RESUMO
The objective of the present study was to evaluate the capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) to characterize poultry gut microbiota and the ability of this molecular method to detect modifications related to rearing conditions to be used as an epidemiological tool. The V3 region of the 16S rRNA gene was selected as the PCR target. Our results showed that this method provides reproducible data. The microbiota analysis of individuals showed that variability between individual fingerprints was higher for ileum and cloaca than for ceca. However, pooling the samples decreased this variability. To estimate the variability within and between farms, we compared molecular gut patterns of animals from the same hatchery reared under similar conditions and fed the same diet in 2 separate farms. Total aerobic bacteria, coliforms, and lactic acid bacteria were enumerated using conventional bacteriological methods. A significant difference was observed for coliforms present in the ceca and the cloaca depending on the farm. Ileal contents fingerprints were more closely related to those of cloacal contents than to those of ceca contents. When comparing samples from the 2 farms, a specific microbiota was highlighted for each farm. For each gut compartment, the microbiota fingerprints were joined in clusters according to the farm. Thus, this rapid and potentially high-throughput method to obtain gut flora fingerprints is sensitive enough to detect a "farm effect" on the balance of poultry gut microbiota despite the birds being fed the same regimens and reared under similar conditions.
Assuntos
Bactérias/classificação , Galinhas/microbiologia , Eletroforese Capilar/métodos , Trato Gastrointestinal/microbiologia , Criação de Animais Domésticos , Animais , DNA Bacteriano , Masculino , Polimorfismo GenéticoRESUMO
An increasing incidence of enteric disorders clinically suggestive of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time reverse transcriptase-polymerase chain reaction assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37% of the intestinal samples collected from diseased turkey flocks. The full-length spike (S) gene of these viruses was amplified, cloned and sequenced from three samples. The French S sequences shared 98% identity at both the nucleotide and amino acid levels, whereas they were at most 65% and 60% identical with North American (NA) TCoV and at most 50% and 37% identical with IBV at the nucleotide and amino acid levels, respectively. Higher divergence with NA TCoV was observed in the S1-encoding domain. Phylogenetic analysis based on the S gene revealed that the newly detected viruses form a sublineage genetically related with, but significantly different from, NA TCoV. Additionally, the RNA-dependent RNA polymerase gene and the N gene, located on the 5' and 3' sides of the S gene in the coronavirus genome, were partially sequenced. Phylogenetic analysis revealed that both the NA TCoV and French TCoV (Fr TCoV) lineages included some IBV relatives, which were however different in the two lineages. This suggested that different recombination events could have played a role in the evolution of the NA and Fr TCoV. The present results provide the first S sequence for a European TCoV. They reveal extensive genetic variation in TCoV and suggest different evolutionary pathways in North America and Europe.
Assuntos
Coronavirus do Peru/genética , Enterite Transmissível dos Perus/virologia , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Coronavirus do Peru/isolamento & purificação , Coronavirus do Peru/patogenicidade , Enterite Transmissível dos Perus/epidemiologia , França/epidemiologia , Variação Genética , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Filogenia , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sensibilidade e Especificidade , Alinhamento de Sequência , Análise de Sequência de DNA , Glicoproteína da Espícula de Coronavírus , Perus , Proteínas do Envelope Viral/química , Proteínas Virais/genéticaRESUMO
Four tagged quantitative Real-Time RT-PCRs (qRT-PCRs) were developed to quantify the positive and negative strands of segments A and B of the bisegmented double-stranded RNA (dsRNA) genome of infectious bursal disease virus (IBDV, family Birnaviridae, genus Avibirnavirus). The qRT-PCRs were validated using single-stranded RNAs corresponding to each genomic strand (A+, B+, A-, B-). Specific quantitation proved possible from 5×10(7) to 5×10(2) copies of the template per reaction, with excellent reproducibility and linearity. The methods detected similar amounts of A+ and A- and of B+ and B- in a purified dsRNA viral genome preparation, thus corroborating the accuracy of quantitation. The qRT-PCRs were used to quantify the four strands in CsCl purified virus fractions and in samples collected during propagation of IBDV in cell culture. Purified virus fractions contained similar amounts of A- and B- strands, but also a large and unexplained excess of A+ and even more B+ strands. Results of the in vitro kinetic study showed an early accumulation of positive strands and a more delayed and lower accumulation of the A- and B- strands, both in similar amounts. These results suggest that minus strand synthesis occurs in IBDV after the equimolar packaging of A+ and B+ strands.
