Retrospective study of poxviruses diagnosed in cattle from Goiás State, Brazil (2010-2018) / Estudo retrospectivo de poxviroses diagnosticadas em bovinos no estado de Goiás (2010-2018)

A retrospective study of poxvirus infections diagnosed in cattle from Goiás state (GO), Brazil, from 2010 to 2018, was performed. All cases have been investigated by the GO Official Veterinary Service (Agrodefesa), from which technical forms and protocols of veterinary diagnosis laboratories were reviewed. In most cases, samples of oral or cutaneous tissues and/or swabs were submitted for virological diagnosis by polymerase chain reaction (PCR) and/or virus isolation. Thirty seven outbreaks/cases of vesicular disease were notified in cattle of 25 counties; in 33 cases the animals presented lesions clinically compatible with poxviruses. The etiology of 25 out of 33 outbreaks/cases was confirmed as poxviruses by PCR and/or viral isolation: 13 as bovine vaccinia virus (VACV), six as pseudocowpox virus (PCPV), five as bovine papular stomatitis virus (BPSV) and one coinfection (VACV and an Orf virus-like parapoxvirus). The laboratory confirmed that cases occurred mainly in dairy cattle (19/25) and during the dry season (22/25). In adult cattle, gross changes were observed mainly in the teats and udder and included vesicles, ulcers, crusts, papules and scars and varied of type, severity and affected region, depending on the poxvirus species. In calves, the main lesions were ulcers in the mouth and muzzle. Zoonotic lesions compatible with poxvirus infections were observed for all diagnosed poxviruses, affecting especially the hands of milkers and other farm workers. Our data demonstrate the sanitary and economic relevance of these diseases and the wide circulation of different poxviruses in cattle from GO.

Foi realizado um estudo retrospectivo das infecções por poxvírus diagnosticadas em bovinos do estado de Goiás (GO), entre 2010 e 2018. Todos os casos foram investigados pela Agência Goiana de Defesa Agropecuária (Agrodefesa). Foram revisados formulários técnicos e protocolos de laboratórios de diagnóstico veterinário. Na maioria dos casos, amostras de tecidos orais ou cutâneos e/ou swabs foram encaminhadas para diagnóstico virológico. Foram notificados 37 surtos/casos de doença vesicular em bovinos em 25 municípios; em 33 casos os animais apresentavam lesões clinicamente compatíveis com poxvírus. A etiologia de 25 de 33 surtos/casos foi confirmada como poxvírus por PCR e/ou isolamento viral: 13 como vírus vaccínia (VACV), seis como vírus pseudocowpox (PCPV), cinco como vírus da estomatite papular bovina (BPSV) e um caso de coinfecção (VACV e um parapoxvírus semelhante ao Orf vírus). Os casos confirmados laboratorialmente ocorreram principalmente em bovinos leiteiros (19/25) e durante a estação seca (22/25). Em bovinos adultos, alterações macroscópicas foram observadas principalmente nas tetas e úbere e incluíram vesículas, úlceras, crostas, pápulas e cicatrizes e variaram quanto ao tipo, gravidade e região afetada, dependendo da espécie do poxvírus. Em bezerros, as principais lesões foram úlceras na boca e focinho. Lesões zoonóticas compatíveis com infecção por poxvírus foram observadas em todas as poxviroses diagnosticadas, afetando principalmente as mãos dos ordenhadores e outros trabalhadores rurais. Nossos dados demonstram a relevância sanitária e econômica dessas doenças e a ampla circulação de diferentes poxvírus em bovinos de GO.

Retrospective study of poxviruses diagnosed in cattle from Goiás State, Brazil (2010-2018) / Estudo retrospectivo de poxviroses diagnosticadas em bovinos no estado de Goiás (2010-2018)

A retrospective study of poxvirus infections diagnosed in cattle from Goiás state (GO), Brazil, from 2010 to 2018, was performed. All cases have been investigated by the GO Official Veterinary Service (Agrodefesa), from which technical forms and protocols of veterinary diagnosis laboratories were reviewed. In most cases, samples of oral or cutaneous tissues and/or swabs were submitted for virological diagnosis by polymerase chain reaction (PCR) and/or virus isolation. Thirty seven outbreaks/cases of vesicular disease were notified in cattle of 25 counties; in 33 cases the animals presented lesions clinically compatible with poxviruses. The etiology of 25 out of 33 outbreaks/cases was confirmed as poxviruses by PCR and/or viral isolation: 13 as bovine vaccinia virus (VACV), six as pseudocowpox virus (PCPV), five as bovine papular stomatitis virus (BPSV) and one coinfection (VACV and an Orf virus-like parapoxvirus). The laboratory confirmed that cases occurred mainly in dairy cattle (19/25) and during the dry season (22/25). In adult cattle, gross changes were observed mainly in the teats and udder and included vesicles, ulcers, crusts, papules and scars and varied of type, severity and affected region, depending on the poxvirus species. In calves, the main lesions were ulcers in the mouth and muzzle. Zoonotic lesions compatible with poxvirus infections were observed for all diagnosed poxviruses, affecting especially the hands of milkers and other farm workers. Our data demonstrate the sanitary and economic relevance of these diseases and the wide circulation of different poxviruses in cattle from GO.(AU)

Foi realizado um estudo retrospectivo das infecções por poxvírus diagnosticadas em bovinos do estado de Goiás (GO), entre 2010 e 2018. Todos os casos foram investigados pela Agência Goiana de Defesa Agropecuária (Agrodefesa). Foram revisados formulários técnicos e protocolos de laboratórios de diagnóstico veterinário. Na maioria dos casos, amostras de tecidos orais ou cutâneos e/ou swabs foram encaminhadas para diagnóstico virológico. Foram notificados 37 surtos/casos de doença vesicular em bovinos em 25 municípios; em 33 casos os animais apresentavam lesões clinicamente compatíveis com poxvírus. A etiologia de 25 de 33 surtos/casos foi confirmada como poxvírus por PCR e/ou isolamento viral: 13 como vírus vaccínia (VACV), seis como vírus pseudocowpox (PCPV), cinco como vírus da estomatite papular bovina (BPSV) e um caso de coinfecção (VACV e um parapoxvírus semelhante ao Orf vírus). Os casos confirmados laboratorialmente ocorreram principalmente em bovinos leiteiros (19/25) e durante a estação seca (22/25). Em bovinos adultos, alterações macroscópicas foram observadas principalmente nas tetas e úbere e incluíram vesículas, úlceras, crostas, pápulas e cicatrizes e variaram quanto ao tipo, gravidade e região afetada, dependendo da espécie do poxvírus. Em bezerros, as principais lesões foram úlceras na boca e focinho. Lesões zoonóticas compatíveis com infecção por poxvírus foram observadas em todas as poxviroses diagnosticadas, afetando principalmente as mãos dos ordenhadores e outros trabalhadores rurais. Nossos dados demonstram a relevância sanitária e econômica dessas doenças e a ampla circulação de diferentes poxvírus em bovinos de GO.(AU)

