Abstract
This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10−6, 10−6 and 10−4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.
Acknowledgement.
The authors are thankful to Director, ICAR-IVRI, Izatnagar for providing necessary facilities for completing this research work. Financial assistance received from the NICRA project is duly acknowledged.
References
Abdel Hamid, O.M., Radwan M.E.I., Ali A. 2014. Biochemical changes associated with babesiosis infested cattle. Journal of Applied Chemistry, 7, 87−9210.9790/5736-07618792Search in Google Scholar
Abdullahi S.M., Idris Y., Alhaji M.A., Kabir I., Abubakar S.M. 2014. Prevalence of haemoparasitic infections in dairy cattle (Friesian Breeds) at nagari integrated dairy farms, Gauta-Nike village, Keffi local government area, Nasarawa State, north central of Nigeria. Scientific Journal of Zoology, 3, 17−23. 10.14196/sjz.v3i2.1234Search in Google Scholar
Almeria S., Castella J., Ferrer D., Ortuno A., Estrada-Peña A., Gutierrez J.F. 2001. Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection. Veterinary Parasitology, 99, 249–259. 10.1016/S0304-4017(01)00464-2Search in Google Scholar
Altay K., Dumanli N., Holman P.J., Aktas M. 2005. Detection of Theileria ovis in naturally infected sheep by nested PCR. Veterinary Parasitology 127, 99–104. 10.1016/j.vetpar.2004.09.012Search in Google Scholar PubMed
Ananda K.J., D’Souza P.E., Puttalakshmamma G.C. 2009. Prevalence of haemoprotozoan diseases in crossbred cattle in Banglore north. Veterinary World, 2, 15–1610.5455/vetworld.2009.15-16Search in Google Scholar
Ananyutthawongese C.T., Saengsombut K., Sukhumsirichat K., Uthaisang W.W., Sarataphan N., Chansiri K. 1999. Detection of bovine hemoparasite infection using multiplex polymerase chain reaction. Science Asia, 25, 85–9010.2306/scienceasia1513-1874.1999.25.085Search in Google Scholar
Ashuma, Sharma, A., Kaur, P., Bal, M.S., Singla, L.D. 2014. Application of multiplex PCR for the simultaneous detection of natural infection of theileriosis, babesiosis, and trypanosomosis in cattle. Journal of Veterinary Parasitology, 28, 112–116Search in Google Scholar
Bhatnagar C.S., Bhardawaj B., Sharma D.K., Meena S.K. 2015. Incidence of haemoprotozoan diseases in cattle in Southern Rajasthan, India. International Journal of Current Microbiology and Applied Science, 4, 509–514Search in Google Scholar
Bilgic H.B., Karagenc T., Simuunza M., Shiels B., Tait A., Eren H., Weir W. 2013. Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Experimental Parasitology, 133, 222–229. 10.1016/j.exppara.2012.11.005Search in Google Scholar PubMed PubMed Central
Brown W.C., Norimine J., Goff W.L., Suarez C.E., Mc Elwain T.F. 2006. Prospects for recombinant vaccines against Babesia bovis and related parasites. Parasite Immunology, 28, 315– 327. 10.1111/j.1365-3024.2006.00849.xSearch in Google Scholar PubMed
Edwards M.C., Gibbs R.A. 1994. Multiplex PCR: advantages, development, and applications. Genome Research, 3, 65–S7510.1101/gr.3.4.S65Search in Google Scholar
Eriks I.S., Palmer G.H., McGuire T.C., Barbet A.F. 1989. Detection and quantitation of Anaplasma marginale in carrier cattle by using a nucleic acid probe. Journal of Clinical Microbiology, 27, 279–28410.1128/jcm.27.2.279-284.1989Search in Google Scholar PubMed PubMed Central
Figueroa J., Chievers L., Johnson G., Buening G. 1993. Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood. Veterinary Parasitology, 50, 69–8110.1016/0304-4017(93)90008-BSearch in Google Scholar
