Testicular processing fluid as a useful matrix for the detection of porcine circovirus type 2 DNA and virus-specific antibodies

Introduction: Testicular processing fluid (PF) obtained during boar castration may serve as a diagnostic matrix for monitoring porcine circovirus type 2 (PCV2) presence.Methods: Anti- PCV2 antibodies in PF were detected using an indirect ELISA, and PCV2 DNA was detected by real-time PCR (qPCR), and the effects of sample pooling were evaluated. Paired sera and PF from boars and sera from gilts were tested with commercial ELISA and qPCR kits. PF-specific cut-offs were set by ROC (ELISA OD; qPCR Ct). Pooling was simulated by diluting positive PF samples with negative PF samples at predefined ratios.Results: With manufacturers’ cut-offs, seropositivity was 89.94% (male sera), 87.43% (PF), and 92.81% (gilt sera); differences were observed only between gilt sera and PF. Using the ROC PF cut-off (OD ≥ 0.23), 88.82% PF were positive, and matrices did not differ; diagnostic agreement metrics for PF improved. In qPCR, positivity was 13.02% (boar sera), 16.90% (PF), and 9.36% (gilt sera). A ROC-specific PCR cut-off (Ct < 36.50) improved specificity, predictive values, and agreement without affecting sensitivity; serum and PF Ct values were moderately correlated (ρ = 0.53). Pooling reduced detection of weak positives (most notably in qPCR) while high-positive PF remained detectable at higher dilutions.Discussion and conclusion: These results demonstrate that PF provides a reliable, cost effective alternative to serum for PCV2 surveillance and monitoring when matrix-specific cut-offs are used; however, excessive pooling may lead to false-negative results. This approach may facilitate large-scale herd monitoring while reducing the need for invasive sampling.