Clonality test by PCR - PARR in real time of canine lymphomas

Lymphoma is a neoplasm of hematopoietic origin that affects canines. The proper establishment of prognosis and rapid institution of treatment are essential for a better quality of life, and immunophenotyping is one of the tools used for this purpose. The objective of this study was to perform a clonality test for immunophenotypic characterization of canine lymphomas using the polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) technique in real-time from samples fixed in formalin and embedded in paraffin. The 23 analyzed samples were fixed in formalin and embedded in paraffin canine lymphoma from the collection Laboratory of Histopathology of the Animal Pathology Area of the Departament of Veterinary Medicine - Federal Rural University of Pernambuco (UFRPE). Samples were processed, their DNA was extracted, quantified, diluted, and standardized at a concentration of 50 ng/µL. After extraction, all samples were subjected to conventional PCR for endogenous control (detection of the IgM target region), in which the extracted DNA was amplified in a final volume of 25 µL. The 128 bp amplified product was detected by 1.5% agarose gel electrophoresis. Of the 23 samples analyzed for the detection of the conserved region referring to the endogenous gene, 91.30% (21/23) amplified the conserved region Cµ by conventional PCR, and two samples 8.70% (2/23) were negative. Endogenous control positive samples were subjected to real-time PCR-PARR for detection of IgH Major and IgH Minor for B lymphocytes (LB), and TCRy for lymphocytes T (LT) target regions. All reactions were performed in duplicate to reduce the risk of false-positive or false-negative results due to technical errors. Samples previously confirmed by immunohistochemistry were used as positive controls for T cell and B cell lymphoma, and MilliQ water was used as a negative reaction control. After amplification, the melting curve gradually increased the temperature by 1o C/5 s to 95o C during continuous fluorescence monitoring. Of the 21 samples analyzed, 100.00% (21/21) demonstrated clonal amplification. Of these, 57.15% (12/21) were positive for phenotype B, and 42.85% (9/21) were positive for phenotype T. Due to the importance of researching and confirming samples from files fixed and embedded in paraffin samples in laboratories, PCR-PARR is a good tool for this purpose. In the present study, real-time PCR analysis demonstrated greater sensitivity in the characterization of the immunophenotype of lymphomas from old samples fixed in formalin and embedded in paraffin. The temperature of melting curve analysis may vary depending on the amount of DNA and its quality. In the present study, it was found that the average melting temperature in the samples varied between ± 3o C when compared to that in the control sample for LB and LT, 83.5o C and 80o C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4o C. Real-time PCR-PARR was satisfactory in the characterization of the immunophenotype of canine lymphomas from formalin-fixed and paraffin-embedded samples; therefore, its use is recommended for both retrospective studies. The use of PCR-PARR associated with histopathological and/or cytopathological examination in cases of canine lymphomas strongly helps pathologists, provide a safe establishment of the immunophenotype, minimize errors, and optimize the diagnosis, thus directly contributing to the establishment of the prognosis.(AU)

Topografia e morfometria do timo em aves (Gallus gallus domesticus) da linhagem Ross 308 AP (AP95) / Topography and morphometry of timo in fowl (Gallus domesticus) from Ross 308 AP LINEAGE (AP95)

Objetivou-se descrever a topografia e morfometria do timo em 30 aves domésticas da linhagem Ross 308 AP (AP95), 15 machos e 15 fêmeas, com idade de 14 a 16 semanas e massa corporal média de 2,1 kg e 1,7 kg, respectivamente nos machos e fêmeas. Após a fixação em solução aquosa de formol a 10%, as peças foram dissecadas. Os lobos tímicos localizavam-se desde o terço cranial do pescoço até os ossos claviculares, de forma linear, longitudinal e lateralmente no pescoço, paralelos ao nervo vago, veia jugular externa e artéria comum do nervo vago. Os lobos apresentaram formatos ovalados e triangulares e o número foi de quatro a oito. Agrupando os lobos de acordo com a localização no pescoço, nos terços cranial, médio e caudal, as médias de comprimento x largura x espessura variaram, nessa ordem, de 1,93 a 52,1 mm, 0,98 a 23,15 mm e 0,32 a 8,52 mm. O timo das aves AP95 apresentou características topográficas e morfológicas similares àquelas de outras linhagens, porém, quanto à morfometria, foram observadas características singulares.

The objective of this study was to describe the topography and morphometry of the thymus in 30 domestic fowl of the Ross 308 AP (AP95) lineage, 15 males and 15 females, with an approximate age of 15 weeks and a mean weight of 2.1 kg for males and 1.7 for females. After fixation in 10 % formalin aqueous solution, the specimens were dissected. The thymic lobes were located from the cranial third of neck to clavicular bones, linearly, longitudinally and laterally in neck, parallel to the vagus nerve, external jugular vein and common vagus nerve artery. The lobes presented oval and triangular shapes and the number varies from four to eight. Grouping the thymic lobes according to the location in neck in cranial, middle or caudal thirds, the averages of length x widths x thickness varied from 1.93 to 52.1 mm, 0.98 to 23.15 mm and 0.32 to 8.52 mm. The thymus of AP95 chicken presented topographic and morphological characteristics similar to those of other strains, but, regarding morphometry, unique characteristics were observed.

