Isolation and identification of Leptospira species from bovines by rpoB and LipL41 genes based phylogenetic analysis

Abstract

In this study, in the beginning 582 serum samples were subjected to Microscopic Agglutination Test (MAT) with eight different serovars prevalent in the region to know the seroprevalence of Leptospira in bovines in Karnataka, India. Based on the findings of the MAT, different samples like blood, urine, aborted materials and uterine discharge collected from the MAT positive animals were used for isolation and genomic detection by conventional PCR targeting two lipL32 and seqY genes using specific primers. Out of the 163 MAT positive samples screened 12 samples (including three isolates) were found positive in PCR. Subsequently, to identify the different species prevalent in the geographical region the PCR positive samples were subjected to rpoB and LipL41 gene amplification. and nucleotide sequence analysis of rpo B, it was found that all the samples were belonging to L. interrogans species with overlapping/superimposing L. interrogans and L.borgpetersenii species. Further, the LipL41 gene sequence phylogenetic analysis differentiated these two species clearly. Therefore, it can be concluded that LipL41 gene based phylogenic analysis besides rpoB gene can be effectively utilized to identify different Leptospira species in a geographical niche including the identification of intermediate species. This is first of its kind in India using LipL41 gene based phylogenetic analysis for Leptospira species identification in limited number of samples from bovines, hence the same can be explored on a larger geographical area with more number of samples and even to identify the prevalence or presence of intermediate species in different geographical locations.

Keywords

• Leptospira
• MAT
• LipL41
• rpoB
• Phylogenic analysis