Enteric viruses in turkey flocks: a historic review

In this review, diagnostic techniques and viral agents involved in enteric diseases affecting turkeys are described. Data from field observations and laboratory researches have been reported in turkey flocks for over 70 years, and several viruses have been identified. After a period of 30 years of inoculation experiments and neutralization studies, adequate visualization of the viruses was achieved using electronic microscopy. During the following years, several studies were then conducted to isolate and classify those viruses using cell-culture, embryo-propagation, serological tests, genome electropherotyping by polyacrylamide gel electrophoresis of double-stranded RNA viruses, and recently, nucleic acid studies. Thus, since the 1990s, the nucleic-acid technology has focused on genomic surveys and on the detection of specific segments of the genome of each virus using the polymerase-chain reaction, resulting in several prevalence studies and phylogenetic analyses of different isolates and proper classification of the viruses.

Enteric viruses in turkey flocks: a historic review

In this review, diagnostic techniques and viral agents involved in enteric diseases affecting turkeys are described. Data from field observations and laboratory researches have been reported in turkey flocks for over 70 years, and several viruses have been identified. After a period of 30 years of inoculation experiments and neutralization studies, adequate visualization of the viruses was achieved using electronic microscopy. During the following years, several studies were then conducted to isolate and classify those viruses using cell-culture, embryo-propagation, serological tests, genome electropherotyping by polyacrylamide gel electrophoresis of double-stranded RNA viruses, and recently, nucleic acid studies. Thus, since the 1990s, the nucleic-acid technology has focused on genomic surveys and on the detection of specific segments of the genome of each virus using the polymerase-chain reaction, resulting in several prevalence studies and phylogenetic analyses of different isolates and proper classification of the viruses.(AU)

Pathological and Molecular Analysis of Turkey Coronavirus Replication in Captive Wild Turkey Embryos (Broad-Breasted Bronze Breeder)

The degree of genetic and pathologic variation exhibited by a turkey Coronavirus (TCoV) strain was investigated after nine serial passages in 25-day-old turkey embryos obtained from wild broad-breasted bronze breeders. In spite of spleen, liver, kidneys, cloacal bursa and thymus have been collected and analysed, the main histopathological changes were only documented in the intestine sections. Microscopic lesions were characterized as mild enteritis, low degree of enterocyte vacuolization and detachment of the intestinal villous after five consecutive passages and were considered absent in the last passages. Genealogic analysis based on S1 and S2 DNA sequences suggested that Brazilian isolate might be considered as originated from TCoV strains circulating in the United States, as 100% identity with TCoV-Gl strain. Although S1 S2 sequences from each passage revealed no significant point mutations, and no correlation could be speculate between S2 nucleotide changes and pathologic features in infected embryos. This is the first demonstration of wild turkey embryos as a model for TCoV isolation and propagation.(AU)

Poult Intestinal Organ Culture for Propagation of Turkey Coronavirus and Assessment of Host-virus Interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and -3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteristics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.(AU)

Epidemiological aspects of astrovirus and coronavirus in poults in the South Eastern Region of Brazil

A survey of Turkey Coronavirus (TCoV) and Astrovirus (TAstV-2) prevalence was carried out from February to December during 2006 year in semiarid region of Brazil, from a turkey producer area, localized in South Eastern of Brazil. To asses the risk factor related to clinical material, climatic condition and type of RT-PCR applied, cloacal swabs (CS), faeces, sera, bursa of Fabricius (BF), thymus (TH) and spleen (SP) and ileum-caeca region were collected from 30-day-old poults suffering of enteritis episode characterized as poult enteritis mortality syndrome (PEMS). The PEMS clinical features were characterized by watery to foamy faeces, light brown-yellow in colour and low mortality rate. Meteorological data (rainfall and relative humidity) observed during along the study presented monthly average temperature ranging from 39.3 and 31.2ºC, precipitation in rainy season from 40 to 270.3 mm/month, and no rain during dry season. Simplex RT-PCR gave odds ratio (OR) values suggesting that ileum-caeca region is at higher chance (OR=1.9; p=0.9741) to have both viral RNA than faeces (OR=1.5; p=0.7319). However, multiplex RT-PCR showed 3.98 (p=0.89982) more chance to give positive results in faeces than CS at dry season. The major risk factors seem to be low rate of humidity and high temperatures at winter, probably responsible for spread, easily, the TCoV and TAstv-2 among the flocks. The positive results of both virus suggested that they can play an important role in enteric disorders, associated to low humidity and high temperatures frequently found in tropical countries.