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.(AU)

A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.(AU)

Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests / Expressão e análise antigênica da proteína RTP36 recombinante da amostra São Paulo de Ehrlichia canis para testes sorológicos

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.(AU)

Ehrlichia canis é o principal agente etiológico da erliquiose monocítica canina (EMC), uma doença infecciosa canina globalmente dispersa. No Brasil, a EMC é considerada endêmica, e a infecção pode atingir 65% em cães em alguns estados. O diagnóstico de erliquiose é importante para fins de tratamento e epidemiológicos. A proteína TRP36 de E. canis leva a uma resposta humoral com produção de anticorpos em fase aguda, encontrada durante o curso da doença. O objetivo deste estudo foi obter a proteína TRP36 recombinante da amostra São Paulo de E. canis e avaliar seu potencial como ferramenta para o diagnóstico sorológico da CME. O isolado de E. canis São Paulo foi cultivado em células da linhagem DH82 e o DNA genômico foi obtido. O fragmento de DNA bacteriano que codifica toda a ORF de TRP36 foi clonado no vetor pBAD / Thio-TOPO e transformado em células competentes Escherichia coli DH10B, com o plasmídeo portador de trp36 para expressão de proteínas. Para avaliar a antigenicidade da proteína, 16 amostras de soro canino foram previamente analisadas (por PCR e teste sorológico comercial SNAP®4Dx®). Os resultados estavam de acordo com o teste SNAP®4Dx®. Os experimentos que utilizam essa proteína recombinante como antígeno, visando ao desenvolvimento de um teste sorológico baseado no ELISA, são o próximo passo para produzir um teste de diagnóstico confiável, acessível e útil para o diagnóstico da EMC no Brasil.(AU)

Calcium homeostasis modulator 1 ( CALHM1 ) polymorphisms in cattle

ABSTRACT: The calcium homeostasis modulator 1 gene (CALHM1), which is located on chromosome 10 in humans and on chromosome 26 in cattle, is a transmembrane glycoprotein that controls the cytosolic calcium concentrations. Altered calcium homeostasis has been associated with several neurodegenerative disorders, including Alzheimers disease (AD). In a recent study, single nucleotide polymorphisms (SNPs) of CALHM1 have been associated with sporadic Creutzfeldt-Jakob disease (CJD). The protein sequence of human CALHM1 shows 93% homology with bovine CALHM1. Although SNPs of human CALHM1 have been correlated with human prion disease, polymorphisms of the bovine CALHM1 gene have not been reported in cattle thus far. To investigate polymorphisms of the bovine CALHM1 gene in Korean native cattle, we analyzed the open reading frame (ORF) of this gene in 175 Hanwoo and 141 Holstein cattle. We observed five SNPs: c.219C>T (rs380966453), c.357C>T (rs385969338), and c.869A>G (rs516301908) within the ORF region of two exons; and c.552+92A>G (rs481706737) and c.553-3A>C (rs448524869) in the intron of bovine CALHM1. Among the three SNPs that are in the ORF region of bovine CALHM1, the genotype and allele frequencies of the c.869A>G (p.His290Arg) and c.219C>T (p.Asn73Asn) SNPs were significantly different between Hanwoo and Holstein cattle (P 0.0001). Haplotype analysis showed that haplotypes ht2, ht3 and ht5 were also significantly different in these two cattle breeds. This study provides the first genetic analysis of the bovine CALHM1 gene in cattle.

Calcium homeostasis modulator 1 ( CALHM1 ) polymorphisms in cattle

The calcium homeostasis modulator 1 gene (CALHM1), which is located on chromosome 10 in humans and on chromosome 26 in cattle, is a transmembrane glycoprotein that controls the cytosolic calcium concentrations. Altered calcium homeostasis has been associated with several neurodegenerative disorders, including Alzheimer's disease (AD). In a recent study, single nucleotide polymorphisms (SNPs) of CALHM1 have been associated with sporadic Creutzfeldt-Jakob disease (CJD). The protein sequence of human CALHM1 shows 93% homology with bovine CALHM1. Although SNPs of human CALHM1 have been correlated with human prion disease, polymorphisms of the bovine CALHM1 gene have not been reported in cattle thus far. To investigate polymorphisms of the bovine CALHM1 gene in Korean native cattle, we analyzed the open reading frame (ORF) of this gene in 175 Hanwoo and 141 Holstein cattle. We observed five SNPs: c.219C>T (rs380966453), c.357C>T (rs385969338), and c.869A>G (rs516301908) within the ORF region of two exons; and c.552+92A>G (rs481706737) and c.553-3A>C (rs448524869) in the intron of bovine CALHM1. Among the three SNPs that are in the ORF region of bovine CALHM1, the genotype and allele frequencies of the c.869A>G (p.His290Arg) and c.219C>T (p.Asn73Asn) SNPs were significantly different between Hanwoo and Holstein cattle (P<0.0001). Haplotype analysis showed that haplotypes ht2, ht3 and ht5 were also significantly different in these two cattle breeds. This study provides the first genetic analysis of the bovine CALHM1 gene in cattle.(AU)

