RESUMO: O cão Golden Retriever distrófico (Golden Retriever muscular dystrophy - GRMD) é um dos melhores modelos da distrofia muscular de Duchenne (DMD), com manifestações genotípicas e fenotípicas similares. A proliferação progressiva de tecido conjuntivo no endomísio das fibras musculares ocorre paralelamente ao curso clínico da doença em animais GRMD. Estudos anteriores sugerem uma relação entre os mastócitos e a deposição de tecido fibroso devido à liberação de mediadores que recrutam fibroblastos. O objetivo deste estudo foi avaliar a presença de mastócitos e sua relação com a lesão muscular e fibrose em cães GRMD de diferentes idades. Amostras de grupos musculares de seis GRMD e quatro controles, com idade entre 2 a 8 meses, foram coletadas e analisadas. As amostras foram processadas e coradas com HE, azul de toluidina e tricrômico de Azan. Nossos resultados mostraram que houve um aumento significativo na infiltração de mastócitos em todos os grupos musculares de cães GRMD em comparação com o grupo controle. O número médio de mastócitos, assim como a deposição de tecido fibroso, diminuiu com a idade em cães GRMD. No grupo controle, todos os tipos musculares mostraram um aumento significativo na quantidade de tecido colágeno. Isto sugere o aumento da degranulação de mastócitos em cães GRMD mais jovens, resultando em aumento do espaço intersticial e tecido fibroso no músculo, que então gradualmente diminuiu com o tempo à medida que os cães envelheceram. No entanto, mais estudos são necessários para esclarecer o papel dos mastócitos na patogênese da fibrose.

ABSTRACT: The Golden Retriever muscular dystrophy (GRMD) is one of the best models of Duchenne muscular dystrophy (DMD), with similar genotypic and phenotypic manifestations. Progressive proliferation of connective tissue in the endomysium of the muscle fibers occurs in parallel with the clinical course of the disease in GRMD animals. Previous studies suggest a relationship between mast cells and the deposition of fibrous tissue due to the release of mediators that recruit fibroblasts. The aim of this study was to evaluate the presence of mast cells and their relationship with muscle injury and fibrosis in GRMD dogs of different ages. Samples of muscle groups from six GRMD and four control dogs, aged 2 to 8 months, were collected and analyzed. The samples were processed and stained with HE, toluidine blue, and Azan trichrome. Our results showed that there was a significant increase in infiltration of mast cells in all muscle groups of GRMD dogs compared to the control group. The average number of mast cells, as well as the deposition of fibrous tissue, decreased with age in GRMD dogs. In the control group, all muscle types showed a significant increase in the amount of collagenous tissue. This suggests increased mast cell degranulation occurred in younger GRMD dogs, resulting in increased interstitial space and fibrous tissue in muscle, which then gradually decreased over time as the dogs aged. However, further studies are needed to clarify the role of mast cells in the pathogenesis of fibrosis.

RESUMEN Objetivo. Este estudio evaluó la cinética celular y la formación de granuloma durante la inflamación crónica inducida por el Bacilo Calmette-Guérin (BCG) en el músculo esquelético de Piaractus mesopotamicus, como modelo histopatológico para estudiar la inmunidad innata. Materiales y métodos. Sesenta peces fueron divididos en dos grupos: peces inoculados con BCG y no inoculados y la respuesta inflamatoria analizada en 3, 7, 14, 21 y 33 días post-inóculo (DPI) por medio del análisis histopatológico y tinciones de hematoxilina-eosina y Ziehl-Neelsen. Resultados. 3 DPI de BCG se observó reacción inflamatoria difusa principalmente formada por infiltrado celular mononuclear. Al 7° DPI la inflamación continuaba difusa con inicio de organización celular alrededor del inoculo, que se observó hasta el 14° DPI con discreta presencia de células de tipo epiteliodes con citoplasma acidófilo y cromatina laxa. Para el 21° DPI se observó alta organización celular alrededor del granuloma con intenso infiltrado mononuclear periférico e incremento en el número de fibroblastos y macrófagos. El proceso inflamatorio se tornó menos difuso a los 33 DPI con formación de pequeños granulomas contenidos dentro de uno más grande. Los granulomas formados más rápidamente así como los formados tardíamente, presentaron características típicas alrededor del inóculo compuesta por una camada de células tipo epitelioides, macrófagos, linfocitos y fibroblastos. Conclusiones. Este estudio mostró la viabilidad del uso del P. mesopotamicus para estudiar la respuesta inflamatoria crónica granulomatosa inducida con BCG, caracterizado por la evolución de la cinética de células inflamatorias en el músculo esquelético clasificándolo como de tipo inmune-epitelioide, similar a la inflamación granulomatosa causada por M. marinum en peces teleósteos.

