de Huddleson, positiva hasta 1:1,000. Glóbulos rojos 5,030,000; blancos 11,360. Hemoglobina 101; valor globular 1.01; neutrófilos 69%; linfocitos 23%; eosinófilos 2%; monocitos 6%. El liquido céfalorraquídeo sale a gota rápida al principio pero tiene tendencia a agotarse; color y aspecto normales; débilmente alcalino. Albúmina 2.20 gm por 1,000. Pandy ++++; 12 elementos por mm3 . Wassermann (con 0.5-0.8 y 1 ce) negativa. Mastic curva meningitica. Se piensa en compresión medular. La inyección cisternal del lipiodol demuestra detención casi total a la altura de la 4a. vértebra dorsal, por lo que se decide operarlo. Abierta la piel, se observa adherido a lámina de la 4a dorsal y a las apófisis espinosas, tejido de granulación. Los elementos óseos de las apófisis espinosas abordadas, 4a y 5a dorsal y las láminas correspondientes no son normales, mostrando proceso ósteo-condensante. Abierto el raquis se observa tejido de granulación y leptomeningitis plástica intensa. Se explora el conducto raquídeo: Hacia arriba el canal está libre, permitiendo fácil paso a una sonda Nelaton delgada; hacia abajo el bloqueo es total, siendo imposible insinuar la sonda exploradora por sobre los arcos posteriores del canal raquídeo. Efectuada la laminectomía amplia se suspende la operación retirándose material para estudio anátomopatológico por separado, de fuera del canal raquídeo, de las partes óseas y de dentro del canal raquídeo. Este estudio se encargó al Dr. Julio A. Herrero y su informe dice así: "El examen histopatológico ha demostrado tanto en el material del conducto medular como en el que se encontró por fuera de él, tejido conjuntivo denso con vasos de paredes espesadas, manguitos perivasculares linfocitarios y pequeños focos granulomatosos constituidos por células plasmáticas, linfocitos, fibroblastos y algunos granulocitos. Todos estos granulomas se encuentran centrados por capilares dilatados. Los fragmentos de tejido óseo revelan calcificación irregular." Este caso presenta lesiones neurológicas de compresión medular grave, que pueden invalidarle definitivamente, secundarias a leptomeningitis segmentaria y a lesión de los arcos posteriores. Si la leptomeningitis fué secundaria a la lesión ósea o concomitante con ella, es dificil dilucidarlo hoy; pero la severidad de estas lesiones brucelósicas de arcos y meninges, no puede escapar a nadie. Las compresiones radiculo-neuriticas secundarias a lesiones brucelósicas de la lámina o los arcos vertebrales, las meninges o afecciones del disco y cuerpo vertebral son frecuentes. Quien se encuentre frente a una afección de este tipo, sin etiología precisa, debe investigar brucelosis.

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(c) Formas a localización espondilítica.-Desde nuestras primeras publicaciones hemos destacado la frecuencia de esta localización. A medida que nuestra experiencia se amplía, vamos ratificando estos conceptos. En esos trabajos destacamos la lesión del cuerpo vertebral. Posteriormente, el estudio de las piezas anatómicas y de casos clínicos nos han demostrado que es tan frecuente el ataque al cuerpo como al disco intervertebral; esto tiene marcadísima importancia por las hernias intrarraquideas que condicionan y que pueden dar compresiones medulares o radiculares. La descripción anatómica de las observaciones 2 y 3 son una demostración del gran significado de estas hernias. En la forma crónica de brucelosis, cuando se presentan, pueden condicionar padecimientos radículos neuríticos principalmente. Tres tipos de lesiones del cuerpo nos ha sido dado observar en brucelosis: 1. Una forma ósteomielitica, que puede ser generalizada a un grupo vertebra o localizada a uno o dos cuerpos vertebrales. Poco frecuente, sólo la he observado en la forma aguda septicémica; 2. La forma pseudo-pótica, frecuente en ambas modalidades, aguda y crónica; 3. Las llamadas formas espondilíticas, tipo reumatismo vertebral crónico excesivamente frecuente en la brucelosis crónica. Dentro de las sepsis focales, la brucelosis ocupa uno de los primeros puestos en la etiopatogenia de las lesiones clinico-radiológicas que se designan con el nombre de "reumatismo vertebral crónico". Asimismo hemos observado lesiones de arcos posteriores y láminas de apófisis espinosa y lesión de articulaciones intervertebrales, conduciendo algunas a artritis o espondiloartritis anquilopoyéticas (tipo Strumpell, Pierre Marie). Es un ejemplo de ello: Observaci6n 7.-Trabaja en estancia en el Sudeste de Córdoba; las vacas que cuidaba durante los 5 años que preceden a la iniciación de su enfermedad abortan mucho, y él efectúa extracción manual de placenta. Inicia su afección hace seis años, con dolor lumbar importante al principio, después lumbar y cervical. Se hace tratar durante casi 4 años consecutivos, diagnosticándole "reumatismo crónico" sin pensar en brucelosis. Realizan múltiples tratamientos y extirpación de focos sépticos amigdalinos y dentarios sin ninguna mejoría. Por el contrario existe invalidez cada vez más manifiesta. Cansado el enfermo de sus padecimientos, teniendo presente que en la zona donde vive es frecuente la brucelosis y que su ganado estaba altamente infestado, él, según dice, exige a un médico le realice las pruebas para investigar brucelosis, siendo positiva la de aglutinación y la melitina de reacción necrótica intensa, con gran reacción febril. Hemos repetido la intradermorreacción con solución muy diluida dándonos reacción intensa con linfangitis. La radiografía de región lumbar de este enfermo muestra el proceso inflama-

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torio severo de arcos y articulaciones intervertebrales, soldadura de las apófisis espinosas entre sí y osificación del ligamento anterior. En la vista frontal se podía observar osificación de ligamentos laterales. Similares lesiones se han producido en columna cervical, por lo que hay inmovilización absoluta de columna lumbar y cervical. (d) Afecciones del tipo del reumatismo cronico y lesiones óseas.Tres tipos principales de lesiones articulares hemos observado en nuestros brucelósicos crónicos, a saber: (1) Procesos infiamatorios generalizados a pequeñas y grandes articulaciones. Estos procesos son, a veces, estrictamente articulares, pero con frecuencia también comprometen las partes óseas vecinas, con procesos de condensación y rarefacción del tipo de la ósteomielitis crónica; (2) procesos localizados a grandes articulaciones. El ataque más frecuente suele ser a la articulación sacroilíaca, le sigue en orden de frecuencia la articulación coxo-femoral (seudo-coxalgia del Mediterráneo de los autores franceses) y en tercer lugar la articulación de la rodilla. Suele haber marcada participación ósea en vecindad. Un tipo de este padecimiento fué un veterinario de San Juan; paciente que adquirió su enfermedad mientras efectuaba gran cantidad de autopsia en cabras que morían, comprobándose que padecían brucelosis. Inicia su afección sin padecimiento subjetivo de enfermedad aguda alguna y sin proceso febril significativo. Su enfermedad fué lenta pero progresiva, caracterizándose por dolor e impotencia funcional de ambas extremidades. Consulta muchos médicos realizándosele siempre el diagnóstico semiológico de coxalgia doble, sin investigarse nunca la causa etiológica del mismo. Cuando nos consulta pudimos observar en el paciente severa artropatía en ambas articulaciones coxo-femorales, con acentuadas lesiones de exostosis marginales en cavidad cotiloidea y en parte superficial de cabeza de fémur. Proceso ósteocondensante en cabeza y cuello del fémur, proceso que le había llevado prácticamente a la invalidez. Realizado un diagnóstico diferencial y de exclusión amplio, sólo se comprobó en él1 brucelosis con prueba de aglutinación significativa e intradermorreacción. Tratamiento ortopédico adecuado, realizado por especialista y biólogo (vacunoterapia), mejoraron francamente al paciente. El tercer tipo de lesiones articulares observado por nosotros en la brucelosis crónica es el de las artritis o espondilo-artritis vertebrales ya incluidas al considerar las lesiones espondilíticas. Dentro de las lesiones óseas hemos observado lesiones del tipo de la ósteomielitis crónica en las ramas del pubis, en el húmero, en tibia y en peroné. En dos pacientes que llevaban un proceso ósteomielítico extendido antiguo, tipo enfermedad de Paget, comprobamos sepsis por brucelas, consiguiendo detención de este proceso y disminución franca de sus padecimientos. No creemos improbable que algunos de estos procesos de

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ósteomielitis crónica que conducen a los tipos de enfermedad como el de Paget puedan ser de esta etiología y por eso consignamos este hecho aquí. (e) Forma a localización cardiovascular preponderante.-Nuestros conceptos sobre el ataque al aparato cardiovascular por las brucelas se han modificado fundamentalmente en estos últimos 4 años a raíz de los estudios que sobre el estado del aparato circulatorio de los brucelósicos ha realizado el Dr. Severo Amuchástegui. 4 Conocíamos que en la forma aguda septicémica se producían severas lesiones miocárdicas o endocárdicas, especialmente endocarditis vegetantes. Pero a pesar de haber atendido algunas lesiones valvulares (enfermos con aórticas y mitrales) en formas crónicas de brucelosis creíamos que las cardiopatías en los brucelósicos eran poco frecuentes y sobre todo patrimonio de la forma aguda septicémica. El trabajo de Amuchástegui, realizado sobre enfermos preponderantemente crónicos, nos pone en evidencia la necesidad de revisar nuestros conceptos y de tener presente siempre también esta sepsis, cuando se desee realizar una investigación patogenética, frente a un padecimiento del aparato cardiovascular, especialmente en zonas altamente infectadas por brucelas. El análisis del cuadro de Amuchástegui, que se reproduce, pone de manifiesto la frecuencia de los signos clínicos de lesión del aparato cardiovascular en 116 enfermos de brucelosis de los cuales 61 son adultos y 55 niños y las anomalías electrocardiográficas que se han registrado en los mismos. Adultos Casos

Disturbios Cardiovasculares: Modificaciones de la presión ............ Válvulopatías ........................... Enfermedad miocárdica ................. Enfermedad coronaria .................. Modificaciones EKG: Modificación eje eléctrico ............... Disturbio del ritmo ..................... Disturbio conducción del estímulo ...... Signo de enfermedad miocárdica ........ Signo de enfermedad coronaria .......... Modificación de la onda P ............... Sin modificación ........................

Niños %

Casos

%

7 27 40 17

11.4 44.2 65.5 27.5

1 22 30 5

1.5 40.0 54.0 9.4

23 41 12 38 21 5 5

54.0 67.2 19.6 62.2 36.0 3.1 3.1

21 39 6 31 15 4 3

33.1 70.9 10.9 53.3 27.2 7.2 5.4

Como puede observarse todos los elementos constitutivos del aparato cardiovascular pueden sufrir trastornos funcionales o lesiones orgánicas como consecuencia de la enfermedad brucelósica. (f) Formas a localización bronco-pleuro-pulmonar.-Nuestra observa-

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ción en la forma crónica se refiere especialmente a procesos bronquiales, habiendo observado bronquitis generalizadas, rebeldes y pertinaces, con síntomas funcionales caracterizados por tos, expectoración purulenta y a veces sanguinolenta. Frecuentemente estas bronquitis no son la manifestación única del padecimiento del paciente, sino que pueden estar asociadas a modificación del estado general, pérdida de fuerza, etc., lo que en más de una oportunidad induce a pensar en procesos tuberculosos. Bronquitis localizadas, en general de ramas importantes de 1° y 2° orden. En nuestra casuística tenemos registrados con control broncoscópico y biopsia y estudio lipiodolado formas proliferativas y formas úlcero-destructivas, conduciendo estas últimas a formaciones bronquiectásicas. Dentro de las lesiones parenquimatosas hemos tenido infiltrados de tipos neumónicos y lesiones nodulares a nódulos medianos y finos, estas últimas tipo granulia. En sólo un enfermo crónico tuvimos pleuresía serofibrinosa recidivante, que creemos de etiología brucelar. (g) Procesos a manifestaciones alérgicas.-Las manifestaciones de carácter francamente alérgico (cefaleas, colon irritable y muy especialmente asma nasal o bronquial), son muy frecuentes en la sintomatología que acusan nuestros pacientes afectos de brucelosis crónica. Excepcionalmente constituye el síndrome patológico único que sufre el enfermo; suele haber siempre localizaciones viscerales o tisurales como consecuencia de esta enfermedad. Pero ellas suelen tener una altura y significación preponderante y suelen responder muy bien al tratamiento por vacunas específicas. Habiéndonos ocupado en trabajos anteriores de la discusión de este tema, sólo diremos aquí que consideramos a las brucelas uno de los gérmenes más aptos para producir alergias bacterianas o para despertar condiciones humorales o tisulares que conduzcan a las alergias del tipo alimenticio, polínicas, etc. (h) Los más variados procesos viscerales.-No es excepcional ver enfermos que presentan los cuadros viscerales más variados. Hemos tenido esplenitis, hepatitis, abscesos subfrénicos, orquitis y orcoepididimitis. Se describen pancreatitis crónicas, ovaritis, salpingoovaritis, etc. imputables a sepsis por brucelas. CONCLUSIONES Nuestra experiencia nos ha demostrado que la brucelosis humana a veces evoluciona como una verdadera septicemia, con todas las características clínicas y bacteriológicas y otras veces se comporta como enfermedad sistémica, visceral. Muchas hipótesis podrían formularse sobre la influencia en el determinismo de producción de una u otra de estas modalidades, con relación

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al tipo y virulencia del germen infectante, masividad de la infección, condiciones del sujeto, etc. Es posible también que la pérdida del poder bactericida de la sangre humana frente a las brucelas, estudiado por Signorellis y que se produce en el enfermo, inicialmente pueda en determinadas condiciones, por la intensidad y duración que adquiera, influir en el determinismo de producción de una u otra modalidad. Pero ambas modalidades clínicas "aguda" y "crónica" responden a una misma etiología, cuentan con una anatomía patológica que enriqueciéndose día a día, tiende a mostrarnos también, como la lesión fundamental, el granuloma brucelósico que les es común. En el diagnóstico habrá que tener presente, que si es fácil obtener hemocultivos positivos en la modalidad aguda, septicémica, sólo por excepción se los obtiene a partir de una modalidad crónica. El diagnóstico de la modalidad crónica es esencialmente clínico, afianzado en el diagnóstico de exclusión amplio. Las reacciones de aglutinación, las pruebas intradérmicas y el estudio de las reacciones focales y generales secundarias a las inyecciones endovenosas de antígeno específico, serán de gran importancia para el juicio definitivo. REFERENCES (1) Evans, A. -C.: Jour. Am. Med. Assoc., 103: 655-667, 1934. (2) Poston, M.: Pub. Hlth. Rep., 53: 1, 1938. (3) Roger, H., y Poursines, J.: "Les meningo-neurobrucelloses," Masson et Cie., Paris, 1938. (4) Amuchástegui, S.: Revista de la Asociación Médica Argentina, t. LXII, No. 625-626, 1938. (5) Signorelli, Saverio: "L'infezzione Brucellare nell'Uomo," V. Idelson, Nápoles, 1949. CLINICAL FEATURES OF HUMAN BRUCELLOSIS (Summary) The author reports on 550 cases of brucellosis including their clinical features and the evolutions that they presented. Two fundamental modalities have been observed: the acute and the chronic forms. The acute modality is less frequent; only 80 cases from the 550 patients (14.55%) presented the acute form. The onset is characterized by a typical septicemic period, the toxemia is variable, generally profound; fever, sweating, generalized pain, etc. The hemoculture is positive with septicemic features. In this group of 80 patients with acute brucellosis, 14 died (17.5%), 18 were cured (22.5%) after a long septicemic course; 48 subsided into secondary chronic modalities and many eventually were cured. The chronic modality occurs much more frequently. It is characterized by its localization in systems or tissues. In general it is not septicemic and that is why the positive hemoculture is uncommon. The chronic modality may originate from any of the two following sources: (a) an acute form which subsides into a chronic one (from 80 acute patients 48

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of them (60%) developed a chronic form); (b) a chronic modality from the very beginning, without an acute onset. Four hundred seventy patients presented this form (85.45%). The clinical symptoms and signs of the chronic modality depend upon the principally affected system. The following types of chronic modality are described: (a) indefinite discomfort, and the outstanding symptom seems to be neurasthenia; (b) neurological forms; (c) spondylitical forms; (d) rheumatical forms with bone lesions; (e) cardiovascular forms; (f) broncho-pleuro-pulmonary form; (g) allergic form; and (h) miscellaneous visceral localization. The more frequent oceurrence of the chronic form leads the author to consider brucellosis as a predominantly chronic disease.

PREPARATION AND PROPERTIES OF BRUCELLA ABORTUS VACCINE (STRAIN 19) DESICCATED BY LYOPHILIZATION By W. F.

VERWEY, N. H.

HARRINGTON, AND CLARA MATT

Sharp and Dohme, Incorporated, Glenolden, Pennsylvania Five years have passed since Brucella vaccine desiccated by lyophilization was made available on a commercial scale, and several years of research had gone by prior to that time. I am indebted to Dr. O. E. Herl of the United States Bureau of Animal Industry for the figures relative to the amount of Brucella abortus vaccine that was approved by the Bureau of Animal Industry for distribution from 1945 through 1949. The increasing use of the lyophilized vaccine shown by those figures makes it appropriate to review before this Congress some of the information that is available concerning its preparation and properties. Since much of my discussion will pertain to the effects of various steps in the lyophile process upon dried Brucella vaccine, it seems advisable for orientation purposes to review the current procedure for preparing the vaccine. The influence of various factors in the process upon the viability and stability of the vaccine will be discussed in the sequence in which these factors fit into the normal procedure. Brucella abortus strain 19 is grown on the surface of potato-glycerineagar, and then the organisms are washed off into M/20 phosphate buffer at pH 6.5. This suspension is adjusted by turbidimetric measurement to twice the density desired in the complete liquid vaccine and an equal quantity of skimmed milk is added. The vaccine then is placed in vials and quickly shell-frozen on the inside walls of the vials by revolving them in a mixture of carbon dioxide ice and alcohol at a temperature of approximately -70°C. After freezing, the vials are put into a vacuum chamber and desiccated from the frozen state under controlled conditions of temperature and pressure. After a suitable period of desiccation, they are removed from the vacuum chamber and then re-evacuated and sealed by equipment especially designed for this purpose. Our work has been directed toward determining the effects of various factors in the lyophilization process upon the viability and stability of the material and toward determining the physiologic and bacteriologic properties of the vaccine. Since the number of viable organisms in the vaccine is the only easily accomplished presumptive measurement of its probable antigenicity, conclusions are based on the counts of viable bacteria that are obtained under various circumstances. The development of a suitable suspending and stabilizing menstruum that would protect the organisms from destruction during lyophilization 209

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was a problem of first importance. Several different suspending fluids have been tried, and the results with most of them have been published previously.' The skimmed milk-phosphate buffer solution, in our hands, at least, has been the most generally satisfactory since it permits the recovery of about 40% of the originally viable cells, and vaccines prepared in this mixture have good stability. Suspending agents and lyophilization conditions can be used that produce a higher percentage of viable organisms immediately after desiccation, but these proceclures have resulted in relatively rapid loss of viability during storage. In the practical use of strain 19 vaccine, the percentage of organisms originally present that are killed during lyophilization is of secondary importance since it should be emphasized that the product is required to meet viability standards after all processing losses have taken place. On1 the other hand, loss of viability after final testing occurring during storage and field handling may have a direct effect upon the immunizing capacity of the administered dose. It is this lack of stability that has been a major problem with the liquid vaccine. Lyophilization can be considered as consisting of three distinct steps; first, the freezing, then the desiccation from the frozen state under vacuum, and then the sealing of the containers. The three steps have been studied separately. The results of individual experiments carried out to determine the effect of freezing at -70°C upon bacterial viability have been variable, but an analysis of 100 consecutive production lots indicates that there is a loss of only about 10% of the viable organisms in this procedure. The next step in the lyophilization process is the sublimation of water from the frozen vaccine under reduced pressure. This is the most critical step in the process, and is the point of major loss of viability if conditions are not properly selected. Losses as high as 96% and as low as 10% have been obtained by varying the conditions of this step. Since the desiccation process is directly affected in a highly complex manner by a number of variable factors, it is almost impossible to define conditions for optimal desiccation in terms that may be translated from one design of equipment to another. However, by working with the same piece of equipment and keeping most conditions constant, it has been possible to study the effects of variations in pressure and processing time. Usual lyophilization practice employs residual pressures around 50 to 100 microns, and such low pressures have been very satisfactory for normal human plasma, complement, and penicillin. It has been found that these pressures and the conditions of temperature that they produce are destructive for Brucella abortus. This is accentuated by long periods of processing. High pressure, i.e., poor vacuum, and standard time of processing produced 47% and 33% survivals while low pressure and long time permitted survival of only 7% and 4% of the originally viable bacteria.

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Studies were also made on 100 consecutive lots of vaccine, which were examined in an attempt to correlate the residual moisture present immediately after lyophilization with the percentage recovery of viable organisms. Over the moisture content range of 1.2% to less than 0.1% there was no correlation whatsoever. It would appear, therefore, that moisture within this range has little to do with the number of organisms that survive. As will be mentioned later, however, the same cannot be said for the role of moisture in the stability of the vaccine under storage conditions. Although the factors mentioned so far are concerned with the recovery of viable Brucella organisms, the major problem with Brucella strain 19 vaccine has been its stability under field conditions of transportation, storage, and use. Studies have been made to evaluate the stability of the desiccated vaccine and to determine some of the factors that affect stability. One of the points examined was the relationship between the moisture content immediately after lyophilization and the stability of the vaccine under conditions of storage. It has been difficult for various reasons to assemble any extensive series of figures bearing on this point, but from experience with a limited number of lots it is cIlear that there is a distinct relationship between high initial moisture content and poor stability. A vaccine having a moisture content of 1.5% or more is quite likely to be unstable. Other results indicate that moisture from any source, whether initially present or subsequently accumulated during storage, is deleterious to vaccine stability. This was demonstrated by a study that compared storage in glass sealed vials and in rubber sealed vials having aluminum caps. In this experiment, a single lot of vaccine was distributed into the two types of containers mentioned and after lyophilization and sealing under vacuum, the containers were stored at 5°C, room temperature, and 37°C. Periodic determinations of moisture and viable count were carried out. The number of viable bacteria at the start of the storage period was designated as 100%. There was a gradual accumulation of moisture in the material in the rubber sealed container, whereas there was, of course, little, if any, in the all-glass vial. This change in the rubber sealed vial is slight at 5°C but is considerable at 37°C. Although there is a decrease in viability with time and higher temperatures even in the all-glass container, this is much more marked in the rubber sealed vials that are absorbing moisture. Although the all-glass container provides better conditions for vaccine stability, unfortunately it has been found to be more inconvenient and more costly to use. Therefore, lyophilized vaccine is now supplied universally in the rubber sealed container. However, if there are areas of the world where high temperatures and lack of refrigeration facilities impose conditions that cannot be met by liquid vaccine or even by rubber sealed lyophilized vaccine, the all-glass vial might have decisive advantages in spite of its

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inconvenience. The vaccine in rubber sealed vials, though not as stable as in the glass-sealed vials, is still very good. It will be noted that this vaccine exceeded Bureau of Animal Industry viability requirements even after 24 years of storage at 20 -5°C and still had 30% of its prestorage count after nine months at room temperature. In continuing the discussion of the stability of lyophilized Brucella abortus vaccine, we felt that it might be of interest to examine records of actual production lots that had been tested for viability at various periods after their preparation. These figures do not represent planned stability tests (such information has been published previously .2) but rather, these tests were carried out at irregular times for a variety of reasons. A total of 36 production lots have been tested and of 13 tests that were carried out within the dating period of the vaccine, all indicated viability above the Bureau of Animal Industry's standards. Of 27 tests carried out between 12 and 25 months after the preparation of the vaccine, 19 were above standard and 8 were below. From these figures it would appear that on storage at refrigerator temperatures the vaccine is uniformly satisfactory within its dating period, and the majority of lots would meet the requirements of the Bureau of Animal Industry for at least 2 years. These results are in confirmation of previously published stability tests. Comparatively few samples of production lots of vaccine have been stored at higher temperatures, but the results that are available would indicate that the vaccine can withstand at least 3 months at room temperature and 6 weeks at 37°C. Tests at still higher temperatures have not been carried out with the rubber sealed containers, but previous results with the glass-sealed ampules demonstrated that the vaccine could withstand temperatures of 50°C for at least 2 days, and it is believed that the rubber-sealed containers would behave in a similar manner during such short exposure periods. The fact that lyophilized Brucella abortus vaccine may be stable beyond its dating period and that it is capable of withstanding exposure to higher temperatures should not be taken as an invitation to abuse it. It is our opinion that every effort should be made to maintain the vaccine under the recommended conditions of refrigeration during transportation and storage, but if this cannot be done, the viability of the vaccine will not decrease rapidly. There is another very important question concerning the stability of the lyophilized vaccine that has been investigated. This concerns the vitality of the viable cells after restoration from the desiccated state. 3 Samples of 2 production lots of lyophilized vaccine after refrigerated

storage in the dry state for 3 months were restored to the liquid state and held at refrigerator temperatures for 90 days. At various intervals during this period, viable counts were made to determine the rate at which the organisms died.

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The results obtained showed that there is no rapid decrease in viable count upon restoration, and the vaccine met the stability requirements for fresh liquid vaccine. We believe that these results give good evidence that lyophilization does not cause serious physiologic damage to the organisms that survive the desiccation process. The data suggest further the possibility that if suitable conditions for transportation are available, the lyophilized vaccine could be restored in the office of a veterinarian where aseptic conditions are more easily attained and then transported to its point of use in the liquid state. Such a procedure reduces the bulk of the package that must be transported and permits the more rapid accomplishment of field immunization. One further type of study that should be mentioned has been carried out. The work of Braun4 has indicated that the rough non-antigenic variant of Brucella abortus strain 19 survives lyophilization better than does the smooth antigenic form. Therefore, the lyophilization of a mixed suspension of smooth and rough organisms would have a tendency to increase the proportion of rough cells in the viable bacterial population. Nine lots of lyophilized vaccine were plated out and 100-200 colonies of each lot were examined according to the method of Henry. 5 In 7 of these lots no rough colonies whatsoever were seen. In the other 2 samples, one rough colony was seen in each instance. These observations demonstrate that the selection of rough variants by lyophilization is not a significant factor in the vaccine as it is prepared currently. DISCUSSION

The results of the studies that have been summarized here indicate that the lyophilized Brucella abortus vaccine has a stability very considerably better than the liquid type vaccine. At the present time all the vaccine supplied in this country is in single dose containers. Where a large number of animals are to be immunized this makes it somewhat inconvenient in the field. The objections to supplying Brucella abortus vaccine in a multiple dose container apparently have been based on doubts concerning the stability of vaccine that has been restored to the liquid state and on fears that contaminating organisms would be able to multiply in the containers if a period of time elapsed between the initial and the final use of the material. The data that have been presented on the stability of restored vaccine seems sufficient to remove doubts concerning stability. A consideration of the fundamental requirements for bacterial multiplication suggests that serious over-growth of contaminants is not likely to occur within a few days unless the vaccine is held at room temperature or above. Such treatment is so far out of the range of recommended practice that it would constitute a definite abuse of this product. Therefore, it seems to us that the many who would use the multiple dose container correctly should not be deprived

214

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

of its advantages because of the few who would abuse it. Fairly extensive, full scale production experience makes it clear that it is entirely practical to prepare Brucella vaccine in multiple dose containers and that there is considerable economy in the use of this procedure. REFERENCES (1) Verwey, W. F. (1944): Report of 48th Annual Meeting of the U. S. Livestock Sanitary Association. (2) Verwey, W. F., and Scheidy, S. F. (1946): Jour. Am. Vet. Med. Assoc., 59: 362. (3) Verwey, W. F., and Matt, C. (1950): Jour. Am. Vet. Med. Assoc., 66: 296. (4) Braun, W. (1950): "Brucellosis" Am. Assoc. Adv. of Sc. (5) Henry, B. S. (1933): Jour. Infectious Diseases, 52: 374. PREPARACIóN Y PROPIEDADES DE LA VACUNA DE BRUCELLA ABORTUS (CEPA 19) DESECADA POR LIOFILIZACIóN (Sumario) El hecho de que más de la cuarta parte de la vacuna Brucella abortus (Cepa 19) que se usa en los Estados Unidos es liofilizada, demuestra que es importante conocer algo sobre su preparación y propiedades. Esta investigación tuvo por objeto estudiar los factores que afectan la viabilidad de los microorganismos de Brucella durante el proceso de desecación y que influyen en su estabilidad. El proceso de liofilización consta de tres etapas distintas: a) la congelación rápida la de vacuna, b) la remoción del liquido de la vacuna congelada a presión muy reducida, e) cierre al vacío de los recipientes para evitar la reabsorción de humedad. En el proceso, según se practica en nuestros laboratorios, aproximadamente el 40% de las bacterias originalmente viables sobreviven el proceso de liofilización y el número inicial de bacterias se regula de manera que el número que sobrevive exceda los requisitos mínimos para vacuna de Brucella fijados por la Oficina de Industria Animal. Se han llevado a cabo estudios que indican que la congelación rápida de la vacuna solamente ocasionó pérdidas insignificantes en la viabilidad y que el proceso de desecación era el que ocasionaba las pérdidas mayores. Un examen de este proceso demostró que la desecación a presiones residuales relativamente altas, por periodos minimos de tiempo, producía los resultados más satisfactorios. También se comprobó que los residuos de humedad que fluctuaran entre el 0.1% y el 1.2% no tenían efecto sobre el número de microorganismos que sobrevivian el proceso de desecación. Como los procedimientos que producen el número máximo de bacterias viables después del proceso de liofilización no son los mismos que los que proporcionan la esbilidad máxima durante el tiempo que la vacuna permanece almacenada, se investigaron las condiciones necesarias para obtener una vacuna estable. Se comprobó que la estabilidad durante el periodo de almacenaje y la resistencia a las condiciones adversas de temperatura dependían, en gran parte, de las oportunidades para acción reciproca entre la humedad y la vacuna liofilizada. Por lo tanto, la selección y cierre de los recipientes son probablemente los factores de mayor importancia de los cuales depende la estabilidad prolongada de la vacuna. Aunque una ampolleta de vidrio cerrada al vacio proveía las mejores condi-

PREPARATION OF BRUCELLA ABORTUS VACCINE

215

ciones de estabilidad, ampolletas cerradas con tapones de caucho recubiertos con un casquete de aluminio también ofrecían buenas condiciones de estabilidad y eran más económicas y convenientes. Los resultados obtenidos demostraron que la vacuna de Brucella envasada en tales ampolletas conserva las propiedades mínimas que exige la Oficina de Industria Animal, por lo menos durante un año si se guarda en refrigeradoras adecuadas. La mayoría de los lotes comprobados conservan sus propiedades por lo menos dos años. Aunque relativamente pocas muestras de lotes de vacunas se han guardado en refrigeradoras a temperaturas elevadas, los resultados indican que la vacuna puede conservar sus propiedades por lo menos durante 3 meses a la temperatura ambiente; durante seis semanas a 37°C, y por 2 dias a 50°C. No se recomienda prescindir de refrigeración durante el transporte y almacenaje de la vacuna, pero parece que el grado de estabilidad es adecuada para permitir el uso de la vacuna desecada bajo cualesquiera condiciones que puedan prevalecer en el terreno. También se han realizado estudios para determinar la vitalidad de las células viables cuando se convierten de nuevo al estado liquido. Estos experimentos indican que los organismos que sobreviven el proceso de liofilización no sufren daño fisiológico apreciable y que cuando se reconvierten muestran la misma estabilidad que la vacuna liquida fresca. Esto indica que cuando existen condiciones adecuadas de refrigeración, la vacuna liofilizada puede reconvertirse de antemano para facilitar el trabajo de inmunización. El proceso de liofilización ha contribuido considerablemente a la estabilidad y, por lo tanto, a la eficacia de la vacuna Br. abortus. Además, las propiedades de la vacuna liofilizada son tales, que podría pensarse en la distribución de este material en recipientes de dosis múltiples. Tal procedimiento resultaría conveniente, seguro, y más económico.

