Evaluation of Real-Time PCR and Culturing for the Detection of Leptospires in Canine Samples

Ahmed Ahmed, Henricus L. B. M. Klaasen, Mark van der Veen, Hans van der Linden, Marga G. A. Goris, Rudy A. Hartskeerl

MSD Animal Health, Department Microbiological Research and Development, Boxmeer, The Netherlands.
WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, Department of Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.
WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis,Department of Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.

DOI: 10.4236/aim.2012.22021                

Abstract

Validated real-time PCRs detecting leptospires for veterinary purposes are not readily available. This paper describes the prospective evaluation of a SYBR Green-based real-time PCR on serum samples collected from experimentally infected dogs. Compared to culturing, the assay had a diagnostic sensitivity and specificity of 91.7% and 90.0%, respectively. Culturing for part is complementary to PCR and preferably both should be applied for diagnosis and vaccine challenge experiments. In a subsequent prospective study on the dynamics of experimental infections with serovars Canicola and Copenhageni and serovar complex Bananal-Liangguang in young and adult dogs, the PCR was applied on serum samples in conjunction with culturing on blood, urine and kidney samples with the following results: 1) Leptospires persisted longer in the blood of young dogs than of adult ones; 2) Numbers of viable leptospires in the blood are rapidly reduced but DNA remains occasionally detectable up to 7 days post infection; 3) Appearance of viable leptospires in the urine follows a biphasic dynamics; 4) EDTA hampers effective culturing from blood samples; 5) Serovar Canicola persists longer in the blood of dogs than Copenhageni and Bananal-Liangguang. Together with a markedly higher recovery rate from kidney samples (71% compared to respectively 33% and 0% in young dogs), this probably reflects the adaptation power of Canicola to its canine maintenance host. Appearance of leptospires in the urine samples indicates that experimental infections have been successful. PCR presents a valuable adjunct to the diagnosis of veterinary leptospirosis and the follow-up in vaccine protection experiments.

Keywords

Leptospirosis; Diagnosis; PCR; Evaluation; Canine; Experimental Infection; Challenge; Infection; Kinetics

Conflicts of Interest

The authors declare no conflicts of interest.

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