10.22376/ijpbs.2019.10.1.p1-12 Volume 12 Issue 3 2021 (July-September) Leptospirosis is an infection caused by the spirochete bacteria lessThan i greaterThan Leptospira lessThan /i greaterThan . It is ubiquitous around the world and in India it has been reported from several parts of the country during the rainfall season. In our present work, a detailed cross sectional study with the serum samples from pyrexia of unknown origin (PUO) patients around the poorly studied Gwalior district of the central India region was undertaken to assess the Leptospirosis incidence. Immunological methods such as MAT (Microscopic agglutination test), ELISA (Enzyme linked immune sorbent assay), and WB (Western blotting), along with molecular methods such as PCR and 2D protein analysis were employed and it was found that about 2.82% (n=854) of samples tested were positive for Leptospirosis. The antibody response was obtained against lessThan i greaterThan Leptospira interrogans lessThan /i greaterThan serovars Bataviae, Ballum, Cyanoptera, Grippotyphosa and Ictero-haemorrhagiae copenhageni. The 2-D Gel electrophoresis of the serovar Copenhageni when probed with positive sera in WB identified a major antigen that provoked the immune response in patients. The reaction corresponding to the 32-kDa size protein and an isoelectric point pI value of 6.5 indicated that the immune response was against the outer membrane protein lessThan i greaterThan lip lessThan /i greaterThan L32 of lessThan i greaterThan Leptospira spp lessThan /i greaterThan . Further confirmation with the MALDI TOF analysis of the excised sample from the parallel gel confirmed the tryptic digested products of lessThan i greaterThan lip lessThan /i greaterThan L32. Isolation of leptospires could not be successful; however, the detection of leptospiral DNA by PCR using lessThan i greaterThan lip lessThan /i greaterThan L32 primers in the MAT positive samples confirmed the etiology of the disease. The present study suggests that Leptospirosis could be considered as one of the prime suspects, in the PUO cases, around Gwalior region of Central India. Bharti Mankere, Preeti Gupta, Kundan Tandel, Urmil Tuteja , Mahendra K Gupta and Kulanthaivel Thavachelvum Leptospirosis, PUO cases, MAT, PCR, MALDI –TOF, LipL32. 86-93