Vol.:(0123456789) 1 3 Acta Parasitologica https://doi.org/10.2478/s11686-020-00196-0 ORIGINAL PAPER Coexistence of Multiple Theileria annulata Genotypes Circulating in Neonatal Calves in Semi‑arid India Sanjhi Paliwal 1  · Vikrant Sudan 1  · Daya Shanker 1  · Mukesh Srivastava 1 Received: 3 October 2019 / Accepted: 11 March 2020 © Witold Stefański Institute of Parasitology, Polish Academy of Sciences 2020 Abstract Background Knowledge of local isolates and strains is a prerequisite for the development of either efective mass vaccination strategy or a suitable molecular marker-based diagnostic tool. Purpose The pathogenesis of Bovine tropical theileriosis (BTT), caused by Theileria annulata in susceptible ruminants, is known to vary depending upon the nature of isolate and strain involved. Therefore, RFLP and sequencing-based characteri- zation of Indian isolates of T. annulata were attempted using TAMS gene. Method In the present study, TAMS 1 gene of T. annulata was amplifed from 25 naturally infected calves from the BTT endemic semi-arid zone of Northern India. The amplifed products were then digested with three restrictions enzymes viz., Taq I, Rsa I, and Alu I to fnd out the variations in pattern of restriction digests, so as to have an idea about the various isolates of T. annulata present in the studied area. Around 14 samples covering all the variants (from the PCR–RFLP patterns) were sequenced and submitted in NCBI (MH277607–MH277620). Result Coexistence of 4 variant genotypes was detected upon in-silico analysis of RFLP and sequence variations. Conclusion The nucleotide variations alongside the chromatogram analysis revealed point mutations leading to presence of noticeable genetic diversity among the isolates. Keywords Genetic variations · TAMS 1 gene · Theileria annulata · PCR–RFLP · Sequencing Introduction Bovine tropical theileriosis (BTT) is a serious lymphopro- liferative disease of ruminants [17] inficting signifcant del- eterious efects on health and production of ruminants across the globe [9]. Global annual loss due to BTT is estimated to be around 800 million US$ [2]. The apicomplexan Theileria annulata, with vast antigenic diversity across the globe, is attributed to be the causative agent for BTT in the Indian subcontinent [17]. It is well known that the various isolates and strains of the protozoan result in variable levels of viru- lence in susceptible host population [3, 16]. Hence, it is jus- tifable to state that the pathogenesis of BTT is widely infu- enced by the strain/isolate of parasite involved. Reports of vaccine failure, arising due to use of local strain as schizont vaccine and subsequent challenge by heterologous stains, are well documented from Indian context [5, 13]. Also, strain variation is a constraining factor in the respective PCR-based detection of T. annulata present in diferent geographical domains across the globe due to failure of respective primers to detect amplifcation owing to change in gene [6]. Hence, knowledge of local strains of T. annulata, in the given area, is a basic prerequisite for developing either an efective mass vaccination strategy or a suitable molecular diagnostic tool. One of the most common ways for the detection of the strain variation in a particular parasite is the sequencing of the selective gene involved, although, it is not cost-efec- tive. Restriction digestion of PCR products using selective enzymes is an economic way of screening a large number of samples. In the present study, PCR–RFLP of the TAMS 1 gene was done using three restrictions enzymes, viz., Taq I, Rsa I, and Alu I, to screen various genotypes of T. annu- lata present in young calves reared under semi-arid BTT endemic belt of Northern India. Thereafter, the various iso- lates, found, were sequenced to justify the distinct RFLP patterns obtained in the study. * Vikrant Sudan viks.sudan@gmail.com 1 U P Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura 281001, India