ABSTRACT Objective. This study evaluated the cell kinetic and formation of granuloma during chronic inflammation induced by Bacillus Calmette-Guérin (BCG) in the skeletal muscle of Piaractus mesopotamicus, as a histopathology model to study innate immunity. Materials and methods. Sixty fish were divided in two groups: BCG-inoculated and non-inoculated fish and the inflammatory response analyzed 3, 7, 14, 21 and 33 days post-inoculation (DPI) by histopathology after hematoxylin-eosin and Ziehl-Neelsen staining. Results. 3 DPI of BCG showed a diffuse inflammatory reaction mostly composed by mononuclear cells. The inflammation continued diffuse 7 DPI initiating the cellular organization surrounding the inoculum and have continued at 14 DPI with discrete presence of epithelioid-like type cells with acidophilic cytoplasm and floppy chromatin. Higher cellular organization (21 DPI) surrounding the granuloma with intense peripheral mononuclear inflammatory infiltrate and nevertheless, an increase in the number of fibroblasts and macrophage-like cells was observed. The inflammatory process became less diffuse 33 DPI with formation of small amount of granuloma surrounded by the same type of reaction found in bigger granuloma. Both the young and old granuloma presented typical characteristic around the inoculum composed by a layer of epithelioid-like type cells, besides macrophages, some lymphocytes and abundant fibroblasts. Conclusions. This study showed the feasibility in the use of pacus to study chronic granulomatous inflammatory response induced by BCG, characterized by changes in the kinetics of inflammatory cells in skeletal muscle classifying as immune-epithelioid type, similar to granulomatous inflammation caused by M. marinum in teleost fish.

Objetivo. El presente estudio fue realizado para evaluar por medio de lectinhistoquímica (LHC), la expresión de carbohidratos en granulomas inducidos por el bacilo de Calmette-Guérin (BCG) en músculo de Piaractus mesopotamicus después de 33 días. Materiales y métodos. Cortes histológicos de 3 µm de grosor fueron incubados con las siguientes lectinas: WGA (Wheat germ aglutinin), DBA (Dolichos biflorus agglutin) y HPA (Helix pomatia agglutinin), y los resultados evaluados por medio de microscopia de luz. Resultados. Bacilos ácido resistentes fueron identificados por la tinción de Ziehl Neelsen(ZN). Se observó un marcaje intenso con WGA en el citoplasma de macrófagos. El marcaje con DBA fue intenso en fibroblastos y débil en macrófagos. Con la lectina HPA el marcaje fue intenso en macrófagos, principalmente en los que estaban en estrecho contacto con las células epitelióides, externamente se observó marcaje débil en fibroblastos. Las células epitelióides no fueron marcadas por las lectinas, pero fueron identificadas con la tinción de Hematoxilina-Eosina (HE). Las lectinas tuvieron un tipo de marcaje específico en algunos monosacáridos, como N-acetilglucosamina presente en los bacilos y en macrófagos, y N-acetilgalactosamina en macrófagos. En el grupo control no fue observada inflamación así como tampoco marcaje con las lectinas. Conclusiones. Esta técnica resultó eficiente en la identificación de receptores para las lectinas WGA, DBA y HPA en el granuloma epitelióide inducido por BCG en P. mesopotamicus.

