3-Title: Molecular detection of Theileria annulata and Theileria orientalis in cattle and its prevalence in four agro-climatic zones of Bihar

3-Title: Molecular detection of Theileria annulata and Theileria orientalis in cattle and its prevalence in four agro-climatic zones of Bihar

Authors: Shimpi Kumari, Pallav Shekhar, Pankaj Kumar, Madhurendra Bachan, Ajit Kumar and Pankaj Kumar

Source: Ruminant Science (2022)-11(2):267-274.

How to cite this manuscript: Kumari Shimpi, Shekhar Pallav, Kumar Pankaj, Bachan Madhurendra, Kumar Ajit and Kumar Pankaj (2022). Molecular detection of Theileria annulata and Theileria orientalis in cattle and its prevalence in four agro-climatic zones of Bihar. Ruminant Science 11(2):267-274.


The prevalence of Theileria species in cattle from four different agroclimatic zones of Bihar, India was studied. The randomised stratified sampling was performed for these zones. Based on prefixed criteria, cattle suspected positive with clinical signs and symptoms and the presence of tick infestation or inapparent healthy animals with heavy tick infestation without clinical signs and symptoms were selected for blood sampling. A total of 400 blood samples were collected, fulfilling the above-mentioned criteria. Only those samples were selected for molecular level diagnosis which were found positive or suspected with microscopic examination. Both genus-level and species-level detection were performed. Under microscopy, out of 400 samples, 132 (33%) samples were found positive for Theileria spp. and 57 samples were doubtful. All these samples found positive or remain in doubt (189) were undertaken for PCR assay. For prevalence study on genus ground, amplification of 18s Ribosomal RNA was made by using primer UPTh1&2. A total number of 165 (41.25%) samples out of 189 samples were found positive for Theileria. Out of these positive samples, total number of 101 (25.25%) samples were found positive for Theileria annulata by using primer Tams1 and Tspms1 and 64 (16%) samples were positive for Theileria orientalis by using primer MPSP1&2. The correlation between the host and several environmental factors like various agro-climatic zones, season, age, sex, and the breed was analyzed. The prevalence of theileriosis was more in higher aged groups and crossbred cows. Females were more susceptible in the summer season. The sensitivity and specificity of both microscopical examination and molecular diagnosis were also estimated keeping microscopical examination as the gold standard. The PCR-based diagnosis had 91.11% sensitivity (95% CI-78.78-97.52%) and 100% specificity (95% CI- 92.18-100%) with a positive predictive value of 100% and negative predictive value of 91.84% (95% CI- 81.58-96.68%). Inter-rater agreements for these two tests were also evaluated. Here PCR showed perfect agreement with the microscopic examination (Kappa=0.911).


Abaker IA, Salih DA, EI-Haj LM, Ahmed RE, Osman MM and Ali AM (2017). Prevalence of Theileria annulata in dairy cattle in Nyala, South Darfur State, Sudan. Veterinary World 10:1475-1480.

Acharya AP, Panda SK, Das RK, Panda MR, Das S and Gupta AR (2016). Prevalence of Bovine tropical theileriosis in cattle reared in and around Bhubaneswar. Indian Journal of Veterinary Medicine 36(1):20-22.

Aparna M, Ravindran R, Vimalkumar MB, Lakshmanan B, Rameshkumar P, Ajith Kumar KG, Promod K, Ajithkumar S, Ravishankar C, Devada K, Subramanian H, George AJ and Ghosh S (2011). Molecular characterization of Theileria orientalis causing fatal infection in crossbred adult bovine in South Asia. Parasitol International 60:524-529.

Azeemi TA, Zameer M and Rahman  G (2013). Incidences of theileria and babesia in Nangrahar province of Afghanistan. Ruminant Science 2(2):145-147.

Benjamin M (1978). Outline of Veterinary Clinical Pathology. 3rd Edn, Ames, Iowa USA: The Iowa State University, Press, pp 51-53.

