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Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (10 9 to 10 2 CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (10 2 CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at ؊20°C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement ( ‫؍‬ 0.65) was found between the presence of A. pleuropneumoniae in tonsils and the individual serological status of the animals.

Background: Influenza D virus (IDV), a segmented single stranded negative sense ribonucleic acid (RNA) virus belongs to the new Delta influenza virus genus of the Orthomyxoviridae family.Cattle was proposed as the natural reservoir of IDV in which infection was associated with mild to moderate respiratory clinical signs (i.e. cough, nasal discharge, and dyspnoea). Methods and principal findings: In order to investigate the role of IDV in bovine respiratory disease, during the period 2017-2020, 883 nasal or naso-pharyngeal swabs from Canadian cattle with respiratory signs (cough and/or dyspnoea) were tested by (RT-)qPCR for IDV and other major bovine viral (bovine herpesvirus 1, bovine viral diarrhoea virus, bovine respiratory syncytial virus, bovine parainfluenza virus 3, and bovine coronavirus) and bacterial (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis) respiratory pathogens. In addition, whole genome sequencing and phylogenetic analyses were carried out on five IDV positive samples. The prevalence of IDV RT-qPCR (with cut-off: Cq < 38) at animal level was estimated at 5.32% (95% confidence interval: 3.94-7.02). Positive result of IDV was significantly associated with (RT-)qPCR positive results for bovine respiratory syncytial virus and Mycoplasma bovis. While phylogenetic analyses indicate that most segments belonged to clade D/660, reassortment between clades D/660 and D/OK were evidenced in four samples collected in 2018-2020. Conclusions and significance: Relative importance of influenza D virus and associated pathogens in bovine respiratory disease of Canadian dairy cattle was established. Whole genome sequencing demonstrated evidence of reassortment between clades D/660 and D/OK. Both these new pieces of information claim for more surveillance of IDV in cattle production worldwide.