O presente estudo foi conduzido para avaliar a prevalência do Coronavirus dos perus (TCoV) e Astrovirus tipo 2 (TAstV-2) entre os meses de Fevereiro a Dezembro de 2006, em uma região produtora localizada no semi-árido a Sudeste do Brasil. Os principais fatores de risco associado a prevalência foram material clínico analisado, condições climáticas e tipo de técnica molecular empregada. Os sinais clínicos foram caracterizados como intenso fluido intestinal e baixo crescimento em aves jovens, sendo o material coletado swabs cloacais, fezes, soros, bursa de Fabrícius, segmentos do intestino delgado, timo e baço. Os dados meteorológicos (índice pluviométrico e umidade relativa) desta região, durante o período de estudo, foram de temperatura média mensal variando de 39.3 a 31.2ºC, precipitação na época chuvosa variando de 40 a 270.3mm/mês e ausência de chuva na estação fria e seca. A técnica de simplex RT-PCR resultou em valores de odds ratio (OR) que sugerem que a região do intestino delgado (junção íleo-cecal) possui alta chance (1.9 vezes) de gerar resultados positivos na amplificação de RNA viral que as fezes (1.5 vezes) analisadas. A técnica de multiplex RT-PCR demonstrou ser 3.98 vezes mais eficiente em promover resultados positivos nas fezes que nos swabs cloacais, durante a época de inverno. Os maiores fatores de risco encontrados foram baixa umidade relativa associada a altas temperaturas, durante a estação seca, o que pode permitir uma maior disseminação aérea do ambos os vírus entre os lotes estudados. A alta prevalência detectada para dois vírus sugerem que, no Brasil, estes representam os maiores responsáveis pelos surtos de enterite viral nas regiões semi-áridas, associado a baixas umidades e altas temperaturas típicas de países tropicais.

Isolamento e propagação de Astrovírus, Adenovírus, Coronavírus, Parvovírus, Rotavírus e Reovírus de aves comerciais com problemas entéricos em ovos embrionados / Isolation and propagation of Astrovirus, Adenovirus, Coronavirus, Parvovirus, Rotavirus and Reovirus from commercial poultry with enteric problems in embryonated eggs

