Nodular lymphocytic conjunctivitis in a horse - case report / Conjuntivite linfocítica nodular em equino - relato de caso

The case of an equine with nodular lymphocytic conjunctivitis is described. A 14-year-old crossbred mare was seen presenting with development of a mass in the nasal angle of the right eye, behind the third eyelid. The tutor reported slow growth over 4 years, always accompanied by epiphora, and that no treatment had been performed prior to consultation. The histopathological and immunohistochemical results found a nodular, subepithelial structure, composed predominantly of densely packed small lymphocytes. Through the exams, associated with studies with monoclonal anti B lymphocyte antibodies and polyclonal anti T lymphocyte antibodies, the diagnosis of nodular lymphocytic conjunctivitis was reached. Only clinical pharmacological treatment was chosen, based on the use of topical and intralesional hydrocortisone acetate. After one month of treatment the mass completely disappeared without sequelae.

Descreve-se o caso de um equino com conjuntivite linfocítica nodular. Uma égua, mestiça, de 14 anos, foi atendida apresentando desenvolvimento de uma massa no ângulo nasal do olho direito, atrás da terceira pálpebra. O tutor relatou um crescimento lento durante 4 anos, sempre acompanhado de epífora, para o qual não foi realizado tratamento prévio à consulta. Os resultados histopatológico e imuno-histoquímico constataram uma estrutura nodular e subepitelial, composta predominantemente por linfocitos pequenos densamente agrupados. Por meio dos exames, associados a estudos com anticorpos monoclonais antilinfocitos B e anticorpos policlonais antilinfocitos T, chegou-se ao diagnóstico de conjuntivite linfocítica nodular. Optou-se apenas pelo tratamento clínico farmacológico, baseando-se na utilização de acetato de hidrocortisona tópica e intralesional. Após um mês de tratamento, a massa involuiu por completo, sem deixar sequelas.

Production of polyclonal antibodies anti - Igm of nile tilapia, Oreochromis niloticus for standardization of immunoenzymatic assay / Produção de anticorpos policlonais anti - Igm de tilápia-do-nilo, Oreochromis niloticus para padronização de ensaio imunoenzimático

In this letter, we describe the development of an immunodiagnostic assay capable of detecting specific IgM in the blood circulation of experimental Nile tilapia that received the vaccine against S. agalactiae.(AU)

Production of polyclonal antibodies anti - Igm of nile tilapia, Oreochromis niloticus for standardization of immunoenzymatic assay / Produção de anticorpos policlonais anti - Igm de tilápia-do-nilo, Oreochromis niloticus para padronização de ensaio imunoenzimático

In this letter, we describe the development of an immunodiagnostic assay capable of detecting specific IgM in the blood circulation of experimental Nile tilapia that received the vaccine against S. agalactiae.

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris / Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris

O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.(AU)

Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.(AU)

Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris / Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris

O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.(AU)

Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.(AU)

Anticorpos policlonais para o imunodiagnóstico de vírus bovino / Polyclonal antibodies for the immunodiagnostic of bovine viruses

O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.

The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.

Anticorpos policlonais para o imunodiagnóstico de vírus bovino / Polyclonal antibodies for the immunodiagnostic of bovine viruses

O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.(AU)

The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.(AU)

Evaluation of new antibodies for the detection of rabies virus in formalin fixed brain tissue samples

Rabies is an important zoonosis with impact on livestock production. The diagnosis is usually based on the rabies virus detection in fresh or refrigerated brain samples by direct immunofluorescence (IF) performed on fixed brain smears. The aim of this study was to evaluate the performance of 6 monoclonal and 1 rabbit-polyclonal new antibodies for rabies diagnostic by using immunohistochemistry (IHC), which detects the agent on formalin fixed paraffin embedded tissues samples. Tested with 2 positive and 2 negative cows for rabies at dilutions 1/200 and 1/1000, obtained immunostaining was strong for one monoclonal and weak for the polyclonal at both dilutions. For 3 monoclonals, the immunostaining was weaker at 1/1000 and was negative at both dilutions for 2 monoclonals. Unwanted background was absent and negative samples remained clear for all antibodies. When the first monoclonal was applied on sections of brain of 19 cows with Rabies and 41 control cows at 1/1000 dilution, immunohistochemistry recognized all positive samples and was negative for all control cows. The number of cases analyzed did not allow estimating sensitivity and specificity of the tested assay, but the correlation observed between IF and IHC in both positive and negative samples suggested that accuracy of the test might be good. The results indicated that the tested antibody can detect the rabies virus on formalin fixed tissue samples, and that immunohistochemistry can complement other confirmatory tests when those cannot be performed.

