Molecular epidemiology, associated risk factors, and phylogeny of Theileria annulata infecting buffaloes and cattle from different agro-climatic regions of Punjab, Pakistan

Document Type : Full paper (Original article)


1 Department of Clinical Sciences, College of Veterinary and Animal Sciences, Jhang, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan

2 M. Phil. Student in Clinical Medicine, Department of Clinical Sciences, College of Veterinary and Animal Sciences, Jhang, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan

3 Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad 38000, Pakistan

4 Department of Clinical Sciences, College of Veterinary and Animal Sciences, Jhang, University of Veterinary and Animal Sciences, Lahore, Pakistan; Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan

5 Ph.D. Student in Microbiology, Department of Pathobiology, College of Veterinary and Animal Sciences, Jhang, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan

6 5Higher Institute of Biotechnology of Sidi Thabet, University of Manouba, Manouba 2010, Tunisia, and Laboratory of Microbiology at the National School of Veterinary Medicine of Sidi Thabet, University of Manouba, Manouba 2010, Tunisia


Background: Tropical theileriosis is the most prevalent hemoprotozoan disease in Pakistan. Aims: The study aimed to investigate the epidemiology and evolutionary relationship of Theileria annulata in bovines in diverse agro-climatic regions of Punjab, Pakistan. Methods: 800 blood specimens were collected from asymptomatic cattle (n=480) and buffaloes (n=320) using a multistage sampling method from Sargodha (n=400) and Multan (n=400) districts. The samples were assessed for blood smear microscopy and cytochrome b gene based PCR. Twenty samples were collected from each union council of each district. Results: The overall prevalence of T. annulata infection in bovines was 9% and 17.13% as determined by blood smear analysis and PCR, respectively. The disease positivity in cattle and buffaloes was respectively 10.21% and 20.42% by blood smear screening and 7.19%, 12.19% by PCR. The overall PCR based prevalence in the Sargodha and Multan districts was 19% and 15.25%, respectively. Absence of rural poultry, tick infestation, and a history of tick-borne diseases had significant effect in cattle. Tick infestation and age were the main statistically significant disease determinants in buffaloes. The evolutionary analysis of the cytochrome b gene showed that the Pakistani isolate infecting buffalo was related to those from Iran, India, Egypt, and Sudan. The isolate from cattle was genetically close to those from Pakistan, India, Iran, Iraq, and Turkey. Conclusion: It can be concluded that biotic and abiotic factors contribute to disease occurrence. The current study will help to devise control strategies to prevent substantial economic losses.