BRUCELOSIS CAPRINA EN LA REPIBLICA ARGENTINA Por R. A. MAUBECIN, B. L. MORÁN, F. R. JURADO y V. C. CEDRO Ministerio de Agricultura y Ganaderfa, Buenos Aires, Argentina INTRODUCCIÓN

El noroeste argentino, caracterizado por la sucesión de valles y sierras, constituye la región donde se cría casi exclusivamente el ganado caprino. Esta región presenta una flora típica que responde a la "formación del monte occidental". El sistema de cría del ganado caprino es primitivo y conserva las características impresas por los conquistadores y colonizadores españoles. Consiste en el encierro en un corral y pastoreos en campos abiertos de forrajeras naturales y arbustos espinosos propios de la región del monte. Para abrevar la majada, se dispone de represas que se forman mediante una excavación en terrenos en declive, donde se recogen las aguas de lluvia. Esta represa constituye en muchas explotaciones, la única fuente que abastece de agua de bebida al hombre y a los animales. La forma de su explotación y la idiosincrasia de los criadores por lo común de escasos recursos económicos imprimen a la cría del caprino el carácter de una explotación de muy bajo rendimiento. Sin embargo la cabra es un animal necesario e insustituible en esas extensas regiones áridas y semidesérticas del territorio Argentino. Epizootiologia.-Las primeras comprobaciones de brucelosis caprina, datan del año 1931 en que Sordelli y colaboradores señalan la enzootia en la provincia de Mendoza, a raiz de la comprobación de casos humanos autóctonos; posteriormente Mazza y colaboradores, Pardal y de la Barrera, demuestran la existencia de brucelosis caprina en las provincias del Noroeste Argentino y Córdoba, entre los años 1932-1935 y a partir de 1938 las comprobaciones del personal técnico del Ministerio de Agricultura y Ganadería, permiten identificar la amplia difusión de la enzoosis. Entre 1938 y 1950 se identificaron 412 focos de brucelosis caprina en todo el territorio argentino distribuidos según el Cuadro 1, que corresponden al examen serológico de 7,061 animales. Dentro de este cuadro se destacan dos tipos de infección caprina, una con elevado porcentaje de reaccionantes a las pruebas serológicas y con manifestaciones clínicas (abortos, artritis, etc.) y otra con ausencia o escasa proporción de reaccionantes con variadas manifestaciones clínicas, dominando el síntoma aborto. En la primera forma se comprueban infecciones humanas frecuentes, mientras que en la segunda el contagio humano, si bien presente es menos común. La morbilidad comprobada en la infección natural llega hasta el 80% 216

BRUCELOSIS CAPRINA EN ARGENTINA

217

en algunas majadas. La mortalidad es baja 3 a 4% cuando no concurren factores coadyuvantes (sequías, etc.). La edad en que se manifiesta la brucelosis en los caprinos es preferentemente de seis meses en adelante, siendo evidenciada clínicamente a partir de la primera gestación, por la gran cantidad de abortos que se producen. Se han comprobado dos formas clínicas de brucelosis caprina, una aguda caracterizada por elevado porcentaje de abortos y reducida proporción de reaccionantes serológicos y otra crónica, con elevado índice de reaccionantes serológicos a altos títulos, y menor proporción de abortos, pero con manifestaciones crónicas de brucelosis, artritis, metritis. Esta modalidad de la brucelosis considerada individualmente, tanto en sus manifestaciones clínicas como serológicas, se refleja aproximadamente en el estudio colectivo de conjuntos de majadas, de determinadas regiones del país. A las diferentes manifestaciones regionales de la brucelosis le asignamos el valor de una modalidad propia de la región condicionada posiblemente por las características de la cepa infectante. DIAGNÓSTICO

Las comprobaciones de los focos de brucelosis caprina consignados en el Cuadro 1 que se acompaña, fueron realizados por el personal técnico de las Direcciones Generales de Sanidad Animal e Investigaciones Ganaderas. El diagnóstico se efectuó en base a los síntomas clínicos (abortos, artritis, nefritis, mastritis, orquitis, etc.) por las pruebas serológicas, alérgicas y estudios bacteriológicos. Serológico.-Las pruebas de seroaglutinación se efectuaron de acuerdo a las técnicas de Huddleson y Wright utilizándose los antígenos elaborados por la División Brucelosis del Instituto de Zoonosis. El método rápido de aglutinación resulta sin lugar a dudas un muy eficaz procedimiento de diagnóstico sobre todo para los equipos técnicos que trabajaron en zonas montañosas alejadas de los centros poblados y de difícil comunicación con el laboratorio. Su aplicación ha sido permanente y de positivo valor para la recopilación de datos destinados a la confección de un mapa epizootiológico. Teniendo en cuenta que animales portadores de brucela (con verificación bacteriológica) en algunos casos reaccionaron negativamente a la seroaglutinación y sumando a este hecho las observaciones clínicas realizadas por nosotros, concluimos por adoptar un criterio de interpretación íntimamente vinculado al estado sanitario de cada hato, con preferencia a la significación individual de la prueba, predominando en consecuencia el concepto de hato infectado sobre el de animal infectado. Los títulos en 1:25 asumen significación específica cuando se presentan

218

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

en hatos con animales reactores en títulos altos con o sin manifestaciones clínicas de la enfermedad. El método lento que se empleó en el laboratorio acusó resultados similares a los obtenidos por el método rápido, debiendo señalar que generalmente se montaron las pruebas con sueros inactivados. CUADRO

1.-Focos de brucelosis caprina en la República Argentina

Provincias y territorios nacionales

Número de focos

Buenos Aires .................. 162 Córdoba ....................... Corrientes ..................... 3 Catamarca ..................... Entre Ríos ..................... 57 La Rioja ....................... 4 Mendoza ....................... Salta .......................... 15 San Juan ...................... 15 55 San Luis ....................... Santiago del Estero ............ 98 Santa F ........................ 1 Jujuy .......................... Tucumán ...................... 2 La Pampa ..................... ..Chaco ......................... Misiones ....................... Formosa ....................... Neuquen ....................... Chubut ........................ Santa Cruz .................... Río Negro ..................... . Tierra del Fuego ............... Totales ........................

412

Caprinos examinados

Caprinos positivos

humanos humanos

-

-

-

453

833

-

-

1,837 -

12 -

1,506 25 112 218 1,250 2,090 2 9

7

-

499 20 28 366 305 -

615 750 181 567 585 300

-

---

-

7,061

1,678

*

287

-

-

-

Observaciones

*

-

-

-

-

4,118

* Significa que además de las cifras consignadas como casos humanos, existen informaciones de otros casos sin especificar cantidad.

Alérgico.-La infección brucelosa en la cabra produce una sensibilización específica del organismo que, iniciada poco después de la infección, persiste por mayor tiempo que las aglutininas, pudiendo a veces prolongarse indefinidamente. Este estado de alergia puede ponerse de manifiesto mediante las pruebas alérgicas intradérmicas llevadas a cabo con distintos alergenos. Se ha utilizado la brucelina de Mirri, el glúcido lípido melitensis de Boivin y Mesrobeanu, la melitina de Burnet, el M. B. P. de Morales Otero y el antígeno de Van der Hoeden. La técnica de la prueba consistió en la inoculación intradérmica en el pliegue anocaudal de una dosis

BRUCELOSIS CAPRINA EN ARGENTINA

219

de 0.2 ce del alergeno correspondiente efectuándose la lectura a las 2448 horas, repitiendo las observaciones a las 72 y 96 horas. En orden decreciente de sensibilidad debemos citar el Van der Hoeden, melitina, brucelina, de Mirri, glúcido-lípido melitensis (20 gamas) y el M. B. P. Las reacciones locales más manifiestas e intensas se han logrado con el Van der Hoeden y la melitina, siendo fácilmente perceptible la infiltración edematosa, el calor y el rubor. Las reacciones positivas alcanzaron su mayor intensidad entre las 24 y 48 horas, comenzando a partir de ese momento la regresión de las mismas observándose, sin embargo, que algunas persisten hasta las 96 horas. Ateniéndose a la experiencia recogida, la alergia cutánea constituye un elemento de diagnóstico valioso que complementando las pruebas de seroaglutinación permite establecer un mejor criterio con respecto a la infección brucelósica en el caprino. La inoculación de estos alergenos provocó la aparición de aglutininas en títulos variables en los animales negativos, y en los reactores produjo una elevación de los mismos con carácter transitorio. Bacteriológico.-El aislamiento de las cepas se intentó por métodos directos de cultivo y por inoculación al cobayo a partir de sangre y leche, que fueron los materiales de que más dispusimos, y en pocas oportunidades de feto y envolturas fetales. Los hemocultivos se realizaron en caldo bacto-triptosa que se mantuvieron en la estufa durante 50 días con siembras periódicas en medio Stafseth. Las siembras de material de leche, fetos, envolturas fetales, como asimismo de órganos y médula ósea cuando se dispuso de ellos, se efectuaron sobre medio Stafseth, agar papa y agar bacto-triptosa en cajas de Petri y tubos que se mantenían en observación hasta 10 días. La incubación a 37°C se hizo siempre simultáneamente bajo CO 2 al 10% y en atmósfera común. La inoculación al cobayo se llevó a cabo por vía subcutánea, con sangrías periódicas y sacrificando entre las cuatro y ocho semanas, sembrándose regularmente material de bazo. Las colonias sospechosas se seleccionaban bajo la lupa binocular esteroscópica con el objeto de obtener cultivos puros y proceder a su clasificación mediante el empleo de sueros específicos, pruebas bioquímicas, exigencias de CO2 , producción de H 2S y comportamiento en los medios bacteriostáticos con colorantes. Las cepas aisladas, con excepción de una, fueron identificadas como Brucella melitensis. Las características de las mismas respondieron a las observadas en las formas normales. Las colonias en medios sólidos presentaron apariencia lisa, con bordes netos, superficie convexa, uniforme

220

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

y brillante, forma circular que observadas con pequeño aumento mostraron ligera coloración azulada. Todas las cepas identificadas se manifestaron como cultivos en fase "S" no solamente por el aspecto morfológico de las colonias sino también por su comportamiento frente a sueros anti "S", la prueba de Pampaña, Alessandrini y Sabatucci y termoaglutinación de Burnet. Los cultivos en medios líquidos produjeron turbidez uniforme y formación de sedimento en el fondo del tubo a medida que envejecían. La inoculación al cobayo de las cepas aisladas determinó la aparición de aglutininas específicas en títulos variables y la producción de lesiones macroscópicas características con preferente localización en bazo, hígado y ganglios. En los numerosos intentos de aislamiento realizados sólo pudieron aislarse cepas de brucela en 16 oportunidades y el relativo éxito obtenido se atribuye a que el gérmen debe ser eliminado por la leche en forma intermitente y a que el estado de bacteriemia sería también transitorio. En cuanto a los estudios efectuados sobre material proveniente de abortos, las posibilidades se vieron reducidas por la dificultad en la remisión y estado de conservación del material. Se explica así que en contadisimos casos se dispusiera de ese material no siempre en buenas condiciones y que en sólo dos oportunidades se pudiera aislar el gérmen. A título informativo debemos señalar que en un material de leche remitido desde la localidad de Casas Viejas (Córdoba), se aisló una cepa que respondió a las características de Br. abortus. Esta cepa se recuperó de un cobayo con título aglutinante de 1/500 y el hemocultivo del gérmen exigió la presencia de C02, carácter que mantuvo en trasplantes posteriores. La producción de H 2S se evidenció hasta el cuarto día y su comportamiento en los medios bacteriostáticos coincidió con el común a la especie, siendo inhibido por la tionina. PROFILAXIS

Ensayos experimentales de vacunación fueron realizados en el laboratorio y en el campo. Estos ensayos se llevaron a cabo en cabritos de 2 a 4 meses de edad empleándose la Br. abortus cepa 19 como inmunígeno. El plan experimental se desarrolló en la siguiente forma: Lote A.-Cabritos vacunados sin exposición posterior a la infección con el

objeto de determinar la receptividad de los mismos a la cepa de vacunas. Lote B.-Cabritas vacunadas y expuestas a la infección natural por cohabitación con animales infectados. Lote C.-Cabritas vacunadas y mantenidas en su ambiente natural en un establecimiento infectado. Un lote de 28 cabritas alojadas en el laboratorio fué sometido a prue-

22i

BRUCELOSIS CAPRINA EN ARGENTINA

bas de aglutinación e indice opsonocitofágico previamente a la vacunación, observándose resultados negativos en todos los animales. El 15 de septiembre de 1945 se procedió a vacunar todas las cabras con una dosis de 3 cc por vía subcutánea, comenzándose posteriormente con los contralores serológicos postvacunales (Cuadro 2). El 26 de febrero de 1946 se fraccionó el lote de cabritos en otros dos: Lote A.-Mantenido inicialmente aislado para verificar la inocuidad de la vacuna, integrado también por un macho y tres cabras testigos sin vacunar. CUADRO 2.-Contralores serológicos postvacunales Control

1° 2° 30 40 50

Fecha

nov. nov. dbr. eno. feb.

3-45 26-45 31-45 21-46 18-46

Control No de Fecha Animales reaccionantes en cada título A e Animales Neg. 1/25 1/50 1/100 1/200 1/400

28 26 26 26 26

-

1 1

-

-

-

-

-

1

-

3 3

2 1 8 7 12

1 4 14 14 10

25 21 3 1 -

CUADRO 3.-Lote A-Contralores serológicos Control

60 7° 8° 90 100 11 ° 120

Fecha

abr. 9-46 jul. 24-46 obr. 5-46 eno. 8-47 abr. 11-47 dbr. 4-47 agto. 18-47

Animales reaccionantes en cada título No.de

Animales

10 9 9 8 8 8 8

Neg.

1/25

-

-

7 1 1 1 2 2

1 1 -

1/50

1/100

1 1 1

1 1 3 3 1 3 3

1 1 1

1/200

1/400

6 1 2 1 2 2

2 1 -

A los 10 meses de la vacunación el 77.77% de los animales habla recuperado su negatividad, pero a partir de esa fecha se observó una reaparición de los tftulos que podria atribuirse al hecho de que bovinos reactores cohabitaron desde esa fecha con los caprinos por circunstancias especiales que no pudieron evitarse, y que una vez producida estimamos interesante continuar su observación (Cuadro 3). Los animales testigos reaccionaron siempre negativamente. Las pariciones en vacunadas y testigos fueron normales. Los análisis bacteriológicos llevados a cabo con material proveniente de partos y de leche fueron negativos en todos los casos menos uno. En la cabra 027 que fué sacrificada cuando presentaba un titulo en 1/100 se aisló Brucella abortus, hecho que vendria a apoyar indirectamente el carácter de infectados de los bovinos mencionados. Como puede apreciarse esta circunstancia invalidó el aislamiento a que estuvo sometido este lote con el propósito de verificar la inocuidad de la vacuna.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Lote B.-Mantenido en contacto con animales infectados naturalmente agregándose un lote testigo integrado por 11 animales. Como puede observarse en el Cuadro 4 la presencia de títulos altos fu6 constante, mientras que en el lote testigo reaccionaron sólo cinco animales en títulos de 1:25 y 1:50. En este lote no se observó una regresión a la negatividad tan manifiesta como pudo observarse en el 7° contralor del lote A, dado que en este lote, en ese contralor comprobóse un solo animal negativo lo cual explicaría la aparición de la infección en los vacunados. CUADRO 4.-Lote B-Contralores serológicos Animales reaccionantes en cada título

Control

Fecha

No. de ,ievgo1/25 tivos

6° 70

8° 90 10 ° 11 ° 12 ° 13 °

I/S0

1/100

1/200

1/400

2 2

10 10

3 3

-

jul. 8-46 agto. 25-46

1 1

sept.

2

16 16

6-46

16

1

1

3

4

7

-

nbr. fbro. mayo jul. jul.

18-46 17-47 8-47 11-47 17-48

16 16 16 16 12

1

1 _1 1 2

4 5 3 2 4

8 4

2 7 12 8 -

-

1 1

-

4 5

CUADRO 5.-Lote C-Contralores serológicos Animales reaccionantes en cada título Control

Fecha

No de Animales

Nega-

tniVoeesg 1/25 1° 2° 30 40 50 60 70 8° 90

eno. 24-46 fbro. 28-46 mayo 28-46 sbre. 8-46 fbo. 15-47 mayo 5-47 jul. 1-47 jul. 21-47 jul. 9-48

25 25 25 25 20 18 18 18 18

1/50

1/100

1/200

1/400

1/500

-

-

25 20 -

-

-

-

-

-

-

11 6 5 2 3 3 4

1 5 4 1 10 3 3 10

4 9 3 2 5 9 2

4 2 3 3 5 3 1

4 1 4 6 1 2 -

-

-

2 -

-

-

-

Los análisis bacteriológicos llevados a cabo en vacunados y testigos, permitieron aislar Brucella melitensis en tres de los vacunados y dos testigos, que fueron los únicos que abortaron en ambos lotes. Lote C.-(Animales vacunados y posteriormente alojados en un establecimiento infectado en la localidad de Dean Funes, Prov. de Córdoba). En enero 19 de 1946 se vacunaron 25 cabritos que conjuntamente con un lote testigo de 12 animales fueron controlados serológicamente antes y después de la vacunación, iniciándose la exposición a la infección en un hato infectado a los dos meses de la vacunación.

Según puede observarse en el cuadro de resultados serológicos la

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223

respuesta postvacunal fué intensa en todos los animales que reaccionaron en título de 1/500 a los cinco días de su administración, observándose un indice opsonocitofágico marcado en todos los animales. En el tercer contralor serológico efectuado a los cuatro meses de la vacunación, cuando ya los animales cohabitaban en condiciones naturales con el hato infectado, se observa un descenso de los títulos que ya comienzan posiblemente a ser interferidos por las reacciones de infección de acuerdo a algunos títulos que se manifiestan en los testigos. Con respecto a las pariciones debemos comunicar que en la primera parición comprobóse un 50% de abortos y crías débiles que murieron tanto entre los animales vacunados como testigos. En la segunda parición se comprobaron seis abortos en los vacunados y siete en el lote testigo. En la tercera parición abortaron tres de los vacunados y dos de los testigos. Cabe señalar que durante el curso de esta experiencia desaparecieron extraviados o muertos siete animales vacunados y cinco testigos. Profilaxis en el campo.-Paralelamente a la tarea de identificación en el campo de los focos de brucelosis caprina, se procedió a impartir instrucciones de carácter profiláctico general, orientadas a impedir los contagios humano y animal. Las medidas adoptadas consistieron en aconsejar la destrucción de corrales y construcción de otros nuevos, alejados de la vivienda, la cremación de fetos y envolturas fetales, el aislamiento de cabras durante el parto o después del aborto y la desinfección de corrales. Esta profilaxis general, aunque de difícil aplicación por las dificultades del medio y la idiosincrasia de los pobladores, han dado resultados satisfactorios pero no los suficientes como para confiar en ella la solución del problema. Al mismo tiempo se impartieron indicaciones higiénico-sanitarias entre los criadores tendientes a evitar el contagio humano, tales como el hervido de la leche, la elaboración de queso y quesillos con leche hervida, la desinfección de manos y cara después de las tareas habituales, evitando el manipuleo de fetos o envolturas lo que ha contribuido en algo a elevar la cultura sanitaria de la población y disminuir los casos humanos. Vacunación de Cabrillas con Cepa Br. Abortus 19.-A campo.-Las dificultades encontradas en la aplicación de la profilaxis general, motivaron la utilización de la vacuna cepa 19, conocida por su empleo en terneras, para probar su eficacia en brucelosis caprina. Al igual que en las experiencias de laboratorio ya descritas se utilizaron cabrillas de 2 a 4 meses de edad, libres de la infección. Se inoculó la vacuna por vía subcutánea a dosis de 2 cc. La vacuna utilizada fué elaborada por el Laboratorio de la División de Enfermedades Infecciosas. Los animales vacunados permanecieron el tiempo de la experiencia en contacto con las cabras infectadas de los hatos a los que pertenecían.

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Se intervinieron en estas experiencias de vacunación en masa, 748 establecimientos de cría distribuidos en regiones de intensa explotación caprina, abarcando un total de 30,000 cabrillas inoculadas. Los controles posteriores se realizaron para comprobar los títulos de las reacciones serológicas en las cabrillas vacunadas y los resultados de las pariciones. La inoculación de la vacuna cepa 19 produce en las cabrillas la aparición de aglutininas en títulos elevados que declinan luego de 4 ó 5 meses, hasta convertirse la casi totalidad en negativos. Cuando los animales vacunados mantienen aglutininas por más de 6 meses se consideran infectados. Esto ha ocurrido en casi todos los establecimientos infectados en donde se procedió a la vacunación de cabrillas. En los controles efectuados a los 12 meses de la vacunación se ha comprobado no solamente la persistencia, o reaparición de las aglutininas de infección, sino también el aborto en proporción similar al resto de los animales no vacunados de las majadas. En conclusión, la vacunación de cabrillas de 2 a 4 meses de edad con cepa Br. abortus No. 19, como método de profilaxis contra la brucelosis caprina, no ha dado resultados satisfactorios. En consecuencia los esfuerzos del futuro deben encaminarse hacia la obtención de un inmunfgeno eficaz, perseverando mientras tanto en la aplicación de las medidas de profilaxis general. BRUCELLOSIS IN GOATS IN THE ARGENTINA REPUBLIC (Summary) Brucellosis in goats has been reported in Argentina in 1922 by Dessy and Lorenzelli, in 1931 by Sordelli and collaborators, in 1932-1935 by Mazza and collaborators, Pardal and de la Barrera and since 1938 the Veterinary Service of the Ministry of Agriculture and Livestock. It is understood that the origin of the brucellosis infection in the goat stock of Argentina dates back to the period of the Conquest and to the introduction of goats by the Spaniards. The first cases reported came from the valleys and mountains of the northwest, where goats are raised and bred. In this section of the country, goat breeding is undertaken by poor settlers or farmers, who own small flocks that feed on the natural ranges formed by a xerophic and spinybrush vegetation and water in flat basin dams or streams throughout the sandy semidesert and rough or broken terrains. As a protection during the night the animals are kept in rather primitive corrals or pens near the lodgings. The "corrals" and "dams" are foci of human and animal infection. The economic and social consequences are shown by the loss of kids and also by the spread of the infection to the man. The prevalence of the disease in the northwest provinces ranges from 5 to 80%, according to area. Mass infection of animals in certain areas coincides with a high human incidence in slightly infected areas with slight or without

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human incidence. Two clinical pictures of caprine brucellosis have been reported: acute and chronic, and criterion for diagnosis have been given through clinical (abortion, arthritis, etc.), serological, allergic and bacteriological manifestations. Trial vaccinations were performed with strain 19 in kids, appraising the agglutination curves and the clinical manifestations to interpret its immunity. The economic and social importance of caprine brucellosis makes it imperative to conduct investigation toward obtaining an effective immunogene for its prophylaxis.

CONTROLLING BRUCELLOSIS IN COLORADO GOATS By GEORGE W. STILES, M.D., PH.D., F.A.P.H.A. Director of Laboratory Section, Colorado State Department of Public Health, Denver, Colorado INTRODUCTION

The problem of controlling brucellosis in Colorado goats is intimately linked with the goat industry in the entire southwestern United States. Early settlers doubtless brought with them goats from their native lands, and in those days the question of disease in their flocks, especially what we now call brucellosis, was far from their thinking, as this species of animal was considered "disease resistant" and exceptionally healthy. While the caprine animal is comparatively free from most parasitic and infectious diseases, today we know that goats are quite vulnerable to the organisms of the brucella group, especially Brucella melitensis; considered by many authorities as more virulent than Brucella abortus or Brucella suis for infections in man. This was amply proven by Bruce, Hughes, and many others in their studies on diseased goats on the island of Malta and elsewhere in the world. For more than a half century goat raising has been carried on in southern Colorado, especially around Trinidad, in Las Animas County. Importations of goats into southern Colorado came from Mexico, Texas, New Mexico, Arizona; and doubtless the early immigrants from Spain, Switzerland and other Mediterranean countries brought their goats with them, along with their methods of cheese making, lack of sanitation, and any Brucella infected animal that chanced to be in the herd. Unfortunately, pure bred goats are scarce in this area, the average animal being valued at less than ten dollars per head, and are locally known as "brush goats" or "tree-climbers." They thrive best on the mountainous range and, under normal conditions, give but relatively small quantities of milk. Nevertheless, cheese making during war years became a highly profitable business. COLORADO's GOAT POPULATION

The writer is indebted to Reed 2 1 for the following statistics pertaining to goats in Colorado: Year

Farms Reporting

Number of Goats

1920 1925 1930* 1935 1940* 1945

1281 1101 2129 2233 1498 1362

28,688 21,525 30,512 25,561 20,667 20,090

*April 1. 226

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According to the latest information there has been a marked decline in the number of goats in southern Colorado since the 1945 census. The total number of goats in the United States, Jan. 1, 1945 was 4,265,501 head. Source of information: U. S. Department of Commerce, Bureau of the Census. CHEESE MAKING FROM GOATs' MILK IN COLORADO During the years 1943 and 1944 the writer25 cooperated with Vincent 26 in the examination of cheese manufactured in the Trinidad, Colorado, area. The results were published in the January 1945 issue of the Rocky Mountain Medical Journal, and a partial summary is the following: 1. The manufacture of cheese from goats' milk in the vicinity of Trinidad, Colorado, has been in progress for many years. The annual output of cheese has an estimated value of one-quarter million dollars. 2. The sanitary conditions on the goat ranches and the cheese making plants in southern Colorado had received little attention until the past year. Mr. Wendell Vincent, in charge of the Denver office of the U. S. Food and Drug Administration, conducted a sanitary survey and collected cheese samples for bacteriological examination. 3. Insanitary practices, a polluted water supply, and other features were discovered. Br. militensis organisms were isolated from guinea pigs injected with goat cheese prepared from unpasteurized goats' milk. 4. Of nineteen cheese samples examined, eight proved to be positive for brucellosis when injected into guinea pigs. Four samples were the feta type, made from raw goats' milk, three of the Romano variety made from unpasteurized goats' milk and one was imported yellow cream cheese from cows' milk. 5. The blood testing of 14,339 blood samples, representing 131 herds, in the Bureau's Cooperative Bang's Disease Laboratory (Denver) gave 8.5% reactors in titers of 1-25 or higher. 6. One human case was reported. A man who appraised about 1,000 diseased goats contracted acute infection with clinical symptoms of brucellosis. A positive blood reaction in titers up to 1-640 and 1-1280 by the State and Government laboratories was established. MODES OF INFECTION

During the goat testing period in Colorado, three federal veterinarians contracted clinical symptoms of brucellosis while bleeding and handling thousands of animals in dirty, insanitary goat corrals. In the summer of 1948, the State Laboratory recovered and identified Br. melitensis from a blood clot of a patient in the southern Colorado goat area. The only evidence as to possible source of infection was the frequent drinking of water from an open stream flowing near the goat corrals. The attending physician assumed that the infection came from contaminated water, as the patient denied any contact with goats or their by-products. During the first blood testing program, one of the goat owners in Las

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Animas County had more than 50% of reactors in his herd. This man developed brucellosis apparently from direct contact with the infected animals, and sold the remainder of the herd, fearing that his family might also contract the disease. Several writers suggest "germ laden dust" as a plausible source of human infection by persons engaged in handling goats, especially during the kidding season. At such times the owner's entire family, including the children, spend much time with the does and kids. In addition to consuming raw infected goats' milk and cheese or contact with diseased tissues, a suggested avenue of human infection is through exposure of the nostrils, mouth or eyes, or of minor cuts or abrasions on the unprotected face, arms, or hands to germ laden dust. It is well known that powdered, dry goat manure may harbor, for variable periods of time, virile Brucella organisms which are excreted from diseased animals. Brucella organisms are also known to pass through the unbroken skin. A measure to safeguard Colorado citizens against Brucella infection was the requirement that all fluid milk, including goats' milk sold in this state, be adequately pasteurized. This regulation was promulgated by the Dairy and Livestock Advisory Committee and adopted by the Colorado State Board of Health, August 7, 1947. According to Dr. R. L. Cleere, 2 Executive Director of the Health Department, the regulation is as follows: "From and after June 1, 1949, all milk and milk products sold or offered for sale in the State of Colorado shall be pasteurized in an approved plant, as defined in the regulations of the State Department of Public Health." Doubtless this one important factor has tended to decrease the incidence of brucellosis in humans in Colorado. BACTERIOLOGICAL EXAMINATION OF CHEESE SAMPLES On September 27, 1944, a sample of American type of cheese was delivered to the writer* by Kenneth W. Lloyd, Colorado State Food and Drug Commissioner. This cheese was collected a few days previously from a Colorado creamery, and reported to have been made from unpasteurized "cows' milk". The bacteriological results, including guinea pig inoculation, showed the presence of Br. melitensis organisms in this cheese, despite the statement by the creamery manager, that cows' milk only was used in his plant. It was subsequently learned that the owner of a nearby goat ranch sold raw goats' milk to this creamery at the time the cheese was manu* The examination was made in the Denver Branch, Pathological Laboratory, U. S. Bureau of Animal Industry, while in the service of the Federal Government.

