RFLPs of ITS, ITS1 and hsp70 amplicons and sequencing of ITS1 of recent clinical isolates of Kala-azar from India and Bangladesh confirms the association of L. tropica with the disease

Abstract

Visceral Leishmaniasis or Kala-azar (KA) is a serious health concern in India. In the present study, Restriction Fragment Length Polymorphism (RFLP) of three genetic markers viz., Internal Transcribed Spacer (ITS), ITS1 and heat shock protein 70 (hsp70) have been employed for typing the clinical isolates [n=15] of KA and post Kala-azar Dermal Leishmaniosis (PKDL) collected from India and Bangladesh in the period of 2006-2010. Experimentally, ITS, ITS1 and hsp70 regions of genomes of all the clinical isolates were separately amplified by PCR and then digested with restriction enzymes: ITS with Alu1, EcoR1 and Msp1, ITS1 with Hae III and Rsa1 and hsp70 with Hae III. The resultant fragments were analyzed by agarose gel electrophoresis and the RFLP profiles of the clinical isolates were compared with that of the WHO reference strains for Leishmania donovani (DD8) and Leishmania tropica (K27), respectively. Also, the ITS1 regions of all the clinical isolates along with the two WHO reference strains were sequenced and a phylogram was constructed to ascertain the extent of similarity or dissimilarity. Interestingly, the RFLP profiles of one of the isolates showed a significant homology with K27 and the phylogram revealed its closeness with the same putting credence to our earlier typing of isolates by RAPD method. This observation also supported an earlier report claiming that both the species are responsible for KA in India and thus, emphasizes urgent need for thorough systematic characterization of the clinical isolates of Indian KA as appropriate treatment regime relies primarily on proper diagnosis.