Molecular characterization of internal transcribed spacer 1 (ITS 1) region of different Trypanosoma evansi isolates of India

Six Trypanosoma evansi isolates collected from ponies (PH1 and PK6), camel (CB2), donkeys (DJ3 and DH4) and cattle (CK5) from Haryana, Rajasthan, Uttar Pradesh and Gujarat states of India were used for molecular characterization of internal transcribed spacer 1 (ITS 1). The DNA was isolated from purified trypanosomes of these six isolates after propagation in mice model. ITS1-PCR of purified parasite DNA yielded an amplification product approximately 540 bp in size. Nucleotide sequence of ITS1 gene of CB2 isolate had 530 bp while CK5, DH4, DJ3, and PH1 isolates had 532 bp, whereas, PK6 isolates had 533 bp size. Blast data of the Indian isolates revealed 99 % homology with other available sequences of T. evansi. Multiple alignment of nucleotide sequence of ITS1 gene variants from Indian T. evansi isolates with selected homologous sequences from GenBank revealed that nucleotide substitution mostly occurred at the position of 101–103, 218–223, 243–244, 301–396 and 470–480. The isolates PH1, CK5, DH4 and DJ3 were found more associated with T. evansi isolates from the Philippines, Thailand, Iran, Egypt and China, whereas, PK6 and CB2 isolates were related to each other and were phylogenetically distant from rest of the Indian isolates used in this study. Based on the ITS1 rDNA sequence, the Neighbour–Joining consensus tree indicated clear evidence of existence of genetic diversity among T. evansi isolates from India.