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factured; and furthermore, that the daughter of the goat ranch owner was reported ill with brucellosis. The goats in question were later blood tested by the U. S. Bureau of Animal Industry and several reactors discovered and slaughtered. With the disclosure of Brucella infection in the cheese from the above creamery, an embargo was placed on a carload lot entering interstate commerce by Vincent, 26 and held 120 days before being released. The creamery was ordered to install adequate pasteurizing equipment by the Colorado State Department of Public Health to safeguard the public against further health hazards. During the summer of 1950 the writer examined 10 samples of Colorado cheese made from goats' milk. These specimens were collected by J. E. Dolan, Chief Milk Sanitarian, Sanitation Section, and by Dr. David R. L. Duncan, Director, Huerfano-Las Animas District Health Department. The results follow: 1. Four samples of cheese made during 1949 from raw goats' milk, ranging from 121 to 209 days after manufacture, were all proven negative for Brucella by guinea pig inoculation. The goat herds furnishing milk for this cheese previously harbored Brucella reacting animals. 2. An additional 6 samples of cheese, freshly made from goats' milk, by special request, showed evidence of Brucella infection in only one of the six specimens examined. The cheese sample for which the laboratory findings were positive was prepared from raw goats' milk supplied by a herd of 275 animals with 8 reactors. Three does were dry at testing time. Six of the 8 reactors were purchased a few months previously from an owner whose animals had not been blood tested for several years. A guinea pig injected with this cheese developed a localized abscess at the point of inoculation and showed an enlarged spleen and congested liver; and the blood serum agglutinated in a titer of 1-25 and 1-50 for brucellosis, 6 weeks after injection. Brucella organisms were not recovered. Another guinea pig was inoculated subcutaneously with a second cheese specimen and died 3 days later from septic infection. Three other samples resulted in negative findings. The owners reported that they used raw goats' milk from slightly infected herds but that considerable heat (195°F for 3 hours) was applied in the process of manufacture. The remaining negative cheese specimen was produced from milk supplied by a herd of 310 goats showing only one reactor. This animal was removed from the herd and destroyed, some days before the cheese was made. Commercial cheese now made in Colorado is required to be manufactured from pasteurized dairy products. MEASURES TO PREVENT MOVEMENT OF DISEASED ANIMALS

Regulations to prevent the introduction and spread of infectious and contagious animal diseases into our country from foreign lands are promulgated by the United States Department of Agriculture at various

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times. Bureau of Animal Industry Order 373 deals with the introduction of Rinderpest and foot-and-mouth disease into the United States from nations where these diseases are known to exist. Very few goats are imported from such countries. B. A. I. Order 379, Sections 92.4, 92.5, and 92.11 are especially applicable to goats. These sections should be studied in detail by interested parties, they deal largely with quarantine measures and evidence of freedom from certain diseases. Unfortunately, as Miller' s states, "there is no specific requirement that goats be tested for brucellosis before entering the United States." On the other hand, information submitted by Dr. M. N. Riemenschneider,22 Colorado State Veterinarian, provides that: "All dairy and breeding goats three (3) months of age or over brought into the State of Colorado must be accompanied by a health certificate showing tuberculosis and Bang's tests with negative results within thirty (30) days prior to importation. The Bang's test must be conducted by a laboratory approved by the State of origin. The tuberculosis and Bang's disease test certificates shall contain a list of the individual goats, together with a satisfactory report and description of the test. All certificates and test charts shall be attached to the bill of lading and must be submitted at any time or place for official inspection." It is the writer's personal opinion that if Colorado can legislate against the introduction of Brucella infected goats into the State, the United States should also prevent diseased goats from entering this country. With regard to the goat blood testing and slaughter program in Colorado the last General Assembly, in Chapter 73, Colorado Session Laws of 1949, provides an indemnity payment of $5.00 per head for each Brucella reacting goat. Such animals are required to be slaughtered and either burned or deeply buried on the premises, and are not allowed to be sent to slaughter houses. DIAGNOSTIC

BLOOD TESTING OF COLORADO GOATS FOR BRUCELLOSIS

The lion's share of credit should go to veterinarians employed in the U. S. Bureau of Animal Industry service stationed in Denver, for making approximately 75,000 blood tests on Colorado goats during the period from 1944 to 1950. These men include Dr. Edgar Heiny"2 who retired July 1, 1949, and was succeeded by Dr. H. E. Schaulis,23 and his assistant Dr. L. H. Smith 2 4; and also other Government and private veterinarians who drew blood samples at various times. Dr. C. E. Moorman is in charge of the Federal-State Cooperative Brucellosis Laboratory. From 1944 through 1948, the number of goats tested for brucellosis in Colorado averaged 14,100 a year, including an average of about 12,000 a year in Las Animas County, where the goat cheese industry of the state is centered. In 1948 the tests were made only in Las Animas County

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and an adjacent county, Huerfano. In 1949 only a few tests were made, by local practitioners. In 1950, however, 4,011 goats were tested in Las Animas County. Tabulations of the laboratory results of the blood tests according to year show that the percentage of reactors decreased very sharply from 1944 to 1945 and continued to decline, but less steeply, thereafter. This is true both of the entire area included in the testing program each year and of Las Animas County as a separate study area, except for an increase in the percentage of reactors among the goats tested in Las Animas County in 1950. Las Animas County only

Testing Area Reactors

Reactors Goats Tested

Goats Tested

1944 1945 1946 1947 1948 1949

18,609 19,152 11,165 11,209 10,354* 0

1950

4,011

Number

Per cent

1,450 444 166 146 126 -

7.99 2.32 1.49 1.30 1.25 -

16,463 15,984 8,911 8,744 * 0

61

1.52

4,011

Number

Per cent

1,368 420 125 73 * -

8.31 2.63 1.40 0.83 * -

61

1.52

*Statistics for Las Animas and Huerfano Counties combined.

The increased proportion of positive reactors in 1950 can be partly accounted for by the limited testing in 1949 and changes in the composition of the herds. Negative results were found in all of the tests made for herds in many of the numerous counties included in the program from 1944 through '1947, as follows: Counties with No Reactors Number

Goats Tested

10 18 13 14

107 126 172 106

1944 1945 1946 1947

In the blood tests of 10,354 goats representing 80 herds in Las Animas and Huerfano Counties in 1948, all of the results were negative for 2,509 animals representing 23 herds. LABORATORY

TECHNIQUES

Blood testing of Colorado goats for brucellosis is conducted in a manner similar to that for millions of cattle by the Bureau of Animal Industry-State Cooperative Plan in operation over the United States.

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The "slide" or "rapid" plate method is used and standard Bureau antigen is applied in making agglutination tests. The antigen was prepared under the supervision of Dr. A. B. Crawford3, Beltsville Research Center, Beltsville, Maryland, who says: "In cattle, a blood titer of 1:100 is indicative of infection; in swine and goats, the agglutination test must be interpreted differently. In the latter, if any animal in the herd shows a titer of 1:100 all swine or goats reacting at 1:50 or 1:25 should also be considered infected, as the titer may be receding although the animal is still harboring the germ, or it may be rising as in recent infection. Repeated tests are sometimes necessary to determine the status of an animal so far as infection is concerned. As the period between exposure and the development of a blood titer may be several months in some instances, some does may be expelling Brucella melitensis in their milk before a positive blood test is obtained." GOAT HERD MANAGEMENT

In the nation-wide plan of brucellosis control in cattle, sanitary measures and good herd management are recognized as highly essential, regardless of other methods being employed. Similar practices of proper caring and handling of goats is likewise necessary to expedite the eradication of the disease in infected herds. The frequency of blood testing is perhaps of first importance to insure success. Long periods of time should not intervene between testings. In checking over his card records, Dr. Schaulis2 3 states that he sees positive proof that the percentage of Brucella reactors is lessened in proportion to the number and frequency of tests applied to infected herds. While funds were provided in Colorado for indemnity of all Brucella infected goats, the owners were not required to have their flocks tested periodically. Consequently comparatively few persons carried through with retests. The program for the eradication of brucellosis among Colorado goats was inaugurated during the autumn of 1944. It was planned to make 30 to 60 day testing periods, but due to shortage of personnel, testing was only done once or twice yearly. The breeding season for southern Colorado goats is usually through September and October. As the gestation period is approximately 5 months,'9 the kidding season generally begins about April 15 and ends about June 1. Goat owners prefer having their flocks tested after kidding, since rough handling before could cause abortion or other damage. With regard to abortion, Dr. Heinyl 2 believes there is a close relationship between the percentage of abortions and the number of reacting animals in the herd. Other observers are of the same opinion. However, the matter of mineral deficiency, low vitamin content in the forage, and other dietetic disturbances, including severe storms and unfavorable weather, are factors to be considered.

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During the first blood testing of goats in Las Animas County (1944), many premature foetuses were reported. The number observed seemed to decrease in proportion to the percentage of reactors discovered and destroyed. On the other hand, during drouth years some herds had an abortion rate of 10% or higher, even when comparatively free from Brucella infection. An undesirable practice found among goat owners in southern Colorado was the pooling of their bucks from many flocks into one large herd for several weeks prior to the breeding season. As the blood testing program revealed many reacting animals among the males, such goats could become potential spreaders of disease. Also, on the ranges, the intermingling of goats from clean herds with those where infection was known to exist was not uncommon. In small herds located in the north central and other sections of the state where owners kept their goats strictly confined to their own premises the year round, without the introduction of outside infected animals, the eradication of brucellosis was accomplished in a comparatively brief period. Blood testing of new goats added to the herd is a factor of vital importance. In his excellent paper Dr. Crawford 3 says: "If you want to introduce new blood into your herd of goats insist on a blood test chart from the vendor signed by a veterinarian or authorized testing laboratory. I would go even further if I were buying from a dealer in States where the disease is known to exist by having the vendor certify that his whole herd is free from disease." Crawford further states: "It is pleasing to learn that many goatkeepers are having periodic tests for brucellosis made of their herds, this in many instances in the absence of state or municipal regulations requiring such tests. I believe such tests should be compulsory in every state. This would show us if infection is present, tend to prevent its spread, and safeguard public health." Boyd' in discussing brucellosis in goats claims: "The subjection of all animals to repeated testing at regular and frequent intervals will quickly determine the extent of infection. Removal of animals showing positive reactions to the agglutination test accompanied by the practice of strict sanitary herd management will result in ultimate control or eradication of the disease." And Gilmanl' contends: "Outbreaks of brucellosis in goats in the United States have been rare. Inasmuch as this species usually becomes infected with Brucella melitensis which is much more infective for man than either Br. abortus or Br. suis, it is necessary

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to resort to prompt recognition and slaughter of infected animals. The application of such measures has stamped out the few outbreaks that have appeared." IMPORTANCE OF STATE AND LOCAL PUBLIC HEALTH ORGANIZATION

IN THE CONTROL PROGRAM

Establishment of a full-time local health unit in Las Animas County in July 1944 was an important factor in the effective, continuing operation of the blood testing and clean-up program started in that goat raising and cheese making area the same year. During the next few years this type of control program was facilitated there and in other counties by close cooperation between the State Department of Public Health and local authorities. In 1947, the functions and regulatory powers of the State Department were greatly strengthened by legislative action, and counties were authorized to form local health districts under organized departments. Soon thereafter Huerfano County joined Las Animas County in establishing a two-county health department, and in 1948 a combined goat testing program was conducted for the two counties through the local department. Local health departments now serve twothirds of the population of the state and are becoming well stabilized agencies for strong disease control and sanitation measures. RECAPITULATION 1. In former years changes in the incidence of brucellosis in southern Colorado goats depended largely on the importation of clean or infected animals, since blood testing was not required. 2. Goats imported into Colorado now must pass adequate blood tests for brucellosis. The author believes similar legislation should be enacted to prevent Brucella-infected goats from entering the United States, since such requirements are not in effect nationally. 3. The goat population of southern Colorado has declined considerably during recent years, and cheese making in the Trinidad area is now less profitable than during war years. 4. Suggested possible modes of human infection include consumption of raw milk or cheese made from unpasteurized milk produced by Brucella infected goat herds; contact with diseased, aborting animals or their tissues; the inhaling of dry infected air from insanitary goat pens; and direct contact of germ-laden dust with the eyes, mouth, hands or other exposed areas of the body. The Brucella organisms are also known to pass through the unbroken skin. 5. The bacteriological examination of cheese made from raw goats' milk, including guinea pig inoculation, gives a fair index as to the probable presence of brucellosis in herds producing the milk. 6. The testing of Colorado goats during the period 1944 to 1950 was done by the U. S. Bureau of Animal Industry-State Cooperative Plan. Some 75,000 blood tests were made during those years. The proportion

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of reactors was less than one-third as large in 1945 as in 1944, or 2.32% compared with 7.99%; and in 1948 the proportion was less than onefifth as large as in 1944, or 1.25%. Very few goats were tested in 1949, but in 1950 about 4,000 goats were tested in Las Animas County, the center of the goat cheese industry. In Las Animas County a rise in the percentage of reactors was found in 1950 as compared with 1948. The increase probably was due to the lapse in the testing program in 1949 and also to changes in the composition and management of the herds. 7. Animals showing an agglutination titer of 1-25 or higher were considered reactors. They were slaughtered on the premises and either deeply buried or burned, and were not allowed to be sent to slaughter houses. Owners of infected goats received an indemnity of $5.00 per head for each reactor. 8. Good management including sanitary practices, frequent blood testing of infected herds, and the destruction of all reactors is essential in the eradication of brucellosis in goats. 9. Quotations from leading authorities show that the frequent, periodic testing of goats for brucellosis, and the removal and destruction of all reactors will eventually eradicate the disease from infected flocks. Full safeguards, however, require that all new additions to clean herds shall be properly blood tested for brucellosis. 10. Since June 1, 1949, all milk or milk products sold or offered for sale in the State of Colorado shall be pasteurized in an approved plant. Doubtless this action of the Colorado State Board of Health has materially reduced the incidence of brucellosis in Colorado. 11. The expansion of local health department organization in the goat raising regions of the state and the increasingly strong advisory and regulatory functions of the State Department of Public Health have been important factors in the brucellosis control programs discussed in this paper. REFERENCES (1) Boyd, W. L.: Brucellosis in animals other than cattle or swine. Brucellosis of goats. 198-199. Symposium on Brucellosis, N. I. H., U. S. Dept. Agric. Nat. Rec. Council, Sept. 22-23, 1949, Bethesda, Md. Pub. A. A. A. S. 1950. (2) Cleere, R. L., M.D., M.P.H., Executive Director, Colorado State Department of Public Hlth. (3) Crawford, A. B., Veterinarian in charge, Animal Disease Station, U. S. Bureau of Animal Industry, Beltsville Research Center, Beltsville, Md. Responsibility of the goatkeeper with relation to brucellosis in goats. Reprint-Better Goatkeeping, Ipswich, Mass., Oct.-Nov., 1948. (4) Evans, Alice C., Brucellosis in the United States. Am. Jour. Public Hlth, 37-139-151, Feb., 1947. (5) Evans, Alice C., The Distribution of Brucella melitensis variety melitensis in the United States. Reprint No. 1807-Pub. Health Reports, 52-1-9, (March 12) 1937.

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(6) Ferenbaugh, T. L., Endemic Mediterranean Fever (Malta Fever) in Southwest Texas. Jour. Am. Med. Assoc. LVII, 730-731, (Aug. 26) 1911. (7) Gentry, E. R. and Ferenbaugh, T. L. Endemic Malta (Mediterranean) Fever in Texas, with the isolation of the Micrococcus melitensis from two patients. Jour. Am. Med. Assoc. LVII, 889-891, (Sept. 9) 1911. (8) Gentry, E. R. and Ferenbaugh, T. L., Endemic Malta (Mediterranean) Fever in Texas. Further notes on its distribution and probable source with report of additional cases. Jour. Am. Med. Assoc. LVII, 1045-1048, (Sept. 23) 1911. (9) Gentry, E. R. and Ferenbaugh, T. L., Malta Fever in Texas, a report on the serum reaction of one hundred and twenty-eight goats in Edwards County. Jour. Am. Med. Assoc. LVII, 1127, (Sept. 30) 1911. (10) Gilman, H. L., The Control of brucellosis in animals employing test and slaughter methods. Goats, 239. Symposium on Brucellosis, N. I. H., U. S. Dept. of Agric. Nat. Rec. Council, Sept. 22-23, 1949, Bethesda, Md. Pub. A. A. A. S., 1950. (11) Harris, H. J., Brucellosis (Undulant Fever) clinical and sub-clinical. Paul B. Hoeber, Inc., New York, 1950. (12) Heiny, Edgar, Veterinarian in charge, (retired) Denver, U. S. Bureau of Animal Industry. Personal communications, years 1944-1948. (13) Holt, R. L. and Reynolds, F. H. K., Malta fever and its prevalence along the Mexican border. Mil. Surgeon LVI, 414-424, (Apr.) 1925. (14) Huddleson, I. F., Brucellosis in diseases transmitted from animals to man, by Thomas G. Hull, 94-117. Chas. C. Thomas, Springfield, Ill., 1947. (15) Huddleson, I. F., Brucellosis in man and animals. The Commonwealth Fund, New York, 1943. (16) Lake, G. C., Malta fever in Southwestern United States. Pub. Hlth Reports XXXVI, 2895-2899 (Nov. 24) 1922. (17) Mayer, K. F. and Eddie, B., The Problem of caprine Brucella infections in the United States. Jour. Am. Vet. Med. Assoc. LXXXV, 39, 286-303, March 1935. (18) Miller, Donald, Acting Veterinarian in charge, Inspection and Quarantine Div., Agric. Rec. Adm., B. A. I., U. S. Dept. Agric. Personal communication Aug. 20, 1950. (19) Potter, C. G., and Simmons, V. L., Milk Goats. Farmer's Bulletin, No. 920, 1-35, U. S. Dept. Agric. (20) Rand, Rolle R., Executive Director, Colorado Development Council Inc. Personal communication, Aug. 11, 1950. (21) Reed, F. K., Agric. Statistician in charge, Denver B. A. E., U. S. Dept. Agric. Personal communication, July 19, 1950. (22) Riemenschneider, M. N., Colorado State Veterinarian. Personal communication, Aug. 28, 1950. (23) Schaulis, H. E., Veterinarian in charge, U. S. B. A. I. Personal communications, 1950. (24) Smith, Louis H., Assistant Veterinarian, Denver U. S. B. A. I. Personal communication. (25) Stiles, Geo. W. Brucellosis in goats, recovery of Brucella melitensis from cheese manufactured from unpasteurized goats' milk. Rocky Mt. Med. Jour. 42, 18-25, (Jan) 1945. (26) Vincent, Wendell, Chief, Denver District, U. S. Food and Drug Administration, Federal Security Agency. (27) Watkins, W. W. and Lake, G. C., Malta Fever, with special reference to the

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Phoenix, Ariz. epidemic of 1922. Jour. Am. Med. Assoc. LXXXIX, 1581-1584, (Nov. 5) 1927. (28) Yount, C. E. and Looney, R. N. Malta fever, with preliminary report on cases occurring in Arizona. Ariz. Med. Jour. I, 18-27, (Apr.) 1913. CONTROL DE LA BRUCELOSIS EN LAS CABRAS DEL ESTADO DE COLORADO (Sumario) Durante más de medio siglo se han criado cabras en el sur de Colorado, en particular cerca de Trinidad, Condado de Las Animas. Las cabras fueron traidas de México, Texas, Nuevo México, Arizona, y sin duda los primeros inmigrantes de España, Suiza y otros paises del Mediterráneo trajeron cabras, incluso aquellos animales infectados que por casualidad se encontraban en los rebaños. Los datos publicados acerca de las investigaciones de 1944 demostraron que las pruebas sanguineas de 14,339 cabras acusaron 8.5% de reactores positivos para brucela. Los animales positivos fueron sacrificados, enterrados profundamente o incinerados. Las pruebas subsecuentes practicadas en las cabras de esta zona durante 1945 revelaron una marcada disminución de reactores, pues de 19,152 animales probados 2.32% resultaron infectados con brucelas. Durante 1946, 1947 y 1948 se probaron anualmente unos 10,000 animales, y 1.49, 1.30 y 1.25% resultaron positivos. Estas pruebas sanguíneas fueron ejecutadas por la Oficina de Industria Animal de los Estados Unidos, en cooperación con el Estado de Colorado. El examen bacteriológico de muestras de queso de leche cruda de cabra de la zona de Trinidad, demostró en 1944 la existencia de Brucella melitensis en varios casos. Dichos hallazgos parecen confirmar la existencia de brucelosis en los rebaños de cabras cuya leche se emplea para fabricar queso. Un pequeño número de muestras de queso examinadas en 1950 no reveló brucelosis, excepto en un caso en que resultaron positivas cabras del rebaño y muestras de leche empleada para la fabricación del queso. Estas pruebas se hicieron por solicitud especial para fines de experimentación. Los datos disponibles parecen apoyar la opinión de que las pruebas sanguineas frecuentes, con eliminación de todos los reactores, permitían erradicar con el tiempo la brucelosis de los rebaños infectados. Desde el punto de vista de la salubridad pública, se han inaugurado medidas en Colorado para el control de posibles infecciones humanas. Desde el 1° de julio de 1949 se requiere en Colorado la pasteurización en una planta aprobada de todos los productos vendidos u ofrecidos en venta. Otra medida adicional para la protección de la salud consiste en exigir que todas las cabras enviadas a Colorado sean probadas en busca de brucelosis. Debe promulgarse un reglamento semejante para probar en busca de brucelosis a todas las cabras procedentes de paises extranjeros antes de permitir su entrada a los Estados Unidos.

EL PROGRAMA DE ERRADICACIÓN DE BRUCELOSIS EN PUERTO RICO

Por el Dr. ENRIQUE R. ToRO, JR. Negociado de Industria Animal, San Juan, Puerto Rico Desde el punto de vista económico y también desde el punto de vista de salud pública, la enfermedad de Bang o brucelosis bovina, continúa siendo una de las de mayor importancia en Puerto Rico. Allá tenemos la fortuna de que el Comisionado de Agricultura y el Comisionado de Salud están decididamente interesados en la industria ganadera y especialmente en el control y erradicación de la brucelosis. También tenemos que mencionar al Dr. Alfonso Rivera, Jefe de la División Veterinaria del Departamento de Agricultura de Puerto Rico, quien con su esfuerzo, entusiasmo y sabios consejos, fué uno de los factores principales en la organización de nuestro programa de brucelosis. Esta enfermedad se desconocía en la Isla hasta que en el año 1923 se llamó al Dr. Pablo Morales Otero, antiguo director de la Escuela de Medicina Tropical de Puerto Rico, para investigar una serie de abortos en unos vacunos. Las sospechas del Dr. Morales Otero de que se trataba de una infección producida por brucela, quedaron confirmadas por inoculaciones experimentales. Aunque la infección hoy día no parece tan seria como en el año 1923 debido a que ha perdido algo de la índole explosiva inicial, sin embargo continúapropagándose. Según estudios bacteriológicos hechos por el Dr. Morales Otero se ha revelado que la infección en Puerto Rico es estrictamente de origen bovino. No se ha encontrado indicio alguno que señale el origen como caprino o porcino. Conjuntamente con la propagación de la infección en el ganado, se ha observado un estado mórbido entre los humanos que ha ido en aumento, hasta que hoy día es casi cinco veces más alto que en el año 1930. Alrededor de este punto resulta de interés informar que el por ciento de leche pasteurizada que se consume en Puerto Rico es del 1.5%. La mayor parte de este por ciento pertenece a la zona metropolitana de la capital, San Juan y sus alrededores, la cual consume 75,000 litros diarios, y de ellos el 85 a 90% es leche pasteurizada. En las otras 76 ciudades y pueblos de la Isla, casi toda la leche se vende en su forma cruda. En el año 1942, se empezaron a hacer trabajos preliminares de brucelosis en los bovinos. Durante los años de la segunda guerra mundial la falta de médicos veterinarios obstaculizó el comienzo de un programa de control y erradicación. No fué hasta el mes de abril de 1948 que se organizó, bajo los auspicios del Departamento de Agricultura de Puerto Rico en cooperación con la Oficina de Industria Animal de los Estados Unidos, una campaña obligatoria. En ella se observan las recomenda238

ERRADICACIÓN DE BRUCELOSIS EN PUERTO RICO

239

ciones hechas por el U. S. Livestock Sanitary Association según fueron aprobadas por la Oficina de Industria Animal de los Estados Unidos. Básicamente, el programa consiste de cuatro planes: Plan A.-Aplicación del método de pruebas de aglutinación de suero, con sacrificio inmediato para consumo público de todos los casos positivos. Por cada uno de ellos se paga una indemnización total de $40.00, $20.00 contribuidos por el Gobierno de Puerto Rico y los $20.00 restantes por el Gobierno de los Estados Unidos. En este plan no se vacunan terneras con la vacuna de la Cepa 19. Plan B.-Este es idéntico al anterior, con la única diferencia de que en él1se vacunan terneras de 6 a 8 meses de edad. Plan C.-Aplicación del método de pruebas de aglutinación, permitiendo retener provisionalmente en la manada los casos positivos. Este es un plan restrictivo y se aplica en aquellos hatos donde la infección es tan alta que el sacrificio inmediato de los afectados se convertiría en una pérdida excesiva para el ganadero. En este plan no se paga indemnización, pero se vacunan terneras de 6 a 12 meses con la vacuna Cepa 19. Plan D.-Este consiste en la vacunación de los adultos. S61o se recomienda en aquellos hatos donde la propagación es muy rápida y la infección muy virulenta. Para aplicarlo es necesario hacerse de un permiso por escrito extendido por las agencias federales e insulares. La prueba de aglutinación usada en nuestro laboratorio es la aprobada por la Oficina de Industria Animal de los Estados Unidos usándose el antígeno estandarizado por dicha agencia. Sobre la vacuna Cepa 19 a que nos hemos venido refiriendo deseamos anotar que en el año 1942 comenzó su aplicación en Puerto Rico. Algunos ganaderos, por su cuenta y riesgo, la usaron indistintamente. Al iniciarse en el año de 1948 el programa oficial sistemático, se hicieron las observaciones de rigor y se llegó a la conclusión de que el uso de dicha vacuna era útil y conveniente. Después de haber transcurrido ocho años de un uso más o menos extenso, encontramos a todos, ganaderos y médicos veterinarios, convertidos en partidarios decididos de ella. Hasta la fecha se han vacunado 23,284 terneras con la vacuna de la Cepa 19. En el mes de abril de 1948 se empezó un reconocimiento y se probaron por la prueba de aglutinación vaquerías autorizadas y clandestinas ubicadas en las 77 municipalidades de Puerto Rico. Se probaron 80,120 vacas y novillas, resultando 2,705 casos positivos o sea un 3.37% de infección. Este 3.37% de infección corresponde únicamente a las ganaderías organizadas para expender leche que es precisamente donde la enfermedad ataca con denuedo; pero se espera que baje aún más cuando se empiece a hacer pruebas por zonas, lo cual exije que todo el ganado vacuno correspondiente a cada municipalidad sea probado. El reconocimiento tomó año y medio en efectuarse e inmediatamente, en septiembre de 1949, se comenzaron a hacer pruebas por zona o municipalidad, con el objeto de declarar como "Zona Certificada-Libre de

Resultado de pruebas de brucelosis hechas durante el reconocimiento que empezó en abril de 1948 en Puerto Rico Municipalidad

Ganado soretido a prueba

Reactores

Porcento de

infección ~~~~~ifcc6

a

Terneras vacunadas uaa

Adjuntas ................... Aibonito .................... Aguada..................... Aguadilla ................... Aguas Buenas ............. Añfasco ..................... Arecibo ..................... Arroyo .....................

219 741 0 722 935 0 1,390 29

7 19 0 1 42 0 18 1

3.2 2.5 0 .13 4.5 0 1.3 3.4

37 8 0 181 36

Barceloneta ................ Barranquitas ............... Bayamón ...................

1,996 173 4,369

11 17 122

.55 9.8 2.7

72 45 1,007

Cabo Rojo ................. Caguas ..................... Camuy ..................... Carolina .................... Cayey ...................... Cataño ..................... Ceiba ..................... Ciales ..................... Cidra ...................... Coamo ..................... Canóvanas ................ Comerio .................... Corozal .....................