Figueroa, J.V., Chieves, L.P., Johnson, G.S. and Buening, G.M. 1992. Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification. Journal of Clinical Microbiology, 30, 2576–258210.1128/jcm.30.10.2576-2582.1992Search in Google Scholar
Gasser R.B. 2006. Molecular tools-Advances, opportunities and prospects. Veterinary Parasitology, 136, 69–89. 10.1016/j.vetpar.2005.12.002Search in Google Scholar
Hoghooghi-Rad N., Ghaemi P., Shayan P., Eckert B., 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 Applied Science Journal, 12, 317–323Search in Google Scholar
Kaur P., Sharma A., Singla L.D., Juyal P.D. 2012. Molecular detection of anaplasmosis and babesiosis by duplex PCR in cattle. Crop Improvement (Sp. Issue): 1395–1396Search in Google Scholar
Kocan K.M., Blouin E.F., Barbet A.F. 2000. Anaplasmosis control, past, present, and future. Annals of the New Yark Academy of Sciences, 916, 501–50910.1111/j.1749-6632.2000.tb05329.xSearch in Google Scholar
Kocan K.M., de la Fuente J., Blouin E.F., Coetzee J.F., Ewing S.A. 2010. The natural history of Anaplasma marginale. Veterinary Parasitology, 167, 95–107. 10.1016/j.vetpar.2009.09.012Search in Google Scholar
Kocan, K. M., de la Fuente, J., 2003. Co-feeding studies of ticks infected with Anaplasma marginale. Veterinary Parasitology, 112, 295–305. 10.1016/j.vetpar.2009.09.012Search in Google Scholar
Kohli S., Atheya U.K., 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, 168–171. 10.14202/vetworld.2014.168-171Search in Google Scholar
Kumar V., Kaur P., Pal H., Kumar P., Sharma H., Wadhawan V.M. 2015. Theileriosis in calves and its successful therapeutic management. Scholars Journal of Agriculture and Veterinary Sciences, 2, 180–181Search in Google Scholar
Lew A.E., Bock R.E., Minchin C.M., Masaka S. 2002. A msp1 alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates. Veterinary Microbiology, 86, 325–335. 10.1016/S0378-1135(02)00017-2Search in Google Scholar
Liu J., Guan G., Liu A., Yin H., Luo J. 2014. A PCR method targeting internal transcribed spacers: the simultaneous detection of Babesia bigemina and Babesia bovis in cattle. Acta Parasitologica, 59, 132–138. 10.2478/s11686-0140222-6Search in Google Scholar
Liu J., Youquan L., Aihong L., Guiquan G., Junren X., Hong Y., Jianxun L. 2015. Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle. Parasitology Research, 114, 2715– 272110.1007/s00436-015-4478-zSearch in Google Scholar PubMed
Markoulatos P., Siafakas N., Moncany M. 2002. Multiplex polymerase chain reaction: a practical approach. Journal of Clinical Laboratory Analysis, 16, 47–5110.1002/jcla.2058Search in Google Scholar
Meenakshisundaram A., Anna T., Malmarugan S. 2014. Concomitant Theileria annulata and Anaplasma marginale infections in cross bred dairy herd. Indian Journal of Veterinary and Animal Science Research, 43, 422–425Search in Google Scholar
Naik B.S., Maiti S.K., Raghuvanshi P.D.S., 2016. Prevalence of tropical theileriosis in cattle in Chhattisgarh state. Journal of Animal Research, 6, 1043–104510.5958/2277-940X.2016.00151.0Search in Google Scholar
Njiiri N.E., Bronsvoort B.M., Collins N.E., Steyn H.C., Troskie M., Vorster I., et al. 2015. The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya. Veterinary Parasitology, 210, 69–76. 10.1016/j.vetpar.2015.02.020Search in Google Scholar
