Immunohistochemistry assay to detect Turkey coronavirus (TCoV) from experimentally infected poults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aplicação da técnica de RT-PCR in situ na detecção da co-infecção pelo Coronavírus grupo 3 (TCoV) e Astrovírus (TAstV-2) em perus com quadro agudo de enterite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pathology and virus tissue distribution of Turkey Coronavirus (TCoV) in experimentally infected chicks and turkey poults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection and Molecular Characterization of Gene 3 and 5 of Turkey Coronavirus from Turkeys with Severe Enteritis in Brazil

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3` UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3` UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Balanço de massa de reatores anaeróbicos de fluxo ascendente com manta de lodo (UASB) tratando águas residuárias de suinocultura

Foi obtido o balanço de massa, a partir dos valores médios das determinações de demanda química de oxigênio (DQO) e produção de metano (CH4), em dois reatores UASB de bancada com volume de 10,5 L tratando águas residuárias de suinocultura, submetidos a condições operacionais distintas no que diz respeito às concentrações de sólidos suspensos totais do afluente (SST de 500; 1.000; 1.500 e 2.000 mg L-1), tempo de detenção hidráulica (TDH de 30; 20; 12 e 8 h), taxas de carregamento orgânico volumétrico (TCOV de 0,8 a 8,0 kg DQO total (m³ d)-1) e temperatura (ambiente e controlada a 25º e 30ºC). Verificou-se que a DQO total removida convertida em CH4 variou de 28 a 51% e a relação DQO-CH4 por DQO dissolvida removida de 0,94 a 2,07; indicando alta participação da remoção física dos sólidos do afluente, de 49 a 72%, na remoção de DQO total nos reatores, a qual variou de 75 a 92%. A concentração de SST do afluente, a temperatura, o TDH e a TCOV influenciaram nesse desempenho dos reatores UASB.

Detection of turkey coronavirus in commercial turkey poults in Brazil

Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabricius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV Histo pathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabricius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.

Pathology and Tissue Distribution of Turkey Coronavirus in Experimentally Infected Chicks and Turkey Poults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Balanço de massa de reatores anaeróbicos de fluxo ascendente com manta de lodo (UASB) tratando águas residuárias de suinocultura

Foi obtido o balanço de massa, a partir dos valores médios das determinações de demanda química de oxigênio (DQO) e produção de metano (CH4), em dois reatores UASB de bancada com volume de 10,5 L tratando águas residuárias de suinocultura, submetidos a condições operacionais distintas no que diz respeito às concentrações de sólidos suspensos totais do afluente (SST de 500; 1.000; 1.500 e 2.000 mg L-1), tempo de detenção hidráulica (TDH de 30; 20; 12 e 8 h), taxas de carregamento orgânico volumétrico (TCOV de 0,8 a 8,0 kg DQO total (m³ d)-1) e temperatura (ambiente e controlada a 25º e 30ºC). Verificou-se que a DQO total removida convertida em CH4 variou de 28 a 51% e a relação DQO-CH4 por DQO dissolvida removida de 0,94 a 2,07; indicando alta participação da remoção física dos sólidos do afluente, de 49 a 72%, na remoção de DQO total nos reatores, a qual variou de 75 a 92%. A concentração de SST do afluente, a temperatura, o TDH e a TCOV influenciaram nesse desempenho dos reatores UASB.

Validation of an immunohistochemistry assay to detect turkey coronavirus: A rapid and simple screening tool for limited resource settings

The objective of the present study was to develop and apply the direct immunohistochemistry (D-IHC) assay to search for turkey coronavirus (TCoV) antigens in formalin-fixed embedded-paraffin tissues by the use of biotin-labeled polyclonal antibody. Twenty-eight-day-old embryonated turkey eggs (n = 50) were inoculated with TCoV-purified virus, and 3 d after inoculation, sections from ileum, ileum-cecal junction, and ceca were harvested, fixed in neutral formalin, and embedded in paraffin blocks and used as positive control. In addition, a total of 100 field samples from ileum, ileum-cecal junction, and ceca, collected from 30 to 45-d-old turkeys poults experiencing an outbreak of acute enteritis, were used to search for TCoV by the same D-IHC. All results were compared with those obtained by conventional RT-PCR and indirect fluorescent antibody assay (IFA) for all tested samples. Turkey coronavirus was detected in experimentally infected embryo tissues and also in field samples in 100% of ileum-cecal junction and ceca by the 3 detection procedures. With IFA as a reference assay, sensitivity and specificity of D-IHC were 98 and 58%, whereas sensitivity and specificity of reverse transcription-PCR were 96 and 66%, calculated from the total of tested samples from experimental infection. Each of the examined procedures was highly specific (D-IHC, 93%; RT-PCR, 90%), sensitive (D-IHC, 85%; RT-PCR, 86%), and agreement of both D-IHC and RT-PCR was 99 and 100%, respectively, compared with IFA results obtained from all the field samples. These findings demonstrated the utility of D-IHC for direct detection of TCoV from field samples and considering the sensitivity and specificity found here, can be used as an alternative technique.

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 characteri tics 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.

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.

Aspectos da patogenia e análise da variabilidade da glicoproteína S do Coronavirus dos Perus isolado no Brasil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)