Topografia e morfometria do timo em aves (Gallus gallus domesticus) da linhagem Ross 308 AP (AP95) / Topography and morphometry of timo in fowl (Gallus domesticus) from Ross 308 AP LINEAGE (AP95)

Objetivou-se descrever a topografia e morfometria do timo em 30 aves domésticas da linhagem Ross 308 AP (AP95), 15 machos e 15 fêmeas, com idade de 14 a 16 semanas e massa corporal média de 2,1 kg e 1,7 kg, respectivamente nos machos e fêmeas. Após a fixação em solução aquosa de formol a 10%, as peças foram dissecadas. Os lobos tímicos localizavam-se desde o terço cranial do pescoço até os ossos claviculares, de forma linear, longitudinal e lateralmente no pescoço, paralelos ao nervo vago, veia jugular externa e artéria comum do nervo vago. Os lobos apresentaram formatos ovalados e triangulares e o número foi de quatro a oito. Agrupando os lobos de acordo com a localização no pescoço, nos terços cranial, médio e caudal, as médias de comprimento x largura x espessura variaram, nessa ordem, de 1,93 a 52,1 mm, 0,98 a 23,15 mm e 0,32 a 8,52 mm. O timo das aves AP95 apresentou características topográficas e morfológicas similares àquelas de outras linhagens, porém, quanto à morfometria, foram observadas características singulares.(AU)

The objective of this study was to describe the topography and morphometry of the thymus in 30 domestic fowl of the Ross 308 AP (AP95) lineage, 15 males and 15 females, with an approximate age of 15 weeks and a mean weight of 2.1 kg for males and 1.7 for females. After fixation in 10 % formalin aqueous solution, the specimens were dissected. The thymic lobes were located from the cranial third of neck to clavicular bones, linearly, longitudinally and laterally in neck, parallel to the vagus nerve, external jugular vein and common vagus nerve artery. The lobes presented oval and triangular shapes and the number varies from four to eight. Grouping the thymic lobes according to the location in neck in cranial, middle or caudal thirds, the averages of length x widths x thickness varied from 1.93 to 52.1 mm, 0.98 to 23.15 mm and 0.32 to 8.52 mm. The thymus of AP95 chicken presented topographic and morphological characteristics similar to those of other strains, but, regarding morphometry, unique characteristics were observed.(AU)

Multicentric lymphoma in sheep (case report) / Linfoma multicêntrico em ovino (relato de caso)

The present study describes a case report in sheep with multicentric lymphoma. Clinical examination revealed the animal presented tachycardia, tachypnea, congested ocular mucous membranes, fever, cachexia, mild dehydration, decreased ruminal motility, difficulty in standing, and dysuria. Supportive treatment and complementary examination (complete blood count, radiography, ultrasound and abdominocentesis) were performed. The results showed neutrophilic leukocytosis, persistent lymphocytosis, exudate with high cell concentration, and imaging examination showed abundant fluid in the abdominal cavity, and enlarged iliac and mesenteric lymph nodes. Due to the severe clinical condition, the animal died and referred for necropsy. Samples from different organs were collected for histopathological analysis. At necropsy, abundant light brown exudate was observed in the thoracic and abdominal cavities, multiple white lobulated masses in different organs, in addition to pink, irregular, multifocal and coalescent nodules in the omentum, with neoplastic cells arranged in cords and cloak of cells supported by a thin fibrovascular stroma. Macroscopic and microscopic findings are characteristic of multicentric lymphoma.(AU)

O presente trabalho descreve um relato de caso em ovino. No exame clínico o animal apresentou taquicardia, taquipnéia, mucosas oculares congestas, febre e caquexia, grau de desidratação leve, diminuição de motilidade ruminal, dificuldade em se manter em estação, além de disúria. Realizou-se tratamento de suporte e exames complementares (hemograma, radiografia, ultrassonografia e abdominocentese). Os resultados dos exames complementares demostraram leucocitose por neutrofilia e linfocitose persistente, presença de exsudato contendo elevada concentração celular, e exames de imagem indicando presença abundante de líquido na cavidade abdominal, e presença de linfonodos ilíacos e mesentéricos em tamanho aumentado. O animal veio a óbito em decorrência da severidade do quadro clínico, sendo encaminhado para necrópsia. Foram coletados fragmentos de diversos órgãos para análise histopatológica. Na necrópsia, observou-se grande quantidade de exsudato pardo-claro nas cavidades abdominal e torácica, múltiplas massas brancacentas e lobuladas em diferentes órgãos, além de nódulos rosados, irregulares, multifocais e coalescentes no omento com células neoplásicas que arranjavam-se em cordões e manto e, eram sustentadas por um fino estroma fibrovascular. Os achados macroscópicos e microscópicos são característicos de linfoma multicêntrico.(AU)

Multicentric lymphoma in sheep (case report) / Linfoma multicêntrico em ovino (relato de caso)

The present study describes a case report in sheep with multicentric lymphoma. Clinical examination revealed the animal presented tachycardia, tachypnea, congested ocular mucous membranes, fever, cachexia, mild dehydration, decreased ruminal motility, difficulty in standing, and dysuria. Supportive treatment and complementary examination (complete blood count, radiography, ultrasound and abdominocentesis) were performed. The results showed neutrophilic leukocytosis, persistent lymphocytosis, exudate with high cell concentration, and imaging examination showed abundant fluid in the abdominal cavity, and enlarged iliac and mesenteric lymph nodes. Due to the severe clinical condition, the animal died and referred for necropsy. Samples from different organs were collected for histopathological analysis. At necropsy, abundant light brown exudate was observed in the thoracic and abdominal cavities, multiple white lobulated masses in different organs, in addition to pink, irregular, multifocal and coalescent nodules in the omentum, with neoplastic cells arranged in cords and cloak of cells supported by a thin fibrovascular stroma. Macroscopic and microscopic findings are characteristic of multicentric lymphoma.

