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Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts. [ABSTRACT FROM AUTHOR]

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. Author Summary: 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. [ABSTRACT FROM AUTHOR]