ANATOMOPATHOLOGICAL DIAGNOSIS OF MYCOBACTERIOSIS IN BULLFROGS ( Rana catesbeiana Shaw, 1802 ) FROM BRAZILIAN COMMERCIAL FROGFARMS

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1 ARS VETERINARIA, 15(2): , ANATOMOPATHOLOGICAL DIAGNOSIS OF MYCOBACTERIOSIS IN BULLFROGS ( Rana catesbeiana Shaw, 1802 ) FROM BRAZILIAN COMMERCIAL FROGFARMS ( DIAGNÓSTICO ANATOMOPATOLÓGICO DE MYCOBACTERIOSE EM RÃS TOURO-GIGANTE ( Rana catesbeiana, SHAW, 1802 ) DE CRIAÇÕES COMERCIAIS ). J. R. E. MORAES 1, M. L. MARTINS 2, V. N. SOUZA 2, F. R. MORAES 1, JR. F. L. SOUZA 3 SUMMARY During the period of March, 1995 to January, 1996 seven cases of mycobacteriosis in the bullfrog (Rana catesbeiana Shaw, 1802) obtained from commercial frogfarms, were identified by anatomopathological technics. The disease affected reproductive frogs ( g) and from the confinement ( g). Clinical manifestations were apathy, anorexia and thinning. Routine examinations revealed a great number of white opaque nodular lesions, measuring 1.0 to 2.0 mm diameter, firm consistency, with multicentric distribution, especially liver and spleen, but infrequently in the kidney, heart, intestine, estomach, lungs and fat. Fragments from the affected organs were collected and fixed in 10% buffer formalin. The tissue sections were stained as usual with haematoxylin-eosin, Ziehl-Neelsen and Lillie. Microscopically analysis showed chronic granulomatous inflammations at different stages of development. The granuloma showed a small caseous necrosis surrounded by epithelioid cells, macrophages, lymphocytes, small number of trombocytes, eosinophils and some pigmented cells called melanomacrophages. These elements were surrounded by a capsule of fibrocytes, fibroblasts and collagen fiber. Young lesions showed accumulation of macrophages and lymphocytes, while mature lesions presented loosely organized masses surrounded by connective capsule. Ziehl-Neelsen method revealed a great number of acid-fast rod. Such lesions were observed in adult frogs slaughtered for human consumption, but without apparent signals. KEY-WORDS: Rana catesbeiana, frog, Micobacteria, mycobacteriosis. RESUMO Durante o período de março de 1995 a janeiro de 1996 sete casos de micobacteriose em rãs touro gigante (Rana catesbeiana, Shaw, 1802) oriundas de criações comerciais foram identificados por técnicas anatomopatológicas. A enfermidade afetou os reprodutores (280 a 300 g) e os animais de engorda (150 a 190 g). As manifestações clínicas foram de apatia, anorexia e emagrecimento progressivo. Os exames revelaram a presença de elevado número de lesões nodulares, opacas, esbranquiçadas, com 1,0 a 2,0 mm de diâmetro, consistência firme e distribuição multicêntrica, comprometendo 1. Department of Veterinary Pathology, Faculty of Agricultural and Veterinary Science-UNESP, Rod. Carlos Tonanni km 5, , Jaboticabal, SP, Brazil; 2. Aquaculture Center - UNESP, Rod. Carlos Tonanni km 5, , Jaboticabal, SP, Brazil; 3. Department of Parasitology, Institute of Biomedical Sciences, USP, Av. Prof. Lineu Prestes 1374, , SP, Brazil.

