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.