Calcium homeostasis modulator 1 ( CALHM1 ) polymorphisms in cattle

The calcium homeostasis modulator 1 gene (CALHM1), which is located on chromosome 10 in humans and on chromosome 26 in cattle, is a transmembrane glycoprotein that controls the cytosolic calcium concentrations. Altered calcium homeostasis has been associated with several neurodegenerative disorders, including Alzheimer's disease (AD). In a recent study, single nucleotide polymorphisms (SNPs) of CALHM1 have been associated with sporadic Creutzfeldt-Jakob disease (CJD). The protein sequence of human CALHM1 shows 93% homology with bovine CALHM1. Although SNPs of human CALHM1 have been correlated with human prion disease, polymorphisms of the bovine CALHM1 gene have not been reported in cattle thus far. To investigate polymorphisms of the bovine CALHM1 gene in Korean native cattle, we analyzed the open reading frame (ORF) of this gene in 175 Hanwoo and 141 Holstein cattle. We observed five SNPs: c.219C>T (rs380966453), c.357C>T (rs385969338), and c.869A>G (rs516301908) within the ORF region of two exons; and c.552+92A>G (rs481706737) and c.553-3A>C (rs448524869) in the intron of bovine CALHM1. Among the three SNPs that are in the ORF region of bovine CALHM1, the genotype and allele frequencies of the c.869A>G (p.His290Arg) and c.219C>T (p.Asn73Asn) SNPs were significantly different between Hanwoo and Holstein cattle (P<0.0001). Haplotype analysis showed that haplotypes ht2, ht3 and ht5 were also significantly different in these two cattle breeds. This study provides the first genetic analysis of the bovine CALHM1 gene in cattle.(AU)

Associação da ocorrência do ectima contagioso (ORF vírus) em ovinos com os cuidadores desses animais / Occurrence of the Association contagious ecthyma (ORF virus) in sheep with these animals' caregivers

A enfermidade ectima contagioso está difundida em todo o estado de São Paulo. Foram amostrados 42 (8,64%) cuidadores de animais e 444 (91,36%) ovinos (n=486). A prevalência de reagentes para vírus-neutralização foi de 67% (IC95%=62-71%) nos ovinos, e em seus cuidadores de 76% (IC95%=63-89%), sendo P=0,22, ou seja, não houve diferença estatística significativa entre as espécies. A distribuição dos títulos teve diferença estatística significativa entre as espécies, com P=0,0048. As variações de titulação foram de 0,6 a 2,1 tanto nos ovinos quanto nos seus cuidadores. Dentre os 42 cuidadores de ovinos participantes do estudo, 32 apresentaram títulos de anticorpos expressos por log10 acima de 0,6.(AU)

These diseases are all widespread in the State of São Paulo. 42 (8.64%) animal caregivers and 444 (91.36%) sheep (n=486) were sampled. The reagents Prevalence paragraph virus neutralization was 67% (95% CI = 62-71%) in sheep and 76% (95% CI = 63-89%) for caregivers, with P=0.22 not being a statistically significant difference between the species. One of the distribution titles had significant difference between statistics as species with P=0.0048. The titration variations were 0.6 to 2.1, both in sheep and their caregivers. Among the 42 sheep caregivers participating in the study, 32 had antibody securities denominated in log10 above 0.6.(AU)

Associação da ocorrência do ectima contagioso (ORF vírus) em ovinos com os cuidadores desses animais / Occurrence of the Association contagious ecthyma (ORF virus) in sheep with these animals' caregivers

A enfermidade ectima contagioso está difundida em todo o estado de São Paulo. Foram amostrados 42 (8,64%) cuidadores de animais e 444 (91,36%) ovinos (n=486). A prevalência de reagentes para vírus-neutralização foi de 67% (IC95%=62-71%) nos ovinos, e em seus cuidadores de 76% (IC95%=63-89%), sendo P=0,22, ou seja, não houve diferença estatística significativa entre as espécies. A distribuição dos títulos teve diferença estatística significativa entre as espécies, com P=0,0048. As variações de titulação foram de 0,6 a 2,1 tanto nos ovinos quanto nos seus cuidadores. Dentre os 42 cuidadores de ovinos participantes do estudo, 32 apresentaram títulos de anticorpos expressos por log10 acima de 0,6.(AU)

These diseases are all widespread in the State of São Paulo. 42 (8.64%) animal caregivers and 444 (91.36%) sheep (n=486) were sampled. The reagents Prevalence paragraph virus neutralization was 67% (95% CI = 62-71%) in sheep and 76% (95% CI = 63-89%) for caregivers, with P=0.22 not being a statistically significant difference between the species. One of the distribution titles had significant difference between statistics as species with P=0.0048. The titration variations were 0.6 to 2.1, both in sheep and their caregivers. Among the 42 sheep caregivers participating in the study, 32 had antibody securities denominated in log10 above 0.6.(AU)

Outbreak of contagious echtyma in sheep in Rio Grande do Sul, Brazil / Surto de ectima contagioso em ovinos no Rio Grande do Sul, Brasil

Background: Contagious ecthyma is a viral disease caused by a Parapoxvirus, which affects primarily sheep and goats. The disease has a worldwide distribution and is characterized by cutaneous pustules and crusts mainly in the muzzle and lips. Although the disease has a worldwide distribution, there are few reports in the literature of contagious ecthyma outbreaks in Brazil. Moreover, this is an important disease, as well as causing huge economic losses due to high morbidity rates, is also a zoonosis occupational character. This report describes the epidemiological, clinical, and anatomopathological aspects of an outbreak of contagious echtyma in sheep in the State of Rio Grande do Sul, Brazil. Case: Cases were observed on January and February of 2016. Seventeen out of 45 Texel sheep were affected including five 4-6-month-old lambs, three 7-12-month-old male sheep and nine 2-year-old ewes. Before the outbreak, a Texel ram was introduced in the herd as replacement. Clinically, affected sheep had pustules, ulcers, and crusts in the lips, labial commissures, muzzle, and nasal bridge. They also presented dyspnea, submandibular and facial subcutaneous edema. One of the affected sheep was euthanized due to the poor prognosis. At necropsy, the lesions observed clinically were confirmed. Histopathology of the skin in the lips and muzzles showed marked acanthosis of the epidermis, [...]

