Standardisation of 16S rRNA Gene Based Nested PCR as a Sensitive Tool for Canine Leptospirosis Diagnosis

Abstract

Aim: The present study aimed to standardize a 16S rRNA gene-based nested PCR assay for the sensitive detection of leptospirosis in dogs and to compare its diagnostic performance with PCR targeting lipL32 and microscopic agglutination test (MAT). To achieve this, a cross-sectional diagnostic study was conducted on clinically suspected canine cases of leptospirosis. Place and Duration of Study: The study was carried out at the Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Pookode, during November 2024 to August 2025. Methodology: A total of 53 dogs with clinical signs suggestive of leptospirosis were screened. Serum samples were tested by MAT using 13 serovars of Leptospira interrogans. Whole blood was subjected to conventional PCR targeting the lipL32 gene, and nested PCR targeting the conserved region of the 16S rRNA gene. The sensitivity of different diagnostic tests was compared using kappa statistics. Results: Of the 53 suspected dogs, 21 (39.6%) were positive by nested PCR, 14 (26.4%) by lipL32 PCR, and 6 (11.3%) by MAT. The predominant serovar detected was Leptospira interrogans Pomona (50%). Nested PCR showed the highest sensitivity (100%), compared to PCR (66.7%) and MAT (28.6%). Conclusion: The study demonstrates that 16S rRNA gene-based nested PCR is a highly sensitive diagnostic tool for canine leptospirosis, capable of detecting infection even in early stages when antibody response is absent, a finding that is consistent with and supported by existing knowledge. Integration of molecular techniques with serological assays can provide a more reliable diagnostic strategy and enhance surveillance and control of leptospirosis in endemic regions.