Main Subjects

Abaker, IA; Salih, DA; El Haj, LM; Ahmed, RE; Osman, MM and Ali, AM (2017). Prevalence of Theileria annulata in dairy cattle in Nyala, South Darfur State, Sudan. Vet. World. 10: 1475-1480.
Al-Hamidhi, S; Parveen, A; Iqbal, F; Asif, M; Akhtar, N; Elshafie, EI; Beja-Pereira, A and Babiker, HA (2022). Diversity and genetic structure of Theileria annulata in Pakistan and other endemic sites. Pathogens. 11: 334.
Ananda, KJ; D’Souza, PE and Puttalakshmamma, GC (2009). Prevalence of haemoprotozoan diseases in crossbred cattle in Banglore North. Vet. World. 2: 15-16.
Asif, M; Said, MB; Parveen, A; Ejaz, A; Ikram, M; Awais, MM; Ozubek, S; Aktas, M; Baber, M and Iqbal, F (2022). Seasonal survey, risk factor’s analysis and genotyping of Theileria annulata infecting cattle in Punjab province, Pakistan. Acta Trop., 234: 106587.
Aslam, F; Rehman, MU; Saleem, G; Ashraf, K; Hafeez, MA and Saqib, M (2023). Identification and molecular characterization of Theileria annulata with associated risk factors in naturally infected camels from selected districts in Punjab, Pakistan. Pak. Vet. J., 43: 79-84.
Atif, FA; Abbas, RZ; Mehnaz, S; Qamar, MF; Hussain, K; Nazir, MU; Zaman, MA; Khan, AU and Said, MB (2022). First report on molecular surveillance based on duplex detection of Anaplasma marginale and Theileria annulata in dairy cattle from Punjab, Pakistan. Trop. Anim. Health Prod., 54: 1-9.
Atif, FA; Khan, MS; Roheen, T; Muhammad, F; Younus, M; Avais, M and Ullah, S (2013). Seroprevalence of Anaplasma marginale infection among cattle from three districts of the Northern Punjab, Pakistan. J. Anim. Plant Sci., 23: 995-998.
Atif, FA; Muhammad, SK; Hafiz, JI; Ghulam, MA; Ejaz, A and Sami, U (2012). Prevalence of Anaplasma marginale, Babesia bigemina and Theileria annulata infections among cattle in Sargodha District, Pakistan. Afr. J. Agric. Res., 7: 3302-3307.
Atif, FA; Zaman, MA; Hussain, K; Qamar, MF; Sajid, MS; Iqbal, U and Mehnaz, S (2021). First molecular surveillance and estimation of risk factors of Anaplasma marginale infection among indigenous, crossbred and exotic cattle. J. Anim. Plant Sci., 31: 913-917.
Basit, MA; Ijaz, M; Abbas, RZ; Khan, JA and Ashraf, K (2022). First molecular evidence of Ehrlichia infection: An emerging pathogen of small ruminants in Pakistan. Pak. Vet. J., 42: 208-214.
Bilgic, HB; Karagenc, T; Shiels, B; Tait, A; Eren, H and Weir, W (2010). Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals. Vet. Parasitol., 174: 341-347.
Bilgic, HB; Karagenç, T; Simuunza, M; Shiels, B; Tait, A; Eren, H and Weir, W (2013). Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Exp. Parasitol., 133: 222-229.
Caeiro, V (1999). General review of tick species present in Portugal. Parasitologia. 41: 11-15.
Ceylan, O; Uslu, A; Ceylan, C and Sevinc, F (2021). Predominancy of Rhipicephalus turanicus in tick-infested sheep from Turkey: a large-scale survey. Pak. Vet. J., 41: 429-433.
Chatanga, E; Mosssad, E; Abubaker, HA; Alnour, SA; Katakura, K; Nakao, R and Salim, B (2019). Evidence of multiple point mutations in Theileria annulata cytochrome b gene incriminated in buparvaquone treatment failure. Acta Tropica. 191: 128-132.
Farooqi, SH; Ijaz, M; Saleem, MH; Rashid, MI; Ahmad, SS; Islam, S; Aqib, AI; Khan, A; Hussain, K and Khan, NU (2017). Prevalence and molecular diagnosis of Theileria annulata in bovine from three distincts zones of Khyber Pakhtunkhwa province, Pakistan. J. Anim. Plant Sci., 27: 1836-1841.
Ferrolho, J; Antunes, S; Santos, AS; Velez, R; Padre, L; Cabezas-Cruz, A; Santos-Silva, MM and Domingos, A (2016). Detection and phylogenetic characterization of Theileria spp. and Anaplasma marginale in Rhipicephalus bursa in Portugal. Ticks Tick Borne Dis., 7: 443-448.
Ghafar, A; Abbas, T; Rehman, A; Sandhu, ZUD; Cabezas-Cruz, A and Jabbar, A (2020). Systematic review of ticks and tick-borne pathogens of small ruminants in Pakistan. Pathogens. 9: 937.
Ghafar, MW and Amer, SA (2019). A preliminary molecular survey of Babesia divergens and first evidence of Theileria annulata in cattle from Saudi Arabia. Vet. World. 12: 266-270.