Outbreak of contagious echtyma in sheep in Rio Grande do Sul, Brazil / Surto de ectima contagioso em ovinos no Rio Grande do Sul, Brasil

Background: Contagious ecthyma is a viral disease caused by a Parapoxvirus, which affects primarily sheep and goats. The disease has a worldwide distribution and is characterized by cutaneous pustules and crusts mainly in the muzzle and lips. Although the disease has a worldwide distribution, there are few reports in the literature of contagious ecthyma outbreaks in Brazil. Moreover, this is an important disease, as well as causing huge economic losses due to high morbidity rates, is also a zoonosis occupational character. This report describes the epidemiological, clinical, and anatomopathological aspects of an outbreak of contagious echtyma in sheep in the State of Rio Grande do Sul, Brazil. Case: Cases were observed on January and February of 2016. Seventeen out of 45 Texel sheep were affected including five 4-6-month-old lambs, three 7-12-month-old male sheep and nine 2-year-old ewes. Before the outbreak, a Texel ram was introduced in the herd as replacement. Clinically, affected sheep had pustules, ulcers, and crusts in the lips, labial commissures, muzzle, and nasal bridge. They also presented dyspnea, submandibular and facial subcutaneous edema. One of the affected sheep was euthanized due to the poor prognosis. At necropsy, the lesions observed clinically were confirmed. Histopathology of the skin in the lips and muzzles showed marked acanthosis of the epidermis, [...](AU)

Detecção molecular de circovírus em aves de vida livre do Estado de Mato Grosso, Brasil

Circovírus são detectados em diversas espécies de mamíferos, aves, vertebrados inferiores e invertebrados. A família Circoviridae compreende dois gêneros e 101 espécies, das quais 52 são descritas como cyclovírus e 49 como circovírus, sendo que 11 já foram identificadas em aves, tais como psitacídeos, pombos, gansos, canários, cisnes, gaivotas, tentilhões, corvos, estorninhos e patos. A circovirose dos psitacídeos, denominada doença do bico e das penas, tem relatos descritivos desde o final do século XIX, sendo hoje reconhecida mundialmente, assim como a doença do pombo jovem, identificada desde 1993.O circovírus possui genoma de DNA fita simples e circular (1,7-2,3 kb), compreendendo um dos menores vírus que infectam vertebrados. Estes vírus são resistentes a algumas condições físicas e preservam capacidade infectante em fezes e secreções respiratórias do hospedeiro, permanecendo viável no ambiente por muito tempo. O método de diagnóstico mais empregado para a identificação de circovírus em aves é a técnica molecular de PCR e os métodos de controle e terapêuticos envolvem o isolamento do animal, o uso de fármacos antimicrobianos e eutanásia. Circovírus em aves já foram descritos em diversas partes do mundo, no entanto, pouco se sabe sobre sua ocorrência no Brasil. Neste sentido, este trabalho teve por objetivo fazer uma revisão bibliográfica sobre os circovírus que acometem as aves e investigar a sua ocorrência em aves de vida livre do Estado de Mato Grosso. Para tanto, foram colhidas amostras de 67 aves, num total de 37 espécies diferentes entre psitacídeos e não-psitacídeos. As aves foram submetidas à necropsia e o DNA do circovírus foi investigado em amostras de baço e/ou fígado através da técnica de Nested-PCR, previamente descrita para a amplificação parcial do gene Rep (ORF-V1). Foram identificadas sequências parciais do gene Rep de circovírus em seis aves (6/67 8,9%), sendo dois sabiás-laranjeira, um gavião carijó, uma seriema, uma arara canindé e um pombo doméstico. A identidade das sequências foram comparadas com as sequências depositadas no GenBank por meio do programa BLASTn, sendo possível a identificação de resultados inéditos nas amostras dos sabiás laranjeiras, do gavião carijó e da seriema, espécies nativas do Brasil, o que chama atenção sobre os mecanismos ecológicos e evolutivos virais que podem ameaçar estas e outras espécies.

Circoviruses are detected in several species of mammals, birds, lower vertebrates and invertebrates. The Circoviridae family comprises two genera and 101 species, of which 52 are described as cycloviruses and 49 as circoviruses, 11 of which have already been identified in birds, such as parrots, pigeons, geese, canaries, swans, seagulls, finches, crows, starlings and ducks. Parrot circovirus, called beak and feather disease, has descriptive reports since the end of the 19th century and is now recognized worldwide, as well as young pigeon disease, identified since 1993. Circovirus has a single and circular stranded DNA genome (1.7-2.3 kb), comprising one of the smallest viruses that infect vertebrates. These viruses are resistant to some physical conditions and preserve their infective capacity in the host's feces and respiratory secretions, remaining viable in the environment for a long time. The most used diagnostic method for the identification of circoviruses in birds is the molecular PCR technique and the control and therapeutic methods involve the isolation of the animal, the use of antimicrobial drugs and euthanasia Circoviruses in birds have already been described in various parts of the world, however, little is known about its occurrence in Brazil. In this sense, this work aimed to carry out a literature review on circoviruses that affect birds and investigate their occurrence in free-range birds in the State of Mato Grosso. For this purpose, samples of 67 birds were collected, in a total of 37 different species between parrots and non-parrots. The birds were submitted to necropsy and the circovirus DNA was investigated in spleen and/or liver samples using the Nested-PCR technique, previously described for the partial amplification of the Rep gene (ORF-V1). Partial sequences of the Circovirus Rep gene were identified in six birds (6/67 8.9%): two rufous-bellied thrush, one roadside hawk, one seriema, one blue-and-yellow macaw and one domestic pigeon. The identity of the sequences was compared with the sequences deposited in GenBank through the BLASTn program, making it possible to identify new findings by verifying the circovirus DNA in samples belonging to the rufous-bellied thrush, roadside hawk and seriema, species native to Brazil, which draws attention to the viral ecological and evolutionary mechanisms that can threaten these and other species.

