The slide enzyme linked immunosorbent assay (SELISA) was standardized for detection of antibodies specific to Trypanosoma evansi and subsequently used for the screening of naturally infected bovine sera. A novel SELISA, a modification of the standard ELISA technique was used for the detection of antibodies against Trypanosoma evansi in bovines using positive and negative control sera. The test is based on immunostaining of the fixed whole Trypanosoma evansi organisms on microscopic glass slide, incubation with sera, antibovine IgG-HRPO conjugate and substrate Diaminobenzidine tetrahydrochloride (DAB). Finally the reaction was read under oil immersion of microscope. A total of 702 sera samples from bovines in Rayalaseema region of Andhra Pradesh were examined by SELISA and 192 were found positive for Trypanosoma evansi antibodies

Jothi, S. Siva

Rayulu, V. C.

Reddy B., Sudhakara

English

Journal of Advanced Veterinary Research (University Assiut, Egypt)

IntroductionTrypanosoma evansi causes a disease that is knownas surra. Different mammalian species are suscep-tible for this disease like water buffaloes, cattle,horses, camels and pigs. (Herrera et al., 2004). Thedisease has a great economic importance due tohigh degree of morbidity associated with decreasedmilk production, diminished working capacity andmortality of animals in fatal cases. Prevention byvector control and vaccination are not effective andchemotherapy is the only means of control of thedisease at present. Therefore, definitive diagnosisof surra is of utmost importance.  Conventional di-agnosis in peripheral blood smear is difficult by di-rect microscopy because of scanty and irregularparasitaemia in chronically infected animals (Kil-lick-Kendrick, 1968). Slide enzyme linked im-munosorbent asay (SELISA) has been employedfor the diagnosis of Babesia bovis infections andscreening of Babesia specific monoclonal antibod-ies. It was reported to be as sensitive as existingnon enzyme based serological tests for Babesiabovis (Kungu and Goodger, 1990). Additionally ithas been applied for diagnosis of Toxoplasmosis(Kumar, 2004) and Babesia bigemina infections(Ravindran et al., 2007). The test procedure is sim-ple and economical, slides can be kept for at leastone week at room temperature and no need ofELISA reader. Therefore, the present study was un-dertaken to standardize SELISA in bovines.Materials and methodsA total of 702 sera samples (320 cattle 382 buf-faloes) collected from Rayalaseema region ofAndhra Pradesh (India) were subjected to serolog-ical screening for Trypanosoma evansi. The serumsamples collected from known infected animalswere used as control positives where as the sera ob-tained from known negative animals and foetalbovine calf serum constituted the negative controls. An isolate of Trypanosoma evansi from cattle wasmaintained in the rats. Trypanosoma evansi para-sites were separated from infected rat blood byusing DEAE-cellulose anion exchange columnchromatography method as described by Lanhamand Godfrey (1970) with minor modifications de-scribed by Srivastava et al. (1988). The DEAE cel-lulose purified trypanosomes were suspended inPBS, pH 8.2 and thin smears of 5 mm diametermarked using a marker pencil were prepared ongrease free glass slides. The smears were air dried,fixed in chilled acetone for 15 minutes, wrapped in*Corresponding author: Sudhakara Reddy. B.Address: Department of Veterinary Clinical Medicine, College ofVeterinary Science, Sri Venkateswara Veterinary University, IndiaE-mail address: bhavanamvet@gmail.comJournal of Advanced Veterinary ResearchVolume 2 (2012) 15-17Development of Slide Enzyme Linked Immunosorbent Assay (SELISA) forDetection of Trypanosoma evansi Infection in BovinesS. Siva Jothi1, V. C. Rayulu1, Sudhakara Reddy B.2*1Department of Parasitology, 2Department of Veterinary Clinical Medicine, College of Veterinary Science, Sri VenkateswaraVeterinary University, Tirupati, Andhra Pradesh, India.(Recieved 02 August 2011/ Accepted 18 December 2011)AbstractThe slide enzyme linked immunosorbent assay (SELISA) was standardized for detection of antibodies specific to Try-panosoma evansi and subsequently used for the screening of naturally infected bovine sera. A novel SELISA, a modificationof the standard ELISA technique was used for the detection of antibodies against Trypanosoma evansi in bovines usingpositive and negative control sera. The test is based on immunostaining of the fixed whole Trypanosoma evansi organismson microscopic glass slide, incubation with sera, antibovine IgG-HRPO conjugate and substrate Diaminobenzidine tetrahy-drochloride (DAB). Finally the reaction was read under oil immersion of microscope. A total of 702 sera samples frombovines in Rayalaseema region of Andhra Pradesh were examined by SELISA and 192 were found positive for Trypanosomaevansi antibodies.Keywords: Trypanosoma evansi; Bovines; Diagnosis; SELISA; India.Original ResearchISSN: 2090-6277/2090-6269, www.advancedvetresearch.comaluminum foil and stored at 40c till use.The test was performed as described by Kunguand Goodger (1990) for Babesia bovis. The antigencoated slides were thawed at 370C for 10 minutes(or) at room temperature for 30 minutes. Test serawere used as neat samples and about 15 microlitreof sample was applied to antigen coated zones onthe surface of the glass slide. The slides were thenplaced in a moist chamber and incubated at roomtemperature for 90 minutes. The samples werewashed by rinsing the glass slides in PBS, pH 7.1,3 times for five min each and air dried. 