2 111 ARS VETERINARIA, 15(2): , especialmente o fígado e o baço e, menos freqüentemente os rins, o coração, o intestino, o estômago, os pulmões e tecido gorduroso. Fragmentos de órgãos e tecidos afetados foram colhidos e fixados em formalina tamponada a 10%. Após processamento usual em histologia para cortes em parafina, secções de 6,0 μm de espessura foram coradas com hematoxilinaeosina, pelo método de Ziehl-Neelsen e pelo de Lillie. O exame dos nódulos à microscopia de luz revelou inflamação crônica granulomatosa em diferentes estágios de desenvolvimento. Sua estrutura era composta por necrose caseosa circundada por células epitelióides, macrófagos, linfócitos e, em menor número, trombócitos, eosinófilos e macrófagos pigmentados, denominados melanomacrófagos. Tais elementos estavam circundados por cápsula constituída por fibrócitos, fibroblastos e fibras colágenas. As lesões jovens apresentavam acúmulos de macrófagos e linfócitos enquanto que lesões mais antigas eram compostas de massas de tecido necrótico pobremente organizadas, circundadas por cápsula fibrosa. O método de Ziehl-Neelsen revelou presença de grande quantidade de bastonetes alcool-ácido resistentes nas lesões. PALAVRAS-CHAVE: Rana catesbeiana, rã, Micobacteria, mycobacteriosis INTRODUCTION Tuberculosis study in cold-blooded animals was stimulated by the report of BATAILLON et al.(1897) about the disease occurrence in carp reared in a pond contaminated with dejecta from tubercular persons. Later, BATAILLON et al. (1902) identified the causing agent in the carp as Mycobacterium piscium. KUSTER (1905) observed spontaneous tuberculosis in Rana temporaria. Morphological characteristics of such tuberculosis was similar to mammals. Moreover, isolated bacteria from frogs was identified by BERGEY et al. (1923) as M. ranae, non pathogenic to laboratory mammals. By another hand, NIGRELLI (1953) isolated M. fortuitum from Hyphessobrycon innesi and ROOS & BRANCATO (1959) isolated this same pathogenic rod species from the human and cattle lesions in the South America. Other species like M. cheloni (FRIEDMAN, 1903), M. marinum (ARONSON, 1926), M. thamnopheos (ARONSON, 1929), M. avium (SCHWABACHER, 1959) were isolated from reptiles, fishes, reptiles and amphibian respectively. In Brazil, DARZINS (1950, 52) and BARROS et al. (1988) described mycobacteriosis outbreaks in Leptodactylus pentadactylus and Rana catesbeiana, respectively. In the first relate, the frogs were from Bahia state and were captured in ponds and spring water. The lesions had a nodular aspect, white yellowish color with different size and number specially in liver, being observed in lungs, peritoneum and intestine. Young lesions was composed by accumulations of mononuclear cells surrounded by thin connective capsule. Old lesions presented a caseous necrosis surrounded by a greater fibrous developments. Langhan s cells were not observed, but acid-fast rod were present. The authors called it M. giae. In the second case, described by BARROS et al. (1988) the frogs were from Rio de Janeiro state and presented anorexia, thinning and apathy. Similar lesions and clinical signs were observed by BARROS et al. (1988) in Rana catesbeiana and the authors observed the presence of heterophils in the granuloma. The same authors reported that the cultural and biochemical characteristics were the same as M. marinum. Mycobacteriosis in R. catesbeiana from commercial frogfarms, in São Paulo State, was observed by SOUZA (1993). From 470 dissecated animals, 169 presented granulomatous lesions and 61 were positive for the presence of acid-resistent rods. Biochemical analysis revealed M. marinum in 50 samples, M. chelonae abscessus in 2 sample, M. gordonae in one sample and Mycobacterium sp in 3 samples. In the present work, the authors reported the diagnosis of mycobacteriosis in the bullfrog Rana catesbeiana from commercial frogfarms, and described its morphological characteristics. MATERIAL AND METHODS Bullfrogs utilized in this study (Rana catesbeiana Shaw, 1802) were sent to the Pathology Laboratory of Aquatic Organisms at Aquaculture Center of UNESP, Jaboticabal, SP, Brazil, from commercial frogfarms located in Pará state. The animals were collected during a period of 10 