CARACTERÍSTICAS MORFOFISIOLÓGICAS RUMINAIS, ALTERAÇÕES HEPÁTICAS E PERFIL DE ÁCIDOS GRAXOS DA CARNE DE OVINOS ALIMENTADOS COM DIETAS CONTENDO DIFERENTES TEORES DE ÓLEO RESIDUAL DE FRITURA

O uso de produtos alternativos na alimentação de ruminantes pode apresentar características benéficas, tanto do ponto de vista econômico, quanto ambiental. A suplementação de ovinos com óleo residual de fritura (ORF), como estratégia de aumentar o aporte energético dietético pode modular as características da morfofisiologia ruminal, bem como do metabolismo hepático e da carne desses animais. Com isso, objetivou-se avaliar as características morfofisiológicas do rúmen, bem como as eventuais alterações hepáticas e o perfil de ácidos graxos da carne de ovinos em terminação alimentados com diferentes níveis de ORF. Para isso, 24 ovinos, machos, sem padrão racial definidos, foram distribuídos em um delineamento inteiramente casualizado com quatro tratamentos (seis carneiros por tratamento). Os animais foram mantidos a pasto do nascimento até 120 dias e logo após confinados por 60 dias, onde foram submetidos a uma das dietas experimentais, que consistiram em diferentes níveis de inclusão do ORF à dieta (0, 3, 6 e 9%) com base na matéria seca. A suplementação com ORF não influenciou na espessura do epitélio, bem como no índice mitótico. No entanto, animais suplementados com 9% do ORF apresentaram um tamanho de papilas reduzido. O incremento dietético do ORF (6 e 9%) proporcionou um aumentou nos tempos de redução do azul de metileno, sedimentação e flotação no fluido ruminal. O acréscimo da suplementação com ORF promoveu um aumento das lesões hepáticas microscopicamente, acompanhado de uma redução da deposição de glicogênio no parênquima hepático. A carne dos animais alimentados com 9% de inclusão do óleo apresentou valores menores do ácido oleico (C18:1), quando comparados com o grupo controle. Os animais do grupo controle, apresentaram um menor valor do ácido - linolênico (C18:3), quando comparados com os demais grupos. Não foram observadas diferenças nos índices trombogênico e aterogênico. A inclusão de óleo residual de fritura em níveis elevados pode gerar alterações nos padrões de fermentação, bem como na mucosa ruminal e no parênquima hepático, não refletindo no entanto em mudanças substanciais no perfil de ácidos graxos da carne dos animais, constituindo uma alternativa para o aproveitamento desse resíduo da indústria alimentar, desde que respeitados os limites de inclusão.

The use of alternative products in the feeding of ruminants can have beneficial characteristics, both from an economic and environmental point of view. Supplementation of sheep with residual frying oil (RFO), as a strategy to increase the dietary energy supply can modulate the characteristics of ruminal morphophysiology, as well as of the hepatic metabolism and meat of such animals. Thus, the objective was to evaluate the morphophysiological characteristics of the rumen, as well as possible hepatic changes and the fatty acid profile of the meat of finishing sheep fed with different levels of RFO. For this, 24 male sheep, with no defined racial pattern, were distributed in a completely randomized design with four treatments (six rams per treatment). The rams were kept on pasture from birth up to 120 days and then confined for 60 days, where they were submitted to one of the experimental diets, which consisted of different levels of inclusion of RFO in the diet (0, 3, 6 and 9%) with based on dry matter. RFO supplementation did not influence int the thickness of the epithelium and in the mitotic index. However, rams supplemented with 9% of RFO had a reduced papillae size. The dietary increment of the RFO (6 and 9%) provided an increase in the times of reduction of the methylene blue, sedimentation and flotation in the ruminal fluid. The addition of RFO supplementation promoted an increase in microscopic hepatic lesions, accompanied by a reduction in glycogen deposition in the hepatic parenchyma. The meat of rams fed with 9% oil inclusion showed lower values of oleic acid (C18:1), when compared with the control group. The rams in the control group showed a lower value of -linolenic acid (C18:3), when compared with the other groups. There were no differences in thrombogenic and atherogenic indices. The inclusion of residual frying oil at high levels can generate changes in the fermentation patterns, as well as in the ruminal mucosa and hepatic parenchyma, however, not reflecting in substantial changes in the fatty acid profile of the meat of the rams, constituting an alternative for the use of this residue from the food industry, as long as the inclusion limits are respected.

Aspectos epidemiológicos e clínico-patológicos das poxviroses de bovinos no Estado de Goiás (2010-2018) e soroprevalência de vaccínia bovina em bovinos no Distrito Federal

Considerando a importância e a escassa literatura a respeito das poxviroses que acometem bovinos do Centro-Oeste brasileiro, o presente trabalho teve como objetivos determinar a soroprevalência da infecção pelo vírus vaccínia (VACV) em bovinos no Distrito Federal (DF) (2015-2016) e caracterizar os aspectos epidemiológicos e clínico-patológicos de casos oficialmente notificados de poxviroses em bovinos no Estado de Goiás (GO) (2010-2018). As amostras utilizadas neste estudo do DF e de GO foram cedidas pela Secretaria de Agricultura, Abastecimento e Desenvolvimento Rural do DF (Seagri-DF) e pela Agência Goiana de Defesa Agropecuária (Agrodefesa), respectivamente. No primeiro estudo, soros de 312 vacas de 64 rebanhos foram testados por teste de virus-neutralização para VACV. Observou-se prevalência de vaccínia bovina (VB) estimada em 33,3% em rebanhos e de 10,6% em vacas. Nenhum fator de risco de significado biológico foi associado à soropositividade de VB. No segundo estudo, durante o período de avaliação, foram notificados 33 casos suspeitos de doenças vesiculares em bovinos, dos quais 25 foram confirmados como poxviroses: 13 VB, seis pseudocowpox, cinco estomatite papular bovina e uma coinfecção (VACV e parapoxvírus semelhante ao Orf vírus). A maioria dos casos ocorreu no período seco dos anos. As lesões afetaram principalmente as tetas e o úbere de vacas leiteiras, e incluíram vesículas, úlceras, crostas, pápulas e cicatrizes. As principais lesões em bezerros incluíram úlceras na boca e focinho. Na maioria dos casos, houve relatos concomitantes com lesões similares em humanos que lidavam com os bovinos enfermos. Os resultados do estudo demonstraram que diferentes poxviroses, especialmente VB, infectam bovinos em parte do Centro-Oeste do Brasil, com potencial zoonótico e com maior frequência em propriedades leiteiras, mas presente também em algumas criações de corte. Com isso, essas doenças requerem vigilância por parte do serviço veterinário oficial e dos órgãos locais de saúde pública.

