PRESENCE OF ACID-FAST BACTERIA IN WILD AND CULTURED SILVER MULLETS (Mugil curema VAL., 1836) FROM MARGARITA ISLAND, VENEZUELA Aldeima T. Perez T., David A. Conroy and Luis Quiñones

SUMMARY Twenty specimens of silver mullet (Mugil curema Val., 1836) from the La Restinga coastal lagoon complex in Margarita Island, Venezuela, and 20 specimens of the same species cultured in sea water ponds at Boca de Río, in the same locality, were examined for the presence of acid-fast bacteria. Samples from the faeces and intestinal contents, kidney, liver and spleen were treated by Petroff method. Smears were taken from the centrifuged sediment and stained by a modified ZiehlNeelsen technique, and the sediment was cultured on tubes of Löwenstein-Jensen medium. Acid-fast bacteria were detected in smears from five (25%) of the wild mullets, and five (25%) of the cultured mullets. Three strains of mycobacteria were isolated

from the wild fish and one strain from the cultured fish. These four strains were identified as Mycobacterium chelonei subsp. abscessus, M. chelonei subsp. chelonei, M. fortuitum and M. scrofulaceum. The strains were isolated from the faeces and spleens of the infected fish. The presence of mycobacteria as the aetiological agents of tuberculosis in grey mullets and other fish species is discussed in relation to the importance of these organisms as potential human pathogens. It is recommended that populations of mullets used as a source of seed or spawning stock for aquaculture operations be carefully monitored to detect the possible occurrence of latent and/or patent acid-fast bacterial infections.

RESUMEN Se examinaron 20 ejemplares de lisa criolla (Mugil curema Val., 1836) procedentes de la Laguna La Restinga, Isla de Margarita, Venezuela, y 20 ejemplares de la misma especie cultivados en estanques en Boca de Río, en la misma localidad, para determinar la presencia de bacterias ácido-resistentes. Las muestras de heces y contenido intestinal, riñón, hígado y bazo se trataron por el método de Petroff. Se realizaron frotis del sedimento centrifugado, coloreados por la técnica modificada de Ziehl-Neelsen, y el sedimento fue cultivado en el medio de Löwenstein-Jensen. Las bacterias ácido-resistentes se detectaron en cinco (25%) ejemplares silvestres y cinco (25%) cultivados. Se aislaron tres cepas de micobacterias de los peces silvestres y

Introduction The first descriptions of spontaneous infections caused by acid-fast bacteria in species of marine and freshwater fish include those by Bataillon et al. (1897), Alexander (1913), Johnstone (1913, 1927), Sutherland (1922), Aronson (1926), Baker and Hagan (1942) and Winsor (1946).

Nigrelli and Vogel (1963) reported that no less than 150 species of fish, belonging to 84 genera, 34 families, and 10 orders, are susceptible to infection by acid-fast bacteria, and Ghittino (1985) has commented that no fish species resistant to experimental or spontaneous infections by acid-fast bacteria appear to be known. The potential importance of tuberculosis in salt-

una de los peces cultivados. Las cuatro cepas se identificaron como Mycobacterium chelonei subsp. abscessus, M. chelonei subsp. chelonei, M. fortuitum y M. scrofulaceum. Las cepas se aislaron de las heces y bazos de los peces infectados. Se discute la presencia de micobacterias como agentes etiológicos de tuberculosis en éstos y otras especies de peces en relación con la importancia de estos microorganismos como patógenos potenciales en humanos. Se recomienda que las poblaciones de lisas usadas como fuentes de alevines o reproductores en operaciones de acuacultura sean cuidadosamente tratadas para detectar la posible presencia de infecciones, activas o latentes, por bacterias ácido-resistentes.