Evaluation of new antibodies for the detection of rabies virus in formalin fixed brain tissue samples

Rabies is an important zoonosis with impact on livestock production. The diagnosis is usually based on the rabies virus detection in fresh or refrigerated brain samples by direct immunofluorescence (IF) performed on fixed brain smears. The aim of this study was to evaluate the performance of 6 monoclonal and 1 rabbit-polyclonal new antibodies for rabies diagnostic by using immunohistochemistry (IHC), which detects the agent on formalin fixed paraffin embedded tissues samples. Tested with 2 positive and 2 negative cows for rabies at dilutions 1/200 and 1/1000, obtained immunostaining was strong for one monoclonal and weak for the polyclonal at both dilutions. For 3 monoclonals, the immunostaining was weaker at 1/1000 and was negative at both dilutions for 2 monoclonals. Unwanted background was absent and negative samples remained clear for all antibodies. When the first monoclonal was applied on sections of brain of 19 cows with Rabies and 41 control cows at 1/1000 dilution, immunohistochemistry recognized all positive samples and was negative for all control cows. The number of cases analyzed did not allow estimating sensitivity and specificity of the tested assay, but the correlation observed between IF and IHC in both positive and negative samples suggested that accuracy of the test might be good. The results indicated that the tested antibody can detect the rabies virus on formalin fixed tissue samples, and that immunohistochemistry can complement other confirmatory tests when those cannot be performed.(AU)

Detecção de Campylobacter jejuni em produtos de frango utilizando separação imunomagnética / Detection of Campylobacter jejuni in chicken products using immunomagnetic separation

Campylobacter jejuni é o principal causador de gastroenterite bacteriana aguda, e a carne de frango tem se mostrado uma importante fonte de transmissão. Este microrganismo é de difícil isolamento e os métodos convencionais muitas vezes não são eficientes, podendo levar a resultados errôneos. O objetivo deste trabalho foi desenvolver e testar a técnica de separação imunomagnética (IMS) na detecção de C. jejuni em produtos de frango. Micropartículas magnéticas ligadas a anticorpos policlonais anti-C. jejuni foram utilizadas para concentrar C. jejuni antes da semeadura em ágar. O protocolo foi comparado com o método convencional. C. jejuni foi recuperado do alimento experimentalmente contaminado por ambos os métodos, entretanto, quando foi usada a IMS, a presença de microrganismos contaminantes nos meios de cultura foi menor. C. jejuni foi isolado de 7% das amostras de alimento naturalmente contaminadas, usando IMS, e de 3% pelo método convencional. C. coli foi isolado de uma amostra pelo método convencional, mas não foi detectado pelo protocolo com IMS. A técnica de IMS pode ser usada para isolamento de C. jejuni de alimentos, oferecendo a vantagem de detectar em amostras o microrganismo cujo isolamento não é obtido por meio do método convencional.(AU)

Campylobacter jejuni is the main cause of acute bacterial gastroenteritis and chicken meat has shown to be an important source of infection. This microorganism is difficult to isolate and the conventional methods are often inefficient and may lead to erroneous results. This study aimed at developing and testing the technique of immunomagnetic separation (IMS) in the detection of C. jejuni in chicken products. Microparticles magnetically connected anti-C. jejuni polyclonal antibodies were used to concentrate C. jejuni before agar seeding. The protocol was compared with the conventional method. C. jejuni was recovered from experimentally contaminated food for both methods, however, when the IMS was used, the presence of contaminating microorganisms in the means of culture was smaller. C. jejuni was isolated from 7% of samples of food naturally contaminated, using IMS, and 3% by conventional method. C. coli was isolated from a sample by conventional method, but it was not detected by protocol with IMS. The IMS technique can be used for isolation of C. jejuni in food, offering the advantage of detecting the microorganism in samples from which the isolation is not obtained with the use of the conventional method.(AU)

