Molecular and Clinical Study of Bovine Coronavirus and Staphylococcus aureus Co-Infection Associated With Bovine Respiratory Disease in Iraqi Calves.

Bovine respiratory disease (BRD) is a multifactorial syndrome causing major economic losses in cattle production worldwide. The present study investigated the prevalence, molecular characterization, and phylogenetic relationship of bovine coronavirus (BCoV) and Staphylococcus aureus co-infection in calves exhibiting respiratory and enteric signs. A total of 80 nasal and fecal samples were collected from calves aged 2 weeks to 2 months, showing clinical signs ranging from mild to acute disease. Clinical severity was categorized according to fever, dehydration, diarrhea, and respiratory distress. Molecular detection of BCoV was performed using real-time RT-PCR and conventional PCR targeting the N gene, while S. aureus isolates were identified by bacteriological culture, biochemical tests, and 16S rRNA gene amplification. Phylogenetic analysis was conducted using MEGA software and sequence alignment tools. The results showed that BCoV was detected in 35% of examined samples, while S. aureus was identified in 28% of cases. Co-infection was observed in 15% of calves and was significantly associated with severe clinical manifestations (p < 0.05). Acute and moderate cases demonstrated markedly higher BCoV positivity compared with mild cases. Agarose gel electrophoresis confirmed amplification of the BCoV N gene at 205 bp and the S. aureus 16S rRNA gene at approximately 1500 bp. Sequence alignment of local S. aureus isolates revealed highly conserved nucleotide regions, whereas phylogenetic analysis demonstrated genetic diversity among both bacterial and viral isolates. Local BCoV strains showed considerable sequence divergence compared with international reference strains, suggesting ongoing viral evolution. In conclusion, co-infection between BCoV and S. aureus significantly increases disease severity in calves and highlights the importance of integrated molecular diagnostics and continuous phylogenetic surveillance for effective BRD control strategies.