months (March, 1995 to January, 1996). The affected bullfrogs from confinement and reproduction box were analised. After anamnesis and clinical examination the survived animals were slaugthered for post-morten examination. The macroscopic changes were also described. Fragments of changed organs were fixed in 10% buffered formalin for histopathological examination by usual histological technics. Paraphin tissue sections with 6.0 (m thickness were stained with haematoxylin-eosin. We utilized the Ziell-Nielsen technic for acid-resitent rods, Lillie technic for melanin characterization and Van Kossa for calcium characterization. RESULTS Clinical signs Fourty percent dissecated animals (n=54) from

3 ARS VETERINARIA, 15(2): , confinement weighting 150 to 190 g nodular lesions were observed. In this case the owner complained of bad aspect (tubercles and liver color) of the internal organs. A total of fourteen reproducer weighting 280 to 300 g showed apathy signs, anorexia and thinning, that frequently develops to death. Necroscopic examination All reproducers (n=14) and 40% of the confined frogs (n=54) presented lesions similar to mycobacteriosis. Necroscopic examination revealed isolated or agglomerated tubercles measuring 1.0 to 3.0 mm diameter, with firm consistency, white yellowish color and caseous content. The nodules had a multicentric distribution in the internal organs, especially liver and spleen, but infrequently in the kidney, heart, intestine, estomach, lungs and fat. The liver appearance was gray in several cases and the volume of spleen was 3 to 4 times its normal size, with firm consistence. Sometimes the kidney presented increase of size, more frequently pale color. Histopathological examination showed chronic granulomatous inflammatory process in different stages of development at all celomatic organs. Such granuloma were seen alone or agglomerated and in some cases coalescenced. Young lesions showed accumulation of macrophages and lymphocytes. In other developed granuloma, those cells were involved by a slender connective capsule shaped by fibroblasts and collagen fiber. Nevertheless, it was possible to observe some lesions with a little caseation, surrounded by epithelioid cells, melanin pigmented macrophages (melanomacrophages), sometimes without melanin, lymphocytes, trombocytes and a small number of neutrophils. Surrounding or superposing the granulomatous connective capsule, groups of eosinophils were present. In another cases the granuloma presented a loosened organized masses composed by cell debris and involved by a dense capsule shaped by fibrocytes and collagen. Mononuclear cells were observed around them. Ziell-Nielsen method revealed a great number of acid-fast rods stained by fucsin and distributed for all the lesion extension. Calcification was rarely observed and Langhans cells were not detected. DISCUSSION Regarding to the present descriptions about frog mycobateriosis, the lesions were similar to the reported in L. pentadactylus (DARZINS, ) and R. catesbeiana (BARROS et al,. 1988) in Brazil. Mycobacteriosis was described in european frog R. temporaria (KUSTER, 1905; LICHTENSTEIN, 1920), and in toads Xenopus laevis and Bufo bufo (ELKAN, 1960; REICHENBACH-KLINKE & ELKAN, 1965). BARROS et al.(1988) recognized this diffuse form of nodular lesions as visceral tuberculosis. The same authors described the cutaneous form in toads X. laevis that could be seem until ulcers (SCHWABACHER, 1959; REICHENBACH-KLINKE & ELKAN, 1965). In the present work, histological and cellular composition of granuloma was similar to those found in mycobacteriosis and mycosis in the national and international literature. Young granuloma was provided by macrophages and lymphocytes that, in some cases, appeared surrounded by thin connective capsule, constituted by fibroblasts and collagen fibers. Old granuloma presented macrophages, lymphocytes, epithelioid cells, a little number of trombocytes, neutrophils and eosinophils. The connective capsule was dense and provided by fibrocytes and collagen. Caseation was rarely observed. In other cases only a loosely organized masses involved by a fibrous tissue was observed. The composition of mycobacterial lesions is based in the presence