Ochirkhuu N., Konnai S., Mingala C.N., Okagawa T., Villanueva M., Pilapil F.M.I.R., et al. 2015. Molecular epidemiological survey and genetic analysis of vector-borne infections of cattle in Luzon Island, the Philippines. Veterinary Parasitology, 212, 161–167. 10.1016/j.vetpar.2015.05.019Search in Google Scholar
Papadopoulos B., Brossard M., Perié N.M. 1996. Piroplasms of domestic animals in the Macedonia region of Greece. 3 Piroplasms of small ruminants. Veterinary Parasitology, 63, 67–74.10.1016/0304-4017(95)00846-2Search in Google Scholar
Rahman A.S.M.S., Sumon S.M.M.R., Khan M., Islam M.T. 2015. Current status of sub-clinical form of babesiosis and anaplasmosis in cattle at Rangpur district in Bangladesh. Progressive Agriculture, 26, 51–59. 10.3329/pa.v26i1.24516Search in Google Scholar
Reetha T.L., Thomas K.S., Babu M. 2012. Occurrence of haemoprotozoan infection in bovines. International Journal of Applied Bioresearch, 13, 1–2Search in Google Scholar
Rizk M.A., Salama A., El-Sayed S.A., Elsify A., El-Ashkar M., Ibrahim H., et al. 2017. Animal level risk factor associated with Babesia and Theileria infections in cattle in Egypt. Acta Parasitologica, 62, 796–804. 10.1515/ap-20170096Search in Google Scholar
Salih D.A., El Hussein A.M., Singla L.D. 2015. Diagnostic approaches for tick-borne haemoparasitic diseases in livestock. Journal of Veterinary Medicine and Animal Health, 7, 45–5610.5897/JVMAH2014.0345Search in Google Scholar
Shahnawaz S., Ali M., Aslam M.A., Fatima R., Chaudhry Z.I., Hassan M.U., et al. 2011. Study on the prevalence of a tick transmitted pathogen, Theileria annulata, and hematological profile in cattle from Southern Punjab (Pakistan). Parasitology Research, 109, 1155–6010.1007/s00436-011-2360-1Search in Google Scholar PubMed
Sharma A., Singla L.D., Tuli A., Kaur P., Batth B.K., Javed M., Juyal P.D. 2013. Molecular prevalence of Babesia bigemina and Trypanosoma evansi in dairy animals from Punjab, India by duplex PCR: A step forward to detection and management of concurrent latent infections. Biomed Research International vol. 2013, Article ID 893862, 8 pages, 2013. 10.1155/2013/893862Search in Google Scholar PubMed PubMed Central
Singh H., Jyoti, Haque M., Singh N.K., Rath S.S. 2012. Molecular detection of Anaplasma marginale infection in carrier cattle. Ticks and Tick-borne Diseases, 3, 55–5810.1016/j.ttbdis.2011.10.002Search in Google Scholar PubMed
Tuli A., Singla L.D., Sharma A., Bal M.S., Filia G., Kaur P. 2015. Molecular epidemiology, risk factors and hematochemical alterations induced by Theileria annulata in bovines of Punjab (India). Acta Parasitologica, 60, 378–390. 10.1515/ap2015-0053Search in Google Scholar
Velusamy R., Rani N., Ponnudurai G., Harikrishnan T.J., Anna T., Arunachalam K., et al. 2014. Influence of season, age and breed on prevalence of haemoprotozoan diseases in cattle of Tamil Nadu, India. Veterinary World, 7, 574–57810.14202/vetworld.2014.574-578Search in Google Scholar
Zhang X., Liu Z., Yang J., Chen Z., Guan G., Ren Q., et al.2014. Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants. Parasitology Research, 113, 527–53110.1007/s00436-013-3684-9Search in Google Scholar PubMed
Zhou M., Cao S., Sevinc F., Sevinc M., Ceylan O., Moumouni P.F.A., et al. 2016. Molecular detection and genetic identification of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in Turkey. Ticks and Tick-borne Diseases, 7, 126–134. 10.1016/j.ttbdis.2015.09.008Search in Google Scholar PubMed
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