Released August 2007 - The Indian Society for Parasitology

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Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 22-28Original paperJ P DDot-ELISA for the detection of preclinical Haemonchuscontortus infection in sheep by using an adult somaticantigen and an immunoaffinity-purified fractionA. Prasad, A. Nasir and Nem SinghDivision of Parasitology, Indian Veterinary Research Institute, Izatnagar.Received 5 March 2007; accepted 28 April 2007ABSTRACT. Haemonchosis is a major cause of production losses in livestock, particularly sheepand goat, due to the blood-feeding habit of L 4 larvae and adult parasites. In this study, dot-ELISAwas used for the detection of infection in sheep, experimentally-infected with 10,000 L 3 ofHaemonchus contortus. Serum samples from infected sheep were collected at weekly intervals.Adult somatic antigen was prepared from the worms collected from the local abattoir at Bareilly(U.P.). Nitrocellulose membranes (0.2 µm) were dotted with 2–6 µl antigen that contained 2–6 µgprotein, and were incubated at 37°C for 2 h. Non-specific binding sites were blocked with blockingagent casein in phosphate-buffered saline. Positive, negative and uninfected control sera fromsheep, both experimentally- and naturally-infected, were used. Positive reaction with brown soliddot formation was obtained with the sera, as early as first week post-infection, withimmunoaffinity- purified adult somatic antigen.Keywords: dot-ELISA, Haemonchus contortus, immunoaffinity chromatography, SDS-PAGE,somatic antigenINTRODUCTIONsudden death (Soulsby, 1982) within a week (Fraser,1991). During prepatency, the clinical symptoms arenot noticed. Therefore, in the present study, dot-enzyme-linked immunosorbent assay (ELISA) hasbeen utilized to detect the infection during prepatentperiod. The importance of preclinical diagnosisfurther increases because the L 4 stage of this nematodeis pathogenic being a blood sucker and till theinfection is detected by coprological examination,considerable damage is done to the host. Dot-ELISAbeing a field-oriented, simple and easy test has alsobeen used to detect several other helminthicinfections, particularly Fasciola hepatica, F.gigantica and Paramphistomum epiclitum.Gastro-intestinal parasitism represents a severe healthproblem in small ruminant production system, and itsconsequences can be extensive ranging from reducedproductivity to mortality (Skykes, 1994). In India,parasitic gastro-enteritis due to nematode infectionhas been reported as a major constraint to profitablesheep production. Haemonchus contortus is one of themost pathogenic, blood-sucking abomasal nematode,which primarily infects sheep and is responsible foreconomic loss to sheep industry. Due to its blood-feeding habit, H. contortus causes severe anaemia andCorresponding author: Dr. A. Prasad, Network Programme(GIP), Division of Parasitology, Indian Veterinary ResearchInstitute, Izatnagar-243 122, India.E-mail: drarvind@ivri.up.nic.inMATERIALS AND METHODSCoproculture of H. contortus larvae: Coproculture
Dot-ELISA for diagnosis of preclinical haemonchosis23was performed in the laboratory as per the methoddescribed by Sahai (1960). The source of faeces forculture was donor animals which were not allowed tograze, and were maintained in the sheds of Division ofParasitology, Indian Veterinary Research Institute.The larvae (L 3) were stored in distilled water at 4°C.Collection of seracollected and filtered through 0.2 µm filter membrane.The filtrate was stored at -70°C withphenylmethylsulphonylfluoride for further use.Protein estimation: The protein concentration ofadult somatic antigen and immunoaffinity purifiedsomatic antigen was estimated as described (Lowry etal., 1951).Positive control sera: Two rabbits were Immunoaffinity chromatography of somatichyperimmunized with H. contortus somatic antigen. antigen of adult H. contortus: ImmunoaffinityInitially, 500 µg protein of antigen was emulsified purification of the somatic antigen of H. contortus waswith Freund's complete adjuvant, and was injected performed utilizing prepacked column containinginto rabbits by i–m route. After 15 days, first booster amino link as matrix (Pierce). The protocol for affinitydose (750 µg of antigen with incomplete Freund's purification of protein was modified as per theadjuvant) was given to immunised rabbits. Similarly, requirements of immunoaffinity chromatography.second, third and fourth boosters were given along Anti H. contortus antibodies were collected from thewith increasing doses of antigens at the rate of 250 sera of sheep infected with L 3 of H. contortus. Theµg/booster. Finally, test bleed was performed five infection was confirmed on autopsy as well as bydays after the last booster dose, and then blood was obtaining larvae from the coproculture of faeces ofcollected by cardiac puncture. Sera were separated infected animal. The antibody was dissolved inand tested for antibody titer by direct coupling buffer (5 mg antibody/ml gel). The columnimmunodiffusion (DID).was equilibrated to room temperature and washedwith five ml of coupling buffer loaded with antibodyNegative control sera: Zero day lamb serum and and incubated at 4°C with gentle shaking. Theuninfected control animal sera were used as negative supernatant was collected and its O. D. value was readcontrols. at 280 nm. The column was washed with PBS (pH 7.2)Experimental sera of sheep: Eight sheep of same age and the somatic antigen was loaded to the column atgroup (each one year old) and weight were selected for the rate of 5 mg protein/ml gel. The column wasthe production of experimental sera. Six sheep freebykeeping at 4°C with gentle shaking. Afterallowed to embed the antigen into the bed completelyfrom helminthic infections were infected with 10,000L of H. contortus and two sheep were kept as control. incubation, the antigen was eluted. Further, 40 ml3Sera, from experimental as well as control animals,elution buffer (glycine buffer 100 mM, pH 2.5–3.0)strdwas added to the column and allowed to drip throughwere collected at weekly (from 1 week–3 week postslowlyunder gravity. Twenty five aliquots of 1.5 mlinfection) intervals, and were stored frozen at -20°Ceach of unbound and bound fractions were collected.and utilized for dot-ELISA.The collected fractions were neutralized by addingSera of sheep naturally-infected with H. contortus: 100 ml of 1M Tris, (pH 7.5) and the O. D. was taken atSera were collected from local abattoir at Bareilly (U. 280 nm. Each fraction was subjected to sodiumP., India). The sera samples as well as the abomasums dodecyl sulphate-polyacrylamide gel electrophoresisof the same animal were collected. The abomasums (SDS-PAGE) analysis and the bound fractions ofwere examined in the laboratory to check worm interest were pooled.burden.Dot-ELISA: Dot-ELISA was performed with adultNormal sheep sera: Normal, uninfected sheep sera somatic antigen and immunoaffinity purified adultwere collected from abattoir at Bareilly.somatic antigen, with sera of sheep bothexperimental- and naturally-infected with H.Preparation of adult somatic antigen: Adult H. contortus. Positive sera (hyperimmune sera raised incontortus were collected in phosphate-buffered saline rabbits) and negative sera (0 day lamb sera), and(PBS; pH 7.2) from the abomasums of sheep, and were control animals tested through DID were utilized.washed with PBS three-times. The worms were first Antigens containing 2–4 µg H. contortus protein werehomogenized in PBS, sonicated and centrifuged at blotted on 0.2 µm nitrocellulose membrane strips and12,000 rpm at 4°C. The clear supernatant was incubated at 37°C for 1 h. Non-specific sites were
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Released August 2007 - The Indian Society for Parasitology

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