RESUMO
Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.
Assuntos
Doenças dos Bovinos/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Animais , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/veterinária , Bovinos , Fezes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira, were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle.
Assuntos
Aborto Animal/diagnóstico , Doenças dos Bovinos/diagnóstico , Coccidiose/veterinária , Neospora/isolamento & purificação , Complicações Parasitárias na Gravidez/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Feto Abortado/parasitologia , Aborto Animal/parasitologia , Animais , Encéfalo/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/diagnóstico , Coccidiose/parasitologia , Primers do DNA/genética , Sondas de DNA/genética , Feminino , Coração/parasitologia , Neospora/genética , Gravidez , Complicações Parasitárias na Gravidez/diagnóstico , Complicações Parasitárias na Gravidez/parasitologiaRESUMO
We evaluated three DNA extraction methods for confirmation of Mycobacterium avium subspecies paratuberculosis from liquid cultures of bovine feces. Use of DNA Extract All Reagents Kit™ resulted in efficient extraction of amplifiable DNA from higher proportion (96.29%) of known positive samples compared to Chelex-100 resin (25.92%) and polyethylene glycol (0%).
Assuntos
Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/veterinária , DNA/química , Fezes/microbiologia , Conformação Molecular , Mycobacterium avium subsp. paratuberculosis/genética , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Meios de Cultura/química , DNA/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/veterinária , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Sensibilidade e EspecificidadeRESUMO
West Nile virus (WNV) RNA was quantified in WNV infected crows and horses with the help of a real-time reverse transcriptase-PCR assay. A 5' nuclease assay, based on NS5 gene detection with a fluorescent probe was used for quantifying WNV RNA using formalin fixed paraffin embedded tissue specimens. Quantitative detection of WNV RNA showed the presence of a higher amount of the viral RNA in crow tissues compared to equine tissues and these results correlated well with the detection of WNV antigen by immunostaining. In crows, the highest amount of virus was seen in the intestine and in horses in the brain.
Assuntos
Cavalos/virologia , Inclusão em Parafina/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Aves Canoras/virologia , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Antígenos Virais/análise , Doenças das Aves/virologia , Formaldeído , Doenças dos Cavalos/virologia , Imuno-Histoquímica , Especificidade de Órgãos , Fixação de Tecidos , Transcrição Gênica , Febre do Nilo Ocidental/veterinária , Febre do Nilo Ocidental/virologiaRESUMO
Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G2254 constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G2254 nucleotide but with an A2254 nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A2254 and or G2254 strain was detected with real time PCR, and was confirmed with a rapid pyrosequencing technique. Pyrosequencing was useful in at least 2 cases where real time PCR was equivocal in determining the infecting EHV-1 strain type. The strain with G2254 mutation was detected in 9.4% of 21 studied abortion cases, and in 86.6% of 15 neurologic cases.