Characterization of surface antigens of Trypanosoma evansi by monoclonal antibodies and development of suitable assays

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dc.contributor.advisorChaudhri, S.S.
dc.contributor.authorRayulu, V. Chengalva
dc.date.accessioned2017-08-02T07:17:06Z
dc.date.available2017-08-02T07:17:06Z
dc.date.issued2007
dc.description.abstractThe present investigation has been carried out on an economically important vector-borne disease, known as ‘surra’, caused by a hemoprotozoan Trypanosoma evansi in domestic animals, with objectives to characterize the surface antigens of T. evansi Indian isolate by monoclonal and polyclonal antibodies, and to develop and validate sensitive, specific, simple, cost-effective and field-adaptable diagnostic tests. A field isolate of T. evansi collected from infected cattle was propagated in rats and preserved by freezing in liquid nitrogen. Whole cell lysate (WCL), flagellar (FL) and cell membrane (CM) antigens were prepared from DEAE- cellulose column chromatography purified trypanosomes. In SDS–PAGE, 16 polypeptide bands from 11.93 to 91.31 kDa in WCL, 14 bandsfrom 11.23 to 116.42 kDa in FL and 4 bands from 11.93 to 59.68 kDa in CM preparation were resolved. Eight hybridoma clones were produced in two attempts by fusion of B lymphocytes from T. evansi surface antigen immunized BALB/c mice with Sp2/0 Ag14 myeloma cells. One clone, identified as 1D7, showed good titre against surface antigen of T. evansi and therefore amplified both in culture flasks and BALB/c mice. Monoclonal antibody (mAb) from the mice ascites fluid was partially purified by precipitation with 50% saturation of ammonium sulphate and found to be of IgA isotype by isotype-specific indirect ELISA, protein G-agarose chromatography, SDS-PAGE and immunoblotting. HIS recognized 13 antigen bands ranging from 13.89 to 77.51 kDa, while mAbs were strongly reactive with a 54.7 kDa polypeptide in immunoblotting. MAb-based latex agglutination test (LAT) and Ag-ELISA were developed to detect circulating antigens of T. evansi in the sera of domestic animals. The shelf–life of the latex reagent was found to be about eight months. Analytical sensitivity of LAT and Ag-ELISA was 1.0 µg/ml and 100 ng/ml antigen respectively. The diagnostic sensitivity and diagnostic specificity were recorded as 95.38% and 59.74% for LAT and 83.08% and 67.14% for Ag-ELISA respectively using microhematocrit technique (MHCT) as reference test, and 90.33% and 88.30% for LAT using Ag-ELISA as reference test. Monoclonal antibody- based CIC-ELISA was also performed for detection of T. evansi antigens in circulating immune complexes (CIC) in the MHCT-positive, Ag-ELISA-negative bovine sera samples. LAT and Ag-ELISA were found to be more sensitive than wet blood film (WBF) and MHCT. A total of 1538 blood and sera samples from cattle (n=826), buffaloes (n=285), equines (n=395) and camels (n=32) in different places of Haryana during summer, rainy and post-rainy seasons of the years 2005 and 2006 were screened for T. evansi infections by WBF, MHCT, LAT and Ag-ELISA. The overall prevalence of T. evansi infection was 2.47, 4.23, 42.59 and 34.98 per cent by WBF, MHCT, LAT and Ag-ELISA, respectively. Prevalence of T. evansi was 3.03, 6.05, 50.73 and 40.92% in cattle; 0.70, 1.05, 37.19 and 30.18% in buffaloes; 2.78, 3.04, 31.90 and 27.85% in equines, and 0, 0, 12.5 and 12.5% in camels by WBF, MHCT, LAT and Ag-ELISA, respectively. Variations in the prevalence of T. evansi infection in different animal species, seasons and regions of Haryana state were observed and discussed.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/5810027104
dc.keywordsTrypanosoma evansi, Surface antigens, Monoclonal antibodies, Antigen detection latex aggluatination test, ELISA, Surra in India, Prevalenceen_US
dc.language.isoenen_US
dc.publisherLUVASen_US
dc.subVeterinary Parasitologyen_US
dc.themeCharacterization of surface antigens of Trypanosoma evansi by monoclonal antibodies and development of suitable assaysen_US
dc.these.typePh.Den_US
dc.titleCharacterization of surface antigens of Trypanosoma evansi by monoclonal antibodies and development of suitable assaysen_US
dc.typeThesisen_US
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