608 2,774 248 2,867 374 215 124 1,454 1,711 2,125 857 208 2,603

33 140 0 275 4 12 4 10 4 93 77 2 38

5.4 5 0 9.5 1.1 5.5 3.2 .6 .23 4.3 8.9 .96 1.4

149 127 41 236 20 57 4 17 2 489 75 17 40

Dorado .....................

865

59

6.8

83

Fajardo ....................

600

22

3.6

52

Guánica ................... Guayama ................... Guayanilla ................ Guaynabo ................. Gurabo .....................

895 2,505 499 1,792 964

58 40 11 118 22

6.4 1.5 2.2 6.5 2.2

91 70 40 96 112

Hatillo ..................... Hormigueros ................ Humacao ..................

1,851 76 578

8 3 48

.43 3.9 8.3

175 11 66

Isabela .....................

297

18

6

Jayuya ..................... Juana Díaz ................. Juncos .....................

297 312 1,253

18 12 47

6 3.8 3.7

0 129 74

Lajas ....................... Lares ....................... Las Marías ................. Luquillo ....................

1,925 69 0 112

194 0 0 11

10 0 0 9.8

340 9 0 35

240

149 0

0

Resultado de pruebas de brucelosis hechas durante el reconocimiento que empezó en abril de 1948 en Puerto Rico (Cont). Municipalidad

Ganado sometido a prueba

Manatf ..................... Maricao .................... Maunabo ................... Mayagiez .................. Moca ....................... Morovis ....................

2,586 0 729 508 45 2,048

34 0 7 27 0 9

1.3 0 .9 5.5 0 .43

119

Naguabo ................... Naranjito ..................

1,511 36

117 0

7.7 0

155 0

Orocovis ....................

3,528

19

Patillas ..................... Pefuelas ................... Ponce ......................

334 159 2,771

9 4 132

Quebradillas ................

381

1

Rincón ..................... Río Grande ................. Rio Piedras .................

49 108 4,935

0 2 311

0 1.8 6.3

11 25 679

Sábana Grande ............. Salinas ..................... San Germán ................ San Juan ................... San Lorenzo ................ San Sebastián .............. Santa Isabel ................

36 5,525 356 0 79 846 86

1 119 19 0 9 45 1

2.7 2.1 5.3 0 11.4 5.3 1.1

32 216 41 0 39 37 15

Toa Alta ................... Toa Baja ................... Trujillo Alto ...............

930 655 377

34 40 33

3.6 6.1 8.7

191 117 24

Utuado .....................

4,868

7

.14

17

Vega Alta .................. Vega Baja .................. Villalba ....................

1,420 518 1,107

5 2 1

.35 .38 .09

51 54 1

Yabucoa .................... Yauco ......................

1,605 116

89 0

4.2 0

69 12

........ .........

313

26

8.3

0

Culebra ....................

0

0

0

0

80,120

12,705

Vieques

Porciento de infección

ores

.53 2.6 2.5 4.7 .25

1

3.3

Terneras vacunadas

64 59 1 46

21 35 21 384 38

1 6,797

El total de terneras vacunadas es solamente 6,797, ya que solamente vacunamos terneras en hatos donde encontramos infección. No vacunamos terneras en hatos limpios. 241

242

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

Brucelosis" a toda comarca que arrojase menos del 1% de infección. Hasta la fecha se han probado 82,276 hembras en catorce zonas. A la luz de los resultados se han señalado once de ellas como zonas certificadas libres de brucelosis. Solamente tres no se han podido certificar como tales por haberse encontrado más del 1% de infección. Este aspecto del programa sigue desarrollándose en la actualidad. Cuando la infección en un hato es moderada, siempre se le recomienda al dueño que se acoja al Plan B, o sea el plan de prueba con sacrificio inmediato de casos positivos y vacunación de terneras. Hasta la fecha hemos tenido un resultado halagador en la eliminación de la infección en estos hatos. Se ha comprobado que aproximadamente el 72% de ellos se libra de la infección, entre la prueba inicial que elimina los casos positivos y la primera reprueba. Nuestro programa de brucelosis funciona bajo la Ley 181 de los estatutos de Puerto Rico, la cual hace obligatorio el que todo hato de ganado sea sometido a uno de los planes ya descritos. El Departamento de Agricultura y Comercio está encargado de la ejecución de dicha Ley. Además, el Departamento de Salud exige en su reglamento de producción y venta de leche que toda vaquería en Puerto Rico esté sometida a uno de los planes. Esta cláusula nos ha ayudado decididamente a obligar a que pruebe su ganado la pequeña, pero siempre presente minoría de ganaderos opuestos al programa. De esta manera si uno de ellos se niega a someter su ganado a prueba, inmediatamente el Departamento de Salud le revoca la licencia de vaquería y el propietario no puede vender la leche. Lo claro de esta reglamentación evita casos ante los tribunales, los cuales, toman mucho tiempo, ocasionan gastos y entorpecen un trabajo rápido que, naturalmente, se traduce en una multiplicación de la infección. Otra ley aprobada por la Legislatura de Puerto Rico autoriza al Departamento de Salud a reglamentar la importación, producción y venta del antígeno de brucela, evitando así que personas poco escrupulosas se dediquen a hacer pruebas particulares de ganado para brucelosis. La misma reglamenta el uso de cualquier vacuna contra la brucelosis bovina. Al recapitular estos párrafos sobre el trabajo de brucelosis en Puerto Rico nos parece que podemos atribuir el buen éxito de la campaña al hecho de haber tenido el cuidado de aunar en pro de ella tanto a las voluntades oficiales, si se me permite la expresión, como a las particulares. Las leyes, los reglamentos emanados de ellas, los métodos usados, las anotaciones hechas y las experiencias, buenas y malas, que hemos conseguido recopilar en nuestra Isla de Puerto Rico están a la disposición de todos ustedes cuya gentil atención agradezco en todo lo que vale. ERADICATION PROGRAM FOR BRUCELLOSIS IN PUERTO RICO (Summarwy) Bang's disease was unknown in Puerto Rico until 1923. According tb bacteriological studies the infection is strietly of bovine origin. In 1942 preliminary

ERRADICACION DE BRUCELOSIS EN PUERTO RICO

243

studies were begun on brucellosis in bovines. In April 1948, an obligatory campaign was organized under the auspices of the Department of Agriculture of Puerto Rico in cooperation with the U. S. Bureau of Animal Industry. In this campaign the recommendations of the U. S. Livestock Sanitary Association, as approved by the U. S. Bureau of Animal Industry were observed. This program operates under Law 181 of Puerto Rico, and the Department of Agriculture is in charge of its execution. In addition, the Department of Health has milk control laws requiring that each dairy operate under one of the plans. The program consists of four plans: Plan A: Application of serum agglutination tests, with immediate sacrifice for public consumption of all positive cases. A total indemnity of $40 is paid, $20 of which are paid by the government of Puerto Rico and $20 by the U. S. Government. Calves are not vaccinated with Strain 19. Plan B.: This plan is the same as Plan A except that 6 to 8-month old calves are vaccinated. This is applicable when the infection is light. Plan C: This consists of agglutination tests and the provisional retention of reactors in the herd. This is applicable in heavily infected herds. Indemnity is not paid, but calves 6 to 8-months old are vaccinated with Strain 19. Plan D: This consists of the vaccination of adults. It is recommended when the infection is virulent. To use this plan requires the permission of insular and federal agencies. The agglutination test used in our laboratory is the one approved by the U. S. Bureau of Animal Industry; standard BAI antigen is used. Strain 19 vaccine was first used in Puerto Rico in 1942 and it has been found to be both useful and advisable. In April 1948, a survey was begun and agglutination tests were made on both authorized and unauthorized dairies, resulting in a 3.37% infection rate. In September 1949, tests were started on an area or municipality basis, in order that those having less than 1% infection could be declared "brucellosis-free areas". To date, eleven such areas have been certified, and only three have not met certification requirements. This aspect of the program is still being carried on.

CONTROL OF BRUCELLOSIS IN THE UNITED STATES By Dr. B. T. SIMMS Chief, Bureau of Animal Industry, Agricultural Research Administration, United States Department of Agriculture, Washington, D. C. The very fact that we are meeting in this Third Inter-American Congress on Brucellosis is in itself encouraging. It shows that all of us here recognize a common enemy to human and animal health, and that we want to unite in the fight against this enemy. From the long experience in eradicating animal diseases in the United States we came to the conclusion some time ago that the degree of success is directly related to the degree of willingness of livestock producers to participate. There must be a majority opinion that the immediate sacrifice is but small tribute compared with the eventual gains that come with freedom from a particular disease. Successful programs for the control and eradication of transmissible animal diseases such as brucellosis do not, like the goddess of old, spring full-grown from someone's brow. Instead, they develop slowly, with frequent growing pains. Many years of careful planning and proper direction are necessary if such programs are to reach full development and final success. Long before brucellosis eradication was even mentioned, the Bureau of Animal Industry was developing methods and procedures which would make such a program possible. From its beginning this Bureau has held firmly to the principle that, insofar as is possible, transmissible diseases of animals should be eliminated rather than just controlled. Down through the years its research has been directed largely toward finding how to eliminate animal diseases. Its first big undertaking was the eradication of contagious pleuro-pneumonia. As new knowledge developed, such other diseases as sheep and cattle scabies, foot-and-mouth disease, tick fever, dourine, bovine tuberculosis, and fowl pest were either eradicated or brought under control. Brucellosis became of ever-increasing interest and concern as tick fever and tuberculosis came under control. One reason for this was the well established but often unrecognized fact that eradication of one disease tends to lead to the spread of others. Shortly after ticks were eliminated, brucellosis was introduced into many previously tick-infested areas by importation of Brucella-infected cattle. And explosive outbreaks of brucellosis occurred in many herds following the replacement of reactors to tuberculin with tuberculosis-free but brucellosis-infected cows and heifers. Another reason for increasing interest in brucellosis was the discovery that Brucella abortus may cause undulant fever in people. 244

CONTROL OF BRUCELLOSIS IN THE U. S.

245

Consequently brucellosis was recognized as important from both a public health and an economic standpoint. By the early 1920's a few research workers were eradicating brucellosis from individual herds of cattle. They were using the agglutination test for diagnosis, removing reactors from tested herds, cleaning and disinfecting premises, and retesting until no additional reactors were found. Many reports of results of such experiments appeared in both technical journals and agricultural publications. A few States began issuing certificates to owners of brucellosis-free herds, some livestock shows began requiring tests for brucellosis of cattle of breeding age before they could be exhibited, and livestock sanitary officials began discussing the advisability of a nation-wide program for its control and eradication. It was pointed out that brucellosis and tuberculosis are similar in many ways. Both are incurable infectious diseases, a biological test is used for diagnosis of both diseases, and human beings are susceptible to both. In each instance, raw milk from an infected herd is likely to contain living organisms. Many workers thought the tuberculosis-eradication method could be used with equal success against brucellosis. When the Federal Government set up a cattle-reduction program in 1934, it was logical to include elimination of Brucella-infected cattle as a part of it. A program of testing cattle for brucellosis with slaughter of the reactors and Government payment of indemnities to owners was well received. But we should bear in mind that this was not originally a disease-eradication program in itself. However, when the cattle-reduction program ended, such good progress had been made there was no thought of turning back. Now, after 15 years of experience a review of the project including both its pitfalls and its progress seems in order. What were the difficulties encountered? Some of the more important were as follows: 1. Shortage of experienced personnel for both field work and supervision. The Bureau had under way programs for the eradication of both fever ticks and bovine tuberculosis. Many clear-thinking livestock sanitary authorities, although recognizing the threat of brucellosis, believed tackling the problem at that time would spread the forces too thin. Furthermore, the best trained disease-eradication veterinarians among the Bureau forces had had little or no actual experience fighting brucellosis. 2. Rapidity of development of the program. The urgency of the cattle-reduction program made it necessary to develop full-scale field operations in only a few months. There was not time enough to set up a small smooth working organization and expand it gradually. 3. Lack of accurate knowledge of the disease by both veterinarians and cattle owners. Many people, including some veterinarians, believed the agglutination test was unreliable. Some thought a large percentage of infected animals recovered in a year or two following infection. Very few realized that brucellosis was the cause of serious economic losses.

246

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

4. The short incubation period of brucellosis and the rapidity of its spread under

ideal conditions. Many pregnant cows and heifers shed myriads of Brucella organisms within 30 to 45 days following exposure. Most of these animals remain negative to the agglutination test until a few days before they abort or calve and become shedders. In herds where the disease exists this makes retesting at short intervals a fundamental necessity. Our earlier experience with the slower spreading tuberculosis misled some of us in this respect in the beginning of our eradication program. In many instances the 60 to 100-day interval for tuberculosis retesting was simply transferred over to the brucellosis eradication program. As we have come to realize the explosive nature of brucellosis these field practices have been corrected. 5. The widespread incidence of the disease. Brucellosis was a problem in every

State and in almost every county. 6. Increasing movement of animals due to improved transportation methods.

The rapid development of hard-surfaced highways and the improvements in trucks were resulting in movements of livestock on an unprecedented scale. The rubber tire far exceeds the cloven hoof as a means of moving Brucella-infected cattle to previously uninfected farms and ranches. 7. Development of local sales markets. Many farmers use local auction markets

and sales yards as a means of buying breeding and feeder stock. These markets, which have developed considerably in recent years partly because of truck transportation, and which lack the inspection facilities of the larger terminal markets, are in many instances potent sources of Brucella infection. However, many of these local markets are now adopting the disease control methods used in the larger terminal markets. 8. The absence of well marked lesions in affected cattle. In tuberculosis-eradica-

tion work the owner who doubted the efficiency of the test when a fat, sleek animal reacted was convinced of its accuracy when he saw well developed lesions at autopsy. The doubting Thomas still doubted after failing to see lesions in reactors to the test for brucellosis at autopsy. On the other hand some factors, such as the following, favored an eradication program. 1. Many years of administrative experience in cooperative disease control and

eradication.The different States and the Bureau had worked out sound administrative procedures for cooperative disease control and eradication. The same type of administration was adopted for this program. In general it has proved satisfactory. 2. The ease and safety of raising replacements. Since calves born of infected

dams and fed infected milk until they are 5 to 6 months old do not usually become permanently infected unless they are exposed later, most owners found it possible to replace reactors rather easily. On many premises the owners had more than enough unbred heifers and unexposed bred heifers to replace all the infected cattle. 3. The fine support given the program by most public-health officers and workers.

This aided materially in convincing livestock owners that the eradication program should be supported. 4. Confidence of livestock owners and producers in cooperative disease control

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and eradication programs. The successful attacks on other diseases had built up a fine relation between livestock sanitary officials and livestock owners. Even though the dairymen, farmers, and ranchers knew little of the details most of them were willing to cooperate in this work. 5. Laws, rules, and regulations giving necessary authority. Most States already

had laws or regulations which were fairly adequate. Consequently the program was not held up pending legislative action. 6. The financial support from the Congress of the United States and from the different State legislative bodies. RESULTS

During the year ending June 30, 1936, two years after the beginning of the program, approximately 6,600,000 cattle were tested with about 7% reactors. Five years later, or during the year ending June 30, 1941, more than 7,300,000 cattle were tested with only 2.4% reactors. During this period nearly 450 counties were classified as modified brucellosisfree. Most officials and livestock producers believed this was satisfactory progress. STRAIN 19 APPROVED FOR FIELD USE

Strain 19 Brucella abortus vaccine was approved for official use during the fiscal year 1941. Before this was done, extensive laboratory and field experiments had proved (1) it was harmless insofar as setting up a transmissible disease was concerned, and (2) when properly used it produced a marked resistance but not a complete immunity. Strain 19 vaccine proved very popular, especially in those States and areas which had not made good progress in eradicating brucellosis. Field experience soon supported the claims which had been made concerning its efficacy. Good results were obtained in herds in which vaccinates were protected against severe exposure. But many who thought their vaccinates could withstand severe exposure were disappointed when they found this was not the case. THE IMPACT OF WORLD WAR II

When World War II came it was impossible to hold the gains which had been made in the brucellosis-eradication program. Loss of veterinary manpower to the armed forces curtailed disease control activities of both sanitary officials and livestock producers. The amount of testing decreased, high prices and demands for all-out food production caused owners to hold reactors which in ordinary times would have gone to slaughter, and unprecedented demands for dairy and breeding stock resulted in a marked increase in movement of female cattle from farm to farm. In 1946, the first post-war year, about 5% of the 4,900,000 cattle tested reacted.

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Since World War II, slow but steady progress has been made. Each year the number of animals tested has increased and the percentage of reactors found has decreased. In the year ending June 30, 1950, nearly 6,000,000 cattle were tested with about 3.5% reactors. During the past five years, approximately 70% of the reactors have been sold for immediate slaughter each year. Field workers estimate that more than half the remaining 30% are sold for slaughter within a year after they react. It is encouraging to note that since the end of World War II, New Hampshire and Maine have joined North Carolina as modified-accredited brucellosis-free States. During this 5-year period over 20 million doses of vaccine were produced under Bureau of Animal Industry inspection, approximately 5 million doses being produced during the last fiscal year. We have no means of knowing how many heifers were vaccinated during these years. However our best estimate is that between 5 and 10 million of our 40 million cows were vaccinated as heifers. Thus we are "fighting with two fists" as the former Chief of the Bureau, Dr. John R. Mohler, said when Strain 19 was released. With steady reduction in the number of reactors we are decreasing the probability of exposure. And with vaccination we are increasing resistance. THE PRESENT SITUATION

The program continues to gain momentum. Many additional counties are requesting area testing, and the sale of vaccine increases year by year. Shortage of personnel and funds prevents as rapid expansion as the requests and demands justify. Evidence is on every hand to show that cattle owners and producers, veterinarians, public health officials, and the general public are both better informed and more interested than ever before. The recent formation of the National Brucellosis Committee is significant. This was organized by the different segments of the industry with no urging or suggestions from State or Federal regulatory officials. Its sole purpose is to speed up control and eradication of brucellosis. More and more of our States are enacting laws or issuing regulations to prevent the sale, except for slaughter, of untested female cattle of breeding age. This is sound legislation since the infected female is the original source of nearly all infection. The Bureau and cooperating State Officials are putting more emphasis on area work but the fundamental democratic principles are not being disturbed. Each State has the right to accept or reject the offer of cooperation as it sees fit. All police and quarantine measures remain the function of the State rather than the Federal Government. Thus the Bureau is in no way forcing any State or any owner to adopt the program.

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EcONOMICS OF BRUCELLOSIS ERADICATION It is impossible to measure the dollars and cents value of this program but some statistics may not be out of place. Between January 1, 1935 and January 1, 1949, the total number of cattle in this country increased about 14.3%. During this same period, total annual milk production increased about 28% and total beef production increased a little more than 42%. We believe a part of these increases was the result of reduction in the incidence of brucellosis. Pertinent figures on milk production may be of interest. In 1946, Dr. I. F. Huddleson, of Michigan State College of Agriculture, after analyzing available data, reached the conclusion that Brucella-infected cows produce 23% less milk than do brucellosis-free cows. Using this as a basis, we arrive at the conclusion that total annual milk production in 1949 was about 900 million pounds above what it would have been if the incidence of brucellosis had been at the 1936 level of 7%. The market value of these additional 900 million pounds was not less than 25 million dollars. These data support our very strong belief that brucellosis eradication is paying big cash dividends. THE FUTURE

What about the future? Pessimists have always been poor prophets in the United States. We are accustomed to doing difficult jobs that many say can't be done-and we can and will do this one. With close cooperation between livestock sanitary officials and cattle owners, with the same fine support from public health officials and consumers, and with continued appropriations for brucellosis eradication, we can look forward with confidence to full success. RECAPITULATION 1. The different State livestock sanitary officials and the Bureau of Animal Industry have under way a cooperative program for the control and eradication of brucellosis. 2. The program is based on (1) testing for brucellosis and removing reactors from herds, thus preventing exposure of brucellosis-free cattle and (2) vaccination of heifers to increase their resistance. 3. Satisfactory progress is being made. The incidence among tested cattle was 7% in 1936, 5% in 1946, and 3.5% in 1950. 4. Reduction in the incidence of brucellosis is a factor in the great increase in beef and milk production between 1936 and 1949. CONTROL DE LA BRUCELOSIS EN LOS ESTADOS UNIDOS (Sumario) Los métodos y procedimientos laborados en los primeros programas de erradicación sirvieron de modelo para el primer programa nacional de control iniciado en 1934 como parte del programa de reducción del ganado. Se bosquejan los factores que intervienen en pro y en contra de la erradicación de la brucelosis.

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En el lado afirmativo figuraron factores tales como: (1) muchos años de experiencia administrativa en programas cooperativos de control y erradicación de enfermedades; (2) seguridad en el reemplazo de rebaño; (3) amplia cooperación de los funcionarios de salud pública; (4) confianza de los ganaderos en los programas de control y erradicación de enfermedades; (5) leyes y reglamentos que conceden la autoridad necesaria para obrar, y (6) apoyo económico del Gobierno Federal y Estatal. Entre las dificultades más importantes figuran: (1) escasez de personal avezado; (2) tiempo insuficiente para formular planes en las primeras etapas del programa, debido a la urgencia de la reducción del ganado; (3) falta de conocimientos exactos acerca de la brucelosis; (4) el breve periodo de incubación de la brucelosis y la rapidez con que se propaga en condiciones ideales; (5) la gran diseminación de la enfermedad; (6) el aumento en el traslado de animales debido a mejores métodos de transporte; (7) establecimiento de mercados locales sin medios de inspección, y (8) la ausencia ocasional de lesiones bien demarcadas en el ganado, lo que hace surgir dudas en cuanto a la eficiencia de la aglutinorreacción. Los adelantos logrados desde la iniciación del programa corresponden a tres fases bien definidas: el periodo de 1934 hasta el comienzo de la Segunda Guerra Mundial, los años de guerra, y los años de post-guerra. De los 6,600,000 cabezas de ganado probadas durante el año que terminó el 30 de junio de 1936, 7% resultaron positivas. Debido a la escasez de personal veterinario durante la guerra, unido a la producción máxima y a la retención del ganado para dicho fin, aumentó el movimiento del ganado. Como consecuencia de esto, aproximadamente 5% de las 4,900,000 cabezas de ganado comprobadas en 1946 resultaron positivas. Esta tendencia cambió el año siguiente. De 6,000,000 de cabezas de ganado comprobadas durante el año que terminó el 30 de junio de 1950, s61o 3.5% resultaron positivas. Los Estados de Carolina del Norte, New Hampshire y Maine han recibido el calificativo de zonas exentas de brucelosis. En 1941 se aprobó para empleo oficial la Cepa 19 de vacuna de Brucella abortus. Desde la terminación de la guerra, se han producido más de 20,000,000 de dosis de vacuna bajo la supervisión de la Oficina de la Industria Animal, y durante el último año fiscal se produjeron 5,000,000 de dosis. Aunque no se dispone de registros exactos, se calcula de los 40 millones de cabezas de ganado de los Estados Unidos, se han vacunado de 5 a 10 millones de terneros. El Dr. John R. Mohler, antiguo jefe de la Oficina de la Industria Animal, ha declarado: "Combatimos con ambos puños." A medida que disminuye el número de reactores, disminuyen las probabilidades de exposición, y la vacuna está aumentando la resistencia. En la actualidad no pueden atenderse las solicitudes de todos los condados que piden se hagan pruebas en sus zonas. El Comité Nacional de Brucelosis, que representa a todos los segmentos de la industria ganadera, ha vuelto a estimular interés en el control y erradicación de la brucelosis. Aumentan los estados que promulgan leyes y reglamentos para impedir la venta, salvo para el sacrificio, de las hembras no comprobadas en la edad de cría, dado que todas las medidas de inspección y cuarentena siguen siendo funciones de los gobiernos estatales más bien que del Gobierno Federal.

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Los datos disponibles apoyan la opinión muy generalizada de que el control y la erradicación de la brucelosis pagan dividendos a los ganaderos. De continuar la Intima cooperación entre los veterinarios de sanidad y los ganaderos, el amplio apoyo de los veterinarios de sanidad pública y de los consumidores, y la ayuda económica, podemos esperar con el tiempo la erradicación de esta costosa enfermedad.

TREND OF NATION-WIDE BRUCELLOSIS ERADICATION IN THE UNITED STATES By W. D. KNOX Editor, Hoard's Dairyman, Fort Atkinson, Wisconsin Brucellosis control and eradication in the livestock population of the United States is making satisfactory progress. The first nation-wide approach began July 1, 1934 when federal funds were set aside to pay indemnities for the removal of cattle reacting to the agglutination test. The purpose of the original program was twofold. Of primary consideration, in 1934, was the reduction of the so-called surplus of dairy products. The removal of brucellosis infected cattle was considered one approach to reducing the production of milk and cream in the nation. The second purpose of the program was to reduce the incidence of a very serious livestock disease. In view of the information available in the early '30s on the control and eradication of brucellosis, the program adopted, in 1934, was that of herd testing and removal of reactor cattle with payment of indemnities. According to a survey, made in February of 1935 by Hoard's Dairyman, testing during the first three months showed 14.4% reaction to the agglutination test in 981,530 cattle tested. There were 50,642 herds tested, of which 47% contained one or more reactors. In the first official

report of the Bureau of Animal Industry, United States Department of Agriculture, the summary for the fiscal year 1935 showed 11.5% reactors in 3,317,760 cattle tested. This program of brucellosis eradication continued until 1941. In that year 7,465,250 cattle were tested, with a 2.4% reaction. As severe economically as the original program may have been, it is obvious, from an impartial review of the record, that very creditable and desirable progress

was made in freeing the livestock industry of brucellosis. In 1941, however, we became involved in World War II. Veterinarians went into armed service, testing and vaccination were curtailed, and of even greater significance, in our opinion, was the tremendous increase

in the traffic of commercial cattle. This traffic was in response to increased demands for dairy products in our heavily industrialized areas, and in new war production centers. Under conditions existing during the war, it was impossible for state livestock health officials to properly regulate the traffic in cattle often reacting to the disease or exposed and

not yet reacting. Certainly the result was not difficult to foretell. Previously clean herds became infected in great numbers throughout the nation.

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had doubled, or had climbed to 5.0% on an annual test of 4,876,866 head tested. It was at this time that the United States' brucellosis control and eradication effort entered its critical stage. Everyone concerned with the future of a healthy, profitable livestock agriculture-a permanent agriculture-recognized that lost ground must be made up, and brucellosis eradication carried out to its eventual completion. Unfortunately, confusion had grown during the war years, and in 1946 predominated throughout the livestock industry. This was brought about and accentuated by widely divergent control programs among the several states, difficulties in shipping livestock interstate, and, with the advent of vaccination, by a growing belief among livestock owners that vaccination would replace sanitary practices and testing, and eliminate the necessity of working toward complete eradication of the disease. Veterinarians and other professional personnel, as well as laymen in the industry, wrote and spoke against the agglutination test in particular, and the national program in general. Most of the criticisms that were leveled at the agglutination test and at state and national programs originated with the leaders in the livestock industry, the breeders of registered, high-value dairy cattle. These were the men who suffered most heavily from the disease. They felt they could not afford to sacrifice valuable breeding animals in order to clean up their herds in a comparatively short period of time. The greatest factor contributing to the dissatisfaction and unrest, however, was the misinformation and misunderstanding that prevailed among livestock owners. It became obvious that the cooperation of the livestock owner in brucellosis control and eradication would depend not on legislation alone but on an effective, educational program that would result in his being armed with accurate, reliable information proving the desirability and the necessity of living without brucellosis. Furthermore, he must be provided with the reliable, up-to-date information on the tools available to him, and their proper use in the herd. When Dr. B. T. Simms became chief of the Bureau of Animal Industry, United States Department of Agriculture, he brought with him an understanding of the point of view of the livestock owner. One of his first acts was to call a meeting in Washington, D. C., September 1947. To that meeting were invited representatives of livestock organizations, general farm organizations, professional groups, and livestock disease control officials-in other words, a cross section of the entire livestock industry. He laid the problem of brucellosis control and eradication before these industry representatives and requested their counsel and cooperation. The primary request was that of uniform state legislation that would serve not only to facilitate trade within the industry but would give

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unanimity of thought and effort regardless of state borders. Such a recommended uniform plan of control was proposed and submitted to the United States Livestock Sanitary Association at its annual meeting in December of 1947. After considerable discussion and the adoption of certain amendments, the United States Livestock Sanitary Association unanimously approved recommendations for federal and state legislation to control and eradicate brucellosis. Four plans were outlined. These were: Plan A.-Test and slaughter, with or without calf vaccination. Plan B.-Test, calf vaccination, temporary retention of reactors until they can be disposed of for slaughter without excessive loss to the owner under provisions of the law. Plan C.-Calf vaccination without test of any part of the herd, this plan to be confined to those herds restricted in movement except under permit from proper livestock sanitary officials. Plan D.-Adult vaccination, when approval is received in writing from state and federal cooperating agencies. In the 1948 and 1949 annual meetings of the United States Livestock Sanitary Association certain minor amendments were adopted; however, the basic over-all plan remains. The month following the adoption of this control program in 1947, the Purebred Dairy Cattle Association unanimously approved the uniform plan of control. Since the first adoption, too, 42 of our 48 states have filed memorandums of agreement with the Bureau of Animal Industry indicating the states' agreement in principle and their endeavor to work toward the recommended state legislation. It must be appreciated that the United States Livestock Sanitary Association could only make recommendations to the respective states. Brucellosis control was, and is, primarily a state function. As a result, differences still exist today among the various states. However, they are gradually disappearing and unanimity of thought in legislation is growing steadily. We have dealt thus far with the legislative approach only. As we stated earlier, any successful program of control and eradication must be predicated by a sound, long-range, educational program. At the same December 1947 meeting, where the uniform plan of control was recommended, the Association adopted a strong statement on educational policy and it specifically recommended that the president of the United States Livestock Sanitary Association and the chief of the Bureau of Animal Industry appoint a joint committee of research workers and sanitary officials to review the literature on brucellosis and bring forth "a bill of proven facts." This assignment was carried out and the publication is available for distribution. Such was the thinking in late 1947. In June 1948, under the leadership of the Bureau of Animal Industry,

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a Midwest Brucellosis Conference was held in Chicago, which was well attended by livestock owners or their representatives, veterinarians, sanitary officials, and others. This conference unanimously recommended the formation of an independent organization to guide and vigorously promote brucellosis eradication. It was the intent of the conference that this organization be predominately controlled and guided by farmers or their representatives. The chief of the Bureau of Animal Industry was specifically requested to call the formation meeting so that the wheels might be set in motion. In January 1949, a Northeastern States Brucellosis Conference was held in New York City. This group unanimously endorsed the Midwest Brucellosis Conference recommendation. As a result of this expression, the chief of the Bureau of Animal Industry arranged for meeting facilities, and every conceivably interested group was invited to participate in organizing what is now known as the National Brucellosis Committee, March 15, 1949 in Washington, D. C. In our mind, there was no evidence of domination by any group at that meeting. Your speaker was given the title of temporary chairman and was instructed to arrange for the formation meeting and to invite the following organizations to participate: Purebred Dairy Cattle Association National Beef Breeds Association American Medical Association United States Public Health Service American Public Health Association American National Livestock Association United States Bureau of Animal Industry American Veterinary Medical Association United States Livestock Sanitary Association. The National Grange American Farm Bureau Federation National Farmers Union Texas and Southwestern Cattle Raisers Association American Meat Institute National Livestock and Meat Board National Association of Swine Records National Livestock Loss Prevention Board National Wool Growers Association Extension Service, or Association of Land-Grant Colleges and Universities All 19 organizations agreed to participate with the exception of the Extension Service, and its director recommended that the Association of Land-Grant Colleges and Universities act in its place. Since the March 15 meeting, the National Milk Producers Federation, the National Research Council, and the Dairy Industry Committee have been added to the Committee.

