Expression Pattern of Sulf1 and Sulf2 in Chicken Tissues and Characterization of Their Expression During Different Periods in Skeletal Muscle Satellite Cells

Heparan sulfate proteoglycans (HSPGs) are present on the cell surface and in the extracellular matrix in all metazoans. HSPGs interact with growth factors and receptors through heparan sulfate (HS) chains. The sulfation pattern of heparan sulfate chains influences signaling events mediated by heparan sulfate proteoglycans located on the cell surface. SULF1 and SULF2 are two endo-sulfatases that can cleave specific 6-O-sulfate groups within the heparan chains. To determine their possible roles in tissues and satellite cells in vitro, their expression pattern was examined in tissues from 40-day-old chickens and in satellite cells from the breast muscles of 1-week-old and 2-week-old chickens using RT-PCR and immunocytochemistry analyses. The SULF1 and SULF2 transcripts were widely distributed in various tissues. Upon increasing culture times in chicken´s primary skeletal muscle satellite cells, SULF1 and SULF2 expression in 1-week-old chickens was significantly higher than in 2-week-old chickens, suggesting that sulfatases play a key role in satellite cell development. Therefore, our findings increase our knowledge of sulfatase expression diversity and provide a solid basis for further research concerning this molecular mechanism.(AU)

Expression Pattern of Sulf1 and Sulf2 in Chicken Tissues and Characterization of Their Expression During Different Periods in Skeletal Muscle Satellite Cells

Heparan sulfate proteoglycans (HSPGs) are present on the cell surface and in the extracellular matrix in all metazoans. HSPGs interact with growth factors and receptors through heparan sulfate (HS) chains. The sulfation pattern of heparan sulfate chains influences signaling events mediated by heparan sulfate proteoglycans located on the cell surface. SULF1 and SULF2 are two endo-sulfatases that can cleave specific 6-O-sulfate groups within the heparan chains. To determine their possible roles in tissues and satellite cells in vitro, their expression pattern was examined in tissues from 40-day-old chickens and in satellite cells from the breast muscles of 1-week-old and 2-week-old chickens using RT-PCR and immunocytochemistry analyses. The SULF1 and SULF2 transcripts were widely distributed in various tissues. Upon increasing culture times in chicken´s primary skeletal muscle satellite cells, SULF1 and SULF2 expression in 1-week-old chickens was significantly higher than in 2-week-old chickens, suggesting that sulfatases play a key role in satellite cell development. Therefore, our findings increase our knowledge of sulfatase expression diversity and provide a solid basis for further research concerning this molecular mechanism.

Investigations of pathological immunohistochemical and immunocytochemical findings in natural infection with Mycoplasma gallisepticum in laying hens

Background: Mycoplasmosis is an infectious disease caused by Mycoplasma gallisepticum (MG), usually seen in therespiratory system of chickens, chick and turkeys, that causing great economic loss. The disease is characterized by respiratory system lesions such as sinusitis, tracheitis, airsacculitis, pneumonia and other symptoms such as loss of yield, arthritis,tenosynovitis. In this study, it was aimed to investigate diagnose of the disease by pathologic and molecular techniquesin hens that naturally infected with MG as well as the usability of immunocytochemical (ICC) method in diagnose of thedisease.Materials, Methods & Results: For this purpose, 98 hens were collected from 10 different coops that serologically positive.After necropsy, routine pathological procedures were performed to samples taken from nose, sinus, larynx, trachea, lungand air sacs. Scraping samples taken from lungs and tracheas were evaluated by ICC. Immunohistochemical (IHC) stainingwas performed to samples taken from nose, sinus, larynx, trachea, lung and air sacs. Indirect immunoperoxidase methodwas applied in the both IHC and ICC staining. Rabbit polyclonal anti MG antibody was used as primer antibody in theIHC and ICC staining. Additionally, culture and PCR techniques were applied to tracheas of all hens for MG. The GPO3and MGSO genes were made for PCR analysis. In the tracheal examinations, 23 cases were positive for PCR, 17 casesICC positive, 16 cases IHC positive and 10 culture samples found positive. All of culture positive cases were also positivefor other three methods. When findings in all organs were evaluated, in 37 cases were detected positive by IHC (38%) and23 cases were positive by ICC (23.5%). In the IHC positive cases, the first order was trachea in 16 cases followed by in 11cases in sinus, in 8 cases in lung, in 6 cases air sac and 4 cases in nose, respectively. In 8 cases, IHC positivity was foundin at least two organs...

