Molecular characterization and sero surveillance of Bovine Theileriosis using recombinant MPSP based ELISA

Abstract

In the present study, based on cytochrome b gene sequences, the phylogenetic newlinerelationship of various field isolates of T. annulata from in and around Mathura, Uttar newlinePradesh, India along with other data available in Genbank was discussed. Theileria newlineannulata, an intracellular blood protozoan parasite has wide geographical distribution newlineand is a serious economic constraint to the livestock industry. A total of 268 (192 newlinecattle and 76 buffalo) blood samples suspected to T. annulata infection were obtained newlinefrom different region of Mathura. Both microscopic and PCR method using cyt b gene newlineprimer were used for screening of T. annulata samples. Out of 268 samples of newlineexamined blood smears, 24 (8.9%) animals were positive for T. annulata. However, newlinePCR using cytochrome b gene of these 268 blood samples revealed 92 animals newline(34.3%) positive for T. annulata infection, with the amplified PCR product of 1092 newlinebp. Finally, the cytochrome b gene of six T. annulata isolates were amplified, custom newlinesequenced and deposited in GenBank (accession no. MH778940-MH778945). The newlinesequence alignment results indicated that two haplotype were noticed while there was newlinesubstitutions of nucleotide bases at the position 710 (thymine for adenine) and at 1076 newline(cytosine for guanine). The comparison of cyt b gene sequences of six isolates newlinerevealed 99.99-100% identity to each other whereas it was 99.93-100% when newlinecompared across the globe. Phylogenetic tree based on cyt b gene sequences indicated newlinethat the present isolates were found to be closer to isolates from Iran than to other newlineparts of the world. Amongst Indian isolates they were found to be closer to isolates newlinefrom Hapur and Hissar than the isolates from Southern parts of India. A specific and newlinesensitive loop mediated isothermal amplification (LAMP) was developed targeting newlineITS gene for rapid detection of T. annulata infection. Out of 100 bovine samples newlinetested, 23 animals and 27 animals were positive by PCR and LAMP, respectively, newlineindicating that LAMP assay was more sensitive.