Standardisation of a multiplex PCR for detection of circovirus and adenovirus in pigeons

Young Pigeon Disease Syndrome (YPDS) is an emerging condition in pigeons characterised by high morbidity and mortality, with multiple viral agents implicated in its aetiology. Among these, pigeon circovirus (PiCV) and fowl adenovirus (FAdV) are considered important contributors, but reports from India remain limited. This study investigated their presence in YPDS suspect pigeons and standardised a multiplex PCR (mPCR) assay for their simultaneous detection. A total of 75 clinical samples, including tissues (liver, spleen, bursa of Fabricius, intestine, lungs, kidney and brain), feathers and cloacal swabs from 27 affected pigeons, were screened using uniplex PCR assays targeting the PiCV capsid and FAdV hexon genes. Representative amplicons were sequenced, and BLAST analysis confirmed their identity with reference PiCV and FAdV sequences. Uniplex PCR detected PiCV DNA in 4 samples (5.3) and FAdV DNA in 5 tissue samples (23.8). Sequencing of representative amplicons confirmed their identity, showing nucleotid similarities of 95.7-99.5 with published PiCV and FAdV reference strains. Based on these findings, a multiplex PCR (mPCR) assay was standardised with optimised primer concentrations and cycling conditions, which yielded distinct, reproducible amplicons for both viruses without cross-reactivity. The detection of PiCV and FAdV in YPDS suspected pigeons highlighted their role in the syndrome, while the standardised mPCR provided a rapid, reliable and cost-effective diagnostic tool for simultaneous identification of these agents. This method offers clear advantages over uniplex PCR by reducing time and reagent use, and it can support routine diagnosis and epidemiological surveillance of pigeon populations affected by YPDS.