10 mi-crolitre of diluted rabbit antibovine horse radishperoxidase IgG (1:20 in PBS, pH7.4) was placedin each circle and the slides were again incubatedat room temperature for 30 minutes in moist cham-ber. The slides were then washed twice with 0.01M tris HCl (pH 7.6) and were air dried followed byaddition of 10 microlitres of Diaminobenzidinetetrahydrochloride (DAB) to each circle. Finally,the slides were incubated at RT in moist chamberin dark for 10 minutes and the reaction was stoppedby washing the slides with distilled water. Theslides were air dried, mounted with glycerol-PBSand examined under oil immersion objective ofbright field microscope.ResultsThe control positive serum samples revealed typi-cal dark brown coloured Trypanosoma evansi inSELISA (fig.1) where as no coloured Trypanosomaevansi were found in the control negatives (fig.2).Here source of antigen is acetone fixed  DEAE cel-lulose purified Trypanosoma evansi. Then the testwas employed in serum samples of bovines.  Outof 702 test serum samples 240 were found positivewith a percentage of 34.18. Same sero-reactivitywas observed when the test sera were checked byIndirect ELISA.DiscussionBlood smear examination is a practice under fieldcondition but is very less sensitive (Killick-Kendrick, 1968). A sensitive serological test whichis easy to perform and reliable is needed in coun-tries like India where Trypanosomosis continues tobe endemic and an important hindrance to livestockindustry. The existing serological tests suffer fromthe limitation of costly equipments which may notbe available in the field conditions. In the presentstudy, SELISA results can be viewed under ordi-nary light microscope and once standardized thetest can easily be applied to field condition for pri-mary screening of the animals.Another novel application of the test reported inliterature pertains to use of the assay as a screeningtest in hybridoma antibody generation againstBabesia bovis (Kungu and Goodger, 1990). Theseauthors reported that the test was 79.94% and89.47%. Sensitive in detecting Babesia bovis spe-cific antibodies at 12 and 36 months after the singleinfection in cattle maintained under tick-free con-ditions. Although Kungu and Goodger (1990) re-ported that the test slides can be readmicroscopically for least a week at room tempera-ture, but in the present study, the test reaction wasfound to be stable even up to 15 days after the test.Taking into considerations the requirements of thetest, repeatability, simplicity, cost effectiveness andits acceptability in the field conditions, SELISAcould emerge as a test of choice for the detectionof antibodies against T.evansi infection in cattle andbuffaloes in developing countries.16S. Siva Jothi et al. / Journal of Advanced Veterinary Research 2 (2012) 15-17Fig. 1. Positive reaction to SELISAFig. 2. Negative reaction to SELISAReferencesHerrera, H.M., Davila, A.M., Norek, A., Abreu, U.G., Souza,S.S., Andrea, O.S., Jansen, A.M., 2004. Enzootiologyof Trypanosoma evansi in Pantanal, Brazil. VeterinaryParasitology 125, 263-275. Killick-Kendrick, R., 1968. The diagnosis of trypanosomi-asis in livestock: a review of current techniques Vet-erinary Bulletin. 38, 191-197. Kumar, M.U., 2004. Immunological and nucleic acid baseddiagnosis of toxoplasmosis in goats, Ph D thesis, IVRI,Deemed University, Izatnagar, Bareilly, UP, India. Kungu, M.W., Goodger, B.V., 1990. A slide enzyme-linkedimmunosorbent assay (SELISA) for the diagnosis ofBabesia bovis infections and for the screening ofbabesia specific monoclonal antibodies. InternationalJournal of Parasitology 20, 341-345. Lanham, S.M., Godfrey, G.D., 1970. Isolation of salivariantrypanosomes from man and other animals usingDEAE- cellulose. Experimental Parasitology 28, 521-534.Ravindran,R., Mishra,A.K., Rao, J.R., 2007. Slide enzymelinked immunosorbent assay for the diagnosis ofBabesia bigemina infection in bovines, Veterinary Re-search Communications 31, 999-1004. Srivastava, R.V.N., Bansal, G.C., Sharma, N.N., 1988. Sep-aration of Trypanosoma evansi  through Diethyl aminoethyl cellulose columns. Journal of Veterinary Para-sitology 2, 67-70. 17S. Siva Jothi et al. / Journal of Advanced Veterinary Research 2 (2012) 15-17

Development of Slide Enzyme Linked Immunosorbent Assay (SELISA) for Detection of Trypanosoma evansi Infection in Bovines

S. Siva Jothi

V. C. Rayulu

Sudhakara Reddy B.

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Development of Slide Enzyme Linked Immunosorbent Assay (SELISA) for Detection of Trypanosoma evansi Infection in Bovines

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Journal of Advanced Veterinary Research (University Assiut, Egypt)

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