Detecção de Campylobacter jejuni em produtos de frango utilizando separação imunomagnética / Detection of Campylobacter jejuni in chicken products using immunomagnetic separation

Campylobacter jejuni é o principal causador de gastroenterite bacteriana aguda, e a carne de frango tem se mostrado uma importante fonte de transmissão. Este microrganismo é de difícil isolamento e os métodos convencionais muitas vezes não são eficientes, podendo levar a resultados errôneos. O objetivo deste trabalho foi desenvolver e testar a técnica de separação imunomagnética (IMS) na detecção de C. jejuni em produtos de frango. Micropartículas magnéticas ligadas a anticorpos policlonais anti-C. jejuni foram utilizadas para concentrar C. jejuni antes da semeadura em ágar. O protocolo foi comparado com o método convencional. C. jejuni foi recuperado do alimento experimentalmente contaminado por ambos os métodos, entretanto, quando foi usada a IMS, a presença de microrganismos contaminantes nos meios de cultura foi menor. C. jejuni foi isolado de 7% das amostras de alimento naturalmente contaminadas, usando IMS, e de 3% pelo método convencional. C. coli foi isolado de uma amostra pelo método convencional, mas não foi detectado pelo protocolo com IMS. A técnica de IMS pode ser usada para isolamento de C. jejuni de alimentos, oferecendo a vantagem de detectar em amostras o microrganismo cujo isolamento não é obtido por meio do método convencional.(AU)

Campylobacter jejuni is the main cause of acute bacterial gastroenteritis and chicken meat has shown to be an important source of infection. This microorganism is difficult to isolate and the conventional methods are often inefficient and may lead to erroneous results. This study aimed at developing and testing the technique of immunomagnetic separation (IMS) in the detection of C. jejuni in chicken products. Microparticles magnetically connected anti-C. jejuni polyclonal antibodies were used to concentrate C. jejuni before agar seeding. The protocol was compared with the conventional method. C. jejuni was recovered from experimentally contaminated food for both methods, however, when the IMS was used, the presence of contaminating microorganisms in the means of culture was smaller. C. jejuni was isolated from 7% of samples of food naturally contaminated, using IMS, and 3% by conventional method. C. coli was isolated from a sample by conventional method, but it was not detected by protocol with IMS. The IMS technique can be used for isolation of C. jejuni in food, offering the advantage of detecting the microorganism in samples from which the isolation is not obtained with the use of the conventional method.(AU)

Production and evaluation of immunoglobulin Y Anti-Brucella abortus (Vaccinal Strain B19)

Background: The immunoglobulin Y is a principal antibody current in the blood of hens, which are transferred from the maternal blood serum to the egg yolk. The extraction of IgY from the egg yolk apply animal welfare when compared to the extraction of IgG, reducing the number of animals and prevent a bleeding of hens through the extraction of the IgY from eggs, besides that IgY presenting high specificity for antigenic binding. The objective of this study was to produce specific polyclonal IgY antibodies anti-Brucella abortus by immunizing hens with B19 vaccine and evaluate their reactivity through Buffered Acidified Plate Antigen (BAPA), 2-Mercaptoethanol (2-ME) and indirect ELISA diagnostic tests.Materials, Methods & Results: Four 25-week-old White Leghorn hens were immunized, two of them comprising the control group (Group 1) with phosphate-buffered saline (PBS) with adjuvant, and the others two immunized with B19 vaccine (Brucella abortus vaccine strain B19), representing the Group 2. The immunizations occurred six times with a 15-day interval between each. Blood samples were taken biweekly (seven times); and daily, the eggs were collected for 13 weeks, the first collection of blood and eggs, performed one week before the first immunization of each group. The IgY was purified from egg yolk, using the method of dilution in acid water and precipitation with ammonium sulfate for delipidation. BAPA, 2-ME and ELISA tests performed to verify the specificity of IgY confirmed the reactivity of polyclonal antibodies specific to the antigen used both in blood serum samples and in the purified egg yolks. The hens from the control group did not present reactivity in the diagnostic tests used, which was already expected, since no antigen was used in any of their immunizations. Hens immunized with the Brucella abortus B19 vaccine produced detectable reactive antibodies in the three tests used on blood serum and IgY samples extracted from the egg yolk.[...]