O presente trabalho descreve um relato de caso em ovino. No exame clínico o animal apresentou taquicardia, taquipnéia, mucosas oculares congestas, febre e caquexia, grau de desidratação leve, diminuição de motilidade ruminal, dificuldade em se manter em estação, além de disúria. Realizou-se tratamento de suporte e exames complementares (hemograma, radiografia, ultrassonografia e abdominocentese). Os resultados dos exames complementares demostraram leucocitose por neutrofilia e linfocitose persistente, presença de exsudato contendo elevada concentração celular, e exames de imagem indicando presença abundante de líquido na cavidade abdominal, e presença de linfonodos ilíacos e mesentéricos em tamanho aumentado. O animal veio a óbito em decorrência da severidade do quadro clínico, sendo encaminhado para necrópsia. Foram coletados fragmentos de diversos órgãos para análise histopatológica. Na necrópsia, observou-se grande quantidade de exsudato pardo-claro nas cavidades abdominal e torácica, múltiplas massas brancacentas e lobuladas em diferentes órgãos, além de nódulos rosados, irregulares, multifocais e coalescentes no omento com células neoplásicas que arranjavam-se em cordões e manto e, eram sustentadas por um fino estroma fibrovascular. Os achados macroscópicos e microscópicos são característicos de linfoma multicêntrico.

Aspectos patológicos e imuno-histoquímicos da polisserosite em búfalos (Bubalus bubalis) / Histopathological and immunohistochemical study of the polyserositis in buffalo (Bubalus bubalis)

Polisserosites são alterações inflamatórias das serosas viscerais e parietais das cavidades corpóreas. Um tipo especial destas alterações foi identificado em bubalinos abatidos para consumo nos anos 80, sendo associada a infecção por Chlamydia psittaci. Apesar da importância da bubalinocultura no Pará, do caráter zoonótico da C. psittaci e da possibilidade de envolvimento de outros agentes na afecção, são raros trabalhos sobre a enfermidade desde os estudos pioneiros. No presente trabalho casos identificados como polisserosite pelo serviço de inspeção sanitária em búfalos abatidos para consumo foram coletados com objetivo de determinar a frequência e origem dos mesmos e de caracterizar as lesões e pesquisar antígenos de Chlamydia spp. nas mesmas. De um total de 2.887 bubalinos abatidos no período de estudo foram identificados 48 (1,66%) casos. O município de Santa Cruz do Arari na Ilha de Marajó apresentou a maior porcentagem de casos em relação ao número de animais abatidos (6,49%, 5/77). Na macroscopia as lesões se caracterizaram por áreas opacas, branco-amareladas de espessamento das serosas, por vezes com franjas fibrosas na superfície. Os resultados permitem concluir que os casos classificados como polisserosite pelos veterinários da inspeção sanitária nos búfalos abatidos para consumo correspondem na histopatologia a um tipo incomum de polisserosite, caracterizada por infiltrado linfocitário, com ocasionais formações de folículos linfoides terciários ou ectópicos. Foi observado ainda nestas lesões um predomínio de linfócitos T (CD3 positivos) no infiltrado, sendo os linfócitos B (CD79 positivos) presentes em maior número no interior das estruturas foliculares. Não foram demonstrados antígenos de clamídias nas lesões do presente estudo, o que demonstra a necessidade de estudos adicionais relativos a(s) etiologia(s) das lesões.(AU)

Polyserositis are inflammatory changes of the visceral and parietal serous of body cavities. A special type of polyserositis was identified in buffaloes in the 80s, being associated with infection by Chlamydia psittaci. Since these pioneering studies, there are no additional works about the condition. Considering the importance of buffalo in Pará, the zoonotic character of C. psittaci and the possibility of involvement of other agents in polyserositis in buffaloes the present study is proposed. We collected cases identified as polyserositis by sanitary inspection service in buffalo slaughtered for consumption in Belem for a complementary characterization of inflammatory cell and the research of Chlamydia spp antigens in lesions. Of 2.887 buffaloes slaughtered in a period of six months, there were 48 (1.66%) cases of polyserositis and 39 analyzed. Santa Cruz do Arari in Marajó Island was the city with the highest frequency of cases, whereas 6.49% of buffaloes had lesions. However, 50% of the present study cases came from Soure municipality in Marajó Island, which provided about 49% of buffaloes slaughtered in the period. In the macroscopy, there were opaque areas with white-yellow thickening of the serous, sometimes with fibrous fringes on the surface. Histopathology showed connective tissue projections partially lined by cuboid or flattened mesothelial cells. Often in projections there were mononuclear infiltrate of variable intensity, consisting mainly of lymphoid cells, with occasional ectopic or tertiary lymphoid follicles.(AU)

Aspectos patológicos e imuno-histoquímicos da polisserosite em búfalos (Bubalus bubalis) / Histopathological and immunohistochemical study of the polyserositis in buffalo (Bubalus bubalis)

Polisserosites são alterações inflamatórias das serosas viscerais e parietais das cavidades corpóreas. Um tipo especial destas alterações foi identificado em bubalinos abatidos para consumo nos anos 80, sendo associada a infecção por Chlamydia psittaci. Apesar da importância da bubalinocultura no Pará, do caráter zoonótico da C. psittaci e da possibilidade de envolvimento de outros agentes na afecção, são raros trabalhos sobre a enfermidade desde os estudos pioneiros. No presente trabalho casos identificados como polisserosite pelo serviço de inspeção sanitária em búfalos abatidos para consumo foram coletados com objetivo de determinar a frequência e origem dos mesmos e de caracterizar as lesões e pesquisar antígenos de Chlamydia spp. nas mesmas. De um total de 2.887 bubalinos abatidos no período de estudo foram identificados 48 (1,66%) casos. O município de Santa Cruz do Arari na Ilha de Marajó apresentou a maior porcentagem de casos em relação ao número de animais abatidos (6,49%, 5/77). Na macroscopia as lesões se caracterizaram por áreas opacas, branco-amareladas de espessamento das serosas, por vezes com franjas fibrosas na superfície. Os resultados permitem concluir que os casos classificados como polisserosite pelos veterinários da inspeção sanitária nos búfalos abatidos para consumo correspondem na histopatologia a um tipo incomum de polisserosite, caracterizada por infiltrado linfocitário, com ocasionais formações de folículos linfoides terciários ou ectópicos. Foi observado ainda nestas lesões um predomínio de linfócitos T (CD3 positivos) no infiltrado, sendo os linfócitos B (CD79 positivos) presentes em maior número no interior das estruturas foliculares. Não foram demonstrados antígenos de clamídias nas lesões do presente estudo, o que demonstra a necessidade de estudos adicionais relativos a(s) etiologia(s) das lesões.(AU)

