Molecular characterization and serosurvillence of Theileria annulata using recombinant Tams1 protein based ELISA

Abstract

The present study deals with molecular characterization and sero-surveillance of newlinetheileriosis caused by Theileria annulata (T. annulata) using recombinant Tams1 protein newlinebased ELISA. Total 202 blood samples were screened for Bovine Tropical Theileriosis newline(BTT) with blood smear examination and PCR protocol. The best suited primers for PCR newlinebased diagnosis of T. annulata were screened. The primer set NTA F/R was found to be newlinehighly efficient in detecting the infection in comparison to standard N516/517 and Tams newlineF/R primers. Alongside, primer set Tams F/R had better detection efficiency when used newlineas outer primer with NTA F/R as inner primer in nested PCR protocol. A colorimetric newlineLAMP assay for rapid diagnosis of the theileriosis was also standardized. Colorimetric newlineLAMP was found to be at par with nested PCR in detecting theileriosis and was more newlinesensitive than single PCR. Alongside, PCR-RFLP analysis based on Tams1 gene revealed newlinecoexistence of four circulating genotypes of T. annulata, while a uniform restriction newlinepattern was observed with HSP 70 gene in PCR-RFLP analysis. Thereafter, 14 T. newlineannulata samples were characterized through sequencing. Thus obtained sequences were newlinesubmitted in GeneBank and accession numbers MH277607-MH277620 were obtained. newlineThese 14 sequences, covering the five haplotypes, shared 91.3-100% nucleotide newlinehomology within them and varied between 90.3-99.0% with isolates from India whereas newlineit was 86.6-98.2% when compared across the globe. Subsequently, phylogenetic analysis, newlinewere made for various isolates of T. annulata as well as for various Theileria spp. from newlinearound the globe. Present isolates were phylogenetically closer to Haryana and Bareilly newlineisolates amongst the Indian counterparts isolates and globally to Turkey isolate. newlineMerozoite surface protein analysis placed T. annulata with monophyletic cluster adjacent newlineto T. lestoquardi. The recombinant Tams1 protein was cloned and expressed in newlineprokaryotic expression system with pET-30b (+) expression vector. The expressed newlinerecombin