Production and evaluation of immunoglobulin Y Anti-Brucella abortus (Vaccinal Strain B19)

Background: The immunoglobulin Y is a principal antibody current in the blood of hens, which are transferred from the maternal blood serum to the egg yolk. The extraction of IgY from the egg yolk apply animal welfare when compared to the extraction of IgG, reducing the number of animals and prevent a bleeding of hens through the extraction of the IgY from eggs, besides that IgY presenting high specificity for antigenic binding. The objective of this study was to produce specific polyclonal IgY antibodies anti-Brucella abortus by immunizing hens with B19 vaccine and evaluate their reactivity through Buffered Acidified Plate Antigen (BAPA), 2-Mercaptoethanol (2-ME) and indirect ELISA diagnostic tests.Materials, Methods & Results: Four 25-week-old White Leghorn hens were immunized, two of them comprising the control group (Group 1) with phosphate-buffered saline (PBS) with adjuvant, and the others two immunized with B19 vaccine (Brucella abortus vaccine strain B19), representing the Group 2. The immunizations occurred six times with a 15-day interval between each. Blood samples were taken biweekly (seven times); and daily, the eggs were collected for 13 weeks, the first collection of blood and eggs, performed one week before the first immunization of each group. The IgY was purified from egg yolk, using the method of dilution in acid water and precipitation with ammonium sulfate for delipidation. BAPA, 2-ME and ELISA tests performed to verify the specificity of IgY confirmed the reactivity of polyclonal antibodies specific to the antigen used both in blood serum samples and in the purified egg yolks. The hens from the control group did not present reactivity in the diagnostic tests used, which was already expected, since no antigen was used in any of their immunizations. Hens immunized with the Brucella abortus B19 vaccine produced detectable reactive antibodies in the three tests used on blood serum and IgY samples extracted from the egg yolk.[...](AU)

Parotid gland adenocarcinoma in a cow: case report / Adenocarcinoma de glândula parótida em vaca: relato de caso

A 9-year-old Girolando dairy cow, weighing 400kg, with a history of increased volume in the right parotid region, which extended to the submandibular region, was assisted. Fine needle aspiration cytology was performed, and the cytological findings were consistent with malignant neoplasm of epithelial origin (carcinoma). Because of the unfavorable prognosis, the animal was euthanized and submitted to an anatomopathological examination. Samples of the increased parotid and affected lymph nodes were collected for histopathological evaluation. The microscopic changes were accentuated features of anaplasia, moderate cell proliferation, atypical mitotic figures, and necrosis. Stroma ranged from delicate to scirrhous, and the tumor boundaries were not distinct. These findings substantiated the preliminary histomorphological diagnosis of undifferentiated carcinoma with metastasis in lymph nodes. Immunohistochemical tests were performed with anti-CK Pan (clone AE1AE3), anti-CK HMW (clone 34ßE12), anti-CK19 (clone RCK108), anti-vimentin (clone V9), anti-S100 (polyclonal), and anti-androgen (polyclonal) antibodies. The immunophenotype favored the diagnosis of salivary gland adenocarcinoma. Despite the rareness in cattle, salivary gland adenocarcinoma should be considered in the differential diagnosis of diseases that occur with increased volume in the head, lymphadenopathy, drooling, dysphagia, and progressive weight loss.(AU)

Foi atendida uma vaca da raça Girolando, de nove anos de idade, de aptidão leiteira, pesando aproximadamente 400kg e com histórico de aumento de volume na região parotídea e submandibular direita. Diante do prognóstico desfavorável, o animal foi submetido à eutanásia e encaminhado para exame anatomopatológico. Fragmentos da glândula parótida e dos linfonodos alterados foram colhidos e encaminhados para exame histopatológico. À avaliação microscópica, observaram-se acentuada anaplasia, moderada proliferação celular, figuras de mitose atípicas e focos de necrose. O estroma variava de delicado a esquirroso e os limites do tumor eram imprecisos. Esses achados fundamentaram o diagnóstico de carcinoma indiferenciado com metástase em linfonodos. No exame imuno-histoquímico, foram utilizados anticorpos primários monoclonais anti-CK Pan (clone AE1AE3), anti-CK alto peso molecular (clone 34ßE12), anti-CK19 (clone RCK108), antivimentina (clone V9), anti-S100 (policlonal) e antirreceptor de andrógenos (policlonal). As células neoplásicas apresentaram imunomarcação para todos os anticorpos testados, resultado que favorece o diagnóstico de adenocarcinoma de glândula salivar. Embora raro em bovinos, o adenocarcinoma de glândula salivar deve ser considerado no diagnóstico diferencial de doenças que cursam com aumento de volume na cabeça, linfadenopatia salivação, disfagia e emagrecimento progressivo.(AU)