Gomes, J; Salgueiro, P; Inácio, J; Amaro, A; Pinto, J; Tait, A; Shiels, B; da Fonseca, IP; Santos-Gomes, G and Weir, W (2016). Population diversity of Theileria annulata in Portugal. Infect. Genet. Evol., 42: 14-19.
Haji, I; Simuunza, M; Kerario, II; Jiang, N and Chen, Q (2022). Epidemiology of tick-borne pathogens of cattle and tick control practices among mixed farming and pastoral communities in Gairo and Monduli districts, Tanzania. Vet. Parasitol. Reg. Stud., 32: 100738.
Hasan, M; Roohi, N; Rashid, MI; Ali, S and Ul-Rehman, Z (2022). Occurrence of ticks and tick-borne mixed parasitic microbiota in cross-bred cattle in District Lahore, Pakistan. Braz. J. Biol., 82: e266721.
Hassan, MA; Liu, J; Rashid, M; Iqbal, N; Guan, G; Yin, H and Luo, J (2018). Molecular survey of piroplasm species from selected areas of China and Pakistan. Parasit. Vectors. 11: 1-7.
Hoghooghi-Rad, N; Ghaemi, P; Shayan, P; Eckert, B and Sadr-Shirazi, N (2011). Detection of native carrier cattle infected with Theileria annulata by semi-nested PCR and smear method in Golestan Province of Iran. World Appl. Sci. J., 12: 317-323.
Jabbar, A; Abbas, T; Sandhu, ZUD; Saddiqi, HA; Qamar, MF and Gasser, RB (2015). Tick-borne diseases of bovines in Pakistan: major scope for future research and improved control. Parasit. Vectors. 8: 1-13.
Karim, S; Budachetri, K; Mukherjee, N; Williams, J; Kausar, A; Hassan, MJ; Adamson, S; Dowd, SE; Apanskevich, D; Arijo, A and Sindhu, ZU (2017). A study of ticks and tick-borne livestock pathogens in Pakistan. PLoS Negl. Trop. Dis., 11: e0005681.
Khan, A; Ashfaq, K; ud Din, I; ul Haq, R; Jamil, M; Ullah, B; Ullah, S; ur Rehman, H and Ullah, F (2017). Bovine theileriosis: prevalence, estimation of hematological profile and chemotherapy in cattle in Dera Ismail Khan, Khyber Pakhtunkhwa Province, Pakistan. ASRJETS., 32: 8-17.
Khattak, RM; Rabib, M; Khan, Z; Ishaq, M; Hameed, H; Taqddus, A; Faryal, M; Durranis, S; Gillani, QUA; Allahyar, R and Shaikh, RS (2012). A comparison of two different techniques for the detection of blood parasite, Theileria annulata, in cattle from two districts in Khyber Pukhtoon Khwa Province (Pakistan). Parasite. 19: 91-95.
Kose, O; Bilgic, HB; Bakirci, S; Karagenc, T; Adanir, R; Yukari, BA and Eren, H (2022). Prevalence of Theileria/Babesia species in ruminants in Burdur province of Turkey. Acta Parasitol., 67: 723-731.
Mohsin, M; Hameed, K; Kamal, M; Ali, A; Rafiq, N; Usman, T; Khan, W; Abbasi, AA; Khan, RU and Yousafzai, GJ (2022). Prevalence and risk factors assessment of theileriosis in livestock of Malakand Division, Pakistan. J. Saudi Soc. Agric., 21: 242-247.
Okafor, CC; Collins, SL; Daniel, JA; Harvey, B; Coetzee, JF and Whitlock, BK (2018). Factors associated with seroprevalence of bovine anaplasmosis in Texas. Vet. Parasitol. Reg. Stud., 14: 32-40.
Oryan, A; Namazi, F; Sharifiyazdi, H; Razavi, M and Shahriari, R (2013). Clinicopathological findings of a natural outbreak of Theileria annulata in cattle: an emerging disease in southern Iran. Parasitol. Res., 112: 123-127.
Pakistan Livestock Census (2006). Agricultural Census Organization, Statistics division, Government of Pakistan. Available at: agriculture/publications/pakistan-livestock-cencus2006/All %20Pakistan%20Report%20%28Introduction%29.pdf (downloaded on June 18, 2023).
Parveen, A; Alkhaibari, AM; Asif, M; Almohammed, HI; Naqvi, Z; Khan, A; Aktas, M; Ozubek, S; Farooq, M and Iqbal, F (2021a). Molecular epidemiology of Theileria annulata in cattle from two districts in Punjab (Pakistan). Animals. 11: 3443.
Parveen, A; Ashraf, S; Aktas, M; Ozubek, S and Iqbal, F (2021b). Molecular epidemiology of Theileria annulata infection of cattle in Layyah District, Pakistan. Exp. Appl. Acarol., 83: 461-473.
Patil, N and Satbige, AS (2019). Molecular detection, haematological and therapeutic studies on theileriosis in buffaloes. Buffalo Bull., 38: 141-146.
Rehman, A; Conraths, FJ; Sauter-Louis, C; Krücken, J and Nijhof, AM (2019). Epidemiology of tick-borne pathogens in the semi-arid and the arid agro-ecological zones of Punjab province, Pakistan. Transbound. Emerg. Dis., 66: 526-536.