Objective. This study was conducted to evaluate, by means of lectinhistochemistry (LHC), the expression of carbohydrates in granulomas induced by the bacillus Calmette-Guerin (BCG) in muscle tissue of Piaractus mesopotamicus after 33 days. Material and methods. Histological sections with 3 µm thick were incubated with the following lectins :WGA (Wheat germ agglutinin), DBA (Dolichos biflorus agglutinin) and HPA (Helix pomatia agglutinin), and the results were evaluated by light microscopy. Results. Acid fast bacilli were stained by Ziehl Neelsen (ZN) and strong labeled by WGA in the cytoplasm of macrophages. Labeling with DBA was intense in fibroblasts and weak in macrophages. On the other hand, HPA binding was stronger in macrophages, especially in those that were in close contact with epithelioid cells, without evidence of binding to fibroblasts. The epithelioid cells were not labeled by the used lectins, but they were identified by Hematoxilin-Eosin (HE). The lectins labeled specific type saccharides in glycoproteins, as N-acetylglucosamine present in bacilli and macrophages, as well as N-acetyl-galactosamine in macrophages. The control group showed no inflammation or lectin binding. Conclusions. This technique may be useful in identifying receptors for WGA, DBA and the HPA lectins in epithelioid granuloma induced by BCG in P. mesopotamicus.

Amostras de diferentes órgãos de Piaractus mesopotamicus mantidos em criação intensiva foram coletadas e processadas mediante as técnicas histológicas usuais para obtenção de cortes que foram corados com hematoxilina-eosina e pelo método de Ziehl-Neelsen. Ao exame em microscopia de luz foi possível identificar mixosporídeos dos gêneros Henneguya sp. e Myxobolus sp. em brânquia e rim de P. mesopotamicus respectivamente. Plasmódios com esporos imaturos de Henneguya sp. foram localizados ao longo das lamelas secundárias e mensurados (comprimento total 30,45±4,84µm e largura 3,52±0,33µm) e no rim esporos de Myxobolus sp. (comprimento total 8,94±0,82µm e largura 5,59±0,39µm). Na análise histopatológica das brânquias observaram-se plasmódios contendo esporos de Henneguya sp., com localização intralamelar e intravascular, em diferentes estágios de desenvolvimento. No rim identificaram-se esporos de Myxobolus sp., na região peritubular e no interstício e glomérulo, circundados por melanomacrófagos. Em poucos casos foi registrada hemorragia focal. O uso da coloração de Ziehl-Neelsen permitiu identificar particularidades dos esporos, facilitou sua biometria e classificação em comparação com a hematoxilina-eosina, demonstrando sua utilidade no diagnóstico histopatológico da referida parasitose.

Samples of different organs from intensively-reared Piaractus mesopotamicus were collected and processed using routine histological techniques in order to produce thin sections for staining with hematoxylin-eosin and with the Ziehl-Neelsen method. Through examination under an optical microscope, myxosporidians of the genera Henneguya sp. and Myxobolus sp. were identified, respectivelyin the gills and kidneys of P. mesopotamicus. Plasmodia with immature spores of Henneguya sp. were located along the secondary lamellae, with total length of 30.45±4.84µm and width of 3.52±0.33µm. Spores of Myxobolus sp. were located in the kidneys, with total length of 8.94±0.82µm and width of 5.59±0.39µm. Histopathological analysis of the gills showed plasmodia containing spores of Henneguya sp., at intralamellar and intravascular localities, at different stages of development. Spores of Myxobolus sp. were identified in the kidneys, in the peritubular region and in the interstices and glomerulus, surrounded by melanomacrophages. Focal hemorrhage was recorded in a few cases. Ziehl-Neelsen staining allowed to identify particular features of the spores and facilitated biometry and enabled classification in comparison with hematoxylin-eosin, thus demonstrating its usefulness for histopathological diagnosis of the parasitosis.

