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Background  Leptospirosis has long been a major public health concern in the southwestern Indian Ocean. However, in Madagascar, only a few, old studies have provided indirect serological evidence of the disease in humans or animals.    Methodology/Principal Findings  We conducted a large animal study focusing on small-mammal populations. Five field trapping surveys were carried out at five sites, from April 2008 to August 2009. Captures consisted of  Rattus norvegicus  (35.8%),  R. rattus  (35.1%),  Mus musculus  (20.5%) and  Suncus murinus  (8.6%). We used microbiological culture, serodiagnosis tests (MAT) and real-time PCR to assess  Leptospira  infection.  Leptospira  carriage was detected by PCR in 91 (33.9%) of the 268 small mammals, by MAT in 17 of the 151 (11.3%) animals for which serum samples were available and by culture in 9 of the 268 animals (3.3%). Rates of infection based on positive PCR results were significantly higher in Moramanga (54%), Toliara (48%) and Mahajanga (47.4%) than in Antsiranana (8.5%) and Toamasina (14%) (p = 0.001). The prevalence of  Leptospira  carriage was significantly higher in  R. norvegicus  (48.9%),  S. murinus  (43.5%) and  R. rattus  (30.8%) than in  M. musculus  (9.1%) (p<0.001). The MAT detected antibodies against the serogroups Canicola and Icterohaemorrhagiae. Isolates were characterized by serology,  secY  sequence-based phylogeny, partial sequencing of  rrs , multi-locus VNTR analysis and pulsed field gel electrophoresis. The 10 isolates obtained from nine rats were all identified as species  L. interrogans  serogroup Canicola serovar Kuwait and all had identical partial  rrs  and  secY  sequences.    Conclusions/Significance  We present here the first direct evidence of widespread leptospiral carriage in small mammals in Madagascar. Our results strongly suggest a high level of environmental contamination, consistent with probable transmission of the infection to humans. This first isolation of pathogenic  Leptospira  strains in this country may significantly improve the detection of specific antibodies in human cases.

First Isolation and Direct Evidence for the Existence of Large Small-Mammal Reservoirs of Leptospira sp. in Madagascar