Coexistence of Multiple Theileria annulata Genotypes Circulating in Neonatal Calves in Semi-arid India



Knowledge of local isolates and strains is a prerequisite for the development of either effective mass vaccination strategy or a suitable molecular marker-based diagnostic tool.


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 characterization of Indian isolates of T. annulata were attempted using TAMS gene.


In the present study, TAMS 1 gene of T. annulata was amplified from 25 naturally infected calves from the BTT endemic semi-arid zone of Northern India. The amplified products were then digested with three restrictions enzymes viz., Taq I, Rsa I, and Alu I to find 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).


Coexistence of 4 variant genotypes was detected upon in-silico analysis of RFLP and sequence variations.


The nucleotide variations alongside the chromatogram analysis revealed point mutations leading to presence of noticeable genetic diversity among the isolates.

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  1. 1.

    Bose J, Kloesenor MH, Schulte RD (2016) Multiple-genotype infections and their complex effect on virulence. Zoology 119:339–349

    Article  Google Scholar 

  2. 2.

    Brown CGD (1997) Dynamics and impact of tick-borne diseases of cattle. Tropical Anim Health Prod 29(4):1S–3S (10 ref.)

    CAS  Article  Google Scholar 

  3. 3.

    Darghouth MA, Boulter NR, Gharbi M, Sassi L, Tait A, Hall R (2006) Vaccination of claves with an attenuated cell line of Theileria annulata and the sporozoite antigen SPAG-1 produces a synergistic effect. Vet Parasitol 142(1–2):54–62

    CAS  Article  Google Scholar 

  4. 4.

    Ghashgai O, Yakhchali M, Sohrabi S (2015) PCR-RFLP for detecting of Theileria annulata infection in cattle and Hyalomma species in Kermanshah Province Iran. Arch Razi Inst 70:7–12

    Google Scholar 

  5. 5.

    Gill BS, Bansal GC, Bhattacharyullu Y, Kaur D, Singh A (1980) Immunological relationship between strains of Theileria annulata (Dschunkowsky and Luhs 1904). Res Vet Sci 29:93–97

    CAS  Article  Google Scholar 

  6. 6.

    Gubbels MJ, Katzer F, Hide G, Jongejan F, Shiels BR (2000) Generation of a mosaic pattern of diversity in the major merozoite-piroplasm surface antigen of Theileria annulata. Mol Biochem Parasitol 110(1):23–32

    CAS  Article  Google Scholar 

  7. 7.

    Gupta KK, Srivastava M, Sudan V, Singh S, Choudhury S, Shanker D (2018) Variation in cardiac markers and electrocardiographic alterations in young calves naturally infected with bovine tropical theileriosis. Trop Anim Health Prod 50(6):1227–1230.

    Article  PubMed  Google Scholar 

  8. 8.

    Manuja A, Malhotra DV, Sikka VK, Sangwan AK, Sharma R, Kumar B, Mehta BD, Gulati BR, Nichani AK (2006) Isolates of Theileria annulata collected from different parts of India show phenotypic and genetic diversity. Vet Parasitol 137:242–252

    CAS  Article  Google Scholar 

  9. 9.

    Mehlhorn H, Schein E, Ahmed JS (1994) Theileria. In: Kreier JP (ed) Parasitic protozoa, vol 7. Academic Press, pp 217–304

  10. 10.

    Paliwal S, Sanker D, Sudan V, Kumar S, Srivastava M, Gupta KK (2019) Comparison of different PCR protocols and respective primer sets from pool of TAMS 1 gene for diagnosis of calf theileriosis from semi arid India. Biologicals 57:50–54

    CAS  Article  Google Scholar 

  11. 11.

    Paliwal S, Sanker D, Sudan V, Srivastava M (2020) Monotypic PCR-RFLP pattern of circulating Theileria annulata isolates from North India based on HSP 70 gene. Res J Parasitol.

    Article  Google Scholar 

  12. 12.

    Patel G, Shanker D, Jaiswal AK, Sudan V, Verma SK (2013) Prevalence and seasonal variation in ixodid ticks on cattle of Mathura district, Uttar Pradesh. J Parasit Dis 37(2):173–176

    Article  Google Scholar 

  13. 13.

    Subramanian G, Ray D, Naithani RC (1986) In vitro culture and attenuation of macroshizonts of Theileria annulata (Dschunkowsky and Luhs 1904) and in vivo use as vaccine. Indian J Anim Sci 56:174–182

    Google Scholar 

  14. 14.

    Sudan V, Shanker D, Jaiswal AK, Singh A, Pandey V (2017) Standardization and validation of simple PCR, duplex PCR and RAPD in comparison to blood smear examination for diagnosing bovine tropical theileriosis. Biologicals 46:88–91

    CAS  Article  Google Scholar 

  15. 15.

    Tavassoli M, Tabatabaei M, Nejad BE, Tabatabaei MH, Najafabadi A, Pourseyed SH (2011) Detection of Theileria annulata by the PCR-RFLP in ticks (Acari, Ixodidae) collected from cattle in West and North-West Iran. Acta Parasitol 56(1):8–13

    Article  Google Scholar 

  16. 16.

    Taylor LH, Katzer F, Shiels BR, Welburn SC (2003) Genetic and phenotypic analysis of Tunisian Theileria annulata clones. Parasitology 26:241–252

    Article  Google Scholar 

  17. 17.

    Taylor MA, Coop RL, Wall RL (2007) Veterinary parasitology, 3rd edn. Blackwell Publishing Ltd, UK, pp 109–113

    Google Scholar 

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The authors are highly thankful to the Director Research, Dean Biotechnology, Dean CoVSc, and Vice Chancellor, DUVASU, for the facilities provided. The authors also want to acknowledge the various funding agencies like Indian Council of Agricultural Research (ICAR) and Rashtriya Krishi Vikas Yojana (RKVY) for sanctioning various projects to the University for the Procurement of Instruments to carry out such work. The authors also acknowledge Incharge, Central Instrumentation Facility (CIF) for permitting the use of instruments required in the study.

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Correspondence to Vikrant Sudan.

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The blood was collected from the animals in compliance with the ethical standards and guidelines of the Institutional Animal Ethics Committee (IAEC) and permission was granted via. order no IAEC/17/23.

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Paliwal, S., Sudan, V., Shanker, D. et al. Coexistence of Multiple Theileria annulata Genotypes Circulating in Neonatal Calves in Semi-arid India. Acta Parasit. 65, 679–685 (2020).

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  • Genetic variations
  • TAMS 1 gene
  • Theileria annulata
  • Sequencing