Viral tenosynovitis in poultry: Integrating histopathology, in situ hybridization, and quantitative reverse transcriptase-PCR for avian reovirus diagnostic workflow

Avian reovirus (ARV), the etiologic agent of poultry viral arthritis/tenosynovitis, frequently presents diagnostic challenges due to its non-specific lesions, ubiquitous nature, and the lack of standardized diagnostic guidelines. We describe cases of poultry arthritis/tenosynovitis, where ARV was the suspected etiology, and investigate the relationship between lesion severity and viral RNA levels using quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) with RNAScope in situ hybridization (ISH) to support ARV detection and analysis. A total of 51 cases (qRT-PCR positive [<i>n</i> = 38; Ct range = 20.9-35.9] and qRT-PCR negative [<i>n</i> = 13; Ct > 37]) were analyzed, with case selection criteria including clinical lameness, complete histologic examination of the gastrocnemius/digital flexor tendons, and qRT-PCR testing for ARV. A subset of qRT-PCR positive cases (<i>n</i> = 33) and negative cases (<i>n</i> = 8) with the sections showing the most severe histologic lesions were selected for ISH. Histologic scoring of tenosynovitis included inflammation severity; synovial proliferation; and the presence of lymphoid nodules, neovascularization, and fibrosis. The qRT-PCR positive cases had significantly higher histologic scores than negative cases. The ISH detected viral transcripts within synoviocytes and subintimal fibroblasts only in qRT-PCR positive cases (11/33, 33.3%; Ct range = 20.9-32.3). Positive ISH results were also statistically associated with lower Ct values and higher lesion scores. In conclusion, this study aids in ARV diagnostic challenges by linking lesion severity with viral transcription, identifying fibroblasts as ARV-infected cells, and demonstrating ISH as both a valuable diagnostic tool and a means to studying ARV pathogenesis in poultry.