Rehman, A; Nijhof, AM; Sauter-Louis, C; Schauer, B; Staubach, C and Conraths, FJ (2017). Distribution of ticks infesting ruminants and risk factors associated with high tick prevalence in livestock farms in the semi-arid and arid agro-ecological zones of Pakistan. Parasit. Vectors. 10: 1-15.
Rodríguez-Hidalgo, R; Pérez-Otáñez, X; Garcés-Carrera, S; Vanwambeke, SO; Madder, M and Benítez-Ortiz, W (2017). The current status of resistance to alpha-cypermethrin, ivermectin, and amitraz of the cattle tick (Rhipicephalus microplus) in Ecuador. PLoS One. 12: e0174652.
Saitou, N and Nei, M (1987). The Neighbor-Joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4: 406-425.
Sajid, MS; Iqbal, Z; Shamim, A; Siddique, RM; Hassan, MJU and Rizwan, HM (2017). Distribution and abundance of ticks infesting livestock population along Karakorum highway from Mansehra to Gilgit, Pakistan. J. Hellenic Vet. Med. Soc., 68: 51-58.
Sallemi, S; Rjeibi, MR; Rouatbi, M; Amairia, S; Ben Said, M; Khamassi Khbou, M and Gharbi, M (2018). Molecular prevalence and phylogenetic analysis of Theileria annulata and Trypanosoma evansi in cattle in Northern Tunisia. Vet. Med. Sci., 4: 17-25.
Selim, AM; Das, M; Senapati, SK; Jena, GR; Mishra, C; Mohanty, B; Panda, SK and Patra, RC (2020). Molecular epidemiology, risk factors and hematological evaluation of asymptomatic Theileria annulata infected cattle in Odisha, India. Iran. J. Vet. Res., 21: 250-256.
Simuunza, M; Weir, W; Courcier, E; Tait, A and Shiels, B (2011). Epidemiological analysis of tick-borne diseases in Zambia. Vet. Parasitol., 175: 331-342.
Shahedi, A; Habibi, G; Fathi, S; Abdigoudarzi, M; Afshari, A; Bozorgi, S and Chegini, MR (2022). Molecular identification of Theileria spp. in ruminants and ticks from southern littoral of Caspian Sea, Iran. Trop. Anim. Health Prod., 54: 1-9.
Shahnawaz, S; Ali, M; Aslam, MA; Fatima, R; Chaudhry, ZI; Hassan, MU; Ali, M and Iqbal, F (2011). A study on the prevalence of a tick-transmitted pathogen, Theileria annulata, and hematological profile of cattle from Southern Punjab (Pakistan). Parasitol. Res., 109: 1155-1160.
Sharifiyazdi, H; Namazi, F; Oryan, A; Shahriari, R and Razavi, M (2012). Point mutations in the Theileria annulata cytochrome b gene is associated with buparvaquone treatment failure. Vet. Parasitol., 187: 431-435.
Silatsa, BA; Simo, G; Githaka, N; Mwaura, S; Kamga, RM; Oumarou, F; Keambou, C; Bishop, RP; Djikeng, A; Kuiate, JR and Njiokou, F (2019). A comprehensive survey of the prevalence and spatial distribution of ticks infesting cattle in different agro-ecological zones of Cameroon. Parasit. Vectors. 12: 1-14.
Soomro, MH; Soomro, SP; Bhutto, B; Shah, ZA; Yaqoob, M and Arijo, A (2014). Prevalence of ticks in buffaloes in the upper Sindh Pakistan. Buffalo Bull., 33: 323-327.
Tajeri, S; Haidar, M; Sakura, T and Langsley, G (2021). Interaction between transforming Theileria parasites and their host bovine leukocytes. Mol. Microbiol., 115: 860-869.
Tamura, K and Nei, M (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol., 10: 512-526.
Tumer, KC; Dincer, PFP; Babacan, S and Yerlikaya, Z (2023). Urinary neutrophil gelatinase-associated lipocalin, cystatin c and clusterin as biomarkers for acute kidney injury in cattle with tropical theileriosis. Pak. Vet. J., 43: 345-350.
Ullah, R; Shams, S; Khan, MA; Ayaz, S; Akbar, NU; Din, QU; Khan, A; Leon, R and Zeb, J (2021). Epidemiology and molecular characterization of Theileria annulata in cattle from central Khyber Pakhtunkhwa, Pakistan. PLoS One. 16: e0249417.
Yousef, SG; El Balkemy, FA and El Damaty, HM (2020). Mutations in Theileria annulata Cytochrome B gene associated with buparvaquone resistance in cattle, Egypt. Pak. Vet. J., 40: 261-263.
Zaman, MA; Rafique, A; Mehreen, U; Mehnaz, S; Atif, FA; Abbas, A and Omar, M (2022). Epidemiological investigation and development of loop mediated isothermal amplification for the diagnosis of ovine theileriosis. Pak. Vet. J., 42: 370-375.
Zeb, J; Shams, S; Din, IU; Ayaz, S; Khan, A; Nasreen, N; Khan, H; Khan, MA and Senbill, H (2020). Molecular epidemiology and associated risk factors of Anaplasma marginale and Theileria annulata in cattle from North-Western Pakistan. Vet. Parasitol., 279: 109044. https://doi. org/10.1016/j.vetpar.2020.109044.