Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter March 25, 2015

Molecular evidence of Theileria equi infection in Hyalomma anatolicum ticks infested on sero-positive Indian horses

Jai Bhagwan, Ashok Kumar, Rajender Kumar, Liza Goyal, Parveen Goel and Sanjay Kumar
From the journal Acta Parasitologica

Abstract

A sizeable Indian equine population is considered to be pre-immune carrier of Theileria equi infection. In this study we confirmed the presence of T. equi specific DNA in Hyalomma anatolicum ticks which were infested on sero-positive horses. Fifty two Indigenous horses were randomly selected from endemic areas and their blood and tick samples were collected. Tick salivary glands and blood samples were processed for separation of DNA and serum, respectively. Serum samples were analyzed by EMA-2ELISA and nine horses were found positive for T. equi specific antibodies. Species-specific primers were designed from EMA-2 gene of T. equi, so as to amplify 398 bp fragment in PCR. The gene fragment was amplified in PCR on the DNA samples (from blood) from these nine sero-positive horses. Corresponding six tick’s DNA samples collected from these nine seropositive animals were observed positive in PCR. Further, qPCR assay demonstrated presence of T. equi DNA in infected tick’s salivary glands, which was also confirmed by microscopic examination of infected acinar. This study concluded that Hyalomma anatolicum ticks infested on T. equi seropositive horses have sporozoite developmental stage in their salivary glands, which is an evidence for transmitting potential of these tick among Indian horse population.

References

Baldani C.D., Hilario E., Nakaghi A.C., Bertolini M.C., Machado R.Z. 2011. Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from Sao Paulo State, Brazil. Revista Brasileira de Parasitologia Veterinária, 20, 54-60Search in Google Scholar

Bashiruddin J.B., Camma C., Rebelo E. 1999. Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene. Veterinary Parasitology, 84, 75-83Search in Google Scholar

Battsetseg B., Lucero S., Xuan X., Claveria F., Byambaa B., Battur B., Boldbaatar D., Batsukh Z., Khaliunaa T., Battsetseg G., Igarashi I., Nagasawa H., Fujisaki K. 2002. Detection of equine Babesia spp. gene fragments in Dermacentor nuttalli Olenev 1929 infesting mongolian horses, and their amplification in egg and larval progenies. Journal of Veterinary Medical Science, 64,727-30Search in Google Scholar

Battsetseg B., Xuan X., Ikadai H., Bautista J.L., Byambaa B., Boldbaatar D., Battur B., Battsetseg G., Batsukh Z., Igarashi I., Nagasawa H., Mikami T., Fujisaki K. 2001. Detection of Babesia caballi and Babesia equi in Dermacentor nuttalli adult ticks. International Journal for Parasitology, 31, 384-6Search in Google Scholar

Boldbaatar D., Xuan X., Battsetseg B., Igarashi I., Battur B., Batsukh Z., Bayambaa B., Fujisaki K. 2005. Epidemiological study of equine piroplasmosis in Mongolia. Veterinary Parasitology, 127, 29-32Search in Google Scholar

De Waal, D.T. 1992. Equine piroplasmosis: a review. British Veterinary Journal, 148, 6-14 Search in Google Scholar

Friedhoff K.T. and Soule C. 1996. An account of equine babesiosis. Review Scientific et Technique Office Internationale des Epizooties, 15, 1191-1201Search in Google Scholar

Geevarghese G., Fernandes S., Kulkarni S.M. 1997. A checklist of Indian ticks (Acari: Ixodoidea). The Indian Journal of Animal Sciences, 67, 566-574Search in Google Scholar

Haque M., Jyoti, Singh N.K., Rath S.S. 2010. Prevalence of Theileria annulata infection in Hyalomma anatolicum anatolicum in Punjab state, India. Journal of Parasitic Diseases, 34, 48-51Search in Google Scholar

Huang, X., Xuan X., Yokoyama N., Xu L., Suzuki H., Sugimoto C., Nagasawa H., Fujisaki K., Igarashi I. 2003. High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential in immunodiagnosis. Journal of Clinical Microbiology, 41, 1147-51Search in Google Scholar

Hussain M.H., Saqib M., Raza F., Muhammad G., Asi M.N., Mansoor M.K., Saleem M., Jabbar A. 2014. Seroprevalence of Babesia caballi and Theileria equi in five draught equine populated metropolises of Punjab,Pakistan. Veterinary Parasitology, 202, 248-256Search in Google Scholar

Ikadai H., Sasaki M., Ishida H., Matsuu A., Igarashi I., Fujisaki K., Oyamada T. 2007. Molecular evidence of Babesia equi trans mission in Haemaphysalis longicornis. American Journal of Tropical Medicine and Hygiene, 76, 694-7Search in Google Scholar

Katoh K., Misawa K., Kuma K., Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30, 3059-3066Search in Google Scholar

Kim C.M., Blanco L.B.C., Alhassan A., Iseki H., Yokoyama N., Xuan X., Igarashi I. 2008. Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples. Veterinary Parasitology, 151, 158-163Search in Google Scholar