Os ovos embrionados são estruturas complexas compostas pelo embrião e as membranas de suporte (corioalantóide, amniótica, gema). O desenvolvimento embrionário e suas membranas geram diversos tipos de células que são necessárias para a replicação bem sucedida de uma grande variedade de vírus. Dentre os vírus entéricos, os ovos embrionados foram usados como o principal hospedeiro para o isolamento, cultivo e propagação da grande maioria destes. O presente trabalho descreve o isolamento, cultivo e propagação em ovos embrionados livres de patógenos específicos (SPF), assim como a caracterização macroscópica do vírus relacionado com problemas entéricos em lotes de aves comerciais no Brasil. Amostras intestinais provenientes de galinhas ou frangos de corte sadias ou com problemas entéricos (diarreia) e que se apresentaram positivas, através da reação em cadeia pela polimerase (PCR) e da técnica da trancriptase reversa da reação em cadeia pela polimerase (RT-PCR), para Astrovírus de Galinha, Parvovírus de Galinha, Vírus da Nefrite Aviária, Rotavírus Aviário, Reovírus Aviário, Vírus da Bronquite Infecciosa das Galinhas e Adenovírus Aviário Tipo 1 foram selecionadas para a realização do isolamento, cultivo e propagação em ovos embrionados SPF. Mortalidade e lesões macroscópicas como hemorragias, edema, nanismo, enrolamento, aspecto gelatinoso e deformações foram encontradas nos embriões, porém as membranas de suporte do ovo não apresentaram alteração. A confirmação viral nas passagens nos ovos foi realizada mediante o uso da PCR ou RT-PCR. Houve a replicação de Astrovírus de galinha, Vírus da Nefrite Aviária, Adenovírus Aviário do Tipo 1, Vírus da Bronquite Infecciosa das Galinhas, Parvovírus de Galinha e Reovírus Aviário nos ovos embrionados SPF, inoculados via saco da gema, aos sete dias de idade. No entanto, estes vírus também foram isolados em ovos embrionados de 14 dias de idade. A inoculação pelo saco da gema se mostrou uma ótima via para o isolamento viral. Foram isoladas duas amostras de Parvovírus de Galinha, oito amostras de Astrovírus de Galinha, sete amostras do Vírus da Bronquite das Galinhas, dezesseis amostras de Vírus da Nefrite Aviária, onze amostras de Adenovírus Aviário do Tipo 1 e três amostras de Reovírus Aviário. Nenhuma amostra de Rotavírus Aviário foi isolada. As características macroscópicas apresentadas nos embriões fo...

The embryonated eggs are complex structures composed by embryo and support membranes (yolk sac, chorioallantoic membrane, amniotic sac). The embryo development and their membranes produced many kinds of cells that are necessary for successful replication of many viruses. Between the enteric viruses, the embryonated eggs have been used as the principal host for isolation, culture and propagation of many of these viruses. The present work describes the isolation, culture, propagation and macroscopic characterization of astrovírus, adenovirus, coronavirus, parvovirus, reovírus e rotavirus, related to enteric problems in commercial flows at Brazil in chicken embryonated eggs specific pathogens free (SPF). Intestinal samples from hens or chickens health or with enteric problems (diarrhea), but showed positive for chicken astrovírus, chicken parvovirus, avian nephritis vírus, avian rotavirus, avian reovirus, bronchitis infectious vírus and fowl adenovirus type 1, through PCR and RT-PRC reactions, were selected for making of isolation, culture and propagation in chicken embryonated eggs specific pathogens free (SPF). Mortality and macroscopic lesions presented in the embryos characterized by hemorrhages, edemas, stunting, enrollment, gelatin features and deformations, however the support membranes not presented any damaged. The viral confirmation in the eggs passages was carry out using the polymerase chain reaction and transcriptase reverse of the polymerase chain reaction. Chicken astrovírus, avian nephritis vírus, fowl adenovirus type 1, bronchitis infectious vírus, chicken parvovirus and avian reovírus grow well in chickens embryonated eggs specific pathogen free SPF, inoculated in the yolk sac on seven days-old, however these viruses were isolated in embryonated eggs of fourteen days-old, too. The inoculation by yolk sac resulted be a optimal route for viral isolation, getting isolate two samples of chicken parvovirus, eight samples of chicken astrovirus, seven samples of bronchitis infectious vírus, sixteen samples of avian nephritis vírus, eleven samples of fowl adenovirus type one and three samples of avian reovirus, but any samples was isolated for avian rotavirus. The macroscopic characteristics presented in the embryos were similar in all of isolated viruses. Many samples used not present lesions and were negative in the molecular d...