Este estudo descreveu a histopatologia de rim, baço e fígado de Piaractus mesopotamicus, Prochilodus lineatus e Pseudoplatystoma fasciatum, parasitados por mixosporídios, capturados no Rio Aquidauana, MS. Após necropsia, amostras do fígado, rim cefálico e baço foram colhidas, fixadas em formalina a 10 % tamponada e processadas de acordo com a rotina histológica. Os cortes foram feitos à espessura de 5 μm e corados com hematoxilina-eosina. Foram encontrados Myxobolus porofilus em P. lineatus, M. colossomatis em P. mesopotamicus e Myxobolus spp. nas três espécies de hospedeiros. Cistos de mixosporídios no exame histopatológico foram vistos no fígado e baço de P. mesopotamicus. Mais de 50% das amostras de fígado de P mesopotamicus e P lineatus apresentou hepatodistrofia difusa. Mais de 80 % das amostras de fígado de P. fasciatum apresentou formações hialinas concêntricas e esteatose em 50% das amostras. Em 95,23 % das amostras de rins de P. mesopotamicus, foram observadas alterações teciduais, e em mais de 60 % dos casos nefrodistrofia difusa moderada e congestão de sinusóides glomerulares. Alterações teciduais nas amostras de rins de P. lineatus foram observadas em menos de 20 % da amostra. No baço dos peixes ora examinados não foram encontradas lesões dignas de relato.

Histological analysis of kidney, spleen and liver of Piaractus mesopotamicus, Prochilodus lineatus and Pseudoplatystoma fasciatum, infected by myxosporean, caugth in Aquidauana river, MS, was studied. After necropsy, samples of liver, previous kidney and spleen were fixed in 10 % buffered formalin and processed followed histological routine methods. Sections of 5 μm were stained with hematoxylin and eosin. Myxobolusporofilus, M. colossomatis and were found in P. lineatus, in P. mesopotamicus respectively and Myxobolus spp. Were also found in all three species of fish. Myxosporideans cysts in the liver and spleen of P mesopotamicus were also related. Up to 50% of P mesopotamicus and P lineatus liver samples showed diffuse hepatodistrofy. Liver sections also showed concentric hialin structures in over 80 % of samples and esteatosis in 50% of them. In P mesopotamicus kidney, 95.23 % of them showed tissue changes consisted of 60 % with diffuse moderate nefrodistrofy and congestion of glomerular sinusoids. In P lineatus kidney, 20 % of the samples showed tissues changes. No heavy damage was observed in the fish spleen.

O objetivo deste trabalho foi avaliar o número de Rondonia rondoni no intestino de Piaractus mesopotamicus, por meio da diferença entre peso úmido e peso seco das amostras de parasitos para cada hospedeiro, a partir da relação do peso seco e número de parasitos pré-estabelecida. Amostras totais de R. rondoni, de 37 espécimes de Piaractus mesopotamicus, foram medidas para obtenção do peso úmido, desidratadas em estufa com temperatura entre 55ºC e 60ºC e, após 24 h seu peso seco foi determinado. Por meio de uma regra de três simples, calculou-se o número de parasitos a partir da diferença entre o peso úmido e o peso seco, considerando um erro padrão médio de 6,027 para um número médio de 1010 indivíduos, quantificado em ensaio prévio. A equação da regressão linear estimada foi de y = 13,138x - 162,01 e r² = 0,9989, a qual foi significativa (p < 0,01), sendo y o número de parasitos e x o peso seco. A normalidade dos dados foi verificada com o teste de Kolmogorov-Smirnov significativo para p < 0,01.

The presente work evaluated the number of Rondonia rondoni in intestine of Piaractus mesopotamicus, for diference of dry and wet weight of parasites samples by each host, from the relation dry weight and number of parasites. Totals samples of R. rondoni of 37 specimens of P. mesopotamicus for attaiment of wet weight, dehydrated in stove with temperature between 55ºC and 60ºC, the wet weight was measured after 24 h. The number of parasites was calculated with rule of three, considering the average number of 1,010 ± 6.027 specimens quantified in previous assay. The equation of linear regression is y = 13.138x - 162.01 and r² = 0.9989. The normality of the data was proven with Kolmogorov-Smirnov (p < 0.01).

