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Licensed Unlicensed Requires Authentication Published by De Gruyter September 25, 2015

Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids

Jaideep Kumar, Ashok Chaudhury, Bidhan C. Bera, Ritesh Kumar, Rajender Kumar, Utpal Tatu and Suresh Chandra Yadav
From the journal Acta Parasitologica


The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.


Agbo E.C., Majiwa P.A.O., Claassen E.J.H.M., Roos M.H. 2001. Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterisation. Experimental Parasitology, 99, 123-13110.1006/expr.2001.4666Search in Google Scholar

Bannai H., Sakurai T., Inoue N., Sugimoto C., Igarashi I. 2003. Cloning and expression of mitochondrial Heat Shock Protein 70 of Trypanosoma congolense and potential use as a diagnostic antigen. Clinical and Diagnostic Laboratory Immunology, 10, 926-93310.1128/CDLI.10.5.926-933.2003Search in Google Scholar

Borst P., Fase-Fowler F., Gibson W.C. 1987. Kinetoplast DNA of Trypanosoma evansi. Molecular and Biochemical Parasitology, 23(1), 31-3810.1016/0166-6851(87)90184-8Search in Google Scholar

Boulangé A., Katende J., Authié E. 2002. Trypanosoma congolense: expression of a heat shock protein 70 and initial evaluation as a diagnostic antigen for bovine trypanosomosis. Experimental Parasitology, 100, 6-1110.1006/expr.2001.4667Search in Google Scholar

Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248-25410.1016/0003-2697(76)90527-3Search in Google Scholar

Chansiri K., Kawazu S., Kamio T., Terada Y., Fujisaki K., Philippe H., Sarataphan N. 1999. Molecular phylogenetic studies on Theileria parasites based on small subunit ribosomal RNA gene sequences. Veterinary Parasitology, 83, 99-10510.1016/S0304-4017(99)00052-7Search in Google Scholar

Desquesnes M., Holzmuller P., Lai D., Dargantes A., Lun Z.R., Jittaplapong S. 2013. Trypanosoma evansi and Surra: A Review and Perspectives on Origin, History, Distribution, Taxonomy, Morphology, Hosts, and Pathogenic Effects. BioMed Research International, Article ID 194176, in Google Scholar

Desquesnes M., Kamyingkird K., Pruvot M., Kengradomkij C., Bossard G., Sarataphan N., Jittapalapong S. 2009. Antibody- ELISA for Trypanosoma evansi: Application in a serological survey of dairy cattle, Thailand, and validation of a locally produced antigen. Preventive Veterinary Medicine, 90, 233-241 10.1016/j.prevetmed.2009.04.011Search in Google Scholar

de Andrade C.R., Kirchhoff L.V., Donelson J.E., Otsu K. 1992. Recombinant Leishmania Hsp90 and Hsp70 Are Recognized by Sera from Visceral Leishmaniasis Patients but Not Chagas’ Disease Patients. Journal of Clinical Microbiology, 30(2), 330-33510.1128/jcm.30.2.330-335.1992Search in Google Scholar

Eisler M.C., Lessard P., Masake R.A., Moloo S.K., Peregrine A.S. 1998. Sensitivity and specificity of antigen-capture ELISAs for diagnosis of Trypanosoma congolense and Trypanosoma vivax infections in Cattle. Veterinary Parasitology, 79, 187-20110.1016/S0304-4017(98)00173-3Search in Google Scholar

Greiner M., Bhat T.S., Patzelt R.J., Kakaire D., Schares G., Dietz E., Bohning D., Zessin K.H., Mehlitz D. 1997. Impact of biological factors on the interpretation of bovine trypanosomosis serology. Preventive Veterinary Medicine, 30, 61-7310.1016/S0167-5877(96)01088-4Search in Google Scholar

Hamilton P.B., Adams E.R., Njiokou F., Gibson W.C., Cuny G. 2009. Herder S. Phylogenetic analysis reveals the presence of the Trypanosoma cruzi clade in African terrestrial mammals. Infection, Genetics and Evolution, 9, 81-8610.1016/j.meegid.2008.10.011Search in Google Scholar

He L., Liu Q., Quan M., Zhou D., Zhou Y., Zhao J. 2009. Molecular cloning and phylogenetic analysis of Babesia orientalis heat shock protein 70. Veterinary Parasitology, 162, 183-9110.1016/j.vetpar.2009.03.039Search in Google Scholar

Kaufmann S.H.E. 1990. Heat-shock proteins: a missing link in the host-parasite relationship? Medical Microbiology and Immunology, 179, 61-6610.1007/BF00198526Search in Google Scholar

Kumar R., Kumar S., Khurana S.K., Yadav S.C. 2013. Development of an antibody-ELISA for seroprevalence of Trypanosoma evansi in equids of North and North-western regions of India. Veterinary Parasitology, 196, 251-25710.1016/j.vetpar.2013.04.018Search in Google Scholar

Laemmli U.K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680-68510.1038/227680a0Search in Google Scholar

Lai D., Hashimi H., Lun Z., Ayala F.J., Lukes J. 2008. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proceedings of National Academy of Sciences USA, 105 (6), 1999-200410.1073/pnas.0711799105Search in Google Scholar

Lanham S.M., Godfrey D.G. 1970. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology, 28, 521-53410.1016/0014-4894(70)90120-7Search in Google Scholar