Parotid gland adenocarcinoma in a cow: case report / Adenocarcinoma de glândula parótida em vaca: relato de caso

A 9-year-old Girolando dairy cow, weighing 400kg, with a history of increased volume in the right parotid region, which extended to the submandibular region, was assisted. Fine needle aspiration cytology was performed, and the cytological findings were consistent with malignant neoplasm of epithelial origin (carcinoma). Because of the unfavorable prognosis, the animal was euthanized and submitted to an anatomopathological examination. Samples of the increased parotid and affected lymph nodes were collected for histopathological evaluation. The microscopic changes were accentuated features of anaplasia, moderate cell proliferation, atypical mitotic figures, and necrosis. Stroma ranged from delicate to scirrhous, and the tumor boundaries were not distinct. These findings substantiated the preliminary histomorphological diagnosis of undifferentiated carcinoma with metastasis in lymph nodes. Immunohistochemical tests were performed with anti-CK Pan (clone AE1AE3), anti-CK HMW (clone 34ßE12), anti-CK19 (clone RCK108), anti-vimentin (clone V9), anti-S100 (polyclonal), and anti-androgen (polyclonal) antibodies. The immunophenotype favored the diagnosis of salivary gland adenocarcinoma. Despite the rareness in cattle, salivary gland adenocarcinoma should be considered in the differential diagnosis of diseases that occur with increased volume in the head, lymphadenopathy, drooling, dysphagia, and progressive weight loss.(AU)

Foi atendida uma vaca da raça Girolando, de nove anos de idade, de aptidão leiteira, pesando aproximadamente 400kg e com histórico de aumento de volume na região parotídea e submandibular direita. Diante do prognóstico desfavorável, o animal foi submetido à eutanásia e encaminhado para exame anatomopatológico. Fragmentos da glândula parótida e dos linfonodos alterados foram colhidos e encaminhados para exame histopatológico. À avaliação microscópica, observaram-se acentuada anaplasia, moderada proliferação celular, figuras de mitose atípicas e focos de necrose. O estroma variava de delicado a esquirroso e os limites do tumor eram imprecisos. Esses achados fundamentaram o diagnóstico de carcinoma indiferenciado com metástase em linfonodos. No exame imuno-histoquímico, foram utilizados anticorpos primários monoclonais anti-CK Pan (clone AE1AE3), anti-CK alto peso molecular (clone 34ßE12), anti-CK19 (clone RCK108), antivimentina (clone V9), anti-S100 (policlonal) e antirreceptor de andrógenos (policlonal). As células neoplásicas apresentaram imunomarcação para todos os anticorpos testados, resultado que favorece o diagnóstico de adenocarcinoma de glândula salivar. Embora raro em bovinos, o adenocarcinoma de glândula salivar deve ser considerado no diagnóstico diferencial de doenças que cursam com aumento de volume na cabeça, linfadenopatia salivação, disfagia e emagrecimento progressivo.(AU)

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Abstract Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody a potential biomarker of Bothrops jararacussu venom against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.

Immunohistochemical, lectin histochemical and ultrastructural studies of canine transmissible venereal tumor in Brazil

