RESUMEN

Iridoviruses of the Ranavirus genus have been implicated in the decline in amphibians worldwide, capable of affecting animals both in the wild and in captivity. This study aimed to detect iridovirus-like particles from three frog farms in southeastern Brazil using primary polyclonal antibodies, transmission electron microscopy (TEM) and histologic findings. The target organs were liver and kidneys. Sixty adults and sixty tadpoles of bullfrogs (Lithobates catesbeianus) were used in the study. TEM revealed the presence of iridovirus-like particles in hepatic tissue using the negative staining technique. Positive results were also observed by immunoelectron microscopy and immunocytochemistry (ICC). The histological analysis of the samples showed liver hemorrhage and corpuscles inclusion in hepatocytes as well as glomerulotubular degeneration and necrosis in the kidneys. The methods used in this study were highly efficient to detect the presence of iridovirus-like particles and possible infection of ranavirus.

Os iridovirus do gênero Ranavirus têm sido implicados no declínio dos anfíbios em todo o mundo afetando animais de vida livre e aqueles em cativeiro. O objetivo deste estudo foi detectar a presença de partículas semelhantes ao iridovírus em três ranários na região sudeste do Brasil, utilizando anticorpos policlonais primários, microscopia eletrônica de transmissão (MET) e achados histológicos. Os órgãos alvo foram o fígado e os rins. Sessenta rãs-touro adultas (Lithobates catesbeianus) e sessenta girinos da mesma espécie foram usados para o estudo. A MET revelou a presença de partículas semelhantes ao iridovírus em tecido hepático utilizando a técnica de contrastação negativa. Os resultados positivos foram também observados por imunomicroscopia eletrônica e imunocitoquímica. As análises histológicas nas mesmas amostras evidenciaram hemorragia no fígado e corpúsculos de inclusão em hepatócitos, e degeneração glomerulotubular e necrose nos rins. Os métodos usados neste estudo foram altamente eficientes na detecção de partículas semelhantes ao iridovírus e possivel infecção por ranavirus.

Asunto(s)

RESUMEN

Iridoviruses of the Ranavirus genus have been implicated in the decline in amphibians worldwide, capable of affecting animals both in the wild and in captivity. This study aimed to detect iridovirus-like particles from three frog farms in southeastern Brazil using primary polyclonal antibodies, transmission electron microscopy (TEM) and histologic findings. The target organs were liver and kidneys. Sixty adults and sixty tadpoles of bullfrogs (Lithobates catesbeianus) were used in the study. TEM revealed the presence of iridovirus-like particles in hepatic tissue using the negative staining technique. Positive results were also observed by immunoelectron microscopy and immunocytochemistry (ICC). The histological analysis of the samples showed liver hemorrhage and corpuscles inclusion in hepatocytes as well as glomerulotubular degeneration and necrosis in the kidneys. The methods used in this study were highly efficient to detect the presence of iridovirus-like particles and possible infection of ranavirus.(AU)

Os iridovirus do gênero Ranavirus têm sido implicados no declínio dos anfíbios em todo o mundo afetando animais de vida livre e aqueles em cativeiro. O objetivo deste estudo foi detectar a presença de partículas semelhantes ao iridovírus em três ranários na região sudeste do Brasil, utilizando anticorpos policlonais primários, microscopia eletrônica de transmissão (MET) e achados histológicos. Os órgãos alvo foram o fígado e os rins. Sessenta rãs-touro adultas (Lithobates catesbeianus) e sessenta girinos da mesma espécie foram usados para o estudo. A MET revelou a presença de partículas semelhantes ao iridovírus em tecido hepático utilizando a técnica de contrastação negativa. Os resultados positivos foram também observados por imunomicroscopia eletrônica e imunocitoquímica. As análises histológicas nas mesmas amostras evidenciaram hemorragia no fígado e corpúsculos de inclusão em hepatócitos, e degeneração glomerulotubular e necrose nos rins. Os métodos usados neste estudo foram altamente eficientes na detecção de partículas semelhantes ao iridovírus e possivel infecção por ranavirus.(AU)

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RESUMEN

In this study thirty shrimp samples from commercial marine shrimp (L. vannamei) farms of southern region of Brazil were obtained. Hepatopancreas and shell scrapings fragments collected in these animals were processed by transmission electron microscopy using negative staining (rapid preparation), immunoelectron microscopy and immunocytochemistry (immunolabelling with colloidal gold particles) techniques. On the transmission electron microscopy a great number of white spot virus particles, ovoid or bacilliform-to-ellipsoid, measured 230-290 nm in length and 80-160 nm in diameter with intra-nuclear projections were visualized by the negative staining technique in 27 (90 percent) out of 30 samples examined. Using immunoelectron microscopy technique, the anti-VP 664 serum agllutinated a large number of particles formed by antigen-antibody interaction. In the immunocytochemistry technique, the antigen-antibody reaction was styrongly marked by the particles of colloidal gold over the virus. Notably, this is the first report, to our knowledge, describing use of these microscopy techniques to study Brazilian L. vannamei marine shrimp samples; moreover, this methodology also appears to be a viable complementary tool for diagnosing the presence of the white spot virus within shrimp tissues. Importantly, these are the first photoelectron micrographs of the WSSV in Brazil.

Se obtuvieron para el estudio 30 muestras de camarones marinos comerciales (L. vannamei) de las granjas de la región sur de Brasil. Fueron procesados fragmentos de hepatopáncreas y raspados internos del cefalotórax recogidos en estos animales por microscopía electrónica de transmisión con tinción negativa (preparación rápida), inmunomicroscopía y técnicas de inmunocitoquímica (inmunomarcación con partículas de oro coloidal). En la microscopía electrónica de transmisión de un gran número de partículas de virus de la mancha blanca, ovoide o elipsoidal a baciliformes, medían 230-290 nm de longitud y 80-160 nm de diámetro. En 27 (90 por ciento) de las 30 muestras examinadas intra-nuclear proyecciones se visualizaron mediante la técnica de tinción negativa. Utilizando una técnica de inmunomicroscopía electrónica, el anti-suero VP 664 reunió a un gran número de partículas formadas por la interacción antígeno-anticuerpo. En la técnica de inmunocitoquímica, la reacción antígeno-anticuerpo fue fuertemente reforzada por las partículas de oro coloidal en los virus. En particular, en Brasil este es el primer informe, a nuestro entender, que describe el uso de estas técnicas de microscopía en muestras de camarón marino L. vanamei. Además, esta metodología también parece ser una herramienta complementaria viable para diagnosticar la presencia del virus de la mancha blanca en tejidos de camarón. Es importante destacar que estas son las primeras fotos en microscopia electrónica del WSSV obtenidas en Brasil.

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RESUMEN

The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.

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