water fish has been discussed by Conroy (1970). Information on the occurrence of acid-fast bacterial infections in mugilid species, however, has only appeared very recently. Othman (1980) examined 70 specimens of golden mullet (Liza aurata [Risso, 1810]) from the Mediterranean coast of Tripoli, Libya, and isolated seven strains of acid-fast bacteria

from the intestinal contents of 9.99% of these fish. In the Americas, Rodrigues and Fernandes (1983) reported a single spontaneous case of tuberculosis in a wild adult silver mullet (Mugil curema) captured in the Santa Cruz Channel, Itamaracá, Pernambuco State, Brazil, and Couch (1985) detected a case of the disease in a single adult striped mullet (M. cephalus L., 1758) captured

Sección de Patobiología Acuática, Facultad de Ciencias Veterinarias, Universidad Central de Venezuela, Maracay, Venezuela. e-mail: [email protected]

Luis Quiñones. Licenciado en Bioanálisis. Sección de Bacteriología, Instituto Nacional de Tuberculosis “Dr. José Ignacio Baldó”, El Algodonal, Caracas.

KEYWORDS / Mugil curema (Mullet) / Mycobacterium / Recibido: 09/03/2001. Aceptado: 08/05/2001

Aldeima T. Perez T. Licenciada en Ciencias Aplicadas del Mar, Mención Acuacultura Marina. Investigador, Instituto Limnológico de la Universidad de Oriente (UDO), Caicara del Ori-

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noco, Estado Bolívar, Venezuela. e-mail: [email protected] David A. Conroy. Doctor en Ictiopatología. Profesor, Departamento de Ictiopatología,

0378-1844/01/06/252-05 $ 3.00/0

JUN 2001, VOL. 26 Nº 6

RESUMO Foram examinados 20 exemplares de lisa nativa (Mugil curema Val., 1836) procedentes da Lagoa “La Restinga”, Ilha de Margarita, Venezuela, e 20 exemplares da mesma espécie cultivadas em tanques na Boca do Rio, na mesma localidade, para determinar a presença de bactérias ácido-resistentes. As amostras de fezes e conteúdo intestinal, rim, fígado e baço foram tratados pelo método de Petroff. Realizaram-se frote do sedimento centrifugado, coloridos pela técnica modificada de Ziehl-Neelsen, e o sedimento foi cultivado no meio de Löwenstein-Jensen. As bactérias ácido-resistentes foram detectadas em cinco (25%) exemplares silvestres e cinco (25%) cultivados. Isolaram-se três cepas de microbactérias dos peixes silvestres e uma dos peixes

in estuarine environments of the northern Gulf of Mexico. In both of these two cases described from the American continent, the diagnosis was made on the basis of examination of material previously fixed, so that it was not possible to isolate the bacteria. The purpose of the present work was to attempt the detection, isolation and identification of acid-fast bacteria occurring in wild and cultured populations of silver mullet (Mugil curema VAL., 1836; fam. Mugilidae) in waters of Margarita Island, in eastern Venezuela. Materials and Methods A total of 20 wild silver mullet and 20 cultured silver mullet were examined. The wild specimens were caught by means of a cast net from areas known as La Maceta, La Redonda, Paso e’ Caballo and Los Canales, in the La Restinga hypersaline (S= 40‰) Lagoon complex (Figure 1), and the cultured specimens were obtained from sea water ponds of the fish farm of the Universidad de Oriente Scientific Research Center at Boca de Río, both localities corresponding to Margarita Island, Nueva Esparta State, Venezuela. The mullets were transported in closed plastic buckets containing seawater to the laboratory, where they were measured and weighed. They were subsequently subjected to a careful external and internal examination to detect the pres-

ence of any obvious disease signs, parasites or abnormalities. The digestive tract, kidney, liver and spleen were carefully cut and removed with sterilized instruments. A portion of each of these organs was removed and treated according to the Petroff (1915) method, consisting in the homogenization and decontamination of the material to counteract the growth of non-acidfast organisms. Smears were taken from the centrifuged sediment and stained by the Ziehl-Neelsen technique as recommended by Bullock (1971) for piscine acid-fast bacteria, and were examined microscopically using an oil immersion objective. The sediments were aseptically inoculated into tubes of LöwensteinJensen medium and maintained in the horizontal position for 24 hours to allow the bacteria to become fixed to the surface of the medium, after which they were incubated in the vertical position at 250C for 8-12 weeks. All isolates obtained were further cultured on LöwensteinJensen medium, and only pure cultures were taken for strain characterization. The tests carried out for identification included: test for growth rate, pigmentation (Runyon, 1955; Kestle et al., 1967; McClung, 1974), colony morphology on LöwensteinJensen medium (Marks and Trollope, 1960), influence of growth temperature (Kestle et al., 1967), niacin production (Runyon et al., 1959; Kubica,