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The formation meeting of the National Brucellosis Committee was held in Chicago May 10, 1949 at which time Clinton K. Tomson, representing the National Beef Breeds Association, was made permanent chairman, and the speaker secretary. A preliminary program of work was submitted. It included education, research, promotion, and legislation. The last point was almost a stumbling block. Delegates were inclined to duplicate the long, laborious, and trying work of the Brucellosis Committee of the United States Livestock Sanitary Association. The discussion led to an effort to dissolve the committee. It did not prevail, though the meeting broke up after a comparatively short session. The officers were instructed to appoint subcommittees and arrange for the next meeting to be held in Columbus, Ohio, October 11, 1949. That meeting was held with excellent attendance. The National Brucellosis Committee unanimously approved the dissolution of the legislation subcommittee, making clear the division of responsibility it thought desirable between the Brucellosis Committee of the United States Livestock Sanitary Association and the National Brucellosis Committee. The aims, objectives, and work of the National Brucellosis Committee are now well-defined within the fields of education, information, promotion, and research. The subcommittees include education and information, research, procedures, and finance. The officers of the Committee, plus the chairmen of the subcommittees, make up the executive committee or board of directors of the National Brucellosis Committee. A tentative outline of their work follows: EDUCATION AND INFORMATION

1. Impress the livestock owner with the basic desirability and absolute necessity of living without brucellosis in the livestock industry. a. National dollar toll. b. Individual farm examples. c. Health of the farm population-demonstrated contrast in free and infected areas. 2. Disseminate latest reliable findings of research bearing on brucellosis; cite experimental procedures and results, averting vague generalities and minimizing opinions. 3. Relate success of areas eradicating brucellosis, proving it can be done. 4. Recognition and publicizing of individuals and organizations making noted contribution to brucellosis control through education, research, legislation and/or results achieved. 5. School essay and poster contests. Avenues of Approach.-1. National, state and local farmer meetings

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-effective, qualified speakers who have confidence of the farmers. Establish Speakers Bureau. Use visual aids, charts, slides, movies; educational exhibits at meetings and shows. 2. Seek cooperation of practicing veterinarians to assist in greater veterinarian-to-farmer education. Distribution of reliable literature. 3. Farm radio. 4. Farm press. 5. County newspapers. 6. County agents. 7. Vo-ag teachers. 8. 4-H agents. 9. Dairy plant fieldmen. 10. Livestock breed associations and fieldmen. 11. Marketing agencies and representatives. RESEARCH

1. Maintain a current record on all brucellosis research and demonstration projects. 2. Compile or sponsor the compilation of all past research work done on brucellosis; locate and investigate any unpublished work and keep record of it. 3. Encourage expansion of research along lines now considered neglected. Contact institution heads; aid in obtaining finances for project work. 4. Publish regularly a research bulletin, cataloging work in process, projects completed, and findings. A technical report. PROCEDURES

1. Develop suggested, detailed, promotional procedures which may be used at national, state, and local levels to carry out the brucellosis eradication program. Disseminate these procedures to extension workers and other responsible for administration of program of this type. 2. Stimulate the organization of specific state and county committees, or groups, devoted solely to brucellosis control. An extension of program of national committee. Let state and local committees work on details of local legislation and program, using guides offered by the United States Livestock Sanitary Association, and information from the National Brucellosis Committee. 3. Affiliate livestock and public health groups actively with aims and objectives of National Brucellosis Committee. FINANCE

This subcommittee is presently engaged in raising, from voluntary subscriptions, a fund of $40,000 per year with the objective in mind of

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assuring enough funds to guarantee a five-year program of work. A permanent executive secretary or general manager will be employed, along with such necessary help as may be determined essential. Additional funds recruited will be used for special research projects, publications, and promotions. Through this organization, in which we hope farmers will have confidence, our entire energies will be devoted to stimulating the greatest possible voluntary farmer participation in brucellosis control and eradication. Our intent is to build from the soil up rather than from Congress down. We fully appreciate a voluntary program alone is not enough. Although we will not be active legislative-wise, we recognize the need for legislation if eradication is to be realized. Legislation alone, however, will fall far short of doing the job without the cooperation of the livestock owner. If our work were to go no farther than it has to date, a significant contribution would still be recorded. The bitter unrest, criticism, and suspicions that prevailed five years ago are now largely dissipated. Understanding prevails and the livestock industry has rededicated itself to its original principle of controlling and eradicating a serious livestock disease rather than endeavoring to live with it. Within the past few months a new development has appeared on the scene to aid in eradication. The United States Public Health Service has a standard milk ordinance which it recommends to state and city health departments throughout the country. It is rather widely followed in most of our larger metropolitan markets. The United States Public Health Service recently recommended that a maximum period of five years be given all dairy herd owners to come under one of the four plans recommended by the United States Livestock Sanitary Association and the Bureau of Animal Industry. This does not assure brucellosis-free herds in that length of time. It merely provides that all herds supplying these particular markets will be under a plan of control. State and local health departments have indicated that they wish to move more rapidly. In one of our more heavily populated states, the state health department has adopted a milk ordinance which, in effect, requires all herds to be free of the disease by January 1, 1955. This, of course, has given added impetus to the control and eradication effort. Major handicaps standing in the path of progress include the seeming indifference of the beef industry to brucellosis control, and the comparatively ineffective control over the movement of infected or exposed livestock. The beef industry, in our opinion, will come along slowly, and with some reluctance, as soon as some of their unjustified fears are dissipated through our educational and informational program. The principal problem in controlling the movement of infected and exposed livestock may be solved through the licensing of livestock auctions, sales com-

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panies, auctioneers, and livestock truckers. They must be required to live up to state and federal legislation or much of our time, effort, and money spent in brucellosis eradication will be rendered ineffective. It is estimated by the Bureau of Animal Industry that the incidence of brucellosis infection has been reduced approximately 30% in the past four years. This means that our present incidence of infection approximates 3.5%. Please keep in mind that these figures, or incidence, are based solely on tested cattle. We do not have reliable information on the incidence of infection in the entire cattle population. The Bureau of Animal Industry's figures do indicate the trends, however, and we have no hesitation in reporting that optimism now generally prevails throughout the livestock industry. We look forward, with confidence, to a not far distant date when the entire nation may be, for all practical purposes, considered free of brucellosis, as we now consider ourselves largely free of bovine tuberculosis. TENDENCIA DEL PROGRAMA NACIONAL DE ERRADICACION DE LA BRUCELOSIS EN ESTADOS UNIDOS (Sumario) El primer ataque nacional para la erradicación de la brucelosis en los Estados Unidos comenzó el 1° de julio de 1934, fecha en que se asignaron fondos federales para pagar indemnizaciones por el sacrificio de animales con aglutinorreacciones positivas. Durante los primeros tres meses del programa 14.4% del ganado resultó positivo. El programa primitivo tenía un doble fin: primero, reducir el exceso de productos de leche, y segundo, erradicar una enfermedad grave del ganado. La continuación del programa original hizo bajar la frecuencia a 2.4% en 1941, tomando por base la comprobación de 7,465,250 cabezas de ganado. Durante la Segunda Guerra Mundial aumentó al doble la frecuencia de la enfermedad, o sea a 5% en 1946. Ese aumento se debió a escasez de veterinarios, disminución de las pruebas y de las vacunaciones, y aumento en el tráfico del ganado comercial. Se ha intensificado de nuevo la campaña contra la enfermedad, en gran parte como resultado de la adopción de un plan uniforme de control y de un programa amplio de educación e información. Se ha formado un Comité Nacional de Brucelosis, integrado por delegados de 22 organizaciones nacionales de agricultura, ganadería, salubridad pública, investigación y reglamentación. El Servicio de Sanidad Pública de los Estados Unidos impulsó recientemente el programa al recomendar que las ordenanzas para la leche contengan la estipulación de que dentro de cinco años todos los rebaños que envien leche a los mercados sujetos a inspección queden bajo un plan aprobado de control de la brucelosis. Los obstáculos principales comprenden la apatía relativa de la industria de la carne, y el control relativamente ineficaz del transporte del ganado infectado o expuesto. En los últimos cuatro años la frecuencia en todo el país ha bajado aproximadamente 30%. La industria confia que, dentro de un periodo razonable, todo el país pueda considerarse prácticamente libre de brucelosis, en la misma forma que ya nos consideramos en gran parte exentos de tuberculosis bovina.

CHEMOTHERAPY OF EXPERIMENTAL BRUCELLA ABORTUS INFECTION IN GUINEA PIGS AND MICE By A. POMALES-LEBRóN, PH.D., HOWARD E. HALL, M.D. AND CARLOS FERNÁNDEZ*

INTRODUCTION Since the advent of the sulfa drugs and the antibiotics, several reports concerned with the treatment of experimental Brucella infections in animals with these agents have appeared in the literature.'-' ° In the majority of these reports the effectiveness of treatment was measured exclusively by the results obtained with cultures from the spleen and lymph nodes, or the spleen alone. In some instances the result of therapy was also judged by the size of the spleen. The criterion of protection against a lethal inoculum have been utilized only occasionally in the evaluation of therapy. In our studies two criteria have been used to measure the efficacy of treatment: (a) The effect on the number of Brucellae cultured from the spleen and lymph nodes of treated animals as compared with untreated

* From the Department of Microbiology, School of Medicine and School of Tropical Medicine of the University of Puerto Rico, the Tropical Research Medical Laboratory, Fort Brooke, Puerto Rico and the Department of Infectious Diseases, University of Maryland School of Medicine. 1Heilman, F. R.: The effect of combined treatment with aureomycin and dihydrostreptomycin on Brucella infections in mice. Proc. Staff Meetings Mayo Clin, 24: 133, 1949. 2 Carle, B. N. and Larson, C. L.: Chemotherapy of experimental brucellosis in guinea pigs. Brucellosis. A publication of the Am. Assoc. Adv. Sc. 1515 Massachussetts Avenue, N.W., Washington 5, D. C. 1950. a Wilson, G. S. and Maier, I.: The sulfanilamide treatment of guinea pigs infected with Br. abortus. Brit. Med. Jour. 1: 8, 1939. 4 Chinn, B. D.: The use of sulfanilamide in experimental brucellosis. Jour. Infect. Dis. 64: 78, 1939. 6 Menefee, E. E. and Poston, M. A.: Effects of sulfanilamide on Brucella melitensis var. melitensis, abortus and suis. Jour. Bact. 37: 269, 1939. 6 Morales-Otero, P. and Pomales-Lebrón, A.: Effect of sulfanilamide and sulfamethylthiazol on experimental Brucella (var melitensis) infection in mice. Proc. Soc. Exp. Biol. & Med. 45: 512, 1940.

7Hanman, E. E. and Huddleson, I. F.: Effect of sulphapyridine (dagenan) on Brucella abortus in vitro and in vivo. Proc. Soc. Exp. Biol. &Med. 42: 555, 1939. 8 Kolmer, J. A. and Rule, A. M.: The therapeutic activity of sulfanilamide and allied compounds in experimental brucellosis of mice. J. Pharmacol. and Exp. Therap. 68: 406, 1940. 9 Live, I., Sperling, F. G. and Stubbs, E. L.: Effect of streptomycin on experimental brucellosis in guinea pigs. Am. Jour. Med. Sc. 211: 267, 1946. 1' Kelly, E. H. and Thomas, F. H.: Streptomycin in experimental brucellosis. Jour. Bact. 54: 80, 1947. 260

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controls (Experiments 3, 4, 5, 6 and 8) and (b) The protection conferred against an 80% lethal inoculum (Experiment 10). The main purpose of this work has been to compare the results of treatment with the different drugs tested. In order to fulfill this purpose, we have compared the effect of different drugs only when they were tested concomitantly under the same conditions. In Puerto Rico brucellosis is caused, as far as we know, exclusively by Br. abortus; consequently we have limited our experiments to the treatment of abortus infections. MATERIALS AND METHODS Brucella abortus strain No. 1489 (obtained from Dr. I. F. Huddleson), growing only under partial CO 2 tension, and Brucella abortus strains "Sablín" and Rex (obtained by us from the blood of patients in Puerto Rico), growing both aerobically and under C02, were utilized in this work. Saline suspensions were prepared from cultures on tryptose agar slants incubated at 37°C during two or three days. Guinea pigs were inoculated subcutaneously and mice intraperitoneally. Three tenths ml of suspension, prepared with strain No. 1489, matching BaSO 4 standard No. 611 was the inoculum used for protection experiments in mice. It was found by experiment that this dose consistently killed, in one to six days, 70 to 80% of mice weighing approximately twenty gm. It was also found by experiment that 0.1 ml of a suspension matching BaSO 4 standard No. 112 produced a non-lethal infection in mice and guinea pigs of such an intensity that abundant growth was obtained consistently from lymph nodes and spleen. Preliminary experiments showed that mice and guinea pigs thus infected could be used to compare the relative effectiveness of the different drugs tested, which was the main purpose of this study. The following drugs were tested: aureomycin, dihydrostreptomycin, streptomycin, chloramphenicol (chloromycetin), dimazol, terramycin, sulfadiazine, para-aminobenzoic acid (PABA), and para-aminosalicylic acid (PAS).'3

1 Slight adjustments of the suspension were made so as to obtain 64% light transmission with the Coleman Junior spectrophotometer (650 millimicrons, cuvette No. 6-304 C). Plate counts: approximately 4 billion viable organisms per ml. 12 Slight adjustments of the suspensions were made in order to obtain 81% light transmission with the Coleman Junior spectrophotometer (cuvette No. 6 304 C, 650 millimicrons). Plate counts: Approximately 200 million viable organisms per ml.

13(a) Aureomycin hydrochloride capsules-(given by mouth to guinea pigs). Aureomycin hydrochloride, with sodium glycinate (parenteral) Lot 7-9993: Given intravenously to mice. Lederle Laboratories, Pearl River, New York. (b) chloramphenicol, capsules: Given orally to guinea pigs and mice. Chloramphenicol (investigational X 4065) solution: Given intravenously to mice. Chloramphenicol

262

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

When aureomycin and chloromycetin suspensions were administered orally to guinea pigs the suspension was mixed thoroughly and the required amount taken into a one cc tuberculin syringe without a needle and deposited in the back of the mouth. The guinea pigs and mice used in this study varied from 350 to 500 gm and from 16 to 22 gm respectively. Quantitative spleen cultures were made using a surface plate count method.' 4 Qualitative cultures from spleen and lymph nodes were made by gently streaking once the surface of a tryptose agar plate with the cut surface of the tissue. Generally the different lymph nodes from the same animal were cultured separately, but in some instances the lymph nodes from the same animal were pooled and macerated and a loopful of the macerated tissue was streaked on the surface of the agar. EXPERIMENTAL Experiment 1.-The purpose of this preliminary experiment was to determine the pathogenesis, in the guinea pig, of the strictly anaerobic Br. abortus strain No. 1489 and the aerobic strain "Sablín." This was done in order to determine which would be the more suitable strain to use in the evaluation of therapy. Two groups of twelve guinea pigs each were inoculated subcutaneously with 0.1 ml of suspension matching BaSO 4 standard No. 1 of Br. abortus strain No. 1489 and Br. abortus strain Sablin respectively. The animals were killed at regular intervals, beginning two days after inoculation. Cultures were made each time from urine and bile and from different organs and tissues. Six animals died of intercurrent infections and were discarded. The results are shown in Tables 1 a and 1 b. Besides the lymph nodes, spleen and blood, the bone marrow and lungs were the tissues from which the organisms were cultivated more frequently. It must be noted that Brucellae were cultured from the urine of only two animals ("Sacrystals: Given in aqueous solution intramuscularly to mice. Parke, Davis and Co., Detroit. (c) Para-aminobenzoic acid (PABA) tablets. Ground and mixed with food. Given to guinea pigs and mice. (d) Dimazol (Nu. 445) tablets. Control No. N61T-/ 2. Ground and mixed with food. Hoffmann-La Roche Inc., Nutley, N. J. (e) Sulfadiazine, tablets. Ground and mixed with food. Abbot Laboratories, Chicago. (f) Para-aminosalicylic acid (PAS), sodium salt. Powder mixed with food. (g) Dihydrostreptomycin sulfate (serial No. 595-4977) and Streptomycin, in aqueous solution, given intramuscularly to mice and guinea pigs respectively. Abbott Laboratories, Chicago. (h) Terramycin, capsules. Powder mixed with food. Charles Pfeizer & Co., Brooklyn, New York. 14 The spleens were pooled, weighed and thoroughly ground in a mortar to a paste with the help of sterile sand. Sterile chilled distilled water was added, a small portion at a time, while mixing with the pestle until a volume of liquid corresponding to 5 ml per gram of tissue had been added. Ten fold dilutions ranging from 1:10 to 1:1,000,000, were made with sterile distilled water using a different 1 ml pipette for each dilution. Beginning with the highest dilution 0.02 cc amounts were deposited on the surface of a tryptose agar plate using a 0.2 cc pipette. Previous to inoculation the plate was incubated in the inverted position over the partially removed cover so as to make the agar surface more avid for the liquid and prevent excessive spreading. Six dilutions were tested on a single plate. Plate counts were made after five days to one week incubation under 10%o C02.

CHEMOTHERAPY OF BR. ABORTUS IN GUINEA PIGS AND MICE

263

TABLE 1 a.-Distributionof Brucellae in the body of untreated guinea pigs at different

stages after inoculation with Br. abortus (Anaerobic strain No. 1489) Growth on Streak Cultures Serial No.

Days after inoculation

Blood culture

E .

2 7 13 25 28 43 51 73

16 17 15 27 23 22 26 25

Cont. + + Cont. + +

.91

co

6+

P

3+ 3+ 3+ 4+ 6+ 4+ 3+ 2+ 2+ 6+ 2+ 4+ 6+ 3+ 4+ 3+ 1+ 3+ 3+ 4+ 3+ 6+ 3+ 1+ 5+ 1+ 1+ 6+ 1+

5+ 2+ 5+ 4+ 1+ 4+ 4+ 3+ 5+ 4+ 3+ 3+ 3+ 6+ 5+ 5+ 1+ 5Ai 3+

X

X

·

-

X = not done; - = no growth; + =positive;Cont.= contaminated;l+ = one to five colonies on streak; 2+ = six to fifteen colonies; 3+ = sixteen to thirty colonies; 4+ = thirty-one to seventy-five colonies; 5+ = more than seventyfive colonies but growth not confluent; 6-+ = confluent growth. Note: This key will apply to all cases where these symbols appear. TABLE 1 b.-Distributionof Brucellaein the body of untreated guinea pigs at different

Br. abortus (Aerobic strain "Sablin") stages after inoculationwith O Growth on Streak Cultures ·

Serial No.

Days after inoculation

1

Blood

culture

b

m

.4

11

28 19 5 6 13 12 24 11 8 9

2 5 8 15 18 27 28 47 52 75

+

1i

_)

3+ 3+ 5+ 6+ 2+ 2+ 2+ 4+ 6+ 5+ 3+ 3+ 3+ 1+ 1+ 1+

i

.0

O

<

3+

-

6+

-

4+ 2+ 2+ 4+

-

22 5+ 1+ 2+ 6+ 1+ 5+ 2+ X 5+ 1+ 3+ x 1+ 1+ 5+ 1+ X 1+ 5+ 6+ 3+ 2+ 1+ X 3+

bl/n") and the bile of only one ("Sablín"). Positive cultures were obtained from the liver of only eight ("Sablin," two and No. 1489, six) of the eighteen animals studied. Strain No. 1489 was slightly more infective than "Sablín." Strain No. 1489 was therefore selected as the more suitable to be used in this work, and it was 5 utilized in all subsequent experiments unless otherwise stated.' 16 In an experiment carried'out later, the details of which are not recorded in this paper, strain No. 1489 was found to be slightly more virulent for mice than strains "Sablín" or "Rex."

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

264

In all experiments, except No. 10, one tenth ml of a saline suspension matching barium sulfate standard No. 1 was used as the infecting dose for both guinea pigs (subcutaneously) and mice (intraperitoneally). Cultures were made always from both spleen and lymph nodes. Experiment No. 2.-The purpose of this experiment was to determine how long after inoculation with strain No. 1489 guinea pigs remained infected. Twenty-four guinea pigs were infected, the animals being killed in groups of three at monthly intervals thereafter, and cultures were made from each animal. The results are summarized in Table 2. It is apparent that individual animals vary in the rapidity with which they can rid themselves of the infection. The organisms gradually disappeared spontane2.-Duration of Br. abortus infection in untreated guinea pigs as shown by cultures made from spleen and lymph nodes, at monthly intervals, after inoculation with Strain No. 1489

TABLE

Streak Cultures Months after inoculation

From the 1st to the 4th

Number of animals Spleen

Lymph nodes

Three killed each month

+

+

2

+

+

1

_

_

1

+

-

Five Six

2

Seven and eight

Three killed each month

-

+ = Positive; - = No growth. ously until seven months after inoculation Brucellae could no longer be recovered either from the spleen or lymph nodes. Experiment No. 3.-This experiment was intended to test the effect of various drugs as follows: 1. Drugs given alone (Table 3 a). 2. Drugs given in combination concomitantly (Table 3 b). 3. Sulfadiazine and streptomycin given in combination, but alternately as stated in Table 3 c. Seventy-two guinea pigs were inoculated and divided into groups of four animals each. Treatment was started one week after Brucella inoculation and lasted two weeks. Cultures were made two days after cessation of treatment. Streptomycin, aureomycin and chloramphenicol were given four times a day (7:00 AM, 10:00 AM, 1:00 PM and 4:00 PM) in total daily doses of 16 mg (subcutaneously), 16 mg (orally in aqueous suspension) and 12 mg (orally in aqueous suspension) respectively. Sulfadiazine, dimazol and para-aminobenzoic acid were given in the diet in a concentration of one per cent. Results are shown in Tables 3 a, b and c. Cultures from animals treated with para-aminobenzoic acid or chloramphenicol yielded growths as abundant as those obtained from untreated controls.

CHEMOTHERAPY OF BR. ABORTUS IN GUINEA PIGS AND MICE

265

Aureomycin, streptomycin, sulfadiazine and dimazol all caused a moderate or slight decrease of the number of organisms cultured from the spleen and lymph nodes. It is interesting to note that in group 4 (table 3 c) the animals treated with sulfadiazine alone during one week, and then with sulfadiazine plus streptomycin, during another week, gave negative cultures from the spleen while good growth was obtained from the lymph nodes of the same animals. In general, the combined treatment with streptomycin plus sulfadiazine or dimazol was more effective than when each drug was given alone. Aureomycin plus streptomycin was the only combination causing a significant decrease in the number of Brucellae in the spleen and lymph nodes. This is in accordance with TABLE 3 a.-Results of treatment of Br. abortus infection in guinea pigs as shown by streak plate cultures from spleen and lymph nodes Cultures from Group No.

Treatment

1 2

Untreated controls Streptomycin

5

Sulfadiazine

10

Dimazol

19 19a 19b

Chloramphenicol (chloromycetin) Para-aminobenzoic acid Aureomycin

animalsber of

4 2 1 1 1 1 1 1 2 1 1 4 3 4

Spleen

nodes

6+ 3+ 5+ 2+ 1+ 3+ 3+ 4+ 2+ 5+ 4+ 6+ 6+ 4+

6+ 3+ 5+ 5+ 6+ 4+ 3+ 4+ 6+ 5+ 6+ 6+ 6+ 3+

Note: Aureomycin was toxic for the guinea pig. Animals were treated with this drug 6 days only. The amount of growth from the individual animals was not recorded. The average growth is given in this Table. the observation made by Heilman' in mice, and by Carle and Larson in guinea pigs.2 The inguinal, retroperitoneal (lumbar) and axillary lymph nodes were cultured from each animal. Cultures from one node could give abundant growth when very few colonies or no growth was obtained from other lymph nodes of the same animal. Experiment No. 4.-This experiment had two purposes: 1. To corroborate the results obtained with aureomycin and with chloramphenicol in experiment No. 3 (Table 3 a) and 2. To test the effect of these drugs on animals infected with two other abortus strains (Rex and Sablín). Sixty guinea pigs were divided into three groups of twenty each. Groups 1, 2 and 3 were infected with strains No. 1489, "Sablín" and "Rex" respectively. Eight anim.nls in each group were treated with chloramphenicol and eight with

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

266

aureomycin. The other four were left as untreated controls. Treatment was started fifteen to thirty minutes after inoculation. Chloramphenicol was given orally sixty milligrams daily in three doses of 20 mg each at 8:00 AM, 12:00 noon and 4:00 PM. Treatment lasted two weeks. Cultures made one day after cessation of treatment yielded abundant growth TABLE 3 b.-Effect of conibined treatment of Br. abortus infection in guinea pigs as measured by cultures from spleen and lymph nodes. Drugs given concomitantly Cultures from Group No.

Treatment

6

Sulfadiazine and streptomycin

9

Dimazol and streptomycin

13

Aureomycin and sulfadiazine

16

Aureomycin and streptomycin

No. of animals

2+ 3+ 3+ 3+

-

4+ Cont. 5+

Cont. Cont. 5+

1 (7)

-

Cont.

-

1

1+

1+

1+

20

Chloramphenicol and sulfadiazine

21

Chloramphenicol and aureomycin

1 1 (5) 1 (6)

1 1

Chloramphenicol and streptomycin

Lmnodesh

1+ 3+ 2+ 3+ 3+

2 1 1 1 1 1 1 2 (7, 10) 2 (9, 11)

1 1 1 1 1

22

Spleen

1 (14) 1

1 1

1+ 3+ 3+ Cont. 2+ Cont. 1+ 5+ Cont. 4+ 4+ 4+

3+ 4+ 3+ Cont. 2+ 4+ 3+ Cont. Cont. 3+ 2+ Cont.

Note: Numbers in parentheses indicate time of death after treatment was started. Most of these animals died due to the toxicity of aureomycin for guinea pigs. See Table 3a (group 1) for untreated controls. from animals infected with each of the three strains (No. 1489, Sablín and Rex). The abundance of growth was comparable with that obtained from untreated controls. Aureomycin was given orally, 20 mg daily, divided into three equal doses (8:00 AM, 12:00 noon and 4:00 PM). Animals began to show toxic symptoms five days after treatment was started. Treatment lasted one week. Cultures from the spleen and lymph nodes yielded good growths although a moderate reduction in the number of colonies was apparent when growths were compared with those obtaining in untreated controls.

CHEMOTHERAPY OF BR. ABORTUS IN GUINEA PIGS AND MICE 267 Experiment No. 5.-This experiment was intended to test the effect of chloramphenicol when given intramuscularly. Twelve guinea pigs were infected. Six animals were treated (starting immediately after inoculation) during ten days by the daily intramuscular injection of 20 mg of chloramphenicol in two doses of 10 mg each (7:00 AM, 7:00 PM). The other six animals were kept as controls. Cultures made from lymph nodes and spleen one day after cessation of treatment yielded abundant growths comparable with cultures from untreated controls. Brucellae were recovered from the blood of all the treated and untreated animals before they were killed the day after completion of treatment. TABLE 3 c.-Effect of combined treatment, with sulfa drugs and streptomycin, in Br.

abortus infection in guinea pigs, as measured by culture from spleen and lymph nodes. Drugs were given alternately as stated Cultures from Group No.

Treatment

3

Streptomycin 2nd and 3rd week. Sulfadiazine 3rd week.

4

Sulfadiazine 2nd and 3rd week. Streptomycin 3rd week. Streptomycin 2nd and 3rd week. Dimazol 3rd week.

7

8

Dimazol 2nd and 3rd week. Streptomycyn 3rd week.