Lun Z., Desser S.S. 1995. Is the broad range of hosts and georgraphical distribution of Trypanosoma evansi attributable to the loss of maxicircle kinetoplast DNA? Parasitology Today, 11(4), 131-13310.1016/0169-4758(95)80129-4Search in Google Scholar

Manful T., Mulindwa J., Frank F.M., Clayton C.E., Matovu E. 2010. A search for Trypanosoma brucei rhodesiense diagnostic antigens by proteomic screening and targeted cloning. PLoS ONE, 5, e9630. DOI: 10.1371/journal.pone.000963010.1371/journal.pone.0009630Search in Google Scholar

Nantulya V.M., Lindqvist K.J. 1989. Antigen detection enzyme immunoassays for the diagnosis of Trypanosoma vivax, T. congolense and T. brucei infections in cattle. Tropical Medicine and Parasitology, 40, 156-161Search in Google Scholar

Nei M., Kumar S. 2000. Molecular Evolution and Phylogenetics. Oxford University Press, New York Newport G., Culpepper J., Agabian N. 1988. Parasite heat-shock proteins. Parasitology Today, 4, 30610.1016/0169-4758(88)90111-1Search in Google Scholar

OIE terrestrial manual, chapter 2.1.17: pdf Search in Google Scholar

Polla B.S. 1991. Heat shock proteins in host-parasite interactions. Immunololgy Today, 12, A38-4110.1016/S0167-5699(05)80011-8Search in Google Scholar

Rafati S., Gholami E., Hassani N., Ghaemimanesh F., Taslimi Y., Taheri T., Soong L. 2007. Leishmania major heat shock protein 70 (HSP70) is not protective in murine models of cutaneous leishmaniasis and stimulates strong humoral responses in cutaneous and visceral leishmaniasis patients. Vaccine, 25, 4159-416910.1016/j.vaccine.2007.03.006Search in Google Scholar

Ranjithkumar M., Saravanan B.C., Yadav S.C., Kumar R., Singh R., Malik T.A., Dey S. 2013. Neurological trypanosomiasis in quinapyramine sulfate-treated horses - A breach of the bloodbrain barrier? Tropical Animal Health and Production, 46, 371-37710.1007/s11250-013-0498-9Search in Google Scholar

Retamal C.A., Thiebaut P., Alves E.W. 1999. Protein purification from polyacrylamide gels by sonication extraction. Analytical Biochemistry, 268, 15-2010.1006/abio.1998.2977Search in Google Scholar

Reyna-Bello A., Garcia F.A., Rivera M., Sanso B., Aso P.M. 1998. Enzyme-linked immunosorbent assay (ELISA) for detection of anti-Trypanosoma evansi equine antibodies. Veterinary Parasitology, 80, 149-5710.1016/S0304-4017(98)00199-XSearch in Google Scholar

Rodrigues A., Fighera A., Souza T.M., Schild A.L., Barros,C.S.L. 2009. Neuropathology of Naturally Occurring Trypanosoma evansi Infection of Horses. Veterinary Pathology, 46, 251-25810.1354/vp.46-2-251Search in Google Scholar

Roy N., Nageshan R.K., Pallavi R., Chakravarthy H., Chandran S., Kumar R., Gupta A.K., Singh R.K., Yadav S.C., Tatu U. 2010. Proteomics of Trypanosoma evansi Infection in Rodents. PLoS ONE, 5(3), e9796. DOI: 10.1371/journal.pone.000979610.1371/journal.pone.0009796Search in Google Scholar

Sharma Y.D. 1992. Structure and possible function of heat-shock proteins in Falciparum malaria. Comparative Biochemistry and Physiology-B, 102(3), 437-44410.1016/0305-0491(92)90033-NSearch in Google Scholar

Shinnick T.M. 1991. Heat shock proteins as antigens of bacterial and parasitic pathogens. Current Topics in Microbiology and Immunology, 167, 145-16010.1007/978-3-642-75875-1_9Search in Google Scholar

Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731-273910.1093/molbev/msr121Search in Google Scholar

Woo P.T.K. 1977. 7-Salivarian trypanosomes producing disease in livestock outside of sub-saharan Africa. In: Julius Kreir (Ed.), Taxonomy Kinetoplastids and Flagellates of Fish. Academic Press, New York, pp. 269-9610.1016/B978-0-12-426001-6.50013-XSearch in Google Scholar

Yadav S.C., Kumar R., Kumar S., Gupta, A.K. 2011. Identification and characterization of cysteine proteinases of Trypanosoma evansi. Parasitology Research, 109, 559-56510.1007/s00436-011-2284-9Search in Google Scholar

Yadav S.C., Kumar R., Kumar V., Jaideep, Kumar R., Gupta A.K., Bera B.C., Tatu U. 2013. Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines. Research in Veterinary Science, 95, 522-52810.1016/j.rvsc.2013.04.030Search in Google Scholar

Zurita A.I., Rodriguez J., Pinero J.E., Pacheco R., Carmelo E., del Castllo A., Valladares B. 2003. Cloning and characterization of the Leishmania (Viannia) braziliensis Hsp70 gene. Diagnostic use of the C-terminal fragment rLb70 (513-663). Journal of Parasitology, 89, 372-378 10.1645/0022-3395(2003)089[0372:CACOTL]2.0.CO;2Search in Google Scholar

Received: 2014-12-27
Revised: 2015-3-16
Accepted: 2015-5-28
Published Online: 2015-9-25
Published in Print: 2015-12-1

W. Stefański Institute of Parasitology, PAS