Investigations of pathological immunohistochemical and immunocytochemical findings in natural infection with Mycoplasma gallisepticum in laying hens

Background: Mycoplasmosis is an infectious disease caused by Mycoplasma gallisepticum (MG), usually seen in therespiratory system of chickens, chick and turkeys, that causing great economic loss. The disease is characterized by respiratory system lesions such as sinusitis, tracheitis, airsacculitis, pneumonia and other symptoms such as loss of yield, arthritis,tenosynovitis. In this study, it was aimed to investigate diagnose of the disease by pathologic and molecular techniquesin hens that naturally infected with MG as well as the usability of immunocytochemical (ICC) method in diagnose of thedisease.Materials, Methods & Results: For this purpose, 98 hens were collected from 10 different coops that serologically positive.After necropsy, routine pathological procedures were performed to samples taken from nose, sinus, larynx, trachea, lungand air sacs. Scraping samples taken from lungs and tracheas were evaluated by ICC. Immunohistochemical (IHC) stainingwas performed to samples taken from nose, sinus, larynx, trachea, lung and air sacs. Indirect immunoperoxidase methodwas applied in the both IHC and ICC staining. Rabbit polyclonal anti MG antibody was used as primer antibody in theIHC and ICC staining. Additionally, culture and PCR techniques were applied to tracheas of all hens for MG. The GPO3and MGSO genes were made for PCR analysis. In the tracheal examinations, 23 cases were positive for PCR, 17 casesICC positive, 16 cases IHC positive and 10 culture samples found positive. All of culture positive cases were also positivefor other three methods. When findings in all organs were evaluated, in 37 cases were detected positive by IHC (38%) and23 cases were positive by ICC (23.5%). In the IHC positive cases, the first order was trachea in 16 cases followed by in 11cases in sinus, in 8 cases in lung, in 6 cases air sac and 4 cases in nose, respectively. In 8 cases, IHC positivity was foundin at least two organs...(AU)

Patologia morfológica e molecular aplicadas à inovação diagnóstica e vigilância da leishmaniose visceral / Morphological and molecular pathology applied to diagnostic innovation and surveillance of visceral leishmaniasis

O conceito de Saúde Única surgiu para ressaltar a união indissociável entre a saúde animal, humana e ambiental. Nesse contexto, a leishmaniose visceral americana (LVA) é considerada uma importante doença de saúde pública no Brasil, devido a sua crescente expansão geográfica e aumento na incidência de casos humanos. A LVA é uma doença parasitária, zoonótica, causada pela Leishmania (Leishmania) infantum (syn. chagasi) e transmitida por flebotomíneos do gênero Lutzomyia. Os cães são considerados os principais reservatórios do parasito nas áreas urbanas. O diagnóstico da LVA é baseado em aspectos epidemiológicos, clínicos e laboratoriais. A demonstração da presença do parasito através de exames diretos em tecidos biológicos do hospedeiro é o diagnóstico de escolha, principalmente, em municípios em que a transmissão de LVA ainda não tenha sido confirmada. Diversas metodologias podem ser aplicadas com essa finalidade. O objetivo desse trabalho é apresentar as técnicas citológicas, anatomo-patológicas e moleculares em amostras fixadas em formalina e incluídas em parafina para o diagnóstico da leishmaniose visceral em humanos e cães. Esses dados são complementares à apresentação realizada no I Simpósio Internacional de Leishmaniose Visceral, realizado nos dia 23 e 24 de Abril de 2018, e organizado pelo Instituto Adolfo Lutz em São Paulo-SP, Brasil.

The One Health concept emerged to highlight the inseparable link between animal, human and environmental health. In this context, American Visceral Leishmaniasis (AVL) is considered an important public health disease in Brazil, due to its increasing geographic expansion and in the incidence of human cases. AVL is a parasitic and zoonotic disease caused by Leishmania (Leishmania) infantum (syn. chagasi) and transmitted by sandflies of the genus Lutzomyia. Dogs are considered the main reservoirs of the parasite in urban areas. The diagnosis of AVL is based on epidemiological, clinical and laboratory aspects. The demonstration of the presence of the parasite through direct examinations in biological tissues of the host is the diagnosis of choice, mainly in municipalities where the transmission of AVL has not yet been confirmed. Several methodologies can be applied for this purpose. The objective of this work is to present the cytological, anatomopathological and molecular techniques in formalin fixed and paraffin embedded samples for the diagnosis of visceral leishmaniasis in humans and dogs. These data are complementary to the present study at the First International Symposium on Visceral Leishmaniasis, held on April 23 and 24, 2018, and organized by Adolfo Lutz Institute in São Paulo, Brazil.

