Microscopic agglutination test (MAT)

Ellinghausen McCullough Johson and Harris (EMJH) liquid medium was prepared as per the standard protocols using the EMJH base and Enrichment media (BD Difco TM USA) with 200 μg of 5-fluorouracil per ml of medium. MAT was performed as previously described by Assenga et al. [1]. In short, all sera were tested for antibodies against six Leptospira antigens including local serogroups Icterohaemorrhagiae, Ballum, Grippotyphosa, Sejroe and reference serogroups Hebdomadis and Australis

Reference serogroups were kindly provided by Royal Tropical Institute (KIT) Leptospira Reference Center, the Netherlands [22]. All sera were serially diluted to 1:10240 in PBS and tested with antigens suspension to establish the antibody titer. MAT titers ≥ 1:160 were scored as positive; titers between 1:20 and 1:80 were scored as exposed to leptospiral infection and absence of agglutination titers was scored as negative [23–26].