Polyserositis are inflammatory changes of the visceral and parietal serous of body cavities. A special type of polyserositis was identified in buffaloes in the 80s, being associated with infection by Chlamydia psittaci. Since these pioneering studies, there are no additional works about the condition. Considering the importance of buffalo in Pará, the zoonotic character of C. psittaci and the possibility of involvement of other agents in polyserositis in buffaloes the present study is proposed. We collected cases identified as polyserositis by sanitary inspection service in buffalo slaughtered for consumption in Belem for a complementary characterization of inflammatory cell and the research of Chlamydia spp antigens in lesions. Of 2.887 buffaloes slaughtered in a period of six months, there were 48 (1.66%) cases of polyserositis and 39 analyzed. Santa Cruz do Arari in Marajó Island was the city with the highest frequency of cases, whereas 6.49% of buffaloes had lesions. However, 50% of the present study cases came from Soure municipality in Marajó Island, which provided about 49% of buffaloes slaughtered in the period. In the macroscopy, there were opaque areas with white-yellow thickening of the serous, sometimes with fibrous fringes on the surface. Histopathology showed connective tissue projections partially lined by cuboid or flattened mesothelial cells. Often in projections there were mononuclear infiltrate of variable intensity, consisting mainly of lymphoid cells, with occasional ectopic or tertiary lymphoid follicles.(AU)

Gallus gallus domesticus: immune system and its potential for generation of immunobiologics / Gallus gallus domesticus: sistema imunológico e seu potencial para geração de imunobiológicos

Gallus gallus domesticus immune system is a promising tool for generation of antibody-based immunobiologics. Immunoglobulin y (IgY) is extracted from egg yolk and has equivalent functions to mammals igg antibody. Avian immune system can be stimulated to produce a high-quality antibody repertoire. In this review, we present an overview of avian immune system emphasizing igy and its applications as an immunobiologic.(AU)

O sistema imunológico de Gallus gallus domesticus é uma ferramenta promissora para a geração de imunobiológico a partir de anticorpos. A imunoglobulina Y (IgY) é extraída da gema do ovo e apresenta funções equivalentes ao anticorpo IgG dos mamíferos. O sistema imune aviário pode ser estimulado para produzir um repertório de anticorpos de alta qualidade. Nesta revisão apresentamos aspectos gerais do sistema imune aviário enfatizando o IgY e suas aplicações como um imunobiológico.(AU)

Aspectos clínicos do tumor venéreo transmissível canino (Tvtc) linfóide, plasmocitóide e misto / Clinical aspects of lymphoid, plasmocytoid and mixed canine transmissible venereal tumor (TVTc)

The objective of this study was to describe the clinical aspects of the subtypes of Canine Transmissible Venereal Tumor (TVTc). The clinical record of 40 dogs with the cytologic diagnosis of TVTc was evaluated. Of these was selected information about classfication, clinical complaint and affected organs with their specific locations. In this study, the plasmocytoid TVTc prevailed. The predominant clinical complaint was penile bleeding (14/40) and vaginal bleeding (13/40). As to the location, genital (G) (34/40) and extragenital (EG) (17/40) tumors were observed, with TVTcL (G-66,66% and EG-66,66%), TVTcM (G-90% and EG-30%) and TVTcP (G-90,47% and EG-38,08%). Among the genitals, the base, body and gland prevailed, and in the vagina: ventral and dorsal floors. In the extragenital tumors, there were changes in the skin, mouth, subcutaneous, nasal region and eye. It was concluded that the TVTcP prevailed more in the skin, while the TVTcL in the head region.

Aspectos clínicos do tumor venéreo transmissível canino (Tvtc) linfóide, plasmocitóide e misto / Clinical aspects of lymphoid, plasmocytoid and mixed canine transmissible venereal tumor (TVTc)

The objective of this study was to describe the clinical aspects of the subtypes of Canine Transmissible Venereal Tumor (TVTc). The clinical record of 40 dogs with the cytologic diagnosis of TVTc was evaluated. Of these was selected information about classfication, clinical complaint and affected organs with their specific locations. In this study, the plasmocytoid TVTc prevailed. The predominant clinical complaint was penile bleeding (14/40) and vaginal bleeding (13/40). As to the location, genital (G) (34/40) and extragenital (EG) (17/40) tumors were observed, with TVTcL (G-66,66% and EG-66,66%), TVTcM (G-90% and EG-30%) and TVTcP (G-90,47% and EG-38,08%). Among the genitals, the base, body and gland prevailed, and in the vagina: ventral and dorsal floors. In the extragenital tumors, there were changes in the skin, mouth, subcutaneous, nasal region and eye. It was concluded that the TVTcP prevailed more in the skin, while the TVTcL in the head region.(AU)

Morphology And Biometry Of The Thymus In Gallus Gallus Domesticus (Linnaeus, 1758) From The Isa Brow Lineage

