Time-dependent synergistic pathogenicity of sequential avian reovirus and Mycoplasma synoviae co-infection in SPF chickens.

Avian reovirus (ARV) and Mycoplasma synoviae (MS) are major pathogens associated with tarsal synovitis and growth suppression in broilers. Yet the effect of infection order on their combined virulence remains poorly understood. A survey of 202 swollen joint samples from northern Chinese farms (2024-2025) revealed 35.1% ARV-MS co-detection, identifying this dual infection as a frequent co-infection in broiler arthritis. To assess timing effects, we inoculated 240 SPF chickens with ARV, MS, or both simultaneously or sequentially at 12- or 24 h intervals. All co-infection protocols were associated with exacerbated weight loss, tarsal swelling, multi-organ pathology, and ARV loads relative to single infections. The most severe phenotype occurred when ARV preceded MS by 24 h (body weight: 195.8 g; tarsal diameter: 1.37 cm at 30 dpi). Co-infection elevated the MS isolation rate from joint tissues from 36.6% (MS-only) to 76.7%, suggesting that ARV pre-exposure promotes MS colonization. It also extended ARV persistence in the liver, spleen, kidney, thymus, bursa, and joints by 3-7 days and increased shedding in blood and the cloaca. Histopathology revealed hepatocyte necrosis, splenic lymphocyte depletion, renal tubular degeneration, and articular hemorrhage, which were more severe in co-infected birds. Collectively, our data provide evidence for a time-dependent synergy in which prior ARV infection, particularly with a 24 h interval, is associated with enhanced MS pathogenicity and systemic dissemination. Our findings highlight the need to include ARV carriage in MS risk assessments and provide insights for prevention and control.