No. oi Spleen

Lymph nodes

1 1 1 1 4

1+ -

3+ 4+ 4+ 4+

1 1 1 1 1 1 1 1

3+ 4+ 2+ 2+ 3+ 1+ 6+ -

3+ 4+ 2+ 1+ 4+ 3+ 5+ 3+

Note: See Table 3 a (group 1) for untreated controls. In view of the unusual toxicity of aureomycin for the guinea pig and of the therapeutic failure of chloramphenicol in this animal, as evidenced by the abundant growth of Brucellae obtained from the tissues (spleen and lymph nodes) of guinea pigs treated with this drug, we started using mice. Experiments No. 6 a, b, c.-The purpose of these experiments was to test the effect of treatment with chloramphenicol given orally (6 a) intramuscularly (6 b) or intravenously (6 c) to infected mice. The effectiveness of therapy was measured exclusively by the results of qualitative cultures of spleen and lymph nodes. Short summaries of each of these experiments follow: 6 a.-Eighteen infected mice were divided into three groups of six animals each. Groups one and two were put on a diet of powdered feed containing 0.5% and 0.25% of chloramphenicol respectively. Six animals were kept as untreated

268

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

controls. Treatment was started immediately after Brucella inoculation and lasted two weeks. Animals were killed and cultures were made two days after cessation of treatment. Cultures from treated animals yielded abundant growths comparable with those obtained from untreated controls. It was observed that the spleens of the animals treated with 0.5% chloramphenicol in the diet were prominently smaller than those of the controls. This experiment was repeated using twenty infected mice, ten of which were put on a diet containing 1% chloramphenicol. Again abundant growths comparable to those from the ten untreated controls were obtained from the spleens of the treated animals. The spleens from the treated mice were also found to be smaller than those of the controls. This same experiment, using 1% chloramphenicol in the diet, was repeated with eighteen mice in which treatment was started one week after Brucella inoculation and lasted ten days. The same negative results were obtained. It must be noted that in the foregoing experiments with mice the small size of the spleen was not a valid criterion of the effectiveness of treatment. These small spleens always yielded abundant growth of Brucellae. 6 b.-Twenty-four infected mice were divided into two groups of twelve animals each. One group was treated by the daily intramuscular injection of 1 mg of chloramphenicol dissolved in distilled water (two injections of 0.5 mg each given at 7:00 AM and 7:00 PM). Treatment started immediately after Brucella inoculation and lasted two weeks. Twelve animals were kept as untreated controls. Cultures made from spleens and lymph nodes two days after cessation of treatment gave growths comparable to those obtained from the twelve untreated controls. The same experiment was repeated with 18 mice, 12 treated and 6 untreated controls with the exception'that treatment began one week after Brucella inoculation, and again abundant growth from spleen and lymph nodes was obtained from the treated and untreated animals alike. 6 c.-Six mice were treated with 1 mg daily of chloramphenicol given intravenously (two injections of 0.5 mg each given at 8:00 AM and 4:00 PM) treatment being started immediately after Brucella inoculation and lasting one week. Negative results were obtained comparable to those already recorded when the drug was given orally or intramuscularly. Experiment No. 7.-This short experiment was intended to show the effect of chloramphenicol given orally on the size of the spleen of normal mice. Six normal mice were put on a diet of powdered Rockland rat feed plus 1% chloramphenicol. Another group of six normal mice was carried as untreated controls. Animals in both groups were killed after one week treatment. The average weight of spleen per gram of mouse in the treated group was 6 mg as compared with 9.6 mg for the untreated controls. Experiment No. 8.-The main purpose of this experiment was to evaluate therapy as measured by quantitative counts of Brucellae present in the spleen as well as qualitative streak cultures from spleen and lymph nodes. All the

CHEMOTHERAPY OF BR. ABORTUS IN GIUNEA PIGS AND MICE 269 TABLE 4 a.-Comparative effect of therapy, in experimental Br. abortus infection in mice, with chloromycetin, aureomycin and dihydrostreptomycin, when the drugs were given alone No. of mice

5 4

4 5 6

Average weight of spleen in mg.

Treatment

Chloromycetin. One per cent in food Chloromycetin intravenously. One mg. daily in two injections of 0.5 mg each given at 8:00 AM and 4:00 PM Aureomycin Dihydrostreptomycin Untreated controls

No. of organisms per gm. of spleen

No. of organisms per average whole spleen

100

30,000,0001 3,000,000

250

20,000,0001 5,000,000

250 260 333

14,000,000 3,500,000 7,500,000 1,950,000 20,000,000 6,660,000

Average streak plate growth Spleen

Lymph no 4es

6+

4+ 5+

6+ 5+ 6+

2+ 3+ 5+

Note: Aureomycin, with sodium glycinate (intravenously) and dihydrostreptomycin (intramuscular). One mg daily, divided in two injections of 0.5 mg each, given at 8:00 AM and 4:00 PM. Treatment was started immediately after Brucella inoculation and lasted five days. Cultures were made two days after cessation of treatment. TABLE 4 b.-Results of therapy in Br. abortus infection in mice. Comparative effect of aureomycin and terramycin, when given alone and in combination with dihydrostreptomycin as measured by the growth obtained in cultures from the spleen and lymph nodes No. of mice

10 10 6 6 6 6

Treatment

Aureomycin plus dihydrostreptomycin Terramycin plus dihydrostreptomycin Aureomycin Teramycin Dihydrostreptomycin Untreated controls

Average Mg. of spleen weight per gm. of spleen in mg. mouse

No. of organisms per gra. of spleen

No. of organisms per average whole spleen

Average streak plate growth Spleen

Lymph no2+ des

260

12

50,000

13,000

2+

1+

220

10

175,000

38,000

2+

1+

350 380 333 660

16 18 15 30

752,000 2,150,000 3,000,000 1,140,000 X X 16,000,000 10,560,000

5+ 6+ 4+ 6+

4+ 2+ 1+ 5+

Note: Treatment started immediately after Brucella inoculation and was interrupted after five days; it was resumed 48 hrs. after being stopped and was continued for two days. Cultures were made 48 hrs. after cessation of treatment. information concerning this experiment is given in Tables 4 a, b and c, which are self explanatory. The results shown in Tables 4 a, b and c substantiate fully those previously obtained in mice and guinea pigs as measured by qualitative streak cultures. Terramycin, dihydrostreptomycin, aureomycin and choramphenicol when given

270

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

alone failed to cause a marked decrease in the number of organisms in the tissues (spleen and lymph nodes) of infected mice. Combined treatment with aureomycin plus dihydrostreptomycin or terramycin plus dihydrostreptomycin was effective in decreasing significantly the number of colonies in cultures from the spleen of treated animals as compared with cultures from the spleen of untreated controls. In this respect treatment with chloramphenicol alone was the least effective and with aureomycin plus dihydrostreptomycin the most effective. TABLE 4 c.-Results of treatment of experimental Br. abortus infection in mice. Comparative effect of (1) aureomycin, chloromycetin and dihydrostreptomycin when given alone and (2) the combined therapy with (a) terramycin plus dihydrostreptomycin.and (b) aureomycin plus dihydrostreptomycin Average Mg. of No. of oweiget c

4 4 5 10 10 6

Treatment

No. of spleen No. of No. po per organisms per average whole gr. o. of of spleenaveagewhole spleen S mouse

Average streak plate growth gr th

Treatment ~~~mice ~spleen

of in mg.

Aureomycin Chloromycetin Dihydrostreptomycin Terramycin plus dihydrostreptomycin

275 500 300

16 25 15.7

1,075,000 3,500,000 1,875,000

296,000 1,750,000 562,000

5+ 5+ 6+

5+ 5+ 3+

410

21

-

420

19

Cultures contaminated 4,000

2+

Aureomycin plus dihydrostreptomycin Untreated controls

Cultures contaminated 10,000

2+

-

400

Not done

5,825,000

2,319,000

6+

5+

Note: Treatment was started five days after Brucella inoculation and lasted five days. Cultures were made two days after cessation of treatment. Aureomycin, with sodium glycinate (intravenous), chloromycetin (intravenous) and dihydrostreptomycin (intramuscular): 1 mg daily, divided in two injections of 0.5 mg each, given at 8:00 AM and 4:00 PM. Terramycin: 0.5% in food. Treatment with para-aminosalicylic acid 16 during five days to one week (1% powdered feed) was ineffective in decreasing significantly the number of organisms cultured from the spleen and lymph nodes of infected mice. Experiment No. 9.-This experiment was concerned with the determination of chloramphenicol levels in the blood of mice and guinea pigs. Six guinea pigs were bled from the heart two hours after oral administration of 20 mg of chloramphenicol. Another group of six animals were bled two hours after the oral administration of 60 mg of the drug. Negative chloramphenicol determinations were obtained in both instances when pooled samples of serum were tested. 16

Not included in Tables.

CHEMOTHERAPY OF BR. ABORTUS IN GUINEA PIGS AND MICE

271

Three guinea pigs were injected intracardially with chloramphenicol (investigational X 4065) in a proportion of 50 mg/kilo. Levels were determined in pooled serum specimens, urine and peritoneal fluid with the following results: One and two hours after injection serum levels of 66.0 gamma per cc and 5.4 gamma per ce were obtained respectively. Four hours after injection no chloramphenicol could be detected in the blood serum or peritoneal fluid but the urine contained 80 gamma per cc. Eighteen normal mice, inoculated intramuscularly with 1 mg of crystalline chloramphenicol dissolved in 0.5 cc of distilled water, were divided into three groups of six animals each. Pooled blood samples one, two and four hours after the injection of the drug were obtained from groups one, two and three respectively. No chloramphenicol levels could be demonstrated in any of the three blood samples. This experiment was repeated three times with consistently negative results. Experiment No. 10.-The purpose of this experiment was to test the effect of therapy as measured by the ability of different drugs, or combinations of drugs, to protect mice from a lethal inoculum of Brucellae. It was found by experiment that when mice, weighing approximately 20 mgs, were inoculated intraperitoneally with 0.3 ml of a saline suspension (matching BaSO4 standard No. 6) prepared from a 48 hr. growth on tryptose agar, 70 to 80% of the animals died consistently one to six days after inoculation. Using this lethal dose, a protection experiment with small groups of animals was carried out with chloramphenicol, dihydrostreptomycin, terramycin, aureomycin, sulfadiazine, dimazol, aureomycin plus dihydrostreptomycin and terramycin plus dihydrostreptomycin. Treatment was started immediately after Brucella inoculation and lasted one week. Cultures were made also at the time of death or when the survivors were killed one day after cessation of treatment. A summary of the results is given in Table 5. Terramycin, aureomycin, chloramphenicol, dihydrostreptomycin, dimazol and sulfadiazine gave 94%, 91%, 92%, 100%, 44% and 66% protection, respectively, against an inoculum which killed 79% of untreated controls. Good growths were obtained from the tissues of these animals 24 hrs. after the cessation of treatment with these drugs. The combined aureomycin-dihydrostreptomycin as well as terramycin-dihydrostreptomycin treatment gave 100% protection and markedly reduced the number of Brucellae isolated from the tissues. The in vitro susceptibility (as shown by the absence of growth in five cc of tryptose broth after 24 hrs. at 37°C) of Br. abortus strain No. 1489 used in these studies was as follows: (a) Aureomycin with sodium glycinate (Lot No. 7-9993) -0.25 gamma per ml. (b) Dihydrostreptomycin-1 gamma per ml. (c) Chloramphenicol-3 gamma per ml. Chloramphenicol and aureomycin inhibit'ed growth but did not kill the organism in concentration as high as 10 gamma per ml. (Highest concentration tested.) Dihydrostreptomycin killed the Brucellae in concentration of 1.5 gamma per ml. SUMMARY AND CONCLUSIONS

1. In guinea pigs infected with Br. abortus (strains No. 1489 or "Sablin") the blood, spleen and lymph nodes were the only tissues from which

272

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

the organisms were consistently recovered, when cultures were made from five to seventy-five days after inoculation. 2. In guinea pigs infected with Br. abortus strain No. 1489, the Brucellae gradually disappeared spontaneously until the organisms could TABLE

5.-Results of protection experiment in mice against and 80% lethal dose of Br. abortus M,1489

No. of animals

Treatment

No. of survivors after one week

Average growth from all animals in each group Spleen

57 19 11 18 18 10

10 10 25 12

None.

Terramycin, 0.5% in food. Aureomycin (with sodium glycinate). Intravenously, 1 mg daily in two doses of 0.5 mg each at 8:00 AM and 4:00 PM. Dimazol. One and one half per cent in food. Sulfadiazine. One and one half per cent in food. Aureomycin plus dihydrostreptomycin Aureomycin intravenously, 1 mg daily in two injections of 0.5 mg each at 8:00 AM and 4:00 PM respectively. Streptomycin, intrasmucular, 1 mg daily divided in two injections of 0.5 mg each. Terramycin plus dihydrostreptomycin. Terramycin 0.5 in food. Streptomycin intramuscularly 1 mg. daily. Dihydrostreptomycin, intramuscular, 1 mg daily. Chloramphenicol, 0.5% in food. Chloramphenicol, 1 mg daily intramuscularly.

Ly;dph

12 (21%) 18 (94%) 10 (91%)

6+ 6+ 6+

5+ 3+ 4+

12 (66%)

6+

5+

8 (44%)

6+

5+

10 (100%)

2+

10 (100%)

2+

10 (100%)

4+

2+

23 (92%) 12 (100%)

5+ 5+

5+ 5+

Note: Animals inoculated intraperitoneally with 0.3 cc of a 48 hr. growth of Br. abortus suspended in saline to match BaSO 4 standard #6. Treatment started from 15 to 30 minutes after Brucella inoculation and lasted one week. Cultures made at the time of death or when the survivors were killed twenty-four hours after cessation of treatment.

no longer be recovered from the tissues (spleen and lymph nodes) seven or eight months after experimental inoculation. 3. Sulfadiazine and dimazol given in 1% concentration in the diet and 16 mg of streptomycin given daily subcutaneously to guinea pigs resulted in a slight decrease in the number of organisms cultured from the spleen and lymph nodes as compared with normal controls. The combined treatment with streptomycin plus either sulfadiazine or dimazol was more effective than with any of these three drugs alone. It was interesting

CHEMOTHERAPY OF BR. ABORTUS IN GUINEA PIGS AND MICE 273 to note that the decrease in the number of colonies obtained from the spleen of animals treated with sulfadiazine plus dihydrostreptomycin was striking compared with the more abundant growths obtained from the lymph nodes of the same animals. This happened in groups three and four in Table 3c in which animals were treated with either drug during the first week, and with both drugs concomitantly during the second week. The cultivation of both spleen and lymph nodes is desirable in studies of this nature. 4. Chloramphenicol failed in the treatment of experimental Br. abortus infection in guinea pigs and mice, when the ability of the drug to decrease significantly the number of Brucellae in the tissues of the infected animals was taken as the sole criterion of the efficacy of therapy. 5. Chloramphenicol was not detectable in the blood serum of mice and guinea pigs two, four and six hours after the intramuscular injection of 1 mg of the drug and the oral administration of 60 mg respectively. When the drug was introduced, in a proportion of 50 mg/kilo, into the blood of guinea pigs by intracardial injection, it was detected in the blood serum in concentrations of 66.0 gamma per cc. and 5.4 gamma per cc. one and two hours after injection, respectively, but level determinations were negative after four hours. A concentration of 80 gamma per cc. was obtained in the urine but no drug was detectable in peritoneal fluid 4 hours after injection. 6. The spleen of mice treated with the oral administration of chloramphenicol were smaller than normal but yielded large numbers of Brucellae in cultures. This decrease in the size of the spleen was not observed in the animals treated parenterally with chloramphenicol. This shows that the size of the spleen is not always a valid criterion in the evaluation of the effectiveness of treatment. 7. Aureomycin (given intravenously 1 mg daily), terramycin (0.5% in the diet) and dihydrostreptomycin (intramuscularly 1 mg daily) failed to decrease significantly the number of Brucellae in the spleen and lymph nodes of infected mice when given alone. Combined treatment with (2) aureomycin plus dihydrostreptomycin and (b) Terramycin plus dihydrostreptomycin were significantly effective in this respect. The aureomycin-dihydrostreptomycin combination was most effective. 8. Aureomycin, dihydrostreptomycin, terramycin and choramphenicol all gave good protection against a dose of organisms that consistently killed 70% to 80% of untreated mice. Sulfadiazine and dimazol were less effective in this respect. 9. When protection experiments in mice are used to measure the effectiveness of therapy, the results obtained in experimental animals are more in harmony with results obtained in the treatment of brucellosis in man, with the sulfa drugs and the antibiotics.

274

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS ACKNOWLEDGEMENT

The aureomycin, chloramphenicol, dimazol and terramycin used in these studies were kindly supplied by Lederle Laboratories, Parke, Davis & Co., Hoffman-La Roche Inc. and Charles Pfeiser & Co., respectively. QUIMIOTERAPIA DE LA INFECCIÓN EXPERIMENTAL POR BRUCELLA ABORTUS EN COBAYOS Y RATONES (Sumario) Este estudio tuvo por objetivo principal probar la eficacia de varios medicamentos en el tratamiento de la infección experimental por Brucella abortus en cobayos y ratones. Se utilizaron las siguientes normas para evaluar la eficacia del tratamiento: (a) El efecto de la droga sobre el número de microorganismos en los tejidos de los animales infectados; (b) la protección de los ratones inoculados con una dosis letal de Brucella abortus al 80%. La sulfadiazina y el dimazol a concentraciones de 1% en el régimen, y 16 mg diarios de estreptomicina por vía subcutánea produjeron en los cobayos ligera disminución del número de microorganismos cultivados del bazo y nódulos linfáticos, al compararlos con los testigos normales. El tratamiento combinado con estreptomicina más sulfadiazina o dimazol, resultó más eficaz que cualquiera de los tres medicamentos aislados. Si para evaluar la eficacia del tratamiento se toma como única norma la capacidad de la droga para hacer disminuir en forma significativa el número de las brucelas en los tejidos de los animales infectados, el cloranfenicol fracasó en la terapéutica de la infección experimental por Brucella abortus en cobayos y ratones. No pudo descubrirse cloranfenicol en el suero sanguíneo de ratones y cobayos a las dos, cuatro y seis horas de la inyección intramuscular de 1 mg de la droga y de la administración bucal de 60 mg, respectivamente. Cuando se introdujeron 50 mg/kg de la droga en la sangre de los cobayos por via intracardíaca, se descubrió en el suero sanguíneo a concentraciones de 66.0 gamma por ce y 5.4 gamma por ce una y dos horas, respectivamente, después de la inyección, pero no se pudo descubrir el medicamento después de cuatro horas. Se obtuvo una concentración de 80 gamma por ce en la orina, pero en el líquido peritoneal no pudo descubrirse la droga a las cuatro horas de la inyección. El tamaño del bazo de los ratones tratados con cloranfenicol por vía bucal era menor de lo normal, pero los cultivos revelaron grandes cantidades de brucelas. No se observó esa disminución en el tamaño del bazo de los animales tratados con cloranfenicol por via parentérica. Esto demuestra que el tamaño del bazo no constituye una norma válida para evaluar la eficacia del tratamiento. La aureomicina (1 mg diario por via intravenosa), la terramicina (0.5% en el régimen), y la dihidrostreptomicina (1 mg diario por via intramuscular), administradas aisladamente, no hicieron disminuir en forma significativa el número de brucelas en el bazo y nódulos linfáticos de los ratones infectados. En este sentido resultó muy eficaz el tratamiento combinado con (a) aureomicina más dihidroestreptomicina y (b) terramicina más dihidroestreptomicina. La combinación de aureomicina-dihidroestreptomicina resultó más eficaz. Tanto la aureomicina, como la dihidroestreptomicina, terramicina y cloranfenicol protegieron contra una dosis de microorganismos que mataba constante-

CHEMOTHERAPY OF BR. ABORTUS IN GUINEA PIGS AND MICE 275 mente de 70 a 80% de los ratones no tratados. La sulfadiazina y el dimazol resultaron menos eficaces en este sentido. Cuando se utilizan experimentos de protección para evaluar la eficacia de la terapéutica, los resultados obtenidos en los animales de experimentación guardan más relación con los resultados obtenidos en el tratamiento de la brucelosis humana con sulfonamidas y antibióticos.

ESTUDIOS SOBRE TERAPÉUTICA DE LA BRUCELOSIS* Por M. Ruiz CASTAÑEDA, G. GUERRERO IBARRA y C. CARRILLO CÁRDENAS

Departamentode Investigaciones Médicas, Hospital General, México, D. F. Estudios epizootológicos y epidemiológicos han revelado que en la Republica Mexicana, los bovinos y caprinos se encuentran infectados por Brucella abortus y Brucella melitensis, respectivamente, en proporciones que varían de acuerdo con la región y condiciones en que viven esos animales. El hombre adquiere la infección, por los mecanismos usuales, de una u otra fuente. Carecemos de informes sobre si existe o no infección en porcinos aun cuando es de suponerse que esta sea insignificante en vista de las bajas cifras de reactores y la rareza con que la Br. suis ha sido encontrada en cultivos de enfermos de brucelosis. Una característica epidemiológica en México es que, a pesar de la alta incidencia de animales reactores en el ganado bovino, los casos humanos debidos a Br. abortus son sumamente escasos. Aun en personas en quienes el origen de la infección se ha relacionado en forma precisa con el consumo de leche de vaca, la brucela responsable de la infección humana ha sido melitensis, lo que se ha creído puede ser resultado de la convivencia frecuente de ganado lechero con cabras infectadas. Lo antes dicho explica que una cifra superior a 99% de los enfermos a que se refiere este trabajo hayan presentado cultivos de Br. melitensis. De 1946 a la fecha hemos podido observar poco más de 1500 enfermos que pueden clasificarse en los grupos y subgrupos siguientes: 1. Enfermos que sól61o se observaron una sola vez. 2. Enfermos cuya evolución fué seguida por más de 3 meses. Del grupo 2, el diagnóstico fué confirmado por hemocultivos positivos en poco más de 800 casos, habiendo seleccionado 448 cuyo estudio pudo seguirse con regularidad. No se incluyen casos de terminación fatal ni de infecciones muy benignas. Los 448 casos seleccionados se dividen en 5 grupos en la forma siguiente: I. Doscientos treinta y siete enfermos sometidos a tratamiento sintomático asociado a la administración de extractos de brucelas (grupo seleccionado entre los casos registrados antes de la introducción formal de antibióticos). II. Sesenta y un casos tratados con la combinación estreptomicina-sulfadiazina. * Estos trabajos fueron posibles, gracias a la cooperación de las firmas E. R. Squibb and Co., Brooklyn N. Y.; Lederle and Co., Pearl River N. Y. y Chas. Pfizer and Co., Brooklyn, N. Y. 276

TERAPÉUTICA DE LA BRUCELOSIS

277

III. Setenta y cuatro casos tratados con aureomicina por via oral. IV. Cincuenta y seis casos tratados con una combinación de estreptomicinasulfadiazina y aureomicina. V. Veinte casos tratados con terramicina. Desde 1937 observamos el efecto benéfico sobre la evolución de la infección brucelar de la aplicación de extractos de brucelas solubles en solución salina isotónica. Este efecto se manifestaba de preferencia en casos crónicos llegando a observarse notable mejoría clínica en un promedio de 27 días desde que se iniciaba el tratamiento.' Asociado a la terapéutica sintomática este método (que parece actuar por disminución del estado de hipersensibilidad a la brucela o productos de su metabolismo) ha sido el tratamiento antibrucelar en los 237 casos presentados en la Tabla I. Las cifras promedio de esta tabla servirán para TABLA I.-Resultados del tratamiento sintomático en 27 enfermos de Brucelosis con bacteriemia Período febril

Cultivos

Recaídas

Satisfactorios

caso

84 120 24 9 237

Observación desde

No.

Después Primer Ultimo de Antes del tratamiento del tratamiento Negativo Positivo (meses) (meses) (días) (meses)

2 2-4 4-6 6-12

Casos

%

Casos

%

el trata(meses)

64.5 58.8 49.8 62.2

4.3 3.5 3.8 4.2

3.9 3.2 3.1 4.5

17 44 7 4

39.6 36.7 29.2 44.5

67 76 17 5

71.4 63.3 70.8 55.5

6.7 9.0 9.0 10.2

55.7

3.9

3.5

72

30.4

165

69.6

8.3

apreciar los avances hechos recientemente mediante el empleo de antibióticos así como las limitaciones de estas drogas en la terapéutica antibrucelar. Los 237 casos de esta tabla, todos confirmados por el aislamiento de Brucella de la sangre, se clasifican en 4 grupos de acuerdo con la duración de la enfermedad hasta el momento en que se inició el tratamiento. Nótese la escasa diferencia que hay en las cifras promedio obtenidas para los 4 grupos y la representación que del total se indica en la última línea. Por lo que se refiere a la duración del período febril a partir del tratamiento hay que hacer notar que los promedios se obtienen de cifras muy alejadas en los extremos, pero que siendo en minoría, afectan poco ese promedio. Para apreciar la evolución del padecimiento se ha considerado la duración de la fiebre por ser una de las manifestaciones más importantes de la infección; los demás síntomas, así como las complicaciones mejoran con la vuelta a la temperatura normal. Ciertas complicaciones tales como purpura, artritis, espondilitis, etc. ocurrieron principalmente en los 72 casos catalogados en la columna de recaídas, las que

278

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

constituyen el 30% del total. Como puede verse, cerca del 70% de los enfermos evolucionaron satisfactoriamente en un promedio de 56 días en que, con el abatimiento gradual de la temperatura fueron disminuyendo las molestias ocasionadas por la sintomatología asociada. Conviene aclarar que en la designación de período febril se incluyen todas las formas febriles conocidas, desde la continua, la intermitente, etc., hasta la típica ondulante. Estreptomicina-sulfadiazina.-Desde 1938 tuvimos oportunidad de tratar un buen número de casos de brucelosis con sulfa drogas incluyendo 30 enfermos estudiados por Guerrero Ibarra 2 a quienes se aplicó sulfadiazina por vía intravenosa. Nuestras observaciones, comparadas con los casos del grupo I nos dieron la impresión de que el efecto terapéutico de estas drogas es muy limitado. Recientemente se estudió en el Instituto del Seguro Social de México, el método recomendado por Huddleson 3 TABLA II.-Enfermos tratados con la combinación Estreptomicina-Sulfadiazina Periodo febril de

Antes del

casos

tratamiento (meses)

19 22 12 4 4

2 2-4 4-6 6-12 12

61

Cultivos Positivos

No.~~~~~~~~~~~~~~~~ A partir del Antes Dess tratamiento de de (dias) 30 días 30 dias

Recaidas

Casos

%

Satisfactorios s.

Casos

%

Observa

ción desde el tratamiento

17.5 13.3 7.2 9.7 19.7

9 17 0 2 2

11 15 7 2 4

15 15 6 1 4

79.0 68.2 50.0 25.0 100.0

4 7 6 3 0

21.0 31.8 50.0 75.0 0

7.7 5.6 8.5 4.2 16.0

13.6

30

39

41

67.2

20

32.8

7.5

aplicando simultáneamente sulfadiazina y transfusiones. Se tuvo la impresión de que los enfermos presentaban rápida pero pasajera mejoría con ese tratamiento.4 Nuestra experiencia, concordante con la de autores contemporáneos, mostró que tanto la penicilina como la estreptomicina carecían de valor práctico en la terapéutica antibrucelar. Sin embargo, la asociación de la estreptomicina con una dosis adecuada de sulfadiazina. 5 6,7 fué el método terapéutico que por primera vez reveló acción indudable y rápida sobre la evolución clínica y bacteriológica de la brucelosis. En 1948 se llevó a cabo un estudio sobre un grupo relativamente importante de enfermos, para lo cual trabajamos en colaboración con el Dr. W. W. Spink. Sobre los resultados del tratamiento de 61 casos se presentaron 2 informes.8s 9 En la Tabla II se reproducen los datos recogidos, complementándolos con observaciones hechas subsecuentemente en ese grupo. Más tarde aplicamos la misma combinación con dihidroestreptomicina con resultados un poco más satisfactorios ya que pudieron administrarse sin riesgo, dosis superiores a 2 gms durante 10 6 15 dias.