Patologia morfológica e molecular aplicadas à inovação diagnóstica e vigilância da leishmaniose visceral / Morphological and molecular pathology applied to diagnostic innovation and surveillance of visceral leishmaniasis

O conceito de Saúde Única surgiu para ressaltar a união indissociável entre a saúde animal, humana e ambiental. Nesse contexto, a leishmaniose visceral americana (LVA) é considerada uma importante doença de saúde pública no Brasil, devido a sua crescente expansão geográfica e aumento na incidência de casos humanos. A LVA é uma doença parasitária, zoonótica, causada pela Leishmania (Leishmania) infantum (syn. chagasi) e transmitida por flebotomíneos do gênero Lutzomyia. Os cães são considerados os principais reservatórios do parasito nas áreas urbanas. O diagnóstico da LVA é baseado em aspectos epidemiológicos, clínicos e laboratoriais. A demonstração da presença do parasito através de exames diretos em tecidos biológicos do hospedeiro é o diagnóstico de escolha, principalmente, em municípios em que a transmissão de LVA ainda não tenha sido confirmada. Diversas metodologias podem ser aplicadas com essa finalidade. O objetivo desse trabalho é apresentar as técnicas citológicas, anatomo-patológicas e moleculares em amostras fixadas em formalina e incluídas em parafina para o diagnóstico da leishmaniose visceral em humanos e cães. Esses dados são complementares à apresentação realizada no I Simpósio Internacional de Leishmaniose Visceral, realizado nos dia 23 e 24 de Abril de 2018, e organizado pelo Instituto Adolfo Lutz em São Paulo-SP, Brasil.(AU)

The One Health concept emerged to highlight the inseparable link between animal, human and environmental health. In this context, American Visceral Leishmaniasis (AVL) is considered an important public health disease in Brazil, due to its increasing geographic expansion and in the incidence of human cases. AVL is a parasitic and zoonotic disease caused by Leishmania (Leishmania) infantum (syn. chagasi) and transmitted by sandflies of the genus Lutzomyia. Dogs are considered the main reservoirs of the parasite in urban areas. The diagnosis of AVL is based on epidemiological, clinical and laboratory aspects. The demonstration of the presence of the parasite through direct examinations in biological tissues of the host is the diagnosis of choice, mainly in municipalities where the transmission of AVL has not yet been confirmed. Several methodologies can be applied for this purpose. The objective of this work is to present the cytological, anatomopathological and molecular techniques in formalin fixed and paraffin embedded samples for the diagnosis of visceral leishmaniasis in humans and dogs. These data are complementary to the present study at the First International Symposium on Visceral Leishmaniasis, held on April 23 and 24, 2018, and organized by Adolfo Lutz Institute in São Paulo, Brazil.(AU)

Leucemia linfocítica b em cão jovem: relato de caso / B - lymphocytic leukemia in young dog: case report / Leucemia linfocítica b en perro joven: relato de caso

O presente relato teve como objetivo descrever um caso de leucemia linfocítica B em um cão da raça maltês, fêmea, com três anos de idade, demonstrando a metodologia de diagnóstico e a variação da patologia clínica após o tratamento exclusivamente por esplenectomia. A linfocitose acentuada observada no hemograma e presença proliferativa de linfócitos na medula óssea determinou a suspeita da leucemia linfocítica. A leucemia linfocítica B foi diagnosticada por meio da citologia aspirativa da medula óssea, imunocitoquímica e citometria de fluxo. O tratamento foi exclusivamente por esplenectomia e dois anos após a cirurgia o animal não apresentou recidiva da afecção.(AU)

This report aimed to describe a case of B lymphocytic leukemia in maltese female dog, three years old, demonstrating the methodology of diagnostic and variation of clinical pathology after treatment exclusively by splenectomy. The accentuate lymphocytes observed in blood counts and presence of proliferative lymphocytes in the bone marrow were determined to diagnostic of lymphocytic leukemia. The B lymphocytic leukemia was diagnosed by bone marrow aspiration cytology, immunocytochemistry and flow cytometry. The treatment was exclusively by splenectomy and two years after the surgery the animal showed no recurrence of the disease.(AU)