Isolamento e propagação de Astrovírus, Adenovírus, Coronavírus, Parvovírus, Rotavírus e Reovírus de aves comerciais com problemas entéricos em ovos embrionados / Isolation and propagation of Astrovirus, Adenovirus, Coronavirus, Parvovirus, Rotavirus and Reovirus from commercial poultry with enteric problems in embryonated eggs

Os ovos embrionados são estruturas complexas compostas pelo embrião e as membranas de suporte (corioalantóide, amniótica, gema). O desenvolvimento embrionário e suas membranas geram diversos tipos de células que são necessárias para a replicação bem sucedida de uma grande variedade de vírus. Dentre os vírus entéricos, os ovos embrionados foram usados como o principal hospedeiro para o isolamento, cultivo e propagação da grande maioria destes. O presente trabalho descreve o isolamento, cultivo e propagação em ovos embrionados livres de patógenos específicos (SPF), assim como a caracterização macroscópica do vírus relacionado com problemas entéricos em lotes de aves comerciais no Brasil. Amostras intestinais provenientes de galinhas ou frangos de corte sadias ou com problemas entéricos (diarreia) e que se apresentaram positivas, através da reação em cadeia pela polimerase (PCR) e da técnica da trancriptase reversa da reação em cadeia pela polimerase (RT-PCR), para Astrovírus de Galinha, Parvovírus de Galinha, Vírus da Nefrite Aviária, Rotavírus Aviário, Reovírus Aviário, Vírus da Bronquite Infecciosa das Galinhas e Adenovírus Aviário Tipo 1 foram selecionadas para a realização do isolamento, cultivo e propagação em ovos embrionados SPF. Mortalidade e lesões macroscópicas como hemorragias, edema, nanismo, enrolamento, aspecto gelatinoso e deformações foram encontradas nos embriões, porém as membranas de suporte do ovo não apresentaram alteração. A confirmação viral nas passagens nos ovos foi realizada mediante o uso da PCR ou RT-PCR. Houve a replicação de Astrovírus de galinha, Vírus da Nefrite Aviária, Adenovírus Aviário do Tipo 1, Vírus da Bronquite Infecciosa das Galinhas, Parvovírus de Galinha e Reovírus Aviário nos ovos embrionados SPF, inoculados via saco da gema, aos sete dias de idade. No entanto, estes vírus também foram isolados em ovos embrionados de 14 dias de idade. A inoculação pelo saco da gema se mostrou uma ótima via para o isolamento viral. Foram isoladas duas amostras de Parvovírus de Galinha, oito amostras de Astrovírus de Galinha, sete amostras do Vírus da Bronquite das Galinhas, dezesseis amostras de Vírus da Nefrite Aviária, onze amostras de Adenovírus Aviário do Tipo 1 e três amostras de Reovírus Aviário. Nenhuma amostra de Rotavírus Aviário foi isolada. As características macroscópicas apresentadas nos embriões fo...(AU)

The embryonated eggs are complex structures composed by embryo and support membranes (yolk sac, chorioallantoic membrane, amniotic sac). The embryo development and their membranes produced many kinds of cells that are necessary for successful replication of many viruses. Between the enteric viruses, the embryonated eggs have been used as the principal host for isolation, culture and propagation of many of these viruses. The present work describes the isolation, culture, propagation and macroscopic characterization of astrovírus, adenovirus, coronavirus, parvovirus, reovírus e rotavirus, related to enteric problems in commercial flows at Brazil in chicken embryonated eggs specific pathogens free (SPF). Intestinal samples from hens or chickens health or with enteric problems (diarrhea), but showed positive for chicken astrovírus, chicken parvovirus, avian nephritis vírus, avian rotavirus, avian reovirus, bronchitis infectious vírus and fowl adenovirus type 1, through PCR and RT-PRC reactions, were selected for making of isolation, culture and propagation in chicken embryonated eggs specific pathogens free (SPF). Mortality and macroscopic lesions presented in the embryos characterized by hemorrhages, edemas, stunting, enrollment, gelatin features and deformations, however the support membranes not presented any damaged. The viral confirmation in the eggs passages was carry out using the polymerase chain reaction and transcriptase reverse of the polymerase chain reaction. Chicken astrovírus, avian nephritis vírus, fowl adenovirus type 1, bronchitis infectious vírus, chicken parvovirus and avian reovírus grow well in chickens embryonated eggs specific pathogen free SPF, inoculated in the yolk sac on seven days-old, however these viruses were isolated in embryonated eggs of fourteen days-old, too. The inoculation by yolk sac resulted be a optimal route for viral isolation, getting isolate two samples of chicken parvovirus, eight samples of chicken astrovirus, seven samples of bronchitis infectious vírus, sixteen samples of avian nephritis vírus, eleven samples of fowl adenovirus type one and three samples of avian reovirus, but any samples was isolated for avian rotavirus. The macroscopic characteristics presented in the embryos were similar in all of isolated viruses. Many samples used not present lesions and were negative in the molecular d...(AU)