The anatomy of the thymus was described in thirty chickens (Gallus gallus domesticus) of the Isa Brow line. Samples were prepared by fixation and preservation in 10% formalin solution and analyzed by subsequent dissection. The thymus lobes were found along the ventral region of the neck from the third cervical vertebra to the proximity to the clavicular bones in relation to the vagus nerve, the common artery of the vagus nerve and the jugular vein. The number of lobes ranged from four or five to the right antimere and three to five to the left. The mean lobe size was 0.80 x 0.43 x 0.38 cm for the cranial lobe, 1.53 x 0.81 x 0.42 cm for the middle cranial lobe, 2.10 x 0.64 x 0.46 for the middle lobe, 2.29 x 0.89 x 0.59 for the middle caudal lobe and 3.82 x 1.39 x 1.11 cm for the caudal lobe in the right antimere. In the left antimere, 0.80 x 0.41 x 0.37 cm was recorded for the cranial lobe, 1.55 x 0.80 x 0.44 cm for the middle cranial lobe, 2.37 x 0.87 x 0.56 for the middle lobe, 2.37 x 0.85 x 0.60 for the middle caudal lobe and 3.89 x 1.43 x 1.08 cm for the caudal lobe. The thymus of birds from the Isa Brow lineage presented characteristics similar to those found in other lineages with respect to the division of lobes and topography. However, skeletal, syntopic and holotopic disparities were observed that indicate some unique characteristics of the thymus in this lineage.

Morphology And Biometry Of The Thymus In Gallus Gallus Domesticus (Linnaeus, 1758) From The Isa Brow Lineage

The anatomy of the thymus was described in thirty chickens (Gallus gallus domesticus) of the Isa Brow line. Samples were prepared by fixation and preservation in 10% formalin solution and analyzed by subsequent dissection. The thymus lobes were found along the ventral region of the neck from the third cervical vertebra to the proximity to the clavicular bones in relation to the vagus nerve, the common artery of the vagus nerve and the jugular vein. The number of lobes ranged from four or five to the right antimere and three to five to the left. The mean lobe size was 0.80 x 0.43 x 0.38 cm for the cranial lobe, 1.53 x 0.81 x 0.42 cm for the middle cranial lobe, 2.10 x 0.64 x 0.46 for the middle lobe, 2.29 x 0.89 x 0.59 for the middle caudal lobe and 3.82 x 1.39 x 1.11 cm for the caudal lobe in the right antimere. In the left antimere, 0.80 x 0.41 x 0.37 cm was recorded for the cranial lobe, 1.55 x 0.80 x 0.44 cm for the middle cranial lobe, 2.37 x 0.87 x 0.56 for the middle lobe, 2.37 x 0.85 x 0.60 for the middle caudal lobe and 3.89 x 1.43 x 1.08 cm for the caudal lobe. The thymus of birds from the Isa Brow lineage presented characteristics similar to those found in other lineages with respect to the division of lobes and topography. However, skeletal, syntopic and holotopic disparities were observed that indicate some unique characteristics of the thymus in this lineage.(AU)

Rectal biopsy in sheep and goats for monitoring and ante-mortem diagnosis of scrapie: Number of lymphoid follicles in two consecutive collections / Biopsia retal em ovinos e caprinos para monitoramento e diagnóstico ante mortem de scrapie: Número de folículos linfoides em duas colheitas consecutivas

This study aimed to evaluate the amount of lymphoid tissue associated with the rectal mucosa obtained by rectal biopsy and the possibility of two consecutive biopsies at different time intervals, for monitoring and ante-mortem diagnosis of scrapie. Rectal mucosa samples were collected from 56 sheep and 32 goats in two steps. In the first step, on day 0, all animals were tested and, for the second step, the animals were divided into groups and each group was subjected to collection on different dates: for sheep 7, 14, 21, and 28 days after the first one and, for goats, on days 14, 21, and 28. From 176 samples, 151 (85.8%) were collected from the rectal mucosa, and in 25 (14.2%) there was a collection failure. Considering the rectal mucosa samples (151), 56.86% of the sheep samples and 51.61% of the goat samples, on day 0, had more then 3 lymphoid follicles (LF). In the second collection, 58.97% of the sheep samples showed 3 LF and 33.33% of the goat samples. Comparing the number of LF of the same animals between the first and second collections, there was a significant difference (p 0.05) between days 0 and 7 for sheep (with more FL on day 0) and days 0 and 28 (with more LF on day 28) and days 0 and 28 for goats (with more FL on day 0). There was no significant difference in the number of FL assessed on dates 0, 14, and 21 when comparing the different species, sheep and goats. On day 28, sheep samples showed a higher number (p <0.05) of LF than goats. It was concluded that rectal biopsy technique involves useful method for obtaining lymphoid tissue associate to mucosa for immunohistochemistry assessment to monitoring and ante-mortem diagnosis of scrapie in sheep and goats. However, inappropriate sampling or insufficient numbers of FL can generate the necessity to repeat the technique, which could be done 14 days after the first collection, without reduction in the number of the FL.

Este estudo teve por objetivo avaliar a quantidade de tecido linfoide associado à mucosa retal obtido pela técnica de biopsia retal e a possibilidade de se realizarem duas biopsias consecutivas, em diferentes intervalos de tempo, para monitoramento e diagnóstico ante mortem de scrapie. Para isso, foram estudados 56 ovinos e 32 caprinos. No dia zero, todos os animais foram submetidos a biopsias e, posteriormente, divididos em grupos. As colheitas foram realizadas aos dias sete, 14, 21 e 28 para os ovinos, e 14, 21 e 28 para os caprinos. De 176 amostras, 151 (85,8%) foram colhidas da mucosa retal e, em 25 (14,2%), houve falha de colheita. Considerando-se as amostras colhidas da mucosa retal (151), em 56,86% das amostras de ovinos e 51,61% de caprinos, no dia 0, havia 3 folículos linfoides (FL). Na segunda colheita, 58,97% das amostras de ovinos possuíam 3 FL e, para caprinos, 33,33%. Na comparação do número de FL entre a primeira e a segunda colheitas houve diferença (p 0,05) entre os dias 0 e 7 (com mais FL no dia 0) e 0 e 28 (com mais FL no dia 28) para ovinos, e entre os dias 0 e 28 (com mais FL no dia 0) para caprinos. Comparando-se as duas espécies, não houve diferença no número de FL nos dias 0, 14 e 21. No dia 28, a proporção de amostras com 3 FL foi maior nos ovinos (p 0,05) que nos caprinos. Concluiu-se que a técnica de biopsia retal compreende método útil para a obtenção de tecido linfoide associado à mucosa para avaliação imuno-histoquímica voltada ao monitoramento e diagnóstico ante mortem de scrapie em ovinos e caprinos. Porém, a colheita inadequada e a obtenção de número insuficiente de FL podem ocasionar a necessidade de repetição da técnica, o que pode ser realizado após 14 dias da primeira colheita, sem redução no número de FL.

