Use of recombinant calflagin protein as a potential candidate for diagnosis of Trypanosoma evansi infection.

Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies against T. evansi were detected with rCLF antigen in serum samples of experimentally infected equines as early as 10 days and 14 days post infection, using immunoblot and ELISA respectively. No cross-reactivity was observed with rCLF antigen in ELISA with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions ranging from 10 to 28 kDa were detected using distinct T. evansi isolates (pony, cattle, donkey and camel origin) indicating presence of multiple calflagin family members in a single trypanosome. Indirect immunofluorescence antibody test with anti-CLF rabbit hyperimmune serum showed localisation of native immunogenic protein near attachment of flagellum. The rCLF protein was found to be a potential diagnostic candidate for distinguishing T. evansi positive and negative equine serum sample, suggesting that it could be used for serological surveys in animals for surra. In addition, it could be used with other potential diagnostic candidates to improve the diagnostic efficiency.

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.