of macrophages and lymphocytes in vertebrate animals (SPECTOR & MARIANO, 1975; TIMUR et al., 1977; ABERNETHY & LUND, 1978; GIAVENNI & FINAZZI, 1980; CHINABUT et al., 1990; NOGA et al., 1990; MATUSHIMA, 1994). REICHENBACH-KLINKE & ELKAN (1965), DARZINS ( ) and BARROS et al. (1988) reported the rare presence of Langhans giant cells, that was corroborated in the present description. In mammals, the chronic granulomatous inflammations, depending on the irritant agent, cell turnover can occur rapidly and dynamically or slowly, a fact which characterizes the cells involved as high or lowturnover cells (VOLKMAN & GOWANS, 1965; SPECTOR & RYAN, 1969; RYAN & SPECTOR, 1970; SPECTOR & MARIANO, 1975). High-turnover granulomas are induced by toxic substances such as mycobacteria and evolve histologically as epithelioid granuloma (ADAMS, 1983) with the presence of Langhans cells (BIRMAN & MARIANO, 1985). In contrast, lowturnover granulomas are induced by substances such as charcoal and carrageenin, which are of low toxicity and of difficult degradation and evolve histologically as mature granulomas (ADAMS, 1983) In the present study, epithelioid cells were observed, but not Langhans cells. This fact looklike conflicting. However, must be reminded the differences between inflammatory reaction in animals filogenetically different and the reaction is less complexes in lower animals (ROBINS et al., 1994). Moreover, the present observation revealed the presence of mononuclear cells and eosinophils, neutrophils and trombocytes, that were present in the induced granulomatous inflammation by BCG in

4 113 ARS VETERINARIA, 15(2): , Figure 1 - A liver fragment with granulomatous inflammation in different stages of development. HE.100 x Oreochromis niloticus (MATUSHIMA, 1994). Some authors reported the presence of melanin or melanomacrophages in the tuberculous granuloma, corroborated in this study. Melanin have bactericidal property presenting free radical characteristics, forming hydrogen peroxide (EDELSTEIN, 1971), that may contribute to defense mechanisms of the host. KUSTER (1905) described three examined frogs out of 200 with mycobacteriosis. LICHTENSTEIN (1920) found only one infected frog in 40 examined. DARZINS ( ) observed the disease in 100% of 60 dissecated frogs. Nevertheless, 40% of infected frogs is an important found that the profilaxis in the frog culture is forgotten. High stocking densities of frogs in the culture may favor the proliferation of bacteria. The main source of infection was not observed, but the owner have be careful with introduced animals and water flow. Some prophylactic measures can be important to avoid infectious and parasitic diseases in the frogfarms: desinfection of hands and boots, diary clean of excess of food, utilize a broom for each reproductive section, a bucket only to collect dead animals, decrease of stocking densities and isolate the ill and lesioned animals. With this profilaxis the disease occurrence decreased significantly in the studied frogfarms. ACKNOWLEDGEMENTS Figure 2 - A liver fragment with an old granuloma with a dense connective capsule showing eosinophils groups and melanomacrophages. HE. 400 x. Figure 3 - A liver fragment with a young granuloma showing macrophages and lymphocytes accumulation surrounded by a slender connective capsule shaped by fibroblasts and collagen fibers. HE. 250 x. We tank to Mrs. M.I.Y. Campos and Miss F.A. Ardisson for technical assistance. REFERENCES ADAMS, D.O. The biology of the granuloma. In: IOACHIN HL (ED.), Pathology of granulomas. New York: Raven Press, p ABERNETHY, C.S., LUND, J.E. Mycobacteriosis in mountain whitefish (Prosopium williamson) from the Yakina river, Washington. Journal of Wildlife Diseases, v. 14, p.333-6, ARONSON, J.D. Spontaneous tuberculosis in salt water fish. Journal of Infectious Diseases, v.39, p , ARONSON, J.D. Spontaneous tuberculosis in snakes. N. sp. Mycobacterium thamnopheos. Journal of Infectious Diseases, v.44, p , BARROS, G.C., LANGENEGGER, C.H., LANGE- NEGGER, J. PEIXOTO, P.V.. Surto de micobacteriose em criação de rãs (Rana catesbeiana) causado por Mycobacterium marinum. Pesquisa Veterinária Brasileira, v. 8, p , 1988

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