El objetivo de ese relato ha sido describir un caso de leucemia linfocítica B en un perro, raza maltés, hembra, de tres años, demostrando la metodología de diagnóstico y la variación de la patología clínica, después del tratamiento exclusivamente por esplenectomía. La linfocitosis aguda, observada en el hemograma, y la presencia de proliferación de linfocitos en la médula ósea, determinó la sospecha de leucemia linfocítica. La leucemia linfocítica B fue diagnosticada mediante la citología por aspiración de médula ósea, inmunocitoquímica y citometría de flujo. El tratamiento fue exclusivamente por esplenectomía y dos años después de la cirugía el animal no mostró recurrencia de la enfermedad.(AU)

Leucemia linfocítica b em cão jovem: relato de caso / B - lymphocytic leukemia in young dog: case report / Leucemia linfocítica b en perro joven: relato de caso

O presente relato teve como objetivo descrever um caso de leucemia linfocítica B em um cão da raça maltês, fêmea, com três anos de idade, demonstrando a metodologia de diagnóstico e a variação da patologia clínica após o tratamento exclusivamente por esplenectomia. A linfocitose acentuada observada no hemograma e presença proliferativa de linfócitos na medula óssea determinou a suspeita da leucemia linfocítica. A leucemia linfocítica B foi diagnosticada por meio da citologia aspirativa da medula óssea, imunocitoquímica e citometria de fluxo. O tratamento foi exclusivamente por esplenectomia e dois anos após a cirurgia o animal não apresentou recidiva da afecção.

This report aimed to describe a case of B lymphocytic leukemia in maltese female dog, three years old, demonstrating the methodology of diagnostic and variation of clinical pathology after treatment exclusively by splenectomy. The accentuate lymphocytes observed in blood counts and presence of proliferative lymphocytes in the bone marrow were determined to diagnostic of lymphocytic leukemia. The B lymphocytic leukemia was diagnosed by bone marrow aspiration cytology, immunocytochemistry and flow cytometry. The treatment was exclusively by splenectomy and two years after the surgery the animal showed no recurrence of the disease.

El objetivo de ese relato ha sido describir un caso de leucemia linfocítica B en un perro, raza maltés, hembra, de tres años, demostrando la metodología de diagnóstico y la variación de la patología clínica, después del tratamiento exclusivamente por esplenectomía. La linfocitosis aguda, observada en el hemograma, y la presencia de proliferación de linfocitos en la médula ósea, determinó la sospecha de leucemia linfocítica. La leucemia linfocítica B fue diagnosticada mediante la citología por aspiración de médula ósea, inmunocitoquímica y citometría de flujo. El tratamiento fue exclusivamente por esplenectomía y dos años después de la cirugía el animal no mostró recurrencia de la enfermedad.

Imunofenotipagem do linfoma canino pela técnica da imunocitoquímica (cell block).

O linfoma canino é uma das neoplasias hematopoiéticas mais freqüentes na rotina do médico veterinário. Os animais podem apresentar diferentes formas clínicas, estadio e sub-estadio. Considerada uma doença heterogênea, o linfoma é classificado a partir do seu imunofenótipo para em seguida ser sub-classificado. Conhecer o imunofenótipo é importante para o diagnóstico, para o prognóstico e tratamento. A imunofenotipagem na maioria dos casos é realizada pela imunohistoquímica (IHQ), através de um fragmento de tecido obtido pela biopsia. Para a realização da biópsia os pacientes precisam ser submetidos a cirurgia porém nem sempre apresentam condições clínicas. A busca por um diagnóstico precoce, pouco invasivo, e com menor custo tem sido motivada em diversas doenças. Uma modalidade ainda pouco explorada na medicina veterinária é a realização do cell block (CB), técnica que permite a emblocagem do material de origem citológica para a realização de exames complementares, incluindo a imunocitoquímica (ICQ). O objetivo deste trabalho foi realizar a imunofenotipagem do linfoma canino através ICQ pela técnica do CB. Foram utilizadas 54 amostras de cães com linfoma de diferentes apresentações clínicas. As amostras da ICQ foram comparadas com as amostras de IHQ provenientes do mesmo tecido. A ICQ pela técnica do CB apresentou uma acurácia de 87,04%, e o valor de Kappa de 0,6986 (p. <0,001). Na análise específica por classe, as classificações de células T ou B tiveram altos valores de Kappa, acurácia, sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo. Não foram identificados animais do imunofenótipo não T não B (nulo). A utilização do cell block foi considerada uma técnica válida para realização da imunofenotipagem do linfoma canino e auxílio diagnóstico. O CB poderá ser implantado na rotina diagnóstica oferecendo informação prognóstica e melhorando o diagnóstico definitivo de uma maneira rápida, simples, econômica e eficaz.

