RESUMO

Avian coronaviruses, including infectious bronchitis virus (IBV) and turkey coronavirus (TCoV), are economically important viruses affecting poultry worldwide. IBV is responsible for causing severe losses to the commercial poultry sector globally. The objectives of this study were to identify the viruses that were causing outbreaks of severe respiratory disease in chickens in Trinidad and Tobago (T&T) and to characterize the strains. Swab samples were collected from birds showing severe respiratory signs in five farms on the island of Trinidad. Samples were tested for the presence of IBV, as well as avian influenza virus (AIV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) by real-time reverse transcription polymerase chain reaction (qRT-PCR). All samples from the five farms tested negative for AIV, NDV and aMPV; however, samples from clinically affected birds in all five of the farms tested positive for IBV. Genetic data revealed the presence of TCoV in chickens on two of the farms. Interestingly, these two farms had never reared turkeys. Phylogenetic analysis showed that IBV S1 sequences formed two distinct clusters. Two sequences grouped with vaccine strains within the GI-1 lineage, whereas three sequences grouped together, but separately from other defined lineages, forming a likely new lineage of IBV. Pairwise comparison revealed that the three unique variant strains within the distinct lineage of IBV were significantly different in their S1 nucleotide coding regions from viruses in the closest lineage (16% difference) and locally used vaccine strains (>20% difference). Results also suggested that one of the samples was a recombinant virus, generated from a recombination event between a Trinidad virus of the GI-1 lineage and a Trinidad virus of the newly defined lineage. Many amino acid differences were also observed between the S1 coding regions of the circulating field and vaccine strains, indicating that the IBV vaccines may not be protective. Vaccine-challenge studies are however needed to prove this.

Assuntos

RESUMO

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.

RESUMO

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

RESUMO

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)

Assuntos

RESUMO

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)

Assuntos

RESUMO

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.

Assuntos

RESUMO

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.

RESUMO

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.