Desenvolvimento de uma reação em cadeia pela polimerase para a detecção de coronavírus de perus (TCOV), por meio de um fragmento da região S2 de seu genoma / Development of a polymerase chain reaction for turkey coronavirus detection (TCOV), by a fragment from the S2 region of its genome

A partir de amostras de campo de diferentes regiões do país, o TCoV foi identificado em aves de 13-84 dias, em órgãos como intestino, rins e traqueia, por uma reação em cadeia pela polimerase para a região 3\' UTR e o gene 3. A presença deste coronavírus nesses diferentes órgãos, que não o intestino, sugere a presença de uma diferente estirpe, com características fenotípicas mais próximas em relação ao vírus da Bronquite Infecciosa das Galinhas (BIG), o IBV. Ao mesmo tempo, um perfil considerando a idade das aves positivas foi desenhado por um teste T de duas amostras, que mostrou maior positividade em aves mais jovens. Com a finalidade de desenvolver um novo ensaio de PCR, várias sequências do TCoV disponíveis no GenBank foram alinhadas, um segmento gênico foi desenhado e submetido a diferentes condições de reação. Um fragmento de 436 pb foi obtido, sequenciado e apresentou 100% de identidade quando sua sequência foi comparada a sequência completa do TCoV. A reação mostrou boa repetibilidade, bem como boa reprodutibilidade, e algumas amostras de campo não se apresentaram positivas

TCOV was identified from field samples of birds with 13-84 days, from different regions of the country, and from organs such as intestine, kidneys and trachea, by polymerase chain reaction for 3\' UTR region and gene 3. The presence of this coronavirus in these different organs, not intestine, suggest a different strain, with similar phenotype observed in Infectious Bronchitis Virus (IBV). At the same time, a profile considering age of positive birds was designed by a Two sample T-test, which showed higher positivity in younger poults. Several TCOV sequences obtained from GenBank were aligned, and a genic segment was designed and submitted to different conditions in reaction in pursuit to develop a new PCR assay. A 436bp fragment was obtained, sequenced and presented 100% ID when compared to a TCOV complete sequence. The reaction featured good repetition, and reproduction as well, and some field samples did not turn out on positive results

Identification of turkey astrovirus and turkey coronavirus in an outbreak of Poult Enteritis and Mortality Syndrome

This article reports a survey on turkey astrovirus (TAstV) and turkey coronavirus (TCoV) infections with RT-PCR in 17 turkey flocks affected by acute enteritis and two apparently normal turkey flocks located in the Southeastern region of Brazil by PCR (TAstV and TCoV). Seven out of the 17 affected flocks were positive for TAstV and 14 for TCoV, with seven co-infections. In one of the two apparently normal flocks, a TAstV-TCoV co-infection was found. Although a definitive association of these agents and the signs can not be made, the implications of these findings are discussed.

Identification of turkey astrovirus and turkey coronavirus in an outbreak of Poult Enteritis and Mortality Syndrome

This article reports a survey on turkey astrovirus (TAstV) and turkey coronavirus (TCoV) infections with RT-PCR in 17 turkey flocks affected by acute enteritis and two apparently normal turkey flocks located in the Southeastern region of Brazil by PCR (TAstV and TCoV). Seven out of the 17 affected flocks were positive for TAstV and 14 for TCoV, with seven co-infections. In one of the two apparently normal flocks, a TAstV-TCoV co-infection was found. Although a definitive association of these agents and the signs can not be made, the implications of these findings are discussed.