JUN 2001, VOL. 26 Nº 6

cultivados. As quatro cepas foram identificadas como Mycobacterium chelonei subsp. abscessus, M. chelonei subsp. chelonei, M. fortuitum e M. scrofulaceum. As cepas foram isoladas das fezes e baços dos peixes infetados. Discute-se a presença de microbactérias como agentes etiológicos de tuberculose nestes e outras espécies de peixes em relação com a importância destes microorganismos como patógenos potenciais em humanos. Se recomenda que as populações de lisas usadas como fontes de alevines ou reprodutores em operações de aquacultura sejam cuidadosamente tratadas para detectar a possível presença de infecções, ativas ou latentes, por bactérias ácido-resistentes.

Figure 1. Location of sampling stations in La Restinga Lagoon, Margarita Island, Venezuela. Sampling stations: La Maceta (1), La Redonda (2), Paso e’Caballo (3), Los Canales (4).

1964), nitrate reduction (Kubica, 1964), catalase activity (Kubica and Pool, 1960; Kubica et al., 1966), Tween hydrolysis (Wayne et al., 1964), Tellurite reduction (Kilburn et al., 1969), growth on Löwenstein-Jensen medium containing 5% sodium chloride (Kestle et al., 1967), arylsulfatase (Jones et al., 1966), urease (Gordon and Mihm, 1959), growth in MacConkey agar (Jones and Kubica, 1965), iron uptake (Szabó and Bory, 1973) and the citrate utilization test (Gordon and Mihm, 1959). The following positive controls were used: Mycobacterium fortitium for catalase activity, M. kansasii for Tween 80 hydrolysis, M. fortitium or M. intracellulare for Tellurite reduction, and M. scrofula-

ceum or M. fortitium for the urease test. Results and Discussion The examination of stained smears prepared from the centrifuged sediment of the organs treated by the Petroff method revealed the presence of acidfast bacteria in five (25%) of the 20 wild silver mullets, and in five (25%) of the 20 cultured silver mullets. The results of this work provide the first records of the occurrence of acid-fast bacteria infections in species of silver mullets from Venezuelan waters. Three strains of acid-fast bacteria were isolated from the wild specimens, and one strain was isolated from the cultured specimens of silver mullet examined.

253

Citrate utilization

Iron uptake

Growth in MacConkey agar

Urease 3 days

2 weeks

Tellurite Reduction

Tween Hydrolysis

Catalase Activity

Arylsulfatase 7 days

-

3 days

+

5% Sodium Chloride

+

3 days

+

10 days

Mycobacterium scrofulaceum Sm. - Y-Or Or +

5 days

45ºC

9

68°C

40ºC

8

> 45°C

37ºC

7

4

INAH

Niacin 32ºC

Dark

6

1

Nitrate Reduction

Growth Temperature

Pigment production 2 weeks

3

25ºC

Group II

2

5

Character #

Group IV Nonphotochromogens

Light 1 hour

Colony Morphology

TABLE I CHARACTERISTICS OF THE STRAINS OF MYCOBACTERIA ISOLATED FROM WILD AND CULTURED SILVER MULLET (Mugil curema) IN MARGARITA ISLAND, VENEZUELA

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 -

-

-

-

-

-

±

+

(Scotochromogens)

