Comparative Evaluation of Conventional and Real-Time PCR assays for the epidemiological surveillance and molecular characterization of Leptospira in asymptomatic stray dogs

Abstract

This study compared serological and molecular approaches for detecting Leptospira spp. using paired blood and urine samples from 124 asymptomatic stray dogs. Blood sera were screened via the microscopic agglutination test (MAT) for ten serovars. Urine samples were analyzed using four PCR assays: a commercial quantitative PCR (qPCR), a lipL32 qPCR, a conventional rrs PCR, and a multiplex PCR (mPCR). Serological exposure was detected in 26 (20.97%) dogs, while commercial qPCR, rrs PCR, lipL32 qPCR, and mPCR assays yielded positive results in 31 (25.00%), 34 (27.42%), 25 (20.16%), and 23 (18.55%) dogs, respectively. Compared with commercial qPCR, the lipL32 qPCR, rrs PCR, and mPCR demonstrated Positive Percentage Agreement (PPA) values of 80.65%, 87.10%, and 67.74%, and Negative Percentage Agreement (NPA) values of 100.00%, 92.47%, and 97.85%. Nineteen urine samples were positive across all four PCR assays. Overall, 42.74% (53/124) of dogs had at least one positive serological or molecular finding, including nine additional conventional PCR-positive findings without reliable sequence confirmation. Sequencing and phylogenetic analysis of 24 samples identified L. interrogans and L. borgpetersenii, representing the first molecular characterization of Leptospira in Bosnia and Herzegovina. The findings demonstrate the complementary value of serological and molecular approaches for Leptospira surveillance while emphasizing the need for cautious interpretation of unconfirmed molecular findings.