Detecção molecular e diagnóstico diferencial de vírus entéricos em criações de perus comerciais / Molecular detection and differential diagnostic of enteric viruses affecting commercial turkey flocks

Setenta e seis amostras intestinais de lotes de perus apresentando ou não sinais clínicos de enterite foram avaliadas quanto à presença de adenovírus grupo 1 (TAV), vírus da enterite hemorrágica dos perus (HEV), astrovírus tipo 1 e 2 (TAstV-1 e TAstV-2), coronavírus dos perus (TCoV), reovírus, rotavírus e vírus da nefrite aviária (ANV) através da reação em cadeia da polimerase. Os resultados obtidos foram analisados quanto à região geográfica de origem das amostras, idade das aves e presença de sinais clínicos nos lotes. Foi detectada elevada positividade das amostras para pelo menos um vírus (93,4%), e grande número de amostras com associações de mais de um vírus (69,7%). Santa Catarina foi o estado com maior média de número de vírus detectados em associação nas amostras (3,14) e Goiás o estado com menor média (1,73). As amostras provenientes de aves em fase inicial de criação (1 a 4 semanas de idade) tiveram média de 3,20 vírus detectados por amostra, e 85% de detecção de TAstV-1 e TCoV (os mais frequentes), enquanto que na fase de terminação (5 a 18 semanas) foi observada menor média de vírus associados nas amostras (2,41), e os agentes mais detectados foram TAstV-1 (57,1%) e rotavírus (51,8%), no entanto, todos os vírus apresentaram menor frequência na fase de terminação do que na inicial, com exceção do TAV e reovírus. Quando os sinais clínicos estavam presentes todos os vírus foram detectados em maior percentual (sendo TAstV-1, TAstV-2 e TCoV os mais frequentes) do que nos lotes sem sinais clínicos, onde TAstV-1 e rotavírus foram os mais frequentes. Estudos posteriores são necessários para a compreensão do papel de cada vírus no desenvolvimento de enterites

Intestinal samples from seventy-six Brazilian turkey flocks affected or not with intestinal disorders were evaluated for the presence of Adenovirus group 1 (TAV), hemorrhagic enteritis virus (HEV), Astrovirus type 1 and 2 (TAstV-1 e TAstV-2), turkey Coronavirus (TCoV), Reovirus, Rotavirus and avian nephritis virus (ANV) using the polimerase chain reaction. Geographic location, age of the flocks and the presence of clinical signs were analyzed in order to find a relationship with the viral detection. A high frequency of positive samples for at least one virus was observed (93,4%) and 69,7% of the samples showed an association of more than one agent. The highest average number of viruses detected in association (3,14) was found in the state of Santa Catarina and Goias showed the lowest average (1,73). An average of 3,20 viruses per sample was detected in poults in initial phase of the production cycle (1 to 4 weeks of age), and TAstV-1 and TCoV were detected in 85% of the samples (the most frequent viruses), while the terminanting phase (5 to 18 weeks) showed lower average number of viruses in association (2,41), and the most frequent viruses were TAstV-1 (57,1%) and rotavírus (51,8%). However, all the viruses were detected more frequently in the initial phase rather than in the terminating phase, in exception of TAV and reoviruses. A higher detection of the viruses was observed in poults with clinical signs (as TAstV-1, TAstV-2 and TCoV were the most frequent) compared to normal birds (as TAstV-1 and rotavirus were the most frequent). Furher researches should focus on the description and comprehension of the role of each virus, and the different combinations of viruses, in the development of enteric disease

Pathological and molecular analysis of turkey Coronavirus replication in captive wild turkey embryos (broad-breasted bronze breeder)

