Optimizing surveillance: feasibility of pooled sample testing for bovine alphaherpesvirus 1 detection by real-time...

The present study aimed to evaluate the feasibility and cost-effectiveness of pooled sample testing (PST) for the detection of bovine alphaherpesvirus 1 (BoAHV-1) in swab samples collected from animals. A pool of five samples were used and two modifications in the procedure were introduced to increase the sensitivity of the approach. The initial volume of the sample used for DNA extraction was increased from 200 µL to 500 µL and 10 µL of extracted DNA instead of 5 µL in the real-time PCR reaction set-up. In an initial spiking experiment with the BoAHV-1 virus, the modified PST approach resulted in a 1.1 Ct value lower than the conventional individual sample approach, supporting the efficacy of the pool size chosen. Subsequently, screening of 215 pools created from 1073 swab samples, revealed 29 pools (13.5%) positive for BoAHV-1. Further examination of the 145 individual samples contributing to these 29 positive pools revealed at least one positive sample in 22 pools, while 7 pools did not contain any positive samples. The overall percentage positivity of BoAHV-1 shedding detected through pooled sample testing was 2.86% (95% CI: 1.92%-4.08%), closely resembling the result from individual sample testing at 2.80% (95% CI: 1.97-3.96%). This similarity supports the potential use of the pooled sample testing (PST) approach in surveillance studies. In conclusion, the findings of this study demonstrate the feasibility of adopting PST for the detection and confirmation of BoHV-1 infection in animals using a real-time PCR assay.