Embryonic response to Eimeria tenella infection. (1970)

The chorioallantoic membranes (CAM) of chicken embryos were infected with Eimeria tenella by inoculation of sporozoites into the allantoic cavity. Embryonic mortality during the hemorrhagic phase of the infection (4th to 8th days postinoculation) was highly dose dependent. Mortality among embryos produced by strains of fowl differing in resistance to cecal coccidiosis was compared with the susceptibility of 11-day-old chicks produced by the same matings. Although significant differences in embryonic resistance were found among different strains of fowl, in ovo inoculation with E. tenella was an ineffective technique for detecting genetic differences in resistance to cecal coccidiosis. The results of experiments designed to study the effect of in ovo inoculation on the resistance of 11-day-old chicks indicated that mortality following inoculation with 105 E. tenella oocysts was much higher among chicks derived from inoculated eggs than among chicks from noninoculated eggs. Although these results suggested that inoculation of embryonated eggs induced partial immunological tolerance to an antigen(s) associated with the parasite, other possible causes of the observed phenomenon could not be positively ruled out. Eimeria tenella, the parasitic agent of cecal coccidiosis in the fowl, is a rigidly host-specific parasite which confines its endogenous development to the cecal pouches and adjacent areas of the intestine. Long (1965) first demonstrated that E. tenella sporozoites, inoculated into the allantoic cavity of the chick embryo, initiate an apparently normal infection within the chorioallantoic membrane (CAM). Intravenous inoculation failed to produce infections, while intraamniotic inoculations resulted in sudden deaths associated with multiple hemorrhages. Subsequently, four species of Eimeria (E. brunetti, E. mivati, E. necatrix, and E. tenella) were found to develop in the CAM of chicken embryos (Long, 1966). Jeffers and Wagenbach (1969) reported significantly greater mortality among female embryos than among male embryos during the acute hemorrhagic embryonic response to E. tenella infection. The objective of the present study was to further characterize the embryonic response to E. tenella infection. General pathological effects of the infection were examined and a Received for publication 2 January 1970. * Supported in part by Research Grant A1-04101 from NIAID, U. S. Public Health Service, and by North Central Regional Project NC-89. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, College of Agriculture, Madison. t Present address: Parasitology Section, Hess and Clark, Ashland, Ohio 44805. I Present address: Department of Biology, Carleton College, Northfield, Minnesota 55057. dose-response curve was obtained for embryos of a representative strain of fowl. The response of embryos from mating groups selected for differential genetic susceptibility to cecal coccidiosis was studied in order to detect possible associated genetic differences in embryonic susceptibility to in ovo inoculation with E. tenella. A strong positive relationship would suggest that inoculation of embryonated eggs with E. tenella might be a rapid and economical method for detecting genetic differences in the postnatal resistance of different strains of fowl. Other experiments were designed to study the effect of in ovo inoculation on the resistance of chicks to challenge with E. tenella oocysts. Burnet and Fenner (1949) theorized that an individual may become immunologically unresponsive to antigens to which it was exposed during embryonic development. Immunological tolerance to certain bacterial and viral antigens has been induced in the chicken embryo (Buxton, 1954; Friedman and Gaby, 1960; Hanson and Cannefax, 1967; Rubin et al., 1962; Solomon, 1968). MATERIALS AND METHODS The oocyst cultures used for all experiments were obtained through serial passage of a pure line E. tenella strain which has been maintained in this laboratory for many years. Bacteriologically sterile oocyst suspensions were prepared using a Clorox digestion procedure described by Wagenbach et al. (1966). Sporocysts, released from the oocysts by agitation in the presence of sterile 4 mm diameter glass beads, were added to an excysting medium consisting of 0.25% trypsin and 5.0%