Rectal biopsy in sheep and goats for monitoring and ante-mortem diagnosis of scrapie: Number of lymphoid follicles in two consecutive collections / Biopsia retal em ovinos e caprinos para monitoramento e diagnóstico ante mortem de scrapie: Número de folículos linfoides em duas colheitas consecutivas

This study aimed to evaluate the amount of lymphoid tissue associated with the rectal mucosa obtained by rectal biopsy and the possibility of two consecutive biopsies at different time intervals, for monitoring and ante-mortem diagnosis of scrapie. Rectal mucosa samples were collected from 56 sheep and 32 goats in two steps. In the first step, on day 0, all animals were tested and, for the second step, the animals were divided into groups and each group was subjected to collection on different dates: for sheep 7, 14, 21, and 28 days after the first one and, for goats, on days 14, 21, and 28. From 176 samples, 151 (85.8%) were collected from the rectal mucosa, and in 25 (14.2%) there was a collection failure. Considering the rectal mucosa samples (151), 56.86% of the sheep samples and 51.61% of the goat samples, on day 0, had more then 3 lymphoid follicles (LF). In the second collection, 58.97% of the sheep samples showed 3 LF and 33.33% of the goat samples. Comparing the number of LF of the same animals between the first and second collections, there was a significant difference (p 0.05) between days 0 and 7 for sheep (with more FL on day 0) and days 0 and 28 (with more LF on day 28) and days 0 and 28 for goats (with more FL on day 0). There was no significant difference in the number of FL assessed on dates 0, 14, and 21 when comparing the different species, sheep and goats. On day 28, sheep samples showed a higher number (p <0.05) of LF than goats. It was concluded that rectal biopsy technique involves useful method for obtaining lymphoid tissue associate to mucosa for immunohistochemistry assessment to monitoring and ante-mortem diagnosis of scrapie in sheep and goats. However, inappropriate sampling or insufficient numbers of FL can generate the necessity to repeat the technique, which could be done 14 days after the first collection, without reduction in the number of the FL.(AU)

Este estudo teve por objetivo avaliar a quantidade de tecido linfoide associado à mucosa retal obtido pela técnica de biopsia retal e a possibilidade de se realizarem duas biopsias consecutivas, em diferentes intervalos de tempo, para monitoramento e diagnóstico ante mortem de scrapie. Para isso, foram estudados 56 ovinos e 32 caprinos. No dia zero, todos os animais foram submetidos a biopsias e, posteriormente, divididos em grupos. As colheitas foram realizadas aos dias sete, 14, 21 e 28 para os ovinos, e 14, 21 e 28 para os caprinos. De 176 amostras, 151 (85,8%) foram colhidas da mucosa retal e, em 25 (14,2%), houve falha de colheita. Considerando-se as amostras colhidas da mucosa retal (151), em 56,86% das amostras de ovinos e 51,61% de caprinos, no dia 0, havia 3 folículos linfoides (FL). Na segunda colheita, 58,97% das amostras de ovinos possuíam 3 FL e, para caprinos, 33,33%. Na comparação do número de FL entre a primeira e a segunda colheitas houve diferença (p 0,05) entre os dias 0 e 7 (com mais FL no dia 0) e 0 e 28 (com mais FL no dia 28) para ovinos, e entre os dias 0 e 28 (com mais FL no dia 0) para caprinos. Comparando-se as duas espécies, não houve diferença no número de FL nos dias 0, 14 e 21. No dia 28, a proporção de amostras com 3 FL foi maior nos ovinos (p 0,05) que nos caprinos. Concluiu-se que a técnica de biopsia retal compreende método útil para a obtenção de tecido linfoide associado à mucosa para avaliação imuno-histoquímica voltada ao monitoramento e diagnóstico ante mortem de scrapie em ovinos e caprinos. Porém, a colheita inadequada e a obtenção de número insuficiente de FL podem ocasionar a necessidade de repetição da técnica, o que pode ser realizado após 14 dias da primeira colheita, sem redução no número de FL.(AU)

Scrapie diagnosis in a goat and four Santa Inês sheep from the same herd in Brazil / Diagnóstico de scrapie em um caprino e quatro ovinos Santa Inês de um mesmo rebanho no Brasil