TERAPÉUTICA DE LA BRUCELOSIS

279

En esta tabla puede notarse desde luego notable reducción de la duración del período febril a partir de la iniciación del tratamiento. Como hemos indicado en otras ocasiones fué notable el contraste de este método terapéutico con observaciones previas. A pesar de que en un número importante de casos la bacteriemia reincidió y más del 60% de los casos presentaron recaídas clínicas, se tuvo la impresión de que la acción favorable del tratamiento fué indudable, puesto que en 30% de los casos bastó un tratamiento de 2 semanas para conseguir completa eliminación de las manifestaciones clínicas de la brucelosis. Aureomicina.-Con el descubrimiento de la aureomicina y la cloromicetina se consiguió el más impresionante avance en la terapéutica de la infección. Ambas drogas suprimen en forma espectacular la sintomatología de la infección y actuán en forma muy efectiva sobre la evolución de la bacteriemia. Nuestra experiencia se limita al estudio de la aureomicina, refiriendo a quien se interese en los efectos de la cloromicetina a estudios presentados por otros autores.' ° 11, 12 En 1948 recibimos una cantidad de aureomicina suficiente para llevar a cabo estudios bastante completos sobre la acción de esta droga. En colaboración con el Dr. Spink se dieron a conocer los resultados de 25 casos s 9 febriles con bacteriemia. Entre los casos mejor estudiados ulteriormente seleccionamos 49 que agregados a los anteriores completan los 74 casos presentados en la Tabla III. Desde el principio del trabajo se tuvo la impresión de que la aureomicina, aplicada en dosis moderadas por vía oral, provocaba cambios clínicos y bacteriológicos en forma francamente espectacular, pues nunca antes se había notado cambio tan radical y constante en menos de 96 horas. Lo más impresionante de la Tabla III es la duración febril a partir del tratamiento en 71 de los 74 casos estudiados. Esa duración de 56 días en el grupo I, y 14 en el II pasó a un máximo de 96 horas en este grupo. Estos resultados tan prometedores fueron pronto ensombrecidos por la frecuencia de recaídas, las que ocurrieron a partir del primer mes después del tratamiento. Sin embargo, si se considera que 60% de los pacientes se recuperaron rápidamente sin recaídas, reintegrándose a sus labores dentro del término de 1 a 2 meses, resulta en franco contraste favorable con el porcentaje obtenido con tratamiento de estreptomicina-sulfadiazina en el que ese porcentaje fué solamente de 32%. Tratamiento triple.-Tanto Herrell' 3 como nosotros' 4 hemos intentado aprovechar una posible acción sinérgica de la aureomicina con la estreptomicina. Este trabajo se llevó a cabo después de la aplicación de clorhidrato de aureomicina para uso parenteral. Su empleo por vía intravenosa de 150 a 300 mg aplicados diariamente durante un lapso de 7 a 8 días provocaba cambios clínicos y bacteriológicos semejantes a los observados con dosis orales de 2 gm diarios. Se ensayó entonces la administración simultánea de 500 mg de aureomicina por via oral con

280

THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

estreptomicina y sulfadiazina en dosis de 1 y 3 gm respectivamente. El tratamiento se mantuvo por 7 a 8 días. Por su costo reducido, este método ha sido el de elección para nuestros enfermos indigentes. Los resultados pueden considerarse como bastante satisfactorios si se les compara con TABLA III.-Enfermos tratados con aureomicina por vía oral Periodo febril No. de casos

Antes del tratamiento (meses)

los siguientes 15días

Desde el Tratamiento Menos Más

Posi

de 4 días

de 4 días

tivo

Recaídas

Casos

tivo

Altas satisfactorias

Observación desde el tratamiento ( (meses)

%N

6

1

6

0

0

6

0

0

6

100.0

5.5

17 13 21 14 3

1-2 2-4 4-6 6-12 Más de 12

15 13 20 14 3

2 0 1 0 O

2 1 1 0 0

15 12 20 14 3

10 6 10 4 0

58.8 46.2 47.7 28.6 0

7 7 11 10 3

41.2 53.8 53.3 71.4 100.0

6.9 6.5 7.8 8.4 9.0

71

3

4

70

30

40.0

44

60.0

7.4

74

TABLA IV.-Enfermos tratadoscon estreptomicina,sulfadiazina y aureomicina PeríodoPeríodo febril febril No. de

Antes

casos

del trata-

miento (meses)

Desde el tratamiento Menos de 4 días

Cultivo durante los siguinetes 15 días

Recaídas

Altas satisfactorias

Observación desde el tratamiento

Más de 4 días

Positivo

Negativo

Casos

%

Casos

%

(meses)

9

1

6

3

0

9

2

22.3

7

77.7

6.2

15 18 6 8

1-2 2-4 4-6 6-12

15 15 6 7

0 3 0 1

0 2 1 1

15 16 5 7

6 5 3 3

40.0 27.8 50.0 37.5

9 13 3 5

60.0 72.2 50.0 62.5

6.9 7.7 6.5 7.1

49

7

4

52

19

34.2

37

65.8

7.0

56

los grupos II y III. Un resumen de estas observaciones se presenta en la Tabla IV. De 56 enfermos tratados, 49 mostraron franca mejoría clínica con cultivos negativos en 52 casos. Hubo 19 recaídas, pero los porcentajes de recuperación, dentro de un período de observación de 7 meses son comparables a los del grupo tratado con 20 a 40 gm de aureomicina por vía oral. La mayoría de estos pacientes se reintegró a sus ocupaciones antes de un mes después de terminar su tratamiento.

281

TERAPÉUTICA DE LA BRUCELOSIS

Terramicina.*-Recientemente y gracias a gestiones del Dr. V. Knight pudimos llevar a cabo un ensayo relativamente importante en la terapéutica antibrucelar mediante el empleo del nuevo antibiótico: terramicina. Este antibiótico tiene propiedades antibacterianas semejantes a las de la aureomicina y la cloromicetina. Aplicando nuestra experiencia con aureomicina, consideramos apropiado iniciar el tratamiento con dosis pequeñas y no pasar de 2 gm al día durante 10 días. En la mayoría de los enfermos el tratamiento se ha repetido durante otros 5 días después de 5 días de descanso. De febrero del presente año a la fecha en que se escribe esta nota (agosto de 1950) se han tratado 40 enfermos, de los cuales hay 20 en los que la observación TABLA V.-Enfermos tratados con terramicina PeriodoPerodo febril febril No. de casos

Desde el tratamiento

Antes del tratamiento Menos (meses)

2 6 7 2 3 20

1 1-2 2-4 4-6 6-12

deasos

4 días

Más

Cultivo durante los siguientes 5 días Pos

Nega

Recaídas Cas(meses)

%

4 días

1 5 4 2 3

1 1 3 0 0

1 3 0 2 1

1 3 7 0 2

1 5 2 0 0

15

5

7

13

8

Altas satisfactoras

tratamiento Casos

%

1 1 5 2 3 40.0

Observacin desde el

12

4.0 3.0 3.0 4.5 3.3 60.0

3.4

después del tratamiento es mayor de 3 meses. Hay, pues, tiempo suficiente para evaluar los resultados obtenidos y creemos que estos son significativos a pesar de lo restringido de su número. En la Tabla V se presenta un resumen de los resultados. Nótese que éstos se asemejan notablemente a los obtenidos con aureomicina, excepto en que los cultivos practicados durante el tratamiento suelen dar resultados positivos en proporción mucho mayor que en pacientes tratados con dosis iguales de aureomicina. Las recaídas clínicas y bacteriológicas han ocurrido más cercanas a la fecha en que se suspendió la administración de la droga. Desde un punto de vista práctico sin embargo, este nuevo antibiótico actúa sobre la evolución de la infección en forma comparable a la aureomicina con la ventaja de ser mejor tolerada. COMENTARIOS

Al hacer un estudio comparativo de los resultados obtenidos por los diversos métodos terapéuticos a que se refiere esta nota, conviene indicar * Hasta el momento de enviar este trabajo tenemos conocimiento de estudios sobre terapéutica de brucelosis con terramicina, pero las publicaciones correspondientes no están disponibles.

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THIRD INTER-AMERICAN CONGRESS ON BRUCELLOSIS

que la selección hecha de los casos presentados es representativa de un grupo de más de 2000 enfermos estudiados hasta la fecha. La mayorla fué tratada en forma sintomática asociando inyecciones periódicas de extractos brucelares. Un número considerable de enfermos abandonó el servicio antes de poderlos observar por tiempo suficiente para recoger datos de utilidad. Suponemos que una parte se derivó hacia otras clínicas; pero que muchos otros no regresaron por encontrarse en franca mejoría. Los 237 casos seleccionados para formar la Tabla I fueron observados durante varios meses por lo que consideramos este grupo como control satisfactorio para evaluar el efecto terapéutico de los nuevos métodos. Por otra parte, hay que indicar que el grupo II no es representativo de la masa total de nuestros enfermos, pues para este primer ensayo con antibióticos se emplearon de preferencia los casos más severos y rebeldes. TABLA VI.-Resumen de resultados del tratamiento de 448 casos de brucelosis por diversos métodos No. de casos

237 61 74 56 20

Tratamiento

Sintomático EstreptomicinaSulfadiazina Aureomicina EstreptomicinaSulfadiazina y Aureomicina Terramicina

Período febril desde el tratamiento

Recadas caídas

Casos satis-

55.7 días 13.6 días

30.4 67.2

69.6 32.8

8.3 7.5

96 horas 96 horas

40.0 34.2

60.0 67.8

7.4 7.0

96 horas

40.0

60.0

3.4

%

seaci

(mese

443

Es posible que la mayoría de estos casos hubieran quedado incluidos entre los casos no influenciados por el tratamiento sintomático. La alarmente frecuencia de lesiones vestibulares provocadas por la estreptomicina así como la aparición de antibióticos de más fácil manejo nos hizo preferir estos al uso de droga. La acción de la combinación estreptomicina-sulfadiazina, favorable, aunque transitoria para un alto porcentaje de nuestros casos, se incrementa al asociarle una pequeña dosis de aureomicina. Sobre la acción terapéutica de la cloromicetina, nos hemos concretado a seguir los trabajos de otros autores y estudiar la anamnesis de unos 40 casos que recurrieron a nuestro servicio presentando recaídas clínicas y bacteriológicas después de haber sido sometidos a tratamiento a veces masivos con esa droga. Por último, al comparar la terramicina con la aureomicina hemos observado menor influencia de aquella droga sobre el curso de la bacteriemia. En el reducido número de casos tratados con terramicina las

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recaidas clinicas han ocurrido en fechas más cercanas al tratamiento que en casos tratados con aureomicina. CONCLUSIONES 1. Alrededor de 80% de los enfermos de brucelosis no tratados con antibióticos evolucionaron hacia la desaparición de la sintomatología principal en un lapso comprendido entre 2 y 6 meses. 2. En enfermos con brucelosis severa la terapeútica combinada de estreptomicina-sulfadiazina se muestra muy inferior en actividad a la acción de la aureomicina y terramicina. Sin embargo, esa combinación asociada con aureomicina a dosis que por separado han sido insuficientes en otros enfermos, reveló notable actividad terapéutica. 3. El empleo de los antibióticos referidos en esta nota no ha resuelto en definitiva el problema terapéutico de la brucelosis provocada por Br. melitensis, posiblemente por circunstancias especiales en la biología de esta variedad de Brucella y en la patogenesis de la enfermedad. Sin embargo, la acción restrictiva inmediata de esas drogas ha modificado radicalmente la evolución clínica de la enfermedad. REFERENCES (1) Castañeda, M. R. and Carrillo Cárdenas, C.: Treatment of Brucellosis with Brucella antigens. Am. Jour. Trop Med., 21: 185-190, (1941). (2) Guerrero-Ibarra, G.: (Unpublished). Depart. of Med. Research, General Hospital, Mexico City. (3) Huddleson, I. F.: The potentiating action of sulfonamides on the Brucella antibody complement action, Ann. J. Vet. Res. 9: 277-285, (1948). (4) Mendez D.: (From the Seguro Social, Mexico) Personal communication. (5) Pulaski, E. J. and Amspacher, W. H.: Streptomycin therapy in Brucellosis, Army Med. Depart., 7: 221-225, (1947). (6) Eisele, C. W. and McCullough, N. B.: Treatment of Brucellosis, J.A.M.A., 185: 1053-1055, (1947). (7) Spink, W. W., Hall, W. H., Shaffer, K. M., and Braude, A. I.: Human Brucellosis. Its specific treatment with a combination of streptomycin and sulfadiazine. J.A.M.A., 136: 382-387, (1948). (8) Castañeda, M. R., Spink, W. W., Carrillo Cárdenas, C., Guerrero-Ibarra, G., and Silva-Goytia, R.: II Interamerican Congress of Brucellosis, Buenos Aires, Argentina, 1948. Rev. Instituto de Sal. y Enfer. Tropicales, Mexico, 10: 53-68, (1949). (9) Spink, W. W., Braude, A. I., Castañeda, M. R., and Silva Goytia, R.: Aureomycin Therapy in human Brucellosis due to Br. melitensis, J.A.M.A., 138: 1145-1148, (1948). (10) León, A. P., Cano, C., and Bernal, E.: La cloromicetina en el tratamiento de la brucelosis humana, Rev. Inst. Sal. y Enf. Trop., México, 10: 155-165, (1949). (11) Woodward, T. E., Smadel, J. E., Holbrook, W. A. and Raby, W. T.: The Beneficial effect of cloromycetin in brucellosis, J. Clinical Invest., 28: 968-976, (1949). (12) Knight, V., Ruiz Sánchez. F. and McDermont, T. W.: Cloramphenicol in the treatment of the acute manifestations of Brucellosis. Am. Jour. Med. Science., 219: 627-638, (1950).

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(13) Herrell, W. E., and Barker, F. E.: The combined use of aureomycin and dyhidrostreptomycin in the treatment of Brucellosis, Proc. Staff., Meetings, Mayo Clinic, 24: 138-145, (1949). (14) Castafeda, M. R., Carrillo Cárdenas C., and Guerrero Ibarra, G.: Antibiotic in Brucellosis, Report to the Mexican Academy of Medicine, (In press) (1950). EVALUATION OF PRESENT THERAPEUTIC METHODS IN BRUCELLOSIS (Summary) A comparative study of the various methods of treating human brucellosis was undertaken. During the years 1946 and 1947 550 cases were studied. These served as a control group for evaluating different therapeutic products developed after 1947. In 1948 we began to use antibiotics on patients selected from those registered in our Clinic from 1948 to 1950. All patients treated presented positive hemocultures identified as Brucella melitensis. Of 231 cases 61 were treated with a combination of streptomycin-sulfadiazine combination; 74 were treated orally with aureomycin; 56 were treated with a combination of streptomycin-sulfadiazine-aureomycin; 40 cases with terramycin, of which 20 are included in Table V. (The other 20 cases had similar data.) Cases presented in Table I were 237 selected from the control group. In the cases observed prior to the use of antibiotic there was a mortality rate of about 5%, frequently with severe complications. For this reason, the results obtained with the use of the combination streptomycin-sulfadiazine appeared to us to represent a notable improvement in therapy. Aureomycin and terramycin gave good clinical and bacteriological results, but they continued to show high percentages of clinical as well as bacteriological relapses. In addition to chloromycetin, the antibiotic cited notably repress the symptomatology and have reduced to a minimum the mortality caused by the disease. Therefore, in spite of the limitations pointed out, they constitute important progress in brucellosis therapy.

LA ANTIBRUCELINA EN EL TRATAMIENTO DE LA BRUCELOSIS FEBRIL* PARTE I Por los Dres. ENSO CRISCUOLO, FÉLIX RAMACCIOTTI Y ESTHER R. W. DE PAOLASSO ACCIóN DE LA ANTIBRUCELINA COMO ANTIBIÓTICO

Las quinonas pertenecen a la serie cíclica, siendo descubierto su primer compuesto por Woskresensky en 1888 oxidando el ácido quínico contenido en la corteza de la quina con bicromato de potasio y ácido sulfúrico. Este compuesto fué designado Chinoile y corresponde a una parabenzoquinona, substancia tipo de esta serie de compuestos. Berzelius les dió a estas substancias el nombre de quinonas. Pueden considerarse derivados por oxidación de los difenoles. Los fenoles son compuestos que tienen oxígeno en estado de oxihidrilo, unido al núcleo aromático (los difenoles tienen pues dos oxihídrilos); por oxidación de estas funciones se obtienen las quinonas, pero sólo pueden obtenerse a partir de los para (1-4) y orto (1-2) difenoles y no de los meta (1-3) difenoles. Beijerinx las descubrió en cultivos de Strepthotrix cromógenos, que las elabora sólo en determinadas condiciones, para inhibir el desarrollo de la flora banal de la putrefacción. Del Vecchio et al. señalan esta acción benéfica de las quinonas destacando su naturaleza antibiótica. El tiocol aislado del Mycobacterium tuberculosis hominis por Anderson es también un derivado quinónico; la espinulosina aislada del P. spinulosum, la fumigatina del Asp. fumigatus (Raistrik), la clavacina del Asp. clavatus, antibióticos de reciente descubrimiento, tienen también estructura quinónica. Morgan, Cooper, Burtt y Corby estudiaron la acción de algunas quinonas (toluquinona-xiloquinona, timoquinona, canfoquinona, quinhidrona, M. bicloroquinona, tricloroquinona, P-bicloroquinona, P-bibromoquinona, monocloroquinona, ácido bromanílico, ácido cloranílico, alfa y beta naftoquinona) sobre cepas de estafilococos y Escherichia coli destacando que la benzoquinona actúa contra el estafilococo y es fuertemente activa contra la Escherichia coli. Page destacó también la acción de las quinonas sobre el estafilococo. Oxford, que observó que la fumigatina y la espinulosina eran derivados de la toluqinona, preparó 14 derivados de la toluquinona y 7 de la benzoquinona destacando que los más activos fueron la 4-metoxitoluquinona, la 4, 6-dimetoxitoluquinona, y la 3,4, 6-trimetoxitoluquinona que son 10 veces más activas que la fumigatina, la que destruye el estafilococo en diluciones al 1/100,000. Del Vecchio inició sus trabajos en 1944 y Argenziano, su colaborador, * Trabajo de la Cátedra de Clínica de las Enfermedades Infecciosas de la Facultad de Ciencias Médicas de la Universidad de Córdoba, Argentina. 285

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preparó por síntesis o extracción una serie de derivados quinónicos estudiando su acción sobre estafilococos áureos, shigelas, salmonelas, brucelas, etc. Después de numerosas pruebas in vitro observaron que la 2 metil 1,4 naftoquinona, la oxibenzoquinona y otros derivados tenían acción antibiótica sobre las brucelas. Estudiaron luego la vitamina K, (Dam 1929) que es una 2 metil 1,4-naftoquinona, llegando a la conclusión de que la misma tenía acción antibiótica sobre las brucelas aun a diluciones muy grandes (1/800,000). Del Vecchio y colaboradores obtuvieron la 2 metil 6 oxi 1,4-naftoquinona, sal sódica denominada comercialmente antibrucelina, que demostró tener poder bacteriostático aun en diluciones al 1/800,000. Panebianco, Napoli y D'Aniello controlan el poder antibiótico de la antibrucelina con 4 cepas de brucelas melitensis, 1-paramelitensis y 1-parabang con los siguientes resultados. Diluciones

1/100,000

1/200, 000

1/400, ooo0

Cepa No. 1 ......... Cepa No. 2 .........

48 hs. 130 hs.

24 hs. 96 hs.

- de inhibición 48 hs. inhibición

Cepa No. 3.........

Bactericida

192 hs.

96 hs. inhibición

Cepa No. 4 ......... Cepa No. 5 ........

154 hs. 72 hs.

72 hs. 48 hs.

48 hs. inhibición - hs. inhibición

Cepa No. 6 .........

192 hs.

120 hs.

96 hs. inhibición

NUESTRAS INVESTIGACIONES

Para controlar el trabajo de los autores italianos hemos seleccionado 22 cepas lisas de brucelas (20 melitensis, 1 abortus y 1 suis) preparando un cultivo de cada cepa en 2 ce de caldo dejándolo en la estufa 48 horas a 37°C; después de esta incubación se sembró un asa en cada tubo que contenía 2 cc de caldo simple con antibrucelina diluida de modo que la concentración final fuera de 1/100,000, 1/200,000 y 1/400,000 de substancia pura. La observación de estos cultivos efectuada durante 10 días arrojó los siguientes resultados: Todas las cepas fueron sensibles a la dilución de 1/200,000 durante períodos que oscilaron entre 48 y 168 horas, excepto la suis que no fué sensible. Quince cepas fueron sensibles a la dilución de 1/400,000 durante períodos que oscilaron entre 24 y 168 horas. Siete cepas no fueron sensibles a esta dilución. Teniendo en cuenta que la antibrucelina que se emplea en el comercio es una dilución al 3/1,000, controlamos la acción de este producto con

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quince cepas lisas de Brucella melitensis comprobando que este producto en la dilución de 1/100,000 no modificó el desarrollo de 9 de las 15 cepas estudiadas. Seis cepas fueron sensibles a la dilución de 1/200,000 durante períodos que oscilaron entre 24 y 48 horas y ninguna cepa a la dilución de 1/400,000. Controlamos también el producto comercial tal como viene envasado con el método de las estrías (técnica de Vincent y Vincent) en placas de agar-hígado comprobando que puro inhibe entre 4 y 6 mm y al 1/10, 1/100 y 1/1,000 no inhibe el desarrollo de las brucelas. Bacteriostasis Diluciones

1/100,000

1/200,000

horas

Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa Cepa

No. 1........... No. 2........... No. 3........... No. 4........... No. 5........... No. 6........... No. 7........... No. 8........... No. 9........... No. 10 .......... No. 11 .......... No. 12 .......... No. 13 .......... No. 14 .......... No. 16 .......... No. 43 .......... No. 44 .......... No. 45 .......... No. 46 ......... No. 47 .......... abortus .........

Cepa suis.............

120 192 120 168 192 144 168 168 144 192 144 96 120 192 168 144 144 72 192 120 144 -

1/400,000 horas

48 168 120 72 120 72 72 144 120 168 72 72 72 96 96 72 72 48 72 48 48

24 96 48 72 120 72 120 96 168 48 24 72 48 -

-

-

En resumen: todas las cepas fueron sensibles en la dilución de 1/100,000 por tiempos que oscilan entre 72 y 192 horas excepto la suis que no fué sensible.

Con el método de los cilindros de Heatley observamos que la antibrucelina pura produce una inhibición de aproximadamente 18 mm en la dilución de 1/10, 6 a 8 mm y nada en las diluciones de 1/100 y 1/1,000. Usando comparativamente estreptomicina y antibrucelina puede verse que la estreptomicina presenta mayor poder inhibidor que la antibrucelina.

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LA ANTIBRUCELINA Y SU EFICACIA EN LA TERAPEUTICA DE LA BRUCELOSIS HUMANA PARTE II Por los Dres. ENSO CRISCUOLO, HUGO VACCHIANI, FABIO H. BERGAGNA, NESTOR PIERANGELI VERA Y ALBERTO CEBALLOS

El tratamiento de la brucelosis humana ha visto acrecentada su eficacia en forma extraordinaria en estos últimos tiempos gracias al descubrimiento de una serie de antibióticos de acción muy satisfactoria, tales como la combinación estreptomicina, sulfadiazina (Spink et al.), aureomicina (Duggar 1948), cloromicetina (Burkholder-Ehrlich 1948) y últimamente con la terramicina (Finlay, Hobby et al. 1950). No obstante el conocimiento y la práctica obtenidas por nosotros en el tratamiento de esta interesante enfermedad con los antibióticos mencionados, creímos de interés publicar los resultados que hemos obtenido con la antibrucelina, derivado quinónico a que antes hicimos mención. La producción de esta substancia por vía química le asigna en nuestro concepto, un gran interés ya que obtenida de tal modo puede estar más al alcance de nuestros pacientes. Los resultados expuestos en la primera parte de este trabajo nos parecieron muy satisfactorios y, no obstante no haber obtenido resultados favorables en el tratamiento de cuatro pacientes con dosis que oscilan entre 30 y 45 cc por día decidimos efectuar un nuevo contralor sobre 8 pacientes utilizando las dosis últimamente aconsejadas por Del Vecchio y colaboradores. Elegimos al efecto 8 pacientes con brucelosis febril y hemocultivo positivo para Brucella melitensis, por ser la que priva en nuestro medio, y los tratamos con 1½ cc/kg de peso por día durante 30 días, inyectando además por vía intradérmica 0.5 cc de antibrucelina dos veces por día durante el mismo lapso, vía y dosis que ejercen efectos beneficiosos según Panebianco sobre el estado alérgico del paciente. La antibrucelina de Del Vecchio es producida por el Instituto Medicamenta Nova de Salerno, Italia, el que presenta el producto en tres series de ampollas. Serie débil............ Serie media ........... Serie fuerte ...........

6 ampollas de 2 cc de antibrucelina en solución acuosa 6 ampollas de 5 cc de antibrucelina en solución acuosa 3 ampollas de 10 cc de antibrucelina en solución acuosa

Cada cc de antibrucelina de cualquiera de las tres series contiene 3 mg de substancia activa, por consiguiente cada ampolla de la serie débil tiene 6 mg, la de serie media 15 mg, y la fuerte 30 mg de substancia activa. El medicamento se puede aplicar según los autores italianos por cualquier vía, siendo la de elección la endovenosa. Se tolera bien aun por vía

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intrarraquídea por la que se han administrado hasta 12 mg por día

(4 cc). Aconsejan los autores repartir la dosis diaria en dos, una inyección matutina y otra vespertina (endovenosa). Como en el caso de los antibióticos de uso corriente debe continuarse la aplicación de antibrucelina hasta varios días después de haber bajado la temperatura a la normal a fin de evitar recaídas. Puede establecerse que la duración del tratamiento oscila entre 10 y 40 días según los distintos autores. En los trabajos de los autores italianos se ha observado que, al mismo tiempo que la fiebre cae en lisis, después de un tiempo variable de tratamiento los pacientes manifiestan bienestar general, retorno del apetito y de las fuerzas, desaparición de los sudores, de los dolores neuríticos o articulares, reducción o desaparición de la hepato y esplenomegalia, y un hecho muy interesante señalado por Panebianco consistente en la reducción y aun la desaparición de las aglutininas específicas del suero. Este fenómeno no fué observado en todos los casos. F. Panabianco, A. Napole y L. D'Aniello comunicaron 30 casos tratados de los cuales 25 curaron en corto plazo, observando que en 7 se redujeron los títulos de aglutininas y en 6 desaparecieron. Un grupo de autores italianos ha comunicado resultados favorables con esta terapéutica, entre ellos P. Barlotta y Malatesta, T. Mogrovejo, Prof. C. Tripi, R. Virgili y V. Del Vecchio, S. Portaova, M. Baldelli, U. Amodio, etc. (consultar bibliografía adjunta). Veamos los resultados obtenidos en nuestro servicio de la Cátedra de Clínica de las Enfermedades Infecciosas de la Facultad de Medicina de Córdoba, Argentina. Los 8 casos estudiados estuvieron internados no menos de 4 meses, permitiendo este lapso efectuar una observación correcta del efecto del medicamento sobre los siguientes puntos: Efectos de la antibrucelinasobre los cultivos Caso No. 1 Case No. 2 Case No. 3 Caso No. 4 Caso No. 5 Caso No. 6 Caso No. 7 Caso No. 8

Antes

DespuCs

positivo positivo positivo positivo positivo positivo positivo positivo

positivo positivo positivo positivo positivo positivo positivo positivo

Efectos de la antibrucelina sobre la reacción de Huddleson Antes

DespuEs

Caso No. 1 Caso No. 2

1/200 1/1000

1/1000 1/1000

Caso No. 3 Caso No. 4 Caso No. 5

1/2000 1/1000 1/2000

1/10,000 1/1000 1/2000

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Caso No. 6 Caso No. 7 Caso No. 8

Despaus 1/200 1/500 1/500

Efectos de la antibrucelinasobre la curva térmica Caso No. 1 No actuó Caso No. 2 No actuó Caso No. 3 No actuó Caso No. 4 No actuó Caso No. 5 No actuó Caso No. 6 No actuó Caso No. 7 Si actuó Caso No. 8 Si actuó Efectos de la antibrucelinasobre el tamaño del bazo Después

Antes

Caso Caso Caso Caso Caso Caso Caso Caso

No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8

palpable palpable palpable palpable palpable palpable normal normal

no varió no varió no varió no se palpa no varió aumentó de tamaño normal palpable

Efectos de la antibrucelina sobre el tamaño del hígado Antes

Caso Caso Caso Caso Caso Caso Caso Caso

Después

no varió no varió no varió no varió aumentó de tamaño aumentó de tamaño normal no varió

palpable palpable palpable palpable palpable palpable normal palpable

No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No.8

Efectos de la antibrucelinasobre la eritrosedimentación (índice de Kats) Caso Caso Caso Caso Caso Caso Csao Caso

No. No. No. No. No. No. No. No.

1 2 3 4 5 6 7 8

Antes

Después

32.50 63.00 32.50 55.00 27.50 36.00 45.50 26.25

34.50 53.00 34.50 14.00 19.25 48.00 10.00 15.50

Efectos de la antibrucelina sobre el cuadro hemático (eritrocitos y leucocitos) Antes

Caso No. 1 Caso No. 2 Caso No. 3

4,500,000 6,000 3,530,000 5,100 4,300,000 4,800

DespuEs

4,400,000 8,600 3,710,000 7,500 2,950,000 6,300

ANTIBRUCELINA EN BRUCELOSIS FEBRIL Caso No. 4

4,280,000 8,000 3,150,000 7,200 3,310,000 6,500 4,500,000 4,300 4,600,000 4,600

Caso No. 5 Caso No. 6 Caso No. 7 Caso No. 8

291

4,520,000 6,200 3,570,000 4,600 4,200,000 6,400 4,010,000 - 4,500 4,800,000 4,800

Efectos de la antibrucelinasobre la crasis sanguinea Después

Antes

Tiemp. H. 2'45 Tiemp. coag. 8' T.H. 2' T.H. coag. 6' T.H. 2' T.H. coag. 5'30 T.H. 2' T.H. coag. 8' T.H. 3'30 T.H. coag. 10'30 T.H. 2' T.H. coag. 6' T.H. 3'30 T.H. coag. 10'30 T.H. 1'30 T.H. coag. 4'30

Caso No. 1 Caso No. 2 Caso No. 3 Caso No. 4 Caso No. 5 Caso No. 6 Caso No. 7 Caso No. 8

1'30 8' 3' 10' 2' 8' 4'30 10' 2'30 7' 6'30 17' 2' 8'

El efecto sobre la retracción del coágulo fué negativo ya que en todos los casos la misma resultó incompleta tanto antes como después del tratamiento. Efectos de la antibrucelinasobre el índice opsónico Caso Caso Caso Caso Caso Caso Caso

No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7

Caso No. 8

Antes

Desputs

52 54 47 33 48 53 56

55 52 47 40 88 52

-

Efectos sobre las artropatías Antes

Caso Caso Caso Caso Caso Caso Caso Caso

No. No. No. No. No. No. No. No.

1 2 3 4 5 6 7 8

hidrartrosis artralgia artralgia artralgia normal normal artralgia artralgia

Después

hidrartrosis hidrartrosis hidrartrosis normal normal artralgia artralgia artralgia

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Antes

Caso Caso Caso Caso Caso Caso Caso Caso

No. No. No. No. No. No. No. No.

