Extracts of larval and adult Oesophagostomum radiatum failed to protect calves against oral challenge of immunity when the antigens were injected subcutaneously once a week for 3 or 4 weeks. Single intraperitoneal injections of live in-vitro-grown third-stage larvae or a mixture of thirdstage, third-molt, and fourth-stage larvae into cattle provided from 44 to 90% and 36 to 83% protection, respectively. The intraperitoneally inoculated larvae penetrated mesenteric tissue and associated lymph nodes producing lesions identical to those in the ileum and cecum of orally inoculated calves. Lowlevel patent infections developed as a result of the intraperitoneal injections in some of the calves given only third-stage larvae; however, none developed in calves given infections of the mixture of stages. Extracts of killed larval and adult nematodes and metabolic products of live infective larvae and adults have been used with varying degrees of success as immunizing agents in several host-parasite systems (McCoy, 1935; Thorson, 1953; Campbell, 1955; Soulsby, 1957; Wade, Swanson, and Fox, 1961; VanZandt, 1962; Denham, 1969; Cypess, 1970). With the advent of successful in-vitro cultivation of nematode parasites, it was hoped that significant breakthroughs would ensue rapidly in vaccinating domestic animals against helminthic infections. However, there has been meager exploitation of this research approach. Robinson (1967) failed to immunize lambs against Haemonchus contortus when he inoculated them subcutaneously with freshly prepared and lyophilized antigens consisting of in-vitro-grown fourth-stage larvae and the culture medium. Only the studies of Silverman, Poynter, and Podger (1962) and Soulsby (1963) have yielded some fruitful results, but their implications were limited in that the investigations involved abnormal host-parasite systems. The present report describes the results of experiments designed to determine the immunogenicity of killed worm extracts and of intact living in-vitro-grown larval stages of Oesophagostomum radiatum introduced parenterally into cattle. Received for publication 7 May 1973. *Animal Parasitology Institute, ARS, Beltsville, Maryland 20705. MATERIALS AND METHODS Holstein-Friesian steer calves raised helminthfree were used. They were kept under conditions that would preclude extraneous infections and were fed a balanced maintenance ration. Their ages ranged from 1 to 41/2 months old when the experiments were begun, but within experiments all calves were the same age. The strain of 0. radiatum used was an isolate originated at the Animal Parasitology Institute and maintained by continuous passage through helminth-free calves. In-vitro-cultivated parasitic larval stages were obtained from roller cultures as described by Douvres (1970). Experiment 1-killed worm antigens Antigens were derived as follows: Adult worms were recovered from experimentally infected calves, washed repeatedly in normal saline, and blotted dry. A fresh weight total of 5.28 g was homogenized in 20 ml 0.05 M Tris-HCl (pH 8.0) in a Potter-Elvehjem tissue grinder in an ice bath. All s bsequent procedures were effected at 4 C. The homogenate was centrifuged at 17,300 g for 15 min, the supernatant was recovered, and the residue was again extracted. The final residue was discarded, and the supernatants of both extractions were combined and concentrated on an Amicon ultrafiltration apparatus (Antigen A). (Amicon Corporation; Lexington, Massachusetts 02173). A second lot of adult worms was processed as above; the crude extract was gel-filtrated (Sephadex-100), and eluted with 0.05 M Tris-HCl (pH 8.0). The extract (160 mg of protein in 25 ml of buffer) was then subjected to gel filtration using Sephadex G-100 equilibrated with 0.05 M TrisHCl, pH 8.0. The column was 2.5 by 90 cm. The flow rate was 30 ml per hour and 5.2-ml fractions were collected. The extract was resolved into 2 protein peaks which were concentrated with the Amicon ultrafiltration apparatus (Antigens B and C).
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Dec 1, 1977
R J Forrest 
Open Access