Scrapie is a fatal and progressive transmissible spongiform encephalopathy (TSE) of natural occurrence in sheep and goats. The suspicion of scrapie may be based on clinical signs; however, the detection of pathological features of the prionic protein (PrP) in target tissues is necessary to diagnose the disease. The presence of an abnormal protein form (PrPSc) in lymphoreticular and nervous tissues is an important characteristic in diagnosis. This paper reports a case of scrapie in a flock of 55 Suffolk crossbred sheep, 19 Santa Inês sheep and 21 goats in the Mato Grosso state, midwestern Brazil. The animals were euthanized after the confirmation of a scrapie case with clinical signs in a Suffolk sheep in the same farm. Samples of brainstem at the level of the obex and lymphoid issues like palatine tonsils, mesenteric lymph nodes, third eyelid fixed in formalin 10% were processed for histological examination. Histological examination with hematoxylin and eosin did not show any microscopic changes in samples. Immunohistochemistry (IHC) examination to detect anti-prion PrPSc was performed in lymphoid tissues. Scrapie diagnosis was confirmed based on IHC positive results for PrPSc in lymphoid tissues of a crossbreed goat and four Santa Inês sheep, without any clinical scrapie signs. IHC showed positive staining in at least three lymphoid germinal centers in goat mesenteric lymph node, palatine tonsil, and third eyelid samples. The mesenteric lymph node, and tonsil samples of all sheep showed positive immunostaining, and only one sheep showed positive staining in lymphoid follicles in the third eyelid. Scrapie diagnosis using IHC in fixed samples of lymphoreticular tissue is technically feasible to detect the disease in both goats and sheep, as a form of pre-clinical diagnosis. The results indicate that the herd was infected by a sheep coming from another herd where scrapie had been diagnosed before(AU)

Scrapie é uma encefalopatia espongiforme transmissível (EET) progressiva e fatal de ocorrência natural em ovinos e caprinos. A suspeita de scrapie é baseada nos sinais clínicos, porém a manifestação patológica da proteína priônica (PrP) nos tecidos-alvo é necessária para a confirmação da doença. A presença de uma forma anormal da proteína (PrPSc) em tecido linforreticular e tecido nervoso constitui uma característica importante para o diagnóstico. Este trabalho é o relato de um foco de scrapie ocorrido em rebanho com 55 ovinos mistos Suffolk, 21 caprinos e 19 ovinos Santa Inês, na região Centro-Oeste do Brasil. Os animais foram eutanasiados após a confirmação de um caso de scrapie com sinais clínicos em um ovino Suffolk nessa propriedade. Amostras de tronco cerebral na altura do obex e tecidos linfoides, que incluíram tonsilas, linfonodos mesentéricos e tecido linfoide da terceira pálpebra foram processados para exame histológico. O exame histológico utilizando a coloração de hematoxilina e eosina não revelou a presença de alterações microscópicas nos tecidos examinados. O diagnóstico de scrapie foi confirmado com base nos resultados positivos de imuno-histoquímica (IHQ) para PrPSc nos tecidos linfoides de um caprino sem raça definida e quatro ovinos da raça Santa Inês, sem sinais clínicos de scrapie. A IHQ apresentou marcação positiva em pelo menos três centros linfoides na tonsila, terceira pálpebra e linfonodo mesentérico do caprino. Em todos os ovinos, a IHQ revelou marcação positiva nos folículos linfoides da tonsila palatínica e linfonodo mesentérico; a marcação positiva nos folículos linfoides da terceira pálpebra só foi observada em um dos ovinos. Este trabalho demonstra a importância da utilização de tecido linforreticular para o diagnóstico pré-clínico de scrapie através de IHQ e é tecnicamente viável em ovinos e caprinos. [...](AU)

Utilização de biópsias da terceira pálpebra e mucosa retal em ovinos para diagnóstico de scrapie em uma propriedade da região sul do Brasil / Use of third eyelid and rectal mucosa biopsies for diagnosis of sheep scrapie on a farm in southern Brazil

Scrapie é uma encefalopatia espongiforme transmissível (EET) que causa lesões cerebrais degenerativas em ovinos e caprinos. Caracteriza-se pelo acúmulo, no tecido encefálico e linforreticular, da forma anormal da proteína priônica (PrP Sc) que provoca a morte maciça de neurônios e células gliais, além de vacuolização intensa no tecido afetado. Esse trabalho descreve a utilização da técnica de imuno-histoquímica (IHQ) para proteína priônica em tecido linforreticular de biópsias de terceira pálpebra e mucosa retal, como método diagnóstico de scrapie em ovinos. Realizaram-se exames de IHQ para scrapie em amostras de uma propriedade de origem de um ovino com diagnóstico dessa enfermidade. Utilizaram-se anticorpos monoclonais antipríon para diagnóstico ante mortem pela técnica de IHQ. Nas 318 amostras de biópsias analisadas, encontrou-se 19 resultados positivos para PrP Sc nos folículos de terceira pálpebra e não foi obtida marcação no tecido linfático de mucosa retal em nenhuma das amostras coletadas. Realizaram-se 18 necropsias dos animais positivos anteriormente por biópsia e 21 necropsias de ovinos parentes dos positivos de scrapie. Confirmou-se o resultado de scrapie pela IHQ após a necropsia dos animais positivos nas biópsias de terceira pálpebra. Nesses animais, os órgãos com maior número de cortes positivos foram a terceira pálpebra (18/18) e a tonsila (8/18). Nos ovinos com parentesco com os positivos, nenhum resultado de scrapie ocorreu. A utilização de tecidos linfoides no diagnóstico de scrapie por IHQ através de biópsias mostrou-se um método viável e eficaz para o diagnóstico pré-clínico.(AU)

Scrapie, a form of transmissible spongiform encephalopathy (TSEs) is a fatal neurodegenerative disorder that affects sheep and goats. The disease is characterized by an accumulation of the abnormal prionic protein (PrP Sc) in the encephalic and lymphoreticular tissues. This paper describes the use of anti-prionic protein immunohistochemical (IHC) procedure as a method of pre-clinical diagnosis of scrapie.The test was carried out in biopsied lymphoreticular tissues from third eyelid and rectal mucosa. Anti-prion protein monoclonal antibodies F89/160.1.5 and F99/97.6.1 were used. Scrapie diagnosis in lymphoreticular tissues through IHC was achieved when the samples had a minimum of three lymphoid follicles in well delimited germinal centre. Positive immunostaining was identified in 19 out of 318 samples of the third eyelid. Material sampled at post-mortem examination in 18 of these scrapie-positive sheep, which were previously verified by biopsy, and in 21 of its relatives, was confirmed with IHC tests. Positive immunostaining from rectal mucosa tissue was not observed. Third eyelid and tonsil were the organs with the larger amount of positive immunostaining (18/18 and 8/18 respectively) at post-mortem examination. None positive result was obtained along the 21 animals related to the positive ones, and none of the positive cases showed IHC labeling in the brain. The use of lymphoid tissues for scrapie diagnosis by IHC through biopsies showed to be a viable and efficient method for pre-clinical diagnostic.(AU)

