Molecular epidemiological studies related to the phylogenetic characterization of Theileria annulata are important in delineating the evolutionary history of the parasite. In the current study, the Theileria annulata (T. annulata) merozoite surface antigen 1 (TAMS 1) gene from 14 bovine isolates of T. annulata originating from semi-arid zone of northern India were amplified and sequenced. TAMS 1 gene sequences (n= 337) reported from 16 countries were subsequently analyzed for haplotype network along with genetic diversity. A total of five haplotypes out of the 14 sequenced isolates and 92 haplotypes out of 337 worldwide sequences are documented in this study. Phylogenetic and molecular evolutionary analyses based on TAMS 1 gene sequences showed that T. annulata is dissipated across different countries and numerous strains are closely linked, even though they belong to different geographical locations. The nucleotide homology between 14 isolates from northern India varied between 91.3 and 100%, whereas it was between 31.5 and 100% when sequences across the globe were compared. Haplotype 14 was recognized as most widely distributed haplotype, with 46 isolates circulating in 10 countries. Globally, negligible genetic distance (F_ST˂0.15) and very high gene flow (N_m˃1) was found in the five populations of the world (South Asia, East Asia, West Asia, Europe and Africa), supporting the absence of clearly defined subgroups in the phylogenetic analysis. Significant negative values of neutrality tests; Tajima's D (D) and Fu and Li's F (F) provided evidence for recent population expansion through positive selection of advantageous variations.

This study is the first to conduct a sero-surveillance of Bovine Tropical Theileriosis (BTT) caused by the protozoan parasite Theileria annulata (T. annulata) using a recombinant Tams1 protein-based dot-ELISA in cattle, and to compare its efficacy with plate-ELISA, single PCR, nested PCR, and blood microscopy. The goal was to identify the most effective method for the early and accurate detection of theileriosis, which significantly impacts livestock through reduced milk yield and increased mortality. A total of 101 field blood samples were examined using blood smear analysis, single PCR, nested PCR, and dot-ELISA. The recombinant Tams1 protein was successfully cloned and expressed using a pET-30b (+) expression vector in a prokaryotic system. The protein was purified with Ni-NTA chromatography, confirmed for immunoreactivity with T. annulata positive serum via Western blot analysis, and used to optimize both dot-ELISA and plate-ELISA. Both dot-ELISA and plate-ELISA using recombinant Tams1 protein exhibited comparable diagnostic performance, with a kappa value of 0.826 and similar analytical productivity (P = 0.6165). Dot-ELISA revealed a BTT seroprevalence of 58.4% in the cattle population, demonstrating good sensitivity (93.33%) and specificity (90%). The diagnostic performance of dot-ELISA was found to be superior to other molecular techniques, including microscopy, single PCR, and nested PCR. Dot-ELISA is also a sustainable solution in comparison to other laboratory diagnostic techniques with benefits of early diagnosis, reduced waste generation, resource efficiency, cost-effective point of care disease surveillance. With its minimal antigen requirement, Tams1 molecule based dot-ELISA is recommended as an effective tool for epidemiological studies and field surveys of BTT.

Bubaline sarcocystosis, caused by Sarcocystis levinei, is worldwide in distribution. Yet, very limited reports are available across the globe on the phylogenetic aspects of this particular Sarcocystis spp. Virtually no literature is available on its molecular aspects from India. Thus, the present study was designed to phylogenetically characterize the various isolates of S. levinei based on 18S rRNA and cox 1 mitochondrial genes. A total of 11 isolates of S. levinei from Northern India were characterized for 18S rRNA (MG957189-MG957199) and cox 1 (MH255771-MH255781) genes. PCR products were cloned, sequenced, and compared with other sequences across the world. A phylogenetic tree was constructed based on the maximum likelihood (ML) method. Phylogenetic analysis placed S. levinei and S. cruzi into two monophyletic sister groups. An appreciable amount of genetic variability was noticed between various S. levinei sequences for both the gene loci. A total of three haplotypes were noticed for each gene. The generated sequences showed 99.8–100.0% and 99.7–100.0% nucleotide homologies within themselves with regard to the 18S rRNA and cox 1 genes, respectively. Few of the studied sequences showed marked similarity and closeness with the Egyptian sequences than their Indian counterparts. This is the first report of molecular characterization and sequence phylogenetic study of S. levinei from India.