Application and Validation of PFGE for Serovar Identification of Leptospira Clinical Isolates

Abstract

Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT), the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE) is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC) between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78%) were identified to the serovar level by the database, and an additional 27 isolates (15%) have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars. Leptospirosis is an infection caused by Leptospira bacteria, and is probably the most widespread zoonosis in the world. It is carried by a wide range of animals that contaminate the environment by shedding organisms in their urine. Humans become infected when they come into contact with contaminated urine or water in the environment that has been contaminated with the urine of infected animals. Despite its ubiquity, isolates are rarely identified to the serovar level due to the cumbersome, complicated serological methods that are involved. Serovar identification is important for epidemiology and enabling public health interventions. In this study, we employed a molecular method of serovar identification using pulsed-field gel electrophoresis to identify 175 clinical isolates of Leptospira. In order to validate this method for serovar identification, we also performed complex serological testing on a subset of the isolates. The results indicated that pulsed-field gel electrophoresis is an appropriate alternative to serological tests for serovar identification. Serovar identities of the clinical isolates are also discussed. Fifteen percent of the clinical isolates were identified as potentially new serovars and demonstrates the utility of a more rapid, standardized molecular method in order to keep up with the changing taxonomy and epidemiology of Leptospira.