Metazoan fauna from cultivated fishes (N = 433) in two feefishing farm of Franca, State of São Paulo, Brazil, was studied during a period of April 1997 through March 1999. Specimens of piauçú Leporinus macrocephalus Garavello & Britski, 1988 (Anostomidae), pacu Piaractus mesopotamicus Holmberg, 1887 (Characidae), carp Cyprinus carpio Linnaeus, 1758 (Cyprinidae), Tillapia rendalli Boulenger, 1896 (Cichlidae), nile-tilapia Oreochromis niloticus Linnaeus, 1758 (Cichlidae), matrinxã Brycon cephalus Günther, 1869 (Characidae) and tambacu hybrid (male of P. mesopotamicus x female of Colossoma macropomum Cuvier, 1818) were collected. A total of 29.3% was parasitized by monogenean, 9.7% with crustacean (copepodits and adults of Lernaea cyprinacea Linnaeus, 1758 and Argulus sp.) and 1.6% with myxosporean (Henneguya piaractus Martins & Souza, 1997 and Myxobolus colossomatis Moinar & Békési, 1993).The most susceptible species to myxosporean infestation was P. mesopotamicus. L. macrocephalus and P. mesopotamicus that showed a higher susceptibility to Argulus sp infestation. It was not observed significant difference (P > 0.05) in the seasonal variation of L. cyprinacea infection of all species. L. macrocephalus, P. mesopotamicus and tambacu showed a great number of P. pillulare with no significant difference (P > 0.05) between them. Monogenean infections were observed in all examined species, without significant difference (P > 0.05) in the seasonal variation. Nevertheless, occurrence of monogenean was higher in P. mesopotamicus and lower in Nile-tilapia. By the other hand, parasites number collected from P. mesopotamicus was lower in the winter. Copepodits of L. cyprinacea were found in the gills of P. mesopotamicus, tambacu and L. macrocephalus. However, adults of L. cyprinacea were observed only in the P. mesopotamicus and tambacu.

The present work studied protozoan parasites of cultivated fishes (N = 433) from two feefishing farm situated in Franca, São Paulo, Brazil, during a period of April 1997 through March 1999. Specimens of piauçú Leporinus macrocephalus Garavello & Britski, 1988 (Anostomidae), pacu Piaractus mesopotamicus Holmberg, 1887 (Characidae) carp Cyprinus carpio Linnaeus, 1758 (Cyprinidae), Tillapia rendalli Boulenger, 1896 (Cichlidae), nile-tilapia Oreochromis niloticus Linnaeus, 1758 (Cichlidae), matrinxã Brycon cephalus Günther, 1869 (Characidae) and tambacu hybrid (male of P. mesopotamicus x female of Colossoma macropomum Cuvier, 1818) were collected. The fishes were parasitized with protozoans Ichthyophthirius multifiliis Fouquet, 1876 (Protozoa), Trichodina sp. and Piscinoodinium pillulare (Schaperclaus, 1954), Lom, 1981 (Protozoa). In the cold season (autumn and/or winter) all species of fish were infected with I. multifiliis. Higher susceptibility to Trichodina sp. was observed in L. macrocephalus, C. carpio and P. mesopotamicus compared to tambacu, B. cephalus, T. rendalli and 0. niloticus. It was not observed significant difference (P > 0.05) in the seasonal variation of Trichodina sp. and P. pillulare infection of all species. A great number of P. pillulare without significant difference (P > 0.05) was reported to L. macrocephalus, P. mesopotamicus and tambacu.

The mean values of hepatosomatic relation (HSR), splenosomatic relation (SSR) and relative condition factor (Kn) in Nile tilapia (Oreochromis nilolicus Trewavas, 1983); pacu (Piaractus mesopolamicus Holmberg, 1887); piauçu (Lepori-nus macrocephalus Garavello & Britski, 1988) and tambacu hybrid (P. mesopotamicus male x Colossoma macropomum Cuvier, 1818 female) are described. The experiment was carried out from feefishing farm situated in Franca, São Paulo State, Brazil. Nile tilapia showed the greatest HSR compared to pacu, piauçu and tambacu. Although, in the last three fishes the relation was similar. The SSR between tilapia and piausu was similar, but smaller than observed in pacu and tambacu. Nevertheless, SSR in pacu and tambacu was different. The hepatic weight/body weigth relation and hepatic weight/body length relation presented positive correlation in ali studied fishes. The splenic weight/body weigth relation and splenic weight/body length relation were not significam (P>0.05) in Nile tilapia, pacu and tambacu, but highly significant in piauçu (PO.01).