Based on the importance and in the scarce literature about poxviruses in cattle in Brazilian Midwestern, the current study aimed to determine the seroprevalence of vaccinia virus (VACV) infection in cattle of Distrito Federal (DF) (2015-2016) and to characterize the epidemiological, clinical and pathological aspects of officialy notified cases of poxviruses in cattle of Goiás State (GO), Brazil (2010-2018). The samples used in this study from DF and GO were kindly provide by Secretaria de Agricultura, Abastecimento e Desenvolvimento Rural (Seagri-DF) from DF and by Agência Goiana de Defesa Agropecuária (Agrodefesa), respectively. In the first study, sera of 312 cows from 64 herds were tested by virus-neutralizing test (VN) to bovine vaccinia (BV). Estimated prevalences of 33.3% (herds) and 10.6% (cows) were observed. No risk factor with biological relevance was associated to seropositivity of BV. In the second study, 33 suggestive cases of vesicular diseases in cattle were notified. Twenty-five out of these 33 cases were confirmed as poxviruses: 13 BV, 6 pseudocowpox, 5 bovine papular stomatitis and 1 coinfection (VACV and Orf virus-like parapoxvirus). Most cases occurred in the dry season of the years. Main lesions included vesicles, ulcers, crusts, papules and scars. These lesions affected mainly the teats and udder of dairy cows. Main lesions in calves consisted of ulcers in the mouth and muzzle. There were concomitant cases with similar lesions in humans that worked closely with the infected cattle. The results of this study demonstrate that different poxviruses (mainly BV) infect cattle in part of the Midwestern Brazil, with zoonotic potencial, mainly in dairy farms, but also present on beef herds. As a result, these diseases require vigilance by the oficial veterinary and local public health service.

DETECÇÃO MOLECULAR, HISTOLÓGICA E IMUNOHISTOQUÍMICA DE Pestivirus H EM UM BEZERRO NATURALMENTE INFECTADO COM SINAIS NEUROLÓGICOS

Os Pestivírus são membros do gênero Pestivirus, família Flaviviridae e classificados em 11 espécies pelo ICTV (Pestivirus A ao K). Os Pestivirus são vírus com envelope esférico, com polaridade positiva de RNA de fita simples, genoma completo de 12,5 kb, sua ORF codifica uma poliproteína de 3.998 aminoácidos, com dois produtos genéticos únicos (Npro e Erns). Infecções causadas por Pestivirus A, B e H são similares. Pestivirus H já foi detectado por RT-PCR codificando os genes 5´UTR, Npro e E2 em sangue, soro e órgãos de bovinos, contudo, não há associação com sinais neurológicos. O presente trabalho descreve a primeira detecção de Pestivirus H em tecidos do sistema nervoso central de um bezerro com sinais clínicos neurológicos e suas características molecular, histológica e imunohistoquímica. O bezerro apresentava síndrome cerebelar e morreu 20 dias após a internação e foi necropsiado. Fragmentos de SNC, coração, fígado, rim, pulmão, intestino e baço foram submetidos a RT-PCR. Amostras de tecido do SNC e linfonodo mesentérico foram processadas para avaliação histopatológica e imunohistoquímica. Os fragmentos de rim, SNC e intestino foram positivos para a região 5'UTR, SNC e intestino foram positivos para a região Npro. Os melhores amplicons nos ensaios de RT-PCR, 5UTR (SNC) e Npro (SNC e intestino) foram sequenciados e a sequência de nucleotídeos e a análise filogenética para a cepa 5'UTR exibiram 93,6 a 99,4%, 85%, 89,4 a 89,9% e 85,7% de identidade nt com as cepas de Pestivirus H subgrupo a, b, c e d, respectivamente, e 71,5 % e 75,6% de identidade nt com a cepa de Pestivirus A (NADL) e Pestivirus B (890), respectivamente. As cepas Npro mostraram 99,7% de identidade nt entre si e 90,2 a 96,5% de identidade nt com outras cepas de Pestivirus H e 67,3 a 67,5% e 66,3 a 66,5% de identidade nt com cepa de Pestivirus A (NADL) e Pestivirus B (890), respectivamente. As alterações histopatológicas incluíram necrose neuronal no cérebro, e necrose e degeneração de células Purkinje do cerebelo. Houve imunorreatividade para antígenos do BVDV no córtex cerebral, cérebro e medula espinhal. Este estudo fornece informações sobre o Pestivirus H em tecidos do SNC em bezerro com sinais neurológicos, sugerindo a inclusão do Pestivirus H na investigação etiológica de síndromes neurológicas.

Pestiviruses are members of the genus Pestivirus, family Flaviviridae and classified into 11 species recognized by ICTV (Pestivirus A to K). Pestiviruses are spherical enveloped viruses, single-stranded RNA positive-polarity, its complete genome is 12.5 kb and their ORF encodes a 3,998 amino acid polyprotein with two unique genetic products (Npro and Erns). Infections caused by Pestivirus A, B and H are similar. Pestivirus H has already been detected by RT-PCR encoding the 5´UTR, Npro and E2 genes in blood, serum and several bovine organs, however, there are no association with neurological signs. The present study describes the first detection of Pestivirus H in tissues of the central nervous system of a calf with neurological clinical signs, and its molecular, histological and immunohistochemical characteristics. The calf presented cerebellar syndrome and died 20 days after hospitalization, being necropsied. Fragments of CNS, heart, liver, kidney, lung, intestine and spleen were submitted to RT-PCR. Samples of CNS and mesenteric lymph node were processed for histopathological and immunohistochemistry evaluation. The kidney, CNS, and intestine fragments were positive to 5UTR region, CNS and intestine fragments were positive to Npro region. The better amplicons in the 5UTR (CNS) and Npro (CNS and intestine) RT-PCR assays were sequenced and the nucleotide sequence and phylogenetic analysis to the 5UTR strain exhibited 93.6 to 99.4%, 85%, 89.4 to 89.9%, and 85.7% of nt identity with Pestivirus H strains subgroup a, b, c, and d, respectively, and 71.5% and 75.6% of nt identity with Pestivirus A strain (NADL) and Pestivirus B (890), respectively. The Npro strains showed 99.7% of nt identity to each other and 90.2 to 96.5% of nt identity with other Pestivirus H strains and 67.3 to 67.5% and 66.3 to 66.5% of nt identity with Pestivirus A (NADL) and Pestivirus B (890), respectively. The histopathologic alterations included neuronal necrosis at the cerebrum and moderate necrosis and degeneration of Purkinje cells of the cerebellum. There was positive immunoreactivity for antigens of BVDV in cerebral cortex, cerebrum and spinal cord. This study provides information about the Pestivirus H in CNS tissues of a calf with neurological signs, suggesting the inclusion of Pestivirus H in the etiological investigation of neurological syndromes.