Branco S, Orvalho J, Leitão A, Pereira I, Malta M, Mariano I, Carvalho T, Baptista R, Shiels BR and Peleteiro MC (2010). Fatal cases of Theileria annulata infection in calves in Portugal associated with neoplastic-like lymphoid cell proliferation. Journal of Veterinary Science 11:27-34.

Campbell JDM and Spooner RL (1999). Macrophages behaving badly: Infected cells and subversion of immune responses to Theileria annulata. Parasitology Today 15(1):10-15.

Chaudhuri SS, Bisla RS, Bhanot V and Singh H (2013). Prevalence of hemprotozoan infections in pyretic dairy animals of eastern Haryana. Indian Journal of Animal Research 47(4):344-347.

Criado-Fornelio A (2007). A review of nucleic acid-based diagnostic tests for Babesia and Theileria, with emphasis on bovine piroplasms. Parassitologia 49:39-44.

Dobbelaere D and Heussler V (1999). Transformation of leukocytes by Theileria parva and T annulata. Annual Review of Microbiology 53:1-42.

Dschunkowsky E and Luhs J (1904). Die piroplasmosen der Rinder Zentralblatt fur Bakteriologie, Parasitenkunde. Infektionskrankheit und Hygiene 35:486-492.

García-Sanmartín J, Nagore D, García-Pérez AL, Juste RA and Hurtado A (2006). Molecular diagnosis of Theileria and Babesia species infecting cattle in northern Spain using reverse line blot macroarrays. BMC Veterinary Research 2:16.

Government of Bihar (2009). Classification of Agro Climatic Zones, March, wwwkrishibihnicin

Gubbels JM, de-Vos AP, van-der Weide M, Viseras J, Schouls M, DeVries E and Jongejan F (1999). Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridigestion. Journal of Clinical Microbiology 37:1782-1789.

Gurjar VS, Khatoon Sanweer and Singh Rajendra (2020). Studies on prevalence of haemoprotozoa in cattle of southern Rajasthan. Ruminant Science 9(2):267-274.

Hitaishi VN, Lakshmanan Bindu, Jain Jose K, Emmanuel Seph Ross and Shameem H (2021). Molecular detection of Theileria spp. in ticks on goats in Kerala, South India. Ruminant Science 10(2):281-284.

Kakati P, Sarmah PC, Ray D, Bhattacharjee K, Sharma RK, Barkalita M, Sarma DK, Baishya BC, Borah P and Stanley B (2015). Emergence of oriental theileriosis in cattle and its transmission through Rhipicephalus (Boophilus) microplus in Assam, India. Veterinary World 8(9):1099-1104.

Kala S, Deo BG and Kumari N (2018). Epidemiological Aspects of Theileriosis in Cattle in and around Patna, Bihar, India. International Journal of Current Microbiology and Applied Sciences 7(3):1183-1191.

Kamau J, de-Vos AJ, Playford M, Salim B, Kinyajui P and Sugimozo C (2011). Emergence of new type of Theileria orientalis in Australian cattle and possible cause of theileriosis outbreaks. Parasite Vectors 4:22.

Kirvar E, Ilhan T, Katzer F, Wilkie G, Hooshmand-Rad P, Zweygarth E, Gestenberg C, Phipps P and Brown CGD (2000).  Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences. Parasitology 120:245-254.

Kohli S, Atheya UK and Thapliyal A (2014). Prevalence of theileriosis in cross-bred cattle: its detection through blood smear examination and polymerase chain reaction in Dehradun district, Uttarakhand, India. Veterinary World 7(3):168-171.

Langer A, Rani S, Sharma S, Bhojani R and Soren S  (2014). Hemato-biochemical alteration in cows naturally infected with theileria. Ruminant Science 3(1):59-62.

Lawrence JA (1979). The differential diagnosis of the bovine Theileriosis of Southern Africa. Journal of the South African Veterinary Association 50:311-313

Levine ND (1985). Genus Theileria Protozoan parasites of domestic animals and man Minneapolis, Minnesota, USA: Burgess Publishing Company, pp 336-346.

Maharana BR, Tewari AK, Saravanan BC and Sudhakar NR (2016). Important hemoprotozoan diseases of livestock: Challenges in current diagnostics and therapeutics. Veterinary World 9(5):487-495.

