Isolation and Partial Characterization of Exo- and Endoantigens of Cooperia punctata Cultured In vitro

Abstract

Cooperia punctata were cultured in vitro in Ae medium for 21 days, washed in BSSA (Balanced Salt Solution-Antibiotics), and incubated for 72 hr in fresh BSSA. Exoantigens (metabolic antigens of mol wt > 10,000) harvested from incubation fluids and partially purified by ultrafiltration were shown to contain protein. An endoantigen (somatic extract) was prepared of the same worms from which exoantigens were harvested. Immunodiffusion tests made with serum of calves immune to C. punctata and exoor endoantigens gave 2 precipitin bands. Similar tests with mucosal extracts (of a calf immune to C. punctata) showed no activity against the exoantigens; one precipitin band developed using endoantigens. A pattern of complete identity was demonstrated between one component of exoantigens and the endoantigens; a pattern of partial identity developed between another component of the exoantigens and the endoantigens. Disc electrophoresis of the exoantigens resolved 3 distinct and 3 to 4 faint protein bands; the endoantigens were resolved into 10 to 12 reproducible protein bands. Incorporation of the disc gels in immunodiffusion tests indicated two distinct areas of activity when diffused against immune serum in the case of exoantigens and endoantigens whereas the mucosal extract had activity against one of the same areas of protein bands as the immune serum. Both antigens were fractionated by Sephadex G-200 into 3 peaks, and in each case, the first 2 peaks were serologically active; only the first peak of the endoantigens was active against the mucosal extract. Products of metabolism have been suspected as a major source of protective antigens in immunity to parasitic worms. The inability to harvest metabolic antigens of parasitic worms in vivo stimulated interest in in vitro cultivation as a means by which large numbers of parasitic worms and products of their metabolism (of antigenic potential) could be harvested. The in vitro cultivation of parasitic stages of Cooperia punctata was reported by Leland (1961). Later, C. punctata was cultured in vitro from egg to egg (Leland, 1962). Douvres and Alicata (1962) were able to culture artificially ensheathed L3 of C. punctata to sexually immature adults in a vitamin-supplemented medium. The development of C. punctata from L3 to egg-laying or sperm-containing adults in a modification of Ae medium was reported by Eckert (1967). Leland (1967) indicated the final step to completion of the life cycle of C. punctata in culture was in vitro fertilization. In 1971, Zimmerman and Received for publication 12 November 1973. * Contribution No. 178, Department of Infectious Diseases, College of Veterinary Medicine and the Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506. Supported by NSF Research Grant GB 7532 and contributing to Regional Project W-102 Biological Methods of Control for Internal Parasites. Leland reported the completion of the life cycle of C. punctata in vitro. At least the minimum nutritional and physicochemical conditions comparable to those in vivo had been approximated. It could be assumed that in vivo and in vitro metabolisms were comparable and therefore the products of metabolism in both conditions should be similar. The purpose of this study was to isolate and partially characterize antigens of C. punctata cultured in vitro. The term exoantigen is used for products of worm metabolism (i.e., metabolites, secretions, excretions); endoantigen refers to soluble somatic worm material (i.e., extract of worm tissue). MATERIALS AND METHODS A schematic of the procedure for antigen harvest and characterization is given in Figure 1. Cultivation techniques Two thousand infective larvae (L3) were isolated from 14-day-old fecal-vermiculite cultures (made with feces of a donor calf containing a monospecific infection of C. punctata) by the Baermann technique, processed, and inoculated into a culture tube containing 2 ml Ae medium a cording to the procedures of Leland (1963). The number of tubes inoculated at any one time varied from about 50 to over 300, depending on the availability of L3. Incubation was at 38.5 C in roller drums ( 1/ rpm). Worms were washed in BSSA (Balanced Salt Solution-Antibiotics, Leland, 1963) and transferred to tubes of fresh medium weekly except that the first transfer pro-