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Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines

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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.

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References

  • Abdel Hamid, O.M., Radwan M.E.I., Ali A. 2014. Biochemical changes associated with babesiosis infested cattle. Journal of Applied Chemistry, 7, 87–92

    CAS  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. DOI: 10.14196/sjz.v3i2.1234

    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. DOI: 10.1016/ S0304-4017(01)00464-2

    Article  CAS  Google Scholar 

  • Altay K., Dumanli N., Holman P.J., Aktas M. 2005. Detection of Thei-leria ovis in naturally infected sheep by nested PCR. Veterinary Parasitology 127, 99–104. DOI: 10.1016/j.vetpar.2004.09.012

    Article  CAS  Google Scholar 

  • Ananda K.J., D’Souza P.E., Puttalakshmamma G.C. 2009. Prevalence of haemoprotozoan diseases in crossbred cattle in Banglore north. Veterinary World, 2, 15–16

    Article  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, 85–90

    Article  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, 112–116

    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, 509–514

    CAS  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. DOI: 10.1016/j.exppara.2012.11.005

    Article  CAS  Google Scholar 

  • 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. DOI: 10.1111/j.1365-3024.2006.00849.x

    Article  CAS  Google Scholar 

  • Edwards M.C., Gibbs R.A. 1994. Multiplex PCR: advantages, development, and applications. Genome Research, 3, 65–S75

    Article  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, 279–284

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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, 69–81

    Article  CAS  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, 2576–2582

    PubMed  PubMed Central  CAS  Google Scholar 

  • Gasser R.B. 2006. Molecular tools-Advances, opportunities and prospects. Veterinary Parasitology, 136, 69–89. DOI: 10.1016/j.vetpar.2005.12.002

    Article  CAS  Google Scholar 

  • Hoghooghi-Rad N., Ghaemi P., Shayan P., Eckert B., Sadr-Shirazi N. 2011. Detection of native carrier cattle infected with Thei-leria annulata by semi-nested PCR and smear method in Golestan Province of Iran. World Applied Science Journal, 12, 317–323

    CAS  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): 1395–1396

    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, 501–509

    Article  CAS  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. DOI: 10.1016/j.vetpar.2009.09.012

    Article  CAS  Google Scholar 

  • Kocan, K. M., de la Fuente, J., 2003. Co-feeding studies of ticks infected with Anaplasma marginale. Veterinary Parasitology, 112, 295–305. DOI: 10.1016/j.vetpar.2009.09.012

    Article  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. DOI: 10.14202/vetworld.2014.168-171

    Article  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, 180–181

    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. DOI: 10.1016/S0378-1135(02)00017-2

    Article  CAS  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. DOI: 10.2478/s11686-014-0222-6

    Article  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, 2715–2721

    Article  Google Scholar 

  • Markoulatos P., Siafakas N., Moncany M. 2002. Multiplex poly-merase chain reaction: a practical approach. Journal of Clinical Laboratory Analysis, 16, 47–51

    Article  CAS  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, 422–425

    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, 1043–1045

    Article  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. DOI: 10.1016/j.vetpar.2015.02.020

    Article  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. DOI: 10.1016/j.vetpar.2015.05.019

    Article  Google Scholar 

  • Papadopoulos B., Brossard M., Perié N.M. 1996. Piroplasms of domestic animals in the Macedonia region of Greece. 3 Piro-plasms of small ruminants. Veterinary Parasitology, 63, 67–74.

    Article  CAS  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 anaplas-mosis in cattle at Rangpur district in Bangladesh. Progressive Agriculture, 26, 51–59. DOI: 10.3329/pa.v26i1.24516

    Article  Google Scholar 

  • Reetha T.L., Thomas K.S., Babu M. 2012. Occurrence of haemopro-tozoan infection in bovines. International Journal of Applied Bioresearch, 13, 1–2

    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. DOI: 10.1515/ap-2017-0096

    Article  CAS  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, 45–56

    Article  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). Parasitol-ogy Research, 109, 1155–60

    Article  CAS  Google Scholar 

  • 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. DOI: 10.1155/2013/893862

    Google Scholar 

  • 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, 55–58

    Article  CAS  Google Scholar 

  • 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. DOI: 10.1515/ap-2015-0053

    Article  CAS  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, 574–578

    Article  Google Scholar 

  • Zhang X., Liu Z., Yang J., Chen Z., Guan G., Ren Q., et al. 2014. Multiplex PCR for diagnosis of Theileria uilenbergi, Theile-ria luwenshuni, and Theileria ovis in small ruminants. Para-sitology Research, 113, 527–531

    Article  Google Scholar 

  • 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. DOI: 10.1016/j.ttbdis.2015.09.008

    Article  Google Scholar 

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Authors and Affiliations

  1. Division of Parasitology, ICAR-IVRI, Izatnagar, 243122, India

    V. R. Kundave, Hira Ram, Partha S. Banerjee & Rajat Garg

  2. Division of Veterinary Medicine, ICAR-IVRI, Izatnagar, 243122, India

    K. Mahendran

  3. Division of Veterinary Biotechnology, ICAR-IVRI, Izatnagar, 243122, India

    G. V. P. P. S. Ravikumar

  4. Division of Biological Standardization, ICAR-IVRI, Izatnagar, 243122, India

    Ashok K. Tiwari

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  1. V. R. Kundave
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  2. Hira Ram
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  3. Partha S. Banerjee
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  4. Rajat Garg
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  5. K. Mahendran
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  6. G. V. P. P. S. Ravikumar
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Correspondence to Hira Ram.

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Kundave, V.R., Ram, H., Banerjee, P.S. et al. Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines. Acta Parasit. 63, 759–765 (2018). https://doi.org/10.1515/ap-2018-0090

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