Completion of the Life Cycle of Cooperia punctata In vitro

Abstract

L3 Cooperia punctata were cultivated in vitro to adult worms that laid large numbers of eggs; many of these eggs hatched to L1 and L2 and a few of the latter developed to L.. Parasitic stages were cultivated in Ae medium (38.5 C); free-living stages were cultivated in A-s medium at 27 C. Embryonation, hatching, and development initially were observed only in cultures with a population density of fewer than 1,742 worms/2 ml medium. Embryonation was not observed in cultures where 15-day-old worms were combined with worms 29 days old. Embryonation and hatching were detected in 28 of 271 subsequent cultures inoculated with 1,500 larvae. The cultivation of the parasitic stages of Cooperia punctata in vitro, including the production of egg-laying or sperm-containing adults in Ae medium, was reported by Leland (1961). Leland (1962) reported the cultivation of C. punctata from egg to egg in vitro. Douvres and Alicata (1962) cultured C. punctata from artificially exsheathed L3 to sexually immature fifth-stage adults in vitamin-supplemented medium. Eckert (1967) also developed C. punctata from L3 to egg-laying or spermcontaining adults in a modification of Ae medium. Leland (1967) reported the cultivation of C. punctata in vitro from egg to egg (infertile). Nonparasitic stages were cultivated in A-s medium (serum component of Ae replaced with BSSA); parasitic stages were cultivated in Ae medium. Infertile eggs were produced by virgin females and females in male-containing cultures. Leland (1970) reported the development of C. punctata from L3 to egg-laying adults in Ae medium in as few as 21 days. The final portion of the life cycle of C. punctata to be accomplished in vitro was hatching eggs produced by adults reared in vitro. The purpose of this study was to determine if hatching of eggs was not occurring because (1) males and females were maturing at Received for publication 17 September 1970. Contribution VIII No. 149, Department of Infectious Diseases, College of Veterinary Medicine, Kansas Agricultural Experiment Station, Manhattan, 66502. Supported in part by NSF Research Grant GB 7532, the National Defense Education Act program, and contributing to Regional Project W-102-Biological Methods of Control for Internal Parasites. different times or (2) the nematode-population density in culture was not suitable. In the course of this work the hatching of eggs was detected. MATERIALS AND METHODS L3 isolated from 14-day-old vermiculite cultures (made with feces taken by rectal collection from a donor calf containing a monospecific infection with C. punctata) were exsheathed and inoculated according to the procedures of Leland (1963). Larval suspensions used as inoculum contained approximately 200 to 2,000 larvae per 0.2 ml. Media used were Ae (Leland, 1963, 1965) or A-s (Leland, 1963, 1967). A-s was a modification of Ae in which the serum component was replaced with BSSA (Balanced Salt Solution-Antibiotics, Leland, 1963). Incubation was in roller drums (1/5 rpm) at 38.5 C or 27 C. Either weekly or semiweekly, worms were washed in BSSA and transferred to tubes of fresh medium. To combine different aged cultures, worms 15 days old (tubes 190, 191, 192, 193, 194, and 195) were placed respectively in tubes containing worms 29 days old (tubes 200, 201, 202, 203, 204, and 205). This procedure provided an optimum density of approximately 1,500 larvae/2 ml medium (Leland, 1970) in each tube during the precombination period. After combining, the population density was doubled (3,000 worms/2 ml). This was necessary because the in vitro entangling characteristic of the nematode prevented division of the culture without injury to the worms. Worms were cultured until no further worm movement was detected (day 72 after combining cultures; refer to Table I for termination ages of population density cultures). Subsequent culture manipulations were as described above, except for the numbers of larvae inoculated into each of tubes 170, 182 to 189, 196, and 206 to 208; the level of inoculum in them ranged from approximately 200 to 2,100 larvae per culture (Table I). Many L1 and L2 produced by in vitro adults were transferred either to Ae medium at 38.5 C, or to BSSA, Ae, or A-s at 27 C. C. punctata larvae were isolated from calf feces by the Baermann technique after 24 hr (L1), 48 hr