The degree of genetic and pathologic variation exhibited by a turkey Coronavirus (TCoV) strain was investigated after nine serial passages in 25-day-old turkey embryos obtained from wild broad-breasted bronze breeders. In spite of spleen, liver, kidneys, cloacal bursa and thymus have been collected and analysed, the main histopathological changes were only documented in the intestine sections. Microscopic lesions were characterized as mild enteritis, low degree of enterocyte vacuolization and detachment of the intestinal villous after five consecutive passages and were considered absent in the last passages. Genealogic analysis based on S1 and S2 DNA sequences suggested that Brazilian isolate might be considered as originated from TCoV strains circulating in the United States, as 100% identity with TCoV-Gl strain. Although S1 S2 sequences from each passage revealed no significant point mutations, and no correlation could be speculate between S2 nucleotide changes and pathologic features in infected embryos. This is the first demonstration of wild turkey embryos as a model for TCoV isolation and propagation.

Poult intestinal organ culture for propagation of turkey coronavirus and assessment of host-virus interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is onl

Detecção do TCoV (Turkey Coronavirus) a partir de amostras provenientes de perus (Meleagris gallopavo) com quadro agudo de enterite

O Complexo de Enterite de Perus (PEC) tem sido incriminado como uma das maiores causas de perdas econômicas em outros países. Neste estudo, foi demonstrado causando diarréia, perda de peso e na maioria das vezes alta mortalidade em perus acometidos de enterite com 30-120 dias de idade de uma determinada região produtora no Brasil. A RT-PCR foi aplicada em suspensões de intestinos (SI) (n=2), respectivos conteúdos intestinais (CI) (n=2), bursa de Fabrícius (BF), fezes (F) (n=5) e swabs cloacais (SC) (n=44), para amplificar a região conservada 3`UTR e gene do nucleocapsídeo de TCoV. Os exames de histopatologia e imunohistoquímica direta foram realizados para detectar o antígeno TCoV a partir de lâminas de intestinos e bursas infectados. Todos os resultados obtidos nos tecidos marcados, revelaram lesões sugestivas de terem sido causadas pela infecção por TCoV. A marcação positiva da imunohistoquímica direta estava presente em todas as lâminas de intestinos, entretanto, todas as BF analisadas foram negativas. Os achados de RT-PCR foram positivos para TCoV em todas as amostras de fezes e 27,27% das amostras de SC foram positivas para a região 3`UTR e região TCoV nucleocapsídeo. As amostras de soro (n=200), foram positivas para TCoV, com títulos variando de 2.0Log a 8.0Log usando o ELISA comercial IDEEX para IBV. Finalmente, o melhor material de campo para o diagnóstico de TCoV foram as fezes (F) e/ou suspensão de intestinos (SI), resultando na primeira descrição de perus com quadro agudo de enterite acometidos por coronavirus Grupo 3 no Brasil

The Poult Enteritis Complex (PEC) has been incriminated as the major cause of losses in other countries, and especially, in this study, was described causing diarrhea, weight gain and most of the time, high mortality. In this study, it was performed the turkey coronavirus (TCoV) detection from 30-120 day old affected poults from a particular producer region in Brazil. The RT-PCR was applied in intestines suspensions (IS) (n=2), respective intestines contents (IC) (n=2), bursa of Fabrícius (BF), faecal droppings (FD) (n=5) and cloacal swabs (CS) (n=44) to amplify the 3þUTR conserved region and TCoV nucleocapsid gene. The histopathological and direct immunohistochemical examinations were performed to detect the TCoV antigen from infected intestine and bursa slides. All results obtained from stained tissues, revealed lesions described to be caused by TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides, however all BF analysed were negative. RT-PCRs findings were positive for TCoV in all FD samples, and 27,27% of CS analysed were positive for 3þUTR and TCoV nucleocapsid region. The sera samples (n=200) were positive for TCoV, with titers range from d 2.0Log to 8.0Log using the IDEEX commercial ELISA for IBV. Finally, the best field material for TCoV diagnosis was FD and/or intestine suspensions (IS), resulting in a first describe of TCoV affecting poults in Brazil