Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic
Leptospira
Authors: Hamond Camila , LeCount Karen , Browne A. Springer , Anderson Tammy , Stuber Tod , Hicks Jessica , Camp Patrick , Fernandes Luis G. V. , van der Linden Hans , Goris Marga G. A. , Bayles Darrell O. , Schlater Linda K. , Nally Jarlath E. 
Published: October 2023

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with
Leptospira borgpetersenii
serogroup Ballum (
n
= 48) and/or
Leptospira kirschneri
serogroup Icterohaemorrhagiae (
n
= 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them:
L. borgpetersenii
serogroup Ballum in LR1, LR5, and LR37, and
L. kirschneri
serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both
L. borgpetersenii
serogroup Ballum and
L. kirschneri
serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of
Leptospira
concurrently. The identification and characterization of multiple species/serogroups of
Leptospira
from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.

IMPORTANCE

Pathogenic
Leptospira
, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of
Leptospira
may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic
Leptospira
from the same host. The identification and characterization of multiple species/serogroups of
Leptospira
from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.