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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. more...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), besides causing human infection, has been shown to naturally infect several susceptible animal species including large cats (tigers, lions, pumas, spotted leopards), dogs, cats, ferrets, gorillas and minks. Cats and minks are continuing to be the most reported species with SARS-CoV-2 infections among animals but it needs to be investigated further.We report the detection of SARS-CoV-2 from a domestic cat that exhibited respiratory disease after being exposed to SARS-CoV-2 virus from humans in the same household. SARS-CoV-2 RNA was detected in two oropharyngeal swabs collected at two time points, 11 days apart; the first, when the cat was reported to be sick and the second, before euthanasia due to poor prognosis. The viral nucleic acid detected at two time points showed no genomic variation and resembled the clade GH circulating in humans in the United States. Clinical and pathological findings noted in this 16-year-old cat were consistent with respiratory and cardiac insufficiency.SARS-CoV-2 viral infection was likely an incidental clinical finding, as the virus was not detected in fixed lungs, heart, or kidney tissues. Only fresh lung tissue collected at necropsy showed the presence of viral nucleic acid, albeit at a very low level. Further research is needed to clarify the clinical course of SARS-CoV-2 in companion animals of advanced age and underlying cardiac disease. more...

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. more...

Background Highly pathogenic H5N1 avian influenza (AI) poses a grave risk to human health. An important aspect of influenza control is rapid diagnosis. Objectives This study describes the efficiency of AI-RNA extraction utilizing silica-based magnetic beads with robotics and its detection with an influenza A matrix gene real-time RT-PCR from tracheal swabs, and compares it to virus isolation and manual spin column extractions. Study design Analytical sensitivity was assessed by performing dilution analysis and detection of H2N2 AI viral RNA. Diagnostic sensitivity and specificity was assessed by analyzing tracheal swabs collected from H7N2 infected and uninfected chickens. Results Both manual and robotic extractions detected AI virus at 1 log10 EID50/ml. Diagnostic sensitivity and specificity of matrix gene detection with the automated extraction method for chicken tracheal swab specimens was similar to that of virus isolation and the manual extraction method. There were only three discordant results among 212 tested specimens. Conclusion The main advantages of automated robotic viral nucleic acid extraction are high throughput processing; hands-free operation; and reduction in human and technical error. This study demonstrates successful detection of influenza A virus with magnetic beads utilizing the Qiagen MagAttract cell kit on a BioRobot M48 platform. more...