Canine lymphoma is one of the most frequent hematopoietic neoplasms in veterinary routine. Animals may have different clinical presentations, stage and sub-stage. Lymphoma is considered a heterogeneous disease, classified by immunophenotype and then sub-classified. Immunophenotype knowledge is important for diagnosis and prognosis. Immunophenotyping, in most cases, is done by means of immunohistochemistry (IHC), using a tissue fragment obtained from biopsy. Patients are often in poor condition to undergo surgery, so it is not always possible to obtain a tissue sample for biopsy. The search for an early, less invasive, and low-cost diagnosis has been motivated in several pathologies. A modality not yet explored in veterinary medicine is the cell block (CB), a technique that allows a re-embedding of material of cytological origin for complementary exams, including immunocytochemistry (ICQ). The objective of this study was to perform immunophenotyping of canine lymphoma through ICQ using CB technique. Fifty-four samples from dogs with different clinical presentations were used. ICQ samples were compared with IHC samples from the same tissue. ICQ from CB technique had 87.04% accuracy, and Kappa value of 0.6986 (p<0.001). In class-specific analysis, such as T, B or mixed cell classification, ICQ had high values of Kappa, accuracy, sensitivity, specificity, positive predictive value and negative predictive value. No animals of the non-T non-B immunophenotype (null) were identified. Cell block was designed for canine lymphoma immunophenotyping and to aid in diagnosis. CB can be implemented in the diagnostic routine, providing prognostic information and improving definitive diagnosis in a fast, simple, inexpensive and effective way.

Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane / Topografia de ligação de proteínas do plasma seminal à membrana de espermatozoides bovinos epididimários e ejaculados

The present study was designed to investigate the topographical distribution of seminal plasma (SP) proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS) and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.(AU)

Investigou-se a distribuição topográfica da ligação de proteínas seminais à membrana de espermatozoides bovinos epididimários e ejaculados. Utilizando imunocitoquímica e microscopia confocal, avaliaram-se a topografia de ligação das proteínas BSP-A3, albumina, transferrina, prostaglandina D sintetase (PGDS) e nucleobindina 2 (NUC2) à membrana espermática. Os experimentos foram realizados utilizando espermatozoides de cinco touros. Os resultados mostraram que, para espermatozoides epididimários, somente detectou-se a PGDS na crista do acrossomo. Nos espermatozoides ejaculados, a PGDS ligou-se de forma mais intensa à crista acrossômica, enquanto a NUC2 apresentou sinal bastante fraco na peça intermediária e região equatorial. A BSP-A3 ligou-se a todas as regiões estudadas, de forma mais intensa na peça intermediária e acrossomo. Nenhum sinal foi detectado para albumina ou transferrina, seja em espermatozoides epididimários ou ejaculados. Concluiu-se que PGDS, BSP-A3 e NUC2 interagem diretamente com espermatozoides bovinos, e mostrou distribuição topográfica específica. Estes achados permitem melhor compreensão sobre o papel desempenhado por essas proteínas na regulação da função espermática e da fertilidade.(AU)

Immunocytochemical characterization of the cytopathic effect induced by bovine respiratory syncytial virus strain RC 98 on Hep-2 cells / Caracterização imunocitoquímica do efeito citopático induzido pelo vírus respiratório sincicial bovino amostra RC98 em células Hep-2

Caracterizou-se o efeito citopático produzido pela amostra de vírus respiratório sincicial bovino (BRSV) RC-98, isolada na Argentina, por meio de imunocitoquímica em cultivos de células da linhagem Hep-2. Um soro policlonal anti-BRSV foi utilizado para a imunocitoquímica em células Hep-2 infectadas. Sinais específicos do virus foram observados no citoplasma de um grande número de células, consistindo em inclusões citoplasmáticas e células sinciciais. Efeitos citopáticos distintos foram observados, com frequência, no núcleo das células infectadas, aparecendo como sinais específicos fortes, podendo corresponder a inclusões intranucleares. A presença de sinais intranucleares pode consistir uma característica particular da amostra RC98 do BRSV. (AU)