Caracterização molecular de DNA de Delta papillomavirus bovino (BPV1, 2 e 13) em sarcoides equinos / Molecular characterization of bovine Deltapapillomavirus (BPV1, 2, and 13) DNA in equine sarcoids

Sarcoides são tumores fibroblásticos, considerados os tumores de pele mais comuns em pele de equinos e que raramente apresentam regressão espontânea. Papilomavírus bovino (BPV) tipos 1 e 2 são relacionados com a patogenia do sarcoide e, provavelmente, o BPV tipo 13 (BPV13), recentemente descrito, também pode estar associado com a formação dessa lesão. Neste estudo, 20 amostras de lesões cutâneas, sendo 12 constituídas por tecidos frescos e 8 amostras de tecido fixado em formalina e embebido em parafina, provenientes de 15 cavalos foram utilizadas para a identificação do DNA de BPV. A análise histopatológica (HE) confirmou todas as lesões como sarcoide. Para a amplificação do DNA de papilomavírus (PV) foram realizadas três reações de PCR. Como triagem, os primers IFNR2/IDNT2 foram utilizados para amplificar um fragmento da ORF L1 do PV. O segundo par de primersutilizado é complementar a sequência dos genes E5 e L2 de BPVs 1, 2 e 13. O terceiro par de primers(FAP59/FAP64) utilizado tem o gene L1 como alvo. A primeira e a segunda PCRs permitiram amplificar produtos em todas as amostras avaliadas. Entretanto, na terceira reação, na qual foram utilizados os primers FAP, foi possível amplificar produtos com tamanho molecular esperado somente nas amostras constituídas por tecidos frescos. O sequenciamento de nucleotídeos e as análises filogenéticas realizadas nos fragmentos E5L2 resultaram na identificação de BPV1, 2 e 13 em 14 (70%), 2 (10%) e em 4 (20%) amostras de sarcoides, respectivamente. As amostras de sarcoides de um dos animais continha somente o DNA de BPV1. Entretanto, nas amostras provenientes do segundo cavalo foi possível identificar o DNA de três tipos de Deltapapillomavirus bovino (BPV1, 2 e 13) em lesões distintas. Este estudo ratifica a presença do DNA de BPV1, 2 e 13 em lesões de sarcoides em equinos, além de identificar três tipos de BPVs em um mesmo animal e descrever pela primeira vez no Brasil a presença de BPV1 e 2 nesse tipo de lesão.(AU)

Sarcoids are fibroblastic lesions, which are considered as the most common skin tumors of horses; spontaneous regression rarely occurs. The bovine papillomavirus (BPV) types 1 and 2 may be involved in the pathogenesis of sarcoids, and probably the recently described BPV type (BPV13) might be associated with the pathogenesis of this lesion. This study characterized the DNA of BPVs in sarcoids from 15 horses from Brazil by analyzing 20 cutaneous lesions (12 recently collected; 8 from formalin-fixed paraffin-embedded (FFPE) tissues). Histopathology confirmed the proliferative lesions as sarcoids. Three PCRs were performed to amplify papillomavirus (PV) DNA. For screening, the primers IFNR2/IDNT2 were used to amplify a fragment of the PV L1 ORF. The second primer set was complementary to a common sequence of the E5L2 genomic region of BPV1, 2, and 13. The third primer pair (FAP59/FAP64) targeted a fragment of the PVs L1 ORF. The screening and E5L2 PCRs yielded amplicons in all samples evaluated. The FAP amplicons identified BPV1, 2, and 13 only from fresh tissue samples. The phylogenetic analyses of E5L2 resulted in the identification of BPV1, 2, and 13 in 14 (70%), 2 (10%), and 4 (20%) sarcoids, respectively. Two horses demonstrated multiple lesions: the sarcoids of one of these contained only BPV1 DNA and those of the other contained three types of bovine Deltapapillomavirus (BPV1, 2, and 13). This study confirmed the presence of BPV1, 2, and 13 DNA in equine sarcoids. Moreover, these findings represent the first description of three types of BPV diagnosed in the same horse, as well as the first confirmation of BPV1 and 2 in horses from Brazil.(AU)

Molecular Diagnosis of Beak and Feather Disease in Native Brazilian Psittacines

The incidence of the psittacine beak and feather disease virus (BFDV) was investigated in Brazilian native parrots with normal feathering arriving at rescue and triage centers for wild animals (CETAS, IBAMA) in the state of Minas Gerais, Brazil. BFDV DNA was investigated by previously described PCR technique for the partial amplification of BFDV ORF-1 in DNA extracts from blood, cloacal swab or liver of psittacines. Some birds provided more than one sample. Nine species of psittacines were sampled between January 2009 and October 2010. Blood (n=46) or cloacal swab (n=128) samples were obtained from psittacines immediately upon arrival at the triage centers. Liver samples were collected from necropsied birds dead on arrival (n=167). All swab samples were negative, except for one Ara ararauna individual (n=3) which blood presented the BFDV DNA. On the other hand, 11 liver samples were positive for BFDV DNA, with a prevalence of 7.8% in Amazona aestiva (n=140). No BFDV DNA was detected in the liver of Amazona amazonica (n=11), A. vinacea (n=5), A. rhodochorytha (n=4), Anodorhynchus hyacinthinus (n=3), Ara ararauna, (n=3), Aratinga leucophtalma (n=2), Guarouba guarouba (n=1) and Pionus maximiliani (n=1). In most cases, alopecia was not associated with BFDV detection in liver, and liver histopathology was inconclusive. Although all cloacal swab samples were negative, a few psittacines (n=19) that died at CETAS-Belo Horizonte were retested, and 21% were detected as positive in liver. A group of psittacines (n=16) was clinically evaluated, and despite showing feather dystrophy, all birds were negative in the cloacal swabs, except for one, which blood sample was positive (A. ararauna). The obtained sequences of the BFDV strains BH 215 and BH 732 were deposited in the GenBank (JQ649409 and JQ649410). A 98% similarity with strain sequences described in Australia, Japan, and New Zealand was observed. It is possible that these strains arrived in Brazil through the legal and illegal trade of parrots. However, it was not possible to associate BFDV infection with the geographical origin of birds and no local marker was detected. The rates of detection, although similar to other studies, indicate the tendency of a high incidence of the disease, possibly associated with stress, and high bird density and wide transmission in captivity conditions.