1 2 3 4 5 6 7 8

Bradispsiquia clonus pie y rótula Hipersomia-hiperreflexia Hiperreflexia clonus Insomnio normal Esbozo de clonus Paraplejía espast. Normal

sin variantes sin variantes sin variantes normal normal normal sin variante normal

Efectos de la antibrucelinasobre el estado general Caso Caso Caso Caso Caso Caso Caso Caso

No. No. No. No. No. No. No. No.

1 2 3 4 5 6 7 8

Antes

Después

bueno regular regular regular malo malo malo regular

regular malo malo bueno malo malo regular bueno

Fueron estudiadas las pruebas de funcionalismo hepático observando que el tratamiento no influenció el resultado de las mismas; las pruebas utilizadas fueron bilirrubinemia, sulfato de cadmio, test de Hanger, y ácido hipúrico. También se efectuó la prueba de Pyrgalis antes y después del tratamiento, observando que en tres de los pacientes de positiva se volvió negativa. El estudio de la intradermorreacción no mostró variante antes y después del tratamiento. De los cuadros expuestos puede deducirse que este producto no ha redituado en el tratamiento de 8 enfermos brucelósicos los resultados descritos por los autores italianos. Los hemocultivos no fueron modificados, las aglutinaciones, la curva térmica, tamaño del hígado y del bazo, el cuadro hemático, la crasis sanguínea, los dolores y la participación articular, las complicaciones neurológicas, ni el estado general mejoraron con la antibrucelina. Notamos en cambio efectos favorables sobre la eritrosedimentación en 6 casos y sobre el índice opsónico en 3. La impresión que tenemos de este medicamento es que no modifica la evolución del cuadro infeccioso, produciendo en cambio bienestar subjetivo que a menudo los pacientes manifiestan al médico, contrastando este hecho subjetivo con la evolución objetiva siempre desfavorable en los casos por nosotros observados. Inconvenientes que presenta esta terapéutica: (a) En todos los pacientes hemos observado una intensa esclerosis venosa que llegó a inutilizar las venas del pliegue del codo de varios pacientes; (b) las

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inyecciones intradérmicas producen necrosis en las pieles delicadas especialmente en la mujeres. CONCLUSIONES

1. Hemos comprobado el poder bacteriostático de la antibrucelina pura en 20 cepas de brucelas melitensis, abortus y suis. De acuerdo con los resultados consignados en este trabajo, nuestras cepas resultaron más sensibles a un antibiótico estudiado que las cepas italianas. 2. Utilizando el producto comercial tal como se presenta envasado resultó tener menor poder inhibidor que la estreptomicina (in vitro). 3. La antibrucelina de Del Veccehio no modificó el cuadro infeccioso de ninguno de los 8 pacientes tratados. 4. La antibrucelina produce intensa esclerosis venosa y necrosis en las pieles delicadas cuando se utiliza la vía intradérmica. 5. No creemos aconsejable este tratamiento en la brucelosis febril. BIBLIOGRAFIA

i

Amodio, U.: Un caso di Artro-Sinovite, Metastase di Brucellosi, curatto con Antibrucellina. Ig. e San. Pub., Vol. IV; mayo-jun. 1948; NN. 5-6. Baldelli, Mario: Un caso di Brucellosi Trattato con Antibrucellina. Ig. e San. Pub., Vol. V; eno.-fbro. 1949; NN. 1-2. Barlotta, P., y Malatesta, A.: Contributi Clinico Alla Cura Delle Brucellosi con Antibrucellina. Ig. e San. Pub., Vol. V, eno.-fbro. 1949. NN. 1-2. Clemente, D.: Un Caso di Febre Melitense Curato con L'Antibrucellina. Ig. e San. Pub. Vol. V, eno.-fbro. 1949; NN. 1-2. Criscuolo, E.; Ramaciotti, F.; y Paolasso, E. R. de W.: Estudio del poder Bacteriostático y Bactericida de la Antibrucelina. Univ. Nac. de Cór., 1950. Criscuolo, E.; Pierangeli, V. N.; Ceballos, A., y Vacchiani, H.: La Antibrucelina en el Tratamiento de la Brucelosis Humana, Univ. Nac. de Cór., 1950. Del Vecehio, G.: La Chemioterapia delle Brucellosi sul Banco di Prova: L'Azione Elettiva della Vitamina K contro le Brucelle. Ig. e San. Pub. Vol. III; ab.-mayo-jun. 1947; NN. i. 4-5-6. Del Vecchio, G.: Chinoni e Brucelle: L'Antibrucellina, Antibiotico Sintetico Ad Aziones Elettiva contro le Brucelle. Ig. e San. Pub., Vol. IV, mayojun. 1948; NN. 5-6. Del Vecchio, G.; Del Vecchio, V., y Argenziano, R.: Sull'Aziones Antibiotica dei Chinoni. Bol. Soc. Ital. Biol. Sper., Vol. XXIV, fasc. 3; 1948; nota VI. Del Vecchio, G.; Del Vecchio, V., y Qrgenziano, R.: Sull'Aziones Antibiotica dei Chinoni. Bol. Soc. Ital. Biol. Sper., Vol. XXIV, fase. 3; 1948; Nota VII. Del Vecohio, G.; Del Veccehio, V., y Argenziano, R.: Sull'Azione Antibiotica dei Chinoni. Dal. Bol. Soc. Ital. Biol. Sper., Vol. XXIV; fase. 3; 1948, Nota V. Del Vecehio, G.; Del Vecehio, V.; Argenziano, R.; Carratf, C.; Napoli, A.: Sull' Azione Antibiotica dei Chinoni. VIII Ulteriori Ricerche Sull'Attivitá Antibiotica in vitro del 2-Metil-1, 4-Naftochinone Verso le Brucelle. Bol. Soc. Ital. Biol. Sper., Vol. XXIV, fasc. 9-10-11; 1948. Del Vecehio, G.; Del Vecchio, V.; Argenziano, R.; Carratú, C.; Napoli, A.: Sull' Azione Antibiotica dei Chinoni. IX L'Attivitá Antibiotica in vitro

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della 2-metil-1,4-Diossinaftalina Verso Le Brucelle. Bol. Soc. Ital. Biol. Sper., Vol. XXIV, fasc. 9-10-11, 1948. Del Vecchio, G.; Del Vecchio, V.; Argenziano, R.; Carratú, C.; Napoli, A.: Sull'Azione Antibiotica dei Chinoni, X, L'Attivitá Antibiotica in vitro del 2-Metil-1,4 Diacetilnaftoidrochinone Verso Le Brucelle. Bol. Soc. Ital. Biol. Sper., Vol. XXIV, fasc. 9-10-11, 1948. Del Vecchio, G.; Del Vecchio, V.; Argenziano, R.; Carratú, C.; Napoli, A.: Sull'Azione Antibiotica dei Chinoni XI, L'Attivitá Antibiótica in vitro dei 2-Metil-1,4-Disuccinilnaftoidrochinone (Sale Sodico) Verso Le Brucelle. Bol. Soc. Biol. Sper., Vol. XXIV, fasc. 9-10-11, 1948. Del Vecchio, G.; Del Vecchio, V.; Napoli, A.; Biondi, G.: Sull'Azione Antibiótica dei Chinoni. XIV L'Attivitá Antibiótica in virto Dell'Naftochinone Sulle Salmonelle. Bol. Soc. Ital. Biol. Sper., Vol. XXIV, fasc. 12, 1948. Del Vecchio, G.; Del Vecchio, C.; Napoli, A.; Biondi, G.: Sull'Azione Antibiotica dei Chinoni. XIII L'Attivitá Antibiótica in vitro del Benzochinone, Deo Chinidrone e Dell'Idrochinone Verso Le Salmonelle. Bol. Soc. Ital. Biol. Sper., Vol. XXIV, fasc. 12, 1948. Del Vecchio, G.; Del Vecchio, V.; Napoli, A.; Nappi, G.: Sull'Azione Antibiótica dei Chinoni. XII Influenza dei Siero di Sangue Di Diversi Animali Sull'Attivitá Antibiótica in vitro del 2-Metil-1,4-Naftochinone. Bol. Soc. Ital. Biol. Sper. Vol. XXIV, fasc. 9-10-11, 1948. Del Vecchio, V., y Virgili, R.: L'Azione Chemioterapica dell'Antibrucellina (Derivato Della Vitamina K Standard) In un Caso di Meningite Brucellare. Ig. e San. Pub.; Vol. IV, mayo-jun. 1948; NN. 5-6. Mirri, A.: La Vitamina K1 Nella Lotta contro la Brucellosi Caprina. Ig. e San. Pub., Vol. V, Lug.-Ag. 1949. NN. 7-8. Mogrovejo, T.: Contributi Alla Terapia della Brucellosi Umana con L'Antibrucellina. Ig. e San. Pub., Vol. V; eno.-fbro. 1949; NN.-1-2. Napoli, A.: Ricerche Sul potere Antibiotico in vitro Dell'Antibrucellina. Ig. e San. Pub., Vol. IV, mayo-jun. 1948. NN. 5-6. Napoli, A.: Ricerche Sul potere Antibiotico in vitro Dell'Antibrucellina. Ig. e San. Pub., Vol. IV, mayo-jun. 1948. NN. 5-6. Nascimbene, A.: La Terapia della Brucellosis Umana con Sostanze Chinoniche I1 Farmaco. sbre.-obre. 1948, No. 5. Panebianco, F.: Ulteriore Contributo allo Studio della Brucellosi umana con trattamento di Antibrucellina. Ig. e San. Pub., Vol. V, eno.-fbro. 1949; NN. 1-2. Panebianco, F.: Risultati clinico-Terapeutici con L'Antibrucellina Nella Brucellosi umana. Ig. e San. Pub., Vol. IV, mayo-jun. 1948. NN-5-6. Panebianco, F.; Napoli, A., y D'Aniello, L.: Azione dell Antibrucellina Nelle Brucellosi umana e suo potere antibiótico in vitro. Ig. e San. Pub., Vol. IV, mayo-jun. 1948. NN. 5-6. Panebianco, F.; Napoli, A., y D'Aniello, L.: Le sepsi da Tipo-Paratifi trattate con sostanze Chinoniche Rilievi Sperimentali Biologici e Clinici. Ig. e San. Pub., Vol. V, mzo.-ab. 1949. NN. 3-4. Panebianco, F.; Napoli, A., y D'Aniello, L.: Azione dell Antibrucellina nelle Brucellosi umana e suo potere antibiótico in vitro. Ig. e San. Pub., Vol. IV, mayo-jun. 1948. NN.-5-6. Petrone, P.: L'Antibrucellina en 5 Casi de Brucellosi. Ig. e San. Pub., Vol. V, mayo-jun. 1949. NN. 5-6. Portanova, S.: L'Antibrucellina in tre casi di Brucellosi. Ig. e San. Pub., Vol. IV, mayo-jun. 1948; NN. 5-6. Tripi, G.: Antibrucellina e Neurobrucellosi. Ig. e San. Pub., Vol. V, eno.-fbro. 1949. NN. 1-2.

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"ANTI-BRUCELLIN" IN THE TREATMENT OF FEBRILE BRUCELLOSIS (Summary) FIRST PART

The authors, after some considerations about the chemistry composition and the antibiotic nature of the chinones as a rule, have done a control work on "antibrucellina" (2 methyl-1 ,4,-naphtochinone) of Del Vecchio and co-workers. These investigators noted decided inhibiting power on four melitensis strains, one paramelitensisand one parabang,with dilutions from 1/100,000 to 1/800,000 between 48 and 192 hours. The authors employed 20 smooth strains of Brucella melitensis, one abortus and one suis strain. They seeded them in a simple broth with pure antibrucellina in dilutions ranging from 1/100,000 to 1/400,000. All the strains, except the suis strain, were sensitive to the dilution 1/100,000 between 48 and 168 hours and 15 strains were sensitive to the dilution 1/400,000 during the same time. The antibrucellina that we could obtain was in a dilution at 3 per thousand. This product, in dilutions at 1/100,000, did not modify the growth of the 15 strains studied by the authors. Six strains were sensitive in dilutions at 1/200,000 and one suffered modification in dilutions at 1/400,000. In plates of agar-liver, pure antibrucellina inhibits between 4 and 6 mm but on the other hand it has no action in dilutions at 1/10; 1/100, etc. With Heatley's method with cylinders, the authors obtained similar results. Finally, in comparative tests, streptomycin showed a greater inhibiting power than antibrucellina. SECOND PART

Encouraged by the favorable results obtained in vitro, the authors treated 8 patients with febrile brucellosis with positive hemoculture for Brucella melitensis. They used the doses, route and time lately advised by Del Vecchio and coworkers, who reported good clinical results in 25 of the 30 cases treated by them. The results obtained by the authors in 8 patients treated with antibrucellina were as follows: (1) The hemoculture remained positive in all cases; (2) Huddleson's reaction did not show any striking variation; (3) the fever did not change in a noticeable form; (4) spleen enlargement disappeared in one; it did not change in 5 and became greater in two of them; (5) liver enlargement did not change in 6 and became greater in two of them; (6) the blood picture did not improve in any of them; (7) the opsonic index increased in three and did not change in the other three; (8) the arthropathies did not improve; (9) the nervous system improved in two and did not change in the rest of them; (10) the general condition improved in two and did not change in the others; (11) the red cell sedimentation rate became lower in six; (12) for the most part our patients felt generally better. The authors believe that this medicine did not modify the evolution of the infectious picture and that it only produces subjective improvement

TERRAMYCIN, CHLORAMPHENICOL AND AUREOMYCIN IN ACUTE BRUCELLOSIS A Preliminary Report* By JOHN H. KILLOUGH, LT. (MC, USN), GORDON B. MAGILL, LT. (jg, MC, USN) AND RICHARD C. SMITH, LT. (jg, MC, USNR) U. S. Naval Medical Research Unit No. 3, Cairo, Egypt Until the advent of the antibiotics, the therapy of acute and chronic brucellosis was viewed with defeatism.' The introduction of the sulfonamides and then penicillin at first engendered hope for the control of this protein disease. 2 However, it was soon found that these agents were only moderately effective in controlling the acute symptoms and ineffective as curative agents. With the addition of streptomycin, its use alone and combined with a sulfonamide, gave the first genuine promise of effective treatment.2 3'4, 7 Immediate clinical response was good and relapses were significantly reduced. Toxicity, however, was serious. Subsequent studies with aureomycin 2 , 5-13 and with chloramphenicol 2 , 8-11, 14, 15 indicate even more rapid clinical response, and relatively negligible toxic manifestations. Reported relapse rates ranged up to 22%. In a more recent careful study of the use of chloramphenicol in brucellosis, Knight et al.' 6 reported 6 relapses in a series of 13 patients. When terramycint became available, it was tested in our laboratories for its in vitro effectiveness against Brucella melitensis. This new antibiotic was found to be as effective as aureomycin and chloramphenicol. In consequence, it was decided to test the clinical effectiveness of terramycin in acute brucellosis and compare it with aureomycin and chloramphenicol. This paper reports our preliminary results. We have found that terramycin is dramatic in alleviating the clinical signs of the acute disease, and that it is as effective as are the compared drugs in preventing relapses. METHOD OF STUDY Clinical Material.-Thirty-nine cases of systemic brucellosis have been treated. All were male patients ranging from 17 to 50 years of age. The original selection of cases was by means of significantly positive Brucella agglutination titers in patients with a history suggestive of brucellosis and a fever of undetermined origin. None had received previous anti* The opinions or assertions contained herein are the private ones of the writers and are not to be construed as official or refiecting the views of the Navy Department or the naval service at large. t Terramycin has been made available for this study through the courtesy of Charles Pfizer & Co., Inc., Brooklyn, N. Y. 296

ANTIBIOTICS IN ACUTE BRUCELLOSIS

297

biotic therapy. At the time of admission, all patients were febrile and acutely ill. The duration of illness before treatment ranged from 11 to 185 days, with an average of 42 days. The degree of fever before treatment varied from 100.2 to 105.8°F per rectum, with the majority of cases in the upper part of this range. Diagnostic Criteria.-Brucella agglutinations were positive in all the patients, ranging in titer from 1:160 to 1:10,240. A series of blood and urine cultures were taken on all patients before institution of therapy. Positive blood cultures were obtained in 36 of the 39 cases. In 33 of these, Brucella melitensis was isolated; the organisms isolated from the other 3 cases have not as yet been finally identified as to the species of Brucella. In addition, urine cultures were positive in 16 cases Of the three cases without any positive cultures, one had an agglutination titer of 1:320, and the other two had titers of 1:2560. Observations.-Rectal temperatures were taken on all patients, with 100.0 ° F being considered the upper limits of normal. Serial blood and urine cultures for Brucella as well as routine laboratory procedures were followed on all patients before, during and after the course of antibiotic therapy. The patients were closely followed for a period of three weeks on the ward after the completion of treatment. If the patients remained afebrile and asymptomatic and their cultures remained negative, they were discharged home to be followed as outpatients. After discharge the patients were checked at intervals of from 1 to 4 weeks with blood and urine cultures for Brucella, as well as with interval histories, physical examinations, and routine laboratory examinations. All of the cases reported have been followed for from 1 to 6 months since the completion of their first course of therapy. Case Selection.-The cases were treated in chronological groups. The first 12 patients admitted received chloramphenicol; the second 11 patients received aureomycin; and the third group of 16 patients received terramycin. Clinically there was no detectable difference in the severity of illness of the 3 groups studied. The majority of cases in all 3 groups had been ill between 1 and 2 months before treatment, the average for the terramycin group being 52 days; for the chloramphenicol group, 37 days; and for the aureomycin group, 33 days. Dosage.-The daily dosage of antibiotic for all groups was calculated on a weight basis. The doses of chloramphenicol and aureomycin were 50 mg/kg/day administered orally on a 3 hourly schedule. The first 15 patients treated with terramycin were given 75 mg/kg/day; the last case received 100 mg/kg/day. Terramycin was administered orally on a 4 hourly schedule. Treatment was continued in all cases for 7 to 14 days after the patient became afebrile. The first 15 patients in the terramycin group received

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an average of 44 grams over an average period of 11.2 days. The last case in the terramycin group received 62 grams in 13.5 days. The patients in the chloramphenicol and aureomycin groups received averages of 36 and 35 grams over average periods of 12.4 and 12.8 days, respectively. RESULTS

Response to Therapy.-(Table 1). Terramycin was found to be fully as effective in controlling the fever and the acute symptoms of these cases TABLE 1.-Summary of Cases Average No. of cases

Terramycin 15 1 Chloramphenicol 12 Aureomycin 11 Total 39

age mg/kg/day

Average no. of

Average

Average no. o'

total dose when days of disease io grams treatment -g/ g1ram1s yin started

number of days treated

days fever after of start of treatment

75 100

44 62

54 24

11.2 13.5

3.4

50

36

37

12.4

5.1

50

35

33

12.8

3.9

-

-

42

-

-

TABLE 2.-Relapse Appraisal

~Drig ~ Terramycin Chloramphenicol Aureomycin Total

No. of cases treated

16 12 11 39

Clinical

Bacterial

Total

Number

%

Number

%

Number

%

8 6 4 18

50 50 36 46

3 2 4 9

19 17 36 23

11 8 8 27

69 66 73 69

1

1

1

as were chloramphenicol and aureomycin. The patients became afebrile within an average of 3.4 days after institution of therapy in the terramycin-treated group, as compared to averages of 5.1 and 3.9 days in the chloramphenicol and aureomycin groups, respectively. Symptoms such as arthritis, arthralgia, headache and asthenia disappeared more slowly than the fever in all groups. Relapses.-(Table 2). Of the thirty-nine cases treated in these series, 27 (69%) have had at least one relapse following completion of therapy. Eighteen (46%) were clinical relapses, and 9 (23%) were bacterial relapses. Twelve of the 18 clinical relapses were associated with Brucella bacteremia. While the table shows certain minor variations in the relapse rates of

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each of the three drugs, we do not believe these to be significant differences. Twenty of the 27 relapses (74%) occurred during the second and third weeks after completion of therapy. Four cases relapsed during the first week, and the other 3 cases relapsed between 4 and 10 weeks after treatment was completed. The average number of days from completion of therapy to onset of relapse was 16 days. Of the total of 27 relapses, 16 have been retreated with one or another of the antibiotics under investigation, in most cases with higher doses over longer periods of time. Five (31%) of these cases have relapsed after this second course of therapy. This series is too small to permit a comparison of the three antibiotics in the retreated group. Side Effects.-No serious toxic effects were noted in any of the groups, nor did minor toxic symptoms require discontinuation of treatment in any of the cases. In the dosages of the drugs as employed in this series, aureomycin caused more discomfort in the patients than did the other two antibiotics. Terramycin and chloramphenicol were both accompanied by negligible side effects. Recently we have increased the dosage of both terramycin and chloramphenicol to 100 mg/kg/day. It is of interest to note that on this increased dosage, terramycin still has had relatively few toxic effects. A great majority of the patients treated with chloramphenicol on this higher dosage schedule, however, have complained of burning of the tongue and shown various degrees of glossitis and chilitis. These cases and their results will be reported in detail in a later paper. DISCUSSION Although only 16 cases treated with terramycin have been recorded in this preliminary report, we believe that the response to terramycin has been sufficiently good to warrant further clinical trials of this new antibiotic. The indications are that it ranks equally well with both chloramphenicol and aureomycin. Whether increased dosage of antibiotic over a more prolonged period of time will tend to reduce the high relapse rate is under investigation. If so, then terramycin may be more valuable in acute brucellosis than either chloramphenicol or aureomycin since terramycin apparently produces fewer toxic effects at high dosage levels. This study reports a very high relapse rate for all three antibiotics. The rate in our series is much higher than previously reported for both chloramphenicol and aureomycin by most investigators.2 5-15 Our figures closely correspond to those of Knight et al.' 6 Probably several factors are involved in our high relapse rates. A large proportion of our patients were in a relatively early, acute, septicemic phase of their disease. Relapses in such a group might well be expected to be more frequent than in a

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more chronically infected group. Another factor may be that we have been in the fortunate position of being able to observe our patients closely in the hospital for at least 3 weeks after completion of treatment. As high as our relapse rates have been to date, it is to be emphasized that even higher rates are probable as the follow-up period lengthens. CONCLUSIONS 1. In a series of 16 cases of acute brucellosis treated with terramycin there was an excellent initial clinical response in every instance. The results with chloramphenicol in 12 patients and with aureomycin in 11 .cases were equally good. 2. That none of these antibiotics are curative, however, is demonstrated by an over-all clinical and/or bacterial relapse rate of 69%. 3. Larger doses of antibiotic appear to be indicated. 4. On higher dosage schedules aureomycin caused considerable patient discomfort, while intensive administration of chloramphenicol produced glossitis and chilitis. 5. One patient tolerated terramycin in doses of 100 mg/kg/day without any untoward effect. REFERENCES (1) Evans, A. C.: Brucellosis (undulant fever). Miscellaneous publication No. 34, U. S. Public Health Service, 1945. (2) Harris, H. J.: Brucellosis (undulant fever). Second Edition, 1950. Paul B. Hoeber, Inc., New York, N. Y. (3) Molinelli, E. A., Ithurralde, D., Basso, G., Miyara, S., Speroni, A., Vital, V. C., Cabassi, E., and Pandolfo, G.: Recent developments in the therapy of human brucellosis. P. R. J. Pub. Health & Trop. Med. 26: 29, 1949. (4) Spink, W. W., Hall, W. H., Shaffer, J., and Braude, A. I.: Treatment of brucellosis with streptomycin and a sulfonamide drug. Jour. Am. Med. Assoc. 189: 352, 1949. (5) Bryer, M. S., Schoenbach, E. B., Wood, R. M., and Long, P. H.: The treatment of acute brucellosis with aureomycin. Bull. Johns Hopkins Hosp. 84: 444, 1949. (6) Spink, W. W., Braude, A. I., Castafeda, M. R., and Goytia, R. S.: Aureomycin therapy in human brucellosis due to Brucella melitensis. Jour. Am. Med. Assoc. 138: 1145, 1948. (7) Braude, A. I., Hall, W. H., and Spink, W. W.: Aureomycin therapy in human brucellosis due to Brucella abortus. Jour. Am. Med. Assoc. 141: 831, 1949. (8) Harris, H. J.: Aureomycin and chloromycetin in brucellosis. Bull. New York Acad. Med. 256: 458, 1949. (9) Harris, H. J.: Aureomycin and chloramphenicol in brucellosis. Jour. Am. Med. Assoc. 142: 161, 1950. : (10) Ralston, R. J., and Payne, E. H.: Treatment of chronic brucellosis with chloramphenicol and aureomycin. Jour. Am. Med. Assoc. 142: 159, 1950. (11) Woodward, T. E.: Chloromycetin and aureomycin: Therapeutic Results. Ann. Int. Med. 31: 53, 1949.

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(12) Galpine, J. F.: A case of abortus infection treated with "Aureomycin". Lancet 1: 1037, 1949. (13) Rose, H. M., and Kneeland, Y. Jr.: Aureomycin in the treatment of infectious diseases. Am. Jour. Med. 7: 532, 1949. (14) Walloey, J. F. L., and Cooper, T. V.: A case of undulant fever treated by chloramphenicol. Brit. Med. Jour. 2: 265, 1949. (15) Woodward, T. E., Smadel, J. E., Holbrook, W. A., and Raby, W. T.: The beneficial effect of chloromycetin in brucellosis. Jour. Clin. Investigation. 38: 968, 1949. (16) Knight, V., Ruiz-Sánchez, F., and McDermott, W.: Chloramphenicol in the treatment of acute manifestations of brucellosis. Am. Jour. Med. Sc. 219: 627, 1950. CASE REPORTS Case No. 1 (Patient # 183) N.B. This case is presented to illustrate two things: first, the clinical response to terramycin treatment and second, the interesting sequence of events in the relapse, namely, the occurrence of the positive urine culture on the ninth day after treatment, followed by the positive blood cultures on the twenty-second and twenty-third days, followed in turn by the severe clinical relapse on the twenty-ninth day. A 47-year-old Egyptian male was admitted because of a present illness of four months duration consisting of recurrent episodes of fever, headache, weight loss, general prostration and joint pains involving all of the joints of his extremities. Physical examination revealed a very ill man with a rectal temperature of 101.4°F. The other positive findings were palpable generalized tender lymph glands and slight liver enlargement. The patient had a Brucella agglutination titer of 1:1280 and several positive blood cultures for Brucella melitensis. Treatment with terramycin was started in the fourth day with a loading dose of 1.0 gm and continued on the basis of 75 mg/kg/day. The drug was discontinued after seven afebrile days with a total dose of 26 gm. The patient was followed for twenty-three days and then discharged. Subsequently, a urine culture on the ninth day after treatment was reported as positive as were blood cultures drawn on the twenty-second and twenty-third days. The patient returned nine days after discharge with a severe recurrence of all of his symptoms. Due to lack of terramycin, aureomycin was instituted on the basis of 50 mg/ kg/day. The patient became afebrile on the fifth day and the drug was discontinued after fourteen afebrile days, the patient having received a total dose of 56 grams. The patient was discharged twenty-one days later and has been observed for one-and-a-half months as an outpatient. Case No. 2 (Patient #213) N.B. This case is presented because it demonstrates a typical fever response to terramycin and because a bacterial relapse without symptoms occurred following therapy.

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A 25-year-old Egyptian male was admitted with a present illness of 22 days duration consisting of headache, fever, dizziness and incapacitating joint pains in all of his extremities. Physical examination revealed a seriously ill man with a rectal temperature of 103.6°F. Other positive physical findings were generalized slight lymphadenopathy and moderate abdominal tenderness over the liver and spleen. The patient had a positive Brucella agglutination titer of 1:5120 and several blood and urine cultures were positive for Brucella melitensis.

On the fourth hospital day terramycin was started with a loading dose of 1.25 grams and continued on the basis of 75 mg/kg/day. All complaints had disappeared by the third day of treatment and the patient became afebrile on the sixth day. Terramycin blood levels were determined on several occasions; the highest level was 11.9 gamma/cc. Terramycin was discontinued on the fourteenth afebrile day after a total dose of 75 grams. The patient has now been followed for one month and has remained asymptomatic in spite of a slight temperature rise on the eleventh and twelfth days after treatment. Urine cultures taken during this brief febrile episode were again positive for Brucella melitensis; blood cultures were negative. Results of further blood and urine cultures have not yet been reported. LA TERRAMICINA, EL CLORANFENICOL Y LA AUREOMICINA EN LA BRUCELOSIS AGUDA INFORME PRELIMINAR (Sumario)

Treinta y nueve varones de 17 a 50 años, que no habían recibido antes ningún antibiótico, y que manifestaban fiebre y enfermedad aguda de 11 a 185 días de duración, fueron divididos en tres grupos para tratamiento: 16 recibieron terramicina, 12 cloranfenicol, y 11 aureomicina. Todos ellos acusaban aglutinorreacciones positivas para brucelas. En 36 se obtuvieron hemocultivos positivos, y en 33 de éstos se aisló la Brucellamelitensis. No se ha identificado todavia la especie en los otros tres, pero uno reveló aglutinación al 1:320 y los otros dos al 1:2,560. Los cultivos urinarios resultaron positivos en 16. El cloranfenicol y la aureomicina se administraron por vía bucal cada tres horas a razón de 50 mg/kg/día. La terramicina se administró por via bucal cada cuatro horas a razón de 75 mg/kg/dia, excepto en un enfermo que recibió 100 mg/kg/día. El tratamiento se continuó de 7 a 14 dias después de desaparecer la fiebre. Los tres medicamentos resultaron eficaces para controlar la fiebre y los sintomas agudos, pero la terramicina produjo al parecer menos efectos tóxicos. Sin embargo, ninguno de estos medicamentos cura, según demuestra el coeficiente global de recaídas de 69%, distribuidas poco más o menos igualmente en los tres grupos. Se llevan a cabo otros estudios utilizando dosis mayores.