M. fortuitum

Sm. -

-

+

+

+

+

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

M. chelonei subsp. abscessus

Sm. -

-

+

+

+

+

-

-

-

+

+

-

-

+

+

+

+

+

-

+

-

-

M. chelonei subsp. chelonei

Sm. -

-

+

+

+

+

-

-

-

+

+

+

-

-

-

-

-

±

+

+

-

+

Sm.: Smooth; Y: Yellow; Or.: Orange; ±: 50 – 84% positive strains The characteristics of the isolated strains are given in Table I. The organisms were identified as Mycobacterium chelonei subsp. abscessus (one strain), M. chelonei subsp. chelonei (one strain), M. fortuitum (one strain) and M. scrofulaceum (one strain). No strains of Nocardia sp. were isolated from the material examined. The strains identified as M. chelonei subsp. abscessus and M. chelonei subsp. chelonei were isolated from the spleen of an adult wild female (total length: 370mm; weight: 600g) and from the spleen of an adult wild male (total length: 350mm; weight: 430g). The faeces of this same wild adult female also yielded the strain of M. scrofulaceum. The strain of M. fortuitum was isolated from the faeces of a cultured female (total length: 273mm; weight: 190g). The acid-fast bacteria, as a group, have been the objects of exhaustive investigations to clarify aspects of their taxonomy and create an adequate

254

scheme for their classification. Runyon (1955) established such a scheme for the classification of the “atypical mycobacteria”, based on the growth rate and pigment production of the organisms. This practical scheme was used in the present work for the preliminary classification of the acid-fast bacteria isolated from silver mullets. Othman (1980) reported the isolation of seven strains of mycobacteria, identified as M. fortuitum, M. marinum, M. tuberculosis and M. thamnopheos, from the intestinal contents of golden mullets in Libya. Ashburner (1977) isolated M. chelonei as the aetiological agent of a mycobacteriosis affecting cultured chinook salmon (Oncorhynchus tschawytscha Walb.) in Australia. Arakawa and Fryer (1984) studied five strains of mycobacteria isolated from cut throat trout (Salmo clarki [Richardson]), chinook salmon and coho salmon (O. kisutch Walb.) from the States of Montana and Oregon,

U.S.A., which they concluded to represent a new subspecies, termed M. chelonei subsp. piscarium. With reference to the taxonomic status of their isolates of M. chelonei from North America, Arakawa and Fryer (1984) commented on the fact that the five strains were obviously related to M. chelonei subsp. abscessus and M. chelonei subsp. chelonei, but that their biochemical properties were intermediate to those of both of these subspecies, in addition to which all of the salmonid isolates failed to grow at 37 ° C. On this basis, therefore, Arakawa and Fryer proposed the new subspecies piscarium. The present isolates from Venezuelan mugilids, identified as M. chelonei subsp. abscessus and M. chelonei subsp. chelonei, differ from M. chelonei subsp. piscarium in their ability to grow at 37°C. M. chelonei was originally isolated by Friedmann (1903) from a spontaneous case of tuberculosis in a turtle (Caretta caretta = “Chelonei corti-

cata”) in the Berlin Aquarium. M. chelonei, M. fortuitum and M. marinum are generally accepted as being fish pathogens, and that most of the strains isolated from fish usually fall into one of these species groupings (Shotts and Teska, 1989; Belas et al., 1995; Teska et al., 1997; Talaat et al., 1999). M. chelonei subsp. abscessus, for example, has been isolated from the water of the Aquarium at Antwerp Zoo (Pattyn et al., 1971). To illustrate the wide diversity among the strains of acid-fast bacteria associated with fish, the findings of von Kormendy et al. (1979) can be cited. These workers examined 12 specimens of Paradise fish (Macropodus opercularis L.) maintained in aquaria, from the livers and spleens of which they isolated 25 strains of mycobacteria. These isolates were identified as eight strains (32%) of Mycobacterium fortuitum, four (16%) of M. marinum, three (12%) of M. smegmatis, two