Knowles D.P. Jr., Kappmeyer L.S., Stiller D., Hennager S.G., Perryman L.E. 1992. Antibody to a recombinant merozoite protein epitope identified horses infected with Babesia equi. Journal of Clinical Microbiology, 30, 3122-3126Search in Google Scholar

Kumar S., Kumar R. 2007. Diagnosis of Babesia equi infection in equines: An update on the methods available. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 2, 1-14Search in Google Scholar

Kumar S., Malhotra D.V., Sangwan A.K., Goel P., Kumar A., Kumar S. 2007. Infectivity rate and transmission potential of Hyalomma anatolicum anatolicum ticks for Babesia equi infection. Veterinary Parasitology, 144, 338-343Search in Google Scholar

Kumar S., Sugimoto C., Igarashi I. 2005. Equine babesiosis in Asia. In: Asian Parasitology - Toxoplasmosis and Babesiosis in Asia (Vol 4) Yano A, Nam HW, Anuar K, Shen J, Saito A and Igarashi I. Pub The Federation of Asian Parasitologist, Japan pp. 187-195Search in Google Scholar

Kumar S., Kumar R., Gupta A.K., Yadav S.C., Goyal S.K., Khurana S.K., Singh R.K. 2013a. Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population. Veterinary Parasitology, 198, 10-17Search in Google Scholar

Kumar Y., Malhotra D.V., Nichani A.K., Dhar S., Kumar S. 2013b. Immunokinetics of Theileria equi specific antibodies A comparison in serial and single dilution ELISA antibody end titres. Turkish Journal of Veterinary and Animal Sciences, 37, 429-433Search in Google Scholar

Kumar S., Kumar R., Gupta A.K., Dwivedi S.K. 2008. Passive transfer of Theileria equi antibodies to neonate foals of immune tolerant mares. Veterinary Parasitology, 151, 80-85Search in Google Scholar

Lori A., Gabrielli S., Calderini P., Moretti A., Pietrobelli M., Tampieri M.P., Galuppi R., Cancrini G. 2010. Tick reservoirs for piroplasm in central and northern Italy. Veterinary Parasitology, 170, 291-296 Search in Google Scholar

Mehlhorn H., Schein E. 1988. Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998. Parasitology Research, 84, 467-75Search in Google Scholar

Nicolaiewsky T.B., Richter M.F., Lunge V.R., Cunha C.W., Delagostin O., Ikuta N., Fonseca A.S., da Silva S.S., Ozaki L.S. 2001. Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction. Veterinary Parasitology, 101, 9-21Search in Google Scholar

Rampersad J., Cesar E., Campbell M., Samlal M., Ammons D.A. 2003. Field evaluation of PCR for the routine detection of Babesia equi in horses. Veterinary Parasitology, 114, 81-87Search in Google Scholar

Sambrook J., Russell D.W. 2001 Molecular cloning a laboratory manual. Third ed. Cold Spring Harbour Laboratory Press, New York Search in Google Scholar

Seo M.G., Yun S.H., Choi S.K., Cho G.J., Park Y.S., Kwon O.D., Cho K.H., Kim T.H., Jeong K.S., Park S.J., Kwon Y.S., Kwak D. 2011. Seroprevalence of equine piroplasms in the Republic of Korea. Veterinary Parasitology, 179, 224-6Search in Google Scholar

Tanaka T., Xuan X., Ikadai H., Igarashi I., Nagasawa H., Fujisaki K., Mikami T., Suzuki N. 1999. Expression of Babesia equi merozoite antigen-2 by recombinant baculovirus and its use in the ELISA. International Journal for Parasitology, 29, 1803-1808Search in Google Scholar

Tiwari A., Singh N.K., Singh H., Jyoti, Bhat S.A., Rath S.S. 2013. Prevalence of Theileria annulata infection in Hyalomma anatolicum anatolicum collected from crossbred cattle of Ludhiana, Punjab. Journal of Parasitic Diseases. DOI: 10.1007/s12639-013-0279-4 Search in Google Scholar

Untergasser A., Nijveen N., Rao X., Bisseling T., Geurts R., Leunissen J.A.M. 2007. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Research, 35, W71-W74. DOI: 10.1093/nar/gkm306Search in Google Scholar

Xuan X., Larsen A., Ikadai H., Tanaka T., Igarashi I., Nagasawa H., Fujisaki K., Toyoda Y., Suzuki N., Mikami T. 2001. Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay. Journal of Clinical Microbiology, 705-9Search in Google Scholar

Yin H., Lu W., Luo J. 1997. Babesiosis in China. Tropical Animal Health and Production, 4 Suppl, 11S-15S Search in Google Scholar

Received: 2014-6-11
Revised: 2014-12-27
Accepted: 2015-1-21
Published Online: 2015-3-25
Published in Print: 2015-6-1

© 2015 W. Stefański Institute of Parasitology, PAS