Mc Fadden AM, Rawdon TG, Meyer J, Makin J, Clough RR, Tham K, Mullner P and Geysen D (2011). An outbreak of haemolytic anaemia associated with infection of Theileria orientalis in naïve cattle. New Zealand Veterinary Journal 59(2):79-85.

Nair AS, Ravindran R, Lakshmanan B, Kumar SS, Tresamol PV, Saseendranath MR, Senthilvel K, Rao JR, Tewari AK and Ghosh S (2011). Haemoprotozoa of cattle in Northern Kerala, India. Tropical Biomedicine 28:68-75.

Osman SA and Al-Gaabary MH (2007). Clinical, haematological and therapeutic studies on tropical theileriosis in water buffaloes (Bubalus bubalis) in Egypt. Veterinary Parasitology 146(3-4):337-340.

Ota N, Mizuno D, Kuboki N, Igarashi I, Nakamura Y, Yamashina H, Hanzaike T, Fujii K, Onoe S, Hata H, Kondo S, Matsui S, Koga M, Matsumoto K, Inokuma H and Yokoyama N (2009). Epidemiological survey of Theileria orientalis infection in grazing cattle in the eastern part of Hokkaido, Japan. Journal of Veterinary Medical Science 71:937-944.

Panda SK, Sahu B, Rajeev Ranjan, Acharya AP and Rath SK (2011). Prevalence and clinicopathological study of theileriosis in bovine in coastal areas of Orissa. Indian Journal Veterinary Pathology 35(2):128-132.

Patel Bhavika, Patel JS, Kumar Binod, Barad DB and Joseph JP (2018). Clinicotherapeutic management of mixed infection by Actinobacillus lignieressi and Theileria spp. in a bullock. Ruminant Science 7(2): 335-336.

Patel Bhavika R, Kumar Binod and Vagh AA (2019). Clinicotherapeutic management of theileriosis in Gir cows. Ruminant Science 8(2):269-270.

Prajapati AS, Suthar AN, Jadhav KM, Das B and Pathan A (2018). Theileriosis in goat-A case report. Ruminant Science 7(2):325-326.

Raguvaran R, Kundave VR, Aishwarya L and Mondal DB (2016). Therapeutic management of tropical theileriosis in cross bred calf. Ruminant Science 5(1):99-100.

Robinson PM (1982). Theileria annulata and its transmission – A review. Tropical Animal Health and Production 14:3-12.

Saravanan S, Mohanapriya T and Ramprabhu R (2021). Ocular and nervous form of theileriosis in a suckling jersey calf- A case report. Ruminant Science 10(2):443-446.

Shastri UV, Pathak SV, Jadhav KV and Deshpande PD (1988). Occurrence of Theileria orientalis in bovines from Maharashtra state and its transmission by Haemaphysalis bispinosa ticks. Indian Journal of Parasitology 12:173-177.

Silva MG, Marques PX and Oliva A (2010). Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method. Veterinary Parasitology 174:199-205.

Singh A, Singh J, Grewal AS and Brar RS (2001). Studies on blood parameters of cross-bred calves with experimental Theileria annulata infections. Veterinary Research Communications 25:289-300.

Singh J, Acharya AP, Panda SK, Patra BK and Behra K (2017). Theilerial infection in young bovine calves in Odisha India. Journal of Entomology and Zoology Studies 5(5):1201-1204.

Soulsby EJL (1982). Helminths, Arthropods and Protozoa of Domesticated Animals. 7th Edn, Bailliere Tindall.

Srividya AR, Taakore H, Tyagi D, Majumdar P, James Vishnu Varthan VJ, Patel J H and Lad Krunal V (2011). Isolation, quantification and purity estimation of DNA from various sources. Bulletin of Pharmaceutical Research 1(3):4-9.

Theiler A (1904). Rhodesian tick fever. Transvaal Agr, 2:421-438.

Zhang J, Kelly P, Li J, Xu C and Wang C (2015). Molecular detection of Theileria spp in livestock on five Caribbean islands. BioMed Research International 624728.