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Aims In this study, we aimed to isolate and evaluate the efficacy of Bacillus velezensis as a probiotic and to assess its activity towards pigeons infected with pigeon circovirus (PiCV). Methods and Results Bacillus velezensis, isolated from pigeon faeces, was orally administered to pigeons for 60 days. After pigeons were challenged with PiCV, the PiCV viral load and expression of indicator genes for innate immunity were detected in spleen tissue and faeces of pigeons. Bacillus velezensis significantly reduced the PiCV viral load in the faeces and spleen of pigeons 5 days post‐challenge (dpc). The mRNA expression levels of treated pigeons showed that interferon‐gamma (IFN‐γ), myxovirus resistance 1 (Mx1), and signal transducers and activators of transcription 1 (STAT1) genes were upregulated, whereas no expression of interleukin‐4 (IL‐4) was detected. Moreover, toll‐like receptor 2 (TLR2) and 4 (TLR4) were significantly upregulated in probiotic‐treated pigeons (P < 0·05). Conclusions This is the first report showing that probiotic supplementation can effectively enhance the T‐helper type 1 immune response and decrease the PiCV viral loads in pigeons. Significance and Impact of the Study This study proposes that the administration of a probiotic strain, B. velezensis, to pigeons can protect against PiCV infection.

Pigeon racing’s recent upturn in popularity can be attributed in part to the huge prize money involved in these competitions. As such, methods to select pigeons with desirable genetic characteristics for racing or for selective breeding have also been gaining more interest. Polymerase chain reaction—restriction fragment length polymorphism (PCR-RFLP) for genotyping-specific genes is one of the most commonly used molecular techniques, which can be costly, laborious and time consuming. The present study reports the development of an alternative genotyping method that employs Kompetitive Allele Specific Polymerase Chain Reaction (KASP) technology with specifically designed primers to detect previously reported racing performance-associated polymorphisms within the LDHA, MTYCB, and DRD4 genes. To validate, KASP assays and PCR-RFLP assays results from 107 samples genotyped for each of the genes were compared and the results showed perfect (100%) agreement of both methods. The developed KASP assays present an alternative rapid, reliable, and cost-effective method to identify polymorphisms in pigeons.