JUN 2001, VOL. 26 Nº 6

(8%) of M. aquae, two (8%) of M. terrae, one (4%) of M. parafortuitum, and five (20%) which could not be identified at species level. The relationship between M. fortuitum and disease in man is now widely recognized (Talaat et al., 1999). This species has been isolated from localized “cold abscesses”, pus from cervical lymphatic nodules, lungs and sputum in human subjects, and from cattle, soil and water. In view of the wide variety of potential foci of infection, the occurrence of M. fortuitum in water bodies could indicate an inter-relationship between fish (including the grey mullets) and the human population, as was postulated by Othman (1980) and Shih et al. (1997). A similar situation holds true for M. chelonei, the two principal subspecies of which (abscessus and chelonei) are known to cause infections in man (Grange, 1985). The present work also provides the first report on the occurrence of M. scrofulaceum in fish. This species is reported to cause approximately 10% of the cases of tuberculous cervical adenitis detected in human beings, but only rarely does it produce a more generalized infection (Prissick and Masson, 1956; 1957). It has additionally been isolated from sputum, cervical lymphatic nodules in human subjects, soil and water samples (Wayne and Doubek, 1968; Prosser 1989; Falkinham et al., 1989). The sources of infection by mycobacteria in fish have not been precisely determined in all cases. It is recognized, however, that the natural mode of infection is probably by direct ingestion of the organism, and in the case of hatchery-reared salmon and trout it is known that infection can occur when untreated adult salmon offal is used as food (Ross et al., 1959; Wood and Ordal, 1958). Othman (1980) suggested that infection of fish, including grey mullets, by Mycobacte-

rium spp. could occur as a result of: (a) feeding of the fish on snails carrying the mycobacteria; (b) contamination of the water bodies by raw sewage containing such bacteria; and (c) rubbish or sewage discharged into the sea from vessels. In the present instance, it is also considered likely that the infections may have occurred as a result of the seepage of raw sewage and other wastes from nearby communities into the water. It is concluded that more careful attention should be given to the recognition of possible cases of acid-fast bacterial infections in wild populations which are used as a source of seed or spawning stock for silver mullet aquaculture operations. Since infected fish do not always show external clinical signs of tuberculosis, a chronic disease, it is recommended that the Petroff technique be used to detect the presence of acidfast bacteria in the faeces and intestinal contents, and that the material be cultured on Löwenstein-Jensen medium in an attempt to isolate and subsequently characterize such bacteria. ACKNOWLEDGMENTS

Arakawa CK, Fryer JL (1984) Isolation and characterization of a new subspecies of Mycobacterium chelonei infectious for salmonid fish. Helgolander Meeresunters 37: 329-342. Aronson JD (1926) Spontaneous tuberculosis in salt water fish. J. inf. Dis. 39: 315-320. Ashburner LD (1977) Mycobacteriosis in hatchery-confined chinook salmon (Oncorhynchus tshawytscha Walbaum) in Australia. J. Fish Biol. 10: 523-528. Baker JA, Hagan WA (1942) Tuberculosis of the Mexican platyfish (Platypoecilus maculatus). J. inf. Dis. 70: 248-252. Bataillon E, Dubard X, Terre L (1897) Un nouveau type de tuberculose hamaine et de la tuberculose aviaire. Compt. rend. Soc. Biol. 49: 446-449. Belas R, Faloon P, Hannaford A (1995) Potential applications of molecular biology to the study of fish mycobacteriosis. Ann. Rev. Fish. Dis. 5:133173. Bullock GL (1971) The identification of fish pathogenic bacteria. In Snieszko SF, Axelrod HR (Eds) Diseases of Fishes, Book 2B, TFH Publications Inc., Neptune City, U.S.A. 1st. edition. 41 pp. Conroy DA (1970) Piscine tuberculosis in the sea water environment. In Snieszko SF (Ed) A Symposium on the Diseases of Fishes and Shellfishes. Am. Fish. Soc. Special Publn. 5: 273-278.

The authors are grateful to Jesus R. Sosa (Clinical Diagnostic Laboratory, Sanatorio Antituberculoso de Oriente, Cumaná, Venezuela) for his assistance in the identification of the mycobacterial strains isolated.

Couch JA (1985) Prospective study of infectious and noninfectious diseases in oysters and fishes in three Gulf of Mexico estuaries. Dis. aquat. Org. 1: 59-82.

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