Apoptosis detection in embryonic chicken lymphoid tissues infected with attenuated very virulent infectious bursal disease virus (vvIBDV)

We exposed chicken embryos at embryonating day (ED18) to a cell-adapted very virulent strain of IBDV (ca-vvIBDV) and original vvIBDV and examined the apoptosis from infected bursa of Fabricius (BF) and thymus organs. Following ca-vvIBDV exposure, embryonic bursa showed mild cellular destruction, lower rate of apoptosis and presence of viral proteins detectable by immunohistochemistry. In contrary, original vvIBDV exposed embryos had an enhanced detectable changes in the bursa associated to an increase apoptotic events, and most of the times, total destruction of BF follicles. In thymus, viral antigen was detectable until after hatch. Positives cell signals to activated caspase-3 were intensively detect in embryos lymphoid tissues exposed to original vvIBDV observed in BF and less in thymus. No immunoreactive thymocytes were visualized in embryos exposed to ca-vvIBDV. Apoptosis changes, such as chromatin condensation, DNA fragmentation, and the appearance of apoptotic nuclear bodies, were observed in both organs. TUNEL-detected DNA was more intense in original vvIBDV infected lymphoid cells, and less apoptotic cells were detectable in attenuated strain. By sequencing analysis, e attenuation presented amino acid changes at position 222 (A→→P), 256 (I→V) and 279 (D→N). One serine in the serine-rich heptapeptide (position 333) was substituted into other amino acid which is similar to the IBDV vaccine strain. Taken together our results indicate that virus attenuation interferes with caspase-3 apoptotic pathway and may play an important role in switch viral pathogenesis.

Histopathological examination of lymphoid organs in cattle and mice experimentally poisoned by Baccharis coridifolia: immunohistochemical characterization of B and T Lymphocytes / Analise histopatológica de órgãos linfóides de bovinos e camundongos intoxicados experimentalmente por Baccharis coridifolia: caracterização imunoistoquímica de linfócitos B e T

Baccharis coridifolia is one of the most important poisonous plants to cattle in the South of Brazil. The plant provokes necrotic lesions in the lymphoid tissues and in the gastrointestinal tract of cattle. Experimental administration to mice produces most of the lesions seen in the lymphoid tissues of cattle. This study was conducted to search possible differences in the susceptibility of T and B lymphocyte subpopulations. Lymph nodes, spleen, thymus and gut-associated lymphoid tissue (GALT) of cattle and mice experimentally poisoned were evaluated. The results were evaluated based on cell populations affected or remaining in the organs. Immunostaining for B lymphocytes (anti-BLA-36) identified the germinal center of follicles of the lymph node, spleen and GALT in both species. Immunostaining for T lymphocyte (anti-CD3) identified the paracortical area of the germinal centers of the lymph nodes and GALT, the periarteriolar area of the spleen, and the whole thymus both in cattle and mice. Experimentally poisoned cattle and mice shows necrosis of the germinal center of secondary follicles of the lymph nodes, spleen and GALT, where necrotic cells were immunostained for B and less often for T lymphocyte. Necrotic cells in the paracortical region of the lymph node were less often and were not immunostained. Necrotic lesions of the thymus were seen only in mice, with positively stained for T lymphocyte. The distribution of the lesions in the lymphoid tissues and the immunostaining in necrotic cells suggested that the active principles of the plant are cytotoxic to B and T cells.(AU)

Baccharis coridifolia é uma das mais importantes plantas tóxicas para bovinos no Sul do Brasil. A intoxicação pela planta produz lesões necróticas nos tecidos linfóides e no trato gastrointestinal de bovinos. A administração experimental para camundongos produziu a maioria das lesões que ocorrem nos tecidos linfóides de bovinos. Este estudo foi conduzido para detectar as possíveis diferenças na susceptibilidade das populações de linfócitos T e B. Foram utilizados linfonodos, baço, timo e acúmulos linfóides associados ao intestino de bovinos e camundongos experimentalmente intoxicados pela planta. Os resultados foram avaliados com base na população de células lesadas ou por aquela que permaneceram nos tecidos. Em ambas as espécies, na marcação imunoistoquímica para linfócitos B (anti-BLA-36) predominou a região do centro germinativo dos linfonodos, baço e acúmulos linfóides associados ao intestino. A marcação para linfócitos T (anti-CD3) predominou na região paracortical dos linfonodos, acúmulos linfóides associados ao intestino e região periarteriolar do baço e timo de ambas as espécies. Bovinos e camundongos experimentalmente intoxicados demonstraram acentuada necrose do centro germinativo dos folículos secundários dos linfonodos, baço e intestino, onde as células necrosadas foram marcadas pela imunoistoquímica para linfócitos B e em menor freqüência para linfócitos T. Necrose celular na região paracortical do linfonodo foi discreta e sem marcação imunoistoquímica. Células necróticas no timo foram observadas somente em camundongos, com imunoistoquímica positiva para linfócitos T. A distribuição das lesões nos tecidos linfóides associadas à marcação imunoistoquímica das células necrosadas sugerem que o princípio ativo da planta é citotóxico para células T e B.(AU)