Molecular Diagnosis of Beak and Feather Disease in Native Brazilian Psittacines

The incidence of the psittacine beak and feather disease virus (BFDV) was investigated in Brazilian native parrots with normal feathering arriving at rescue and triage centers for wild animals (CETAS, IBAMA) in the state of Minas Gerais, Brazil. BFDV DNA was investigated by previously described PCR technique for the partial amplification of BFDV ORF-1 in DNA extracts from blood, cloacal swab or liver of psittacines. Some birds provided more than one sample. Nine species of psittacines were sampled between January 2009 and October 2010. Blood (n=46) or cloacal swab (n=128) samples were obtained from psittacines immediately upon arrival at the triage centers. Liver samples were collected from necropsied birds dead on arrival (n=167). All swab samples were negative, except for one Ara ararauna individual (n=3) which blood presented the BFDV DNA. On the other hand, 11 liver samples were positive for BFDV DNA, with a prevalence of 7.8% in Amazona aestiva (n=140). No BFDV DNA was detected in the liver of Amazona amazonica (n=11), A. vinacea (n=5), A. rhodochorytha (n=4), Anodorhynchus hyacinthinus (n=3), Ara ararauna, (n=3), Aratinga leucophtalma (n=2), Guarouba guarouba (n=1) and Pionus maximiliani (n=1). In most cases, alopecia was not associated with BFDV detection in liver, and liver histopathology was inconclusive. Although all cloacal swab samples were negative, a few psittacines (n=19) that died at CETAS-Belo Horizonte were retested, and 21% were detected as positive in liver. A group of psittacines (n=16) was clinically evaluated, and despite showing feather dystrophy, all birds were negative in the cloacal swabs, except for one, which blood sample was positive (A. ararauna). The obtained sequences of the BFDV strains BH 215 and BH 732 were deposited in the GenBank (JQ649409 and JQ649410). A 98% similarity with strain sequences described in Australia, Japan, and New Zealand was observed. It is possible that these strains arrived in Brazil through the legal and illegal trade of parrots. However, it was not possible to associate BFDV infection with the geographical origin of birds and no local marker was detected. The rates of detection, although similar to other studies, indicate the tendency of a high incidence of the disease, possibly associated with stress, and high bird density and wide transmission in captivity conditions.(AU)

Production of egg yolk antibody (IgY) against recombinant porcine epidemic diarrhea virus COE protein

Background: Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs, and is characterized by a series ofclinical symptoms, such as severe diarrhea, vomiting and dehydration. Partial protective antigen gene (COE gene) of Sprotein possessing the main B cell epitope, is able to encode proteins with reactogenicity to induce the production of neutralizing antibodies. IgY was found to reduce the mortality in piglets after challenge exposures. Anti-COE IgY antibodyhas never been reported before, here it is described a method for the production of anti-COE IgY, which could be appliedin the treatment for the porcine epidemic diarrhea virus (PEDV) infection.Materials, Methods & Results: A PEDV strain was isolated from a clinical sample. The COE ORF (Open reading frame,ORF)was amplifi ed by PCR and iserted into the pMD18-T clone vector. The isolated was defi ned as Porcine epidemic diarrheavirus strain JS-HZ2012 subtype by sequencing, the clinical sample was defi ned as the nucleic acid sequence has a 99.5%homology with that of PEDV CV777 strain. And then the COE ORF was subcloned into pET-32a by T4 DNA ligase andintroduced into the E.coli Bal21 (DE3). COE protein was produced by the induction of the E.coli Bal21 containing pET32a-COE with isopropyl-β-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant COE protein (rCOE)fused with His-tag was analyzed by SDS-PAGE and detected by western-blotting using anti-His monoclonal antibody.The rCOE was purifi ed by Ni+ affi nity purifi cation chromatography under denature condition and dialyzed against PBS.The concentration of the rCOE was determined by BCA method. After immunnizing the chickens with rCOE , All animalhandling procedures were performed under veterinary supervision and following the recommendations of the local lawsand regulations on Animal Experimentation. Anti-COE IgY was isolated by chloroform extraction and...(AU)

Production of egg yolk antibody (IgY) against recombinant porcine epidemic diarrhea virus COE protein

Background: Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs, and is characterized by a series ofclinical symptoms, such as severe diarrhea, vomiting and dehydration. Partial protective antigen gene (COE gene) of Sprotein possessing the main B cell epitope, is able to encode proteins with reactogenicity to induce the production of neutralizing antibodies. IgY was found to reduce the mortality in piglets after challenge exposures. Anti-COE IgY antibodyhas never been reported before, here it is described a method for the production of anti-COE IgY, which could be appliedin the treatment for the porcine epidemic diarrhea virus (PEDV) infection.Materials, Methods & Results: A PEDV strain was isolated from a clinical sample. The COE ORF (Open reading frame,ORF)was amplifi ed by PCR and iserted into the pMD18-T clone vector. The isolated was defi ned as Porcine epidemic diarrheavirus strain JS-HZ2012 subtype by sequencing, the clinical sample was defi ned as the nucleic acid sequence has a 99.5%homology with that of PEDV CV777 strain. And then the COE ORF was subcloned into pET-32a by T4 DNA ligase andintroduced into the E.coli Bal21 (DE3). COE protein was produced by the induction of the E.coli Bal21 containing pET32a-COE with isopropyl-β-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant COE protein (rCOE)fused with His-tag was analyzed by SDS-PAGE and detected by western-blotting using anti-His monoclonal antibody.The rCOE was purifi ed by Ni+ affi nity purifi cation chromatography under denature condition and dialyzed against PBS.The concentration of the rCOE was determined by BCA method. After immunnizing the chickens with rCOE , All animalhandling procedures were performed under veterinary supervision and following the recommendations of the local lawsand regulations on Animal Experimentation. Anti-COE IgY was isolated by chloroform extraction and...