ABSTRACT: Canine transmissible venereal tumor (CTVT) is a naturally occurring contagious round-cell neoplasia, with poorly understood origin and transmission. This study aims to further investigate the tumor nature through immunohistochemistry, lectin histochemistry and transmission electron microscopy (TEM) analysis, and to provide support for diagnostic and differential diagnoses of CTVT. Immunohistochemistry was performed in 10 genital and six exclusively extragenital tumors, which were previously diagnosed by citology and histopathology. CTVT samples were incubated with biotinylated antibodies to specific membrane and cytoplasmic antigens (anti-lysozyme, anti-macrophage, anti-vimentin, anti-CD18, monoclonal anti-CD117, monoclonal anti-CD3, polyclonal anti-CD117, polyclonal CD3 and anti-CD79a), followed by the avidin-biotin-peroxidase complex technique. The lectins Con A, DBA, SBA, PNA, UEA-1, WGA, sWGA, GSL, JSA, PSA, PHA-L, PHA-E and RCA were additionally tested in four genital CTVTs and TEM was performed in eight genital tumors. The anti-vimentin antibody revealed strong immunoreactivity to neoplastic cells in all the assessed samples (16/16). The polyclonal anti-CD3 antibodies showed moderate to strong immunoreactivity in fourteen (14/16) and the polyclonal anti-CD117 in fifteen cases (15/16). There was no immunoreactivity to anti-lysozyme, anti-macrophage, anti-CD18, monoclonal anti-CD117, monoclonal anti-CD3 and anti-CD79a antibodies. At lectin histochemistry, it was observed strong staining of tumor cells to Con-A, PHA-L and RCA. There was no histopathological and immunoreactivity differences between genital and extragenital CTVTs. These findings do not support the hypothesis of histiocytic origin of CTVT. In contrast, the lectin histochemical results were similar to cells from lymphoid/myeloid origin.

RESUMO: O Tumor Venéreo Transmissível Canino (CTVT) é uma neoplasia de células células redondas, contagiosa, com origem e transmissão ainda mal compreendidas. Com a finalidade de aprofundar a investigação sobre a natureza (origem) do TVTC, bem como fornecer subsídios para o estabelecimento do diagnóstico e diagnóstico diferencial, realizaram-se avaliações imuno-histoquímica, lectino-histoquímica e ultraestrutural de TVTC(s). A avaliação imuno-histoquímica foi feita em 10 TVTCs genitais e em 6 exclusivamente extragenitais previamente diagnosticados através de citologia e da histopatologia. Os TVTCs foram testados para reagentes específicos de antígenos de membrana e citoplasmáticos (anti-lisozima, anti-macrófago, anti-vimentina, anti-CD18, anti-CD3, anti-CD79, anti-CD117) com utilização da técnica complexo avidina-biotina-peroxidase. Adicionalmente, foram utilizadas as lectinas Con A, DBA, SBA, PNA, UEA-1, WGA, sWGA, GSL, SJA, PSA, PHA-L, PHA-E e RCA em quatro TVTCs genitais. Microscopia eletrônica foi realizada em oito TVTC genitais. Em 100% dos tumores testados (16/16) com anticorpo anti-vimentina (mono e policlonal) houve forte imuno-reatividade. Não houve reatividade para os anticorpos anti-lisozima, anti-macrófago, anti-CD18, anti-CD3, anti-CD79a e anti-CD117 quando empregamos anticorpos monoclonais, entretanto, com a utilização de anticorpos policlonais verificou-se marcação dos tumores com os anticorpos anti-CD3 e anti-CD117. Na avaliação lectino-histoquímica foi verificada forte marcação das células tumorais com Con-A, PHA-L e RCA. Não houve diferença histopatológica e de imuno-reatividade entre os TVTCs genitais e extragenitais. Estes achados não corroboram com a hipótese da origem histiocítica do CTVT (ausência de reatividade dos anticorpos anti-lisozima, anti-macrófago e anti-CD18), entretanto, os resultados da avaliação lectino-histoquímica foram em parte similares aos obtidos quando células de origem linfóide/mielóide (ConA, PHA-L e RCA) foram analisadas (Gimeno et al. 1995).

Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom

Abstract Background The five-paced pit viper (Deinagkistrodon acutus), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom. Methods IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with D. acutus venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities of D. acutus venom were conducted on mice. Results For the first time, IgY antibodies against D. acutus venom were raised successfully in egg yolk of chickens injected with D. acutus venom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components of D. acutus venom. Furthermore, immunodiffusion assay showed that anti-D. acutus venom IgY cross-reacted with the venoms of Trimeresurus albolabris and D. saxatilis Emelianov, but did not react to the venoms of Bungarus multicinctus and Naja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutus venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50 of D. acutus venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of D. acutus venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of D. acutus venom. Conclusion Anti-D. acutus venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with D. acutus venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of D. acutus venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.