Hatching In vitro of Oncospheres of Taenia solium

Various authors have studied hatching of cestode oncospheres infecting animals and man (Heath and Smyth, 1970, Parasitology 61: 329343). Silverman (1954, Ann. Trop. Med. Parasitol. 48: 207-215) reported up to 74% activation of oncospheres of Taenia saginata. Jones et al. (1960, J. Parasit. 46: 170-174) described the hatching of T. hydatigena; Berntzen and Voge (1962, J. Parasit. 48: 110-119) reported on the hatching in vitro of Hymenolepis citelli and H. nana. Berntzen and Voge (1965, J. Parasit. 51: 235-242) also described the requirements for hatching of oncospheres of four hymenolepid cestodes and noted that each of the four species of the genus Hymenolepis had slightly different requirements for hatching in vitro. Smyth (1963, J. Parasit. 51: 235-242) proposed the uniformity of requirements to be furnished, not only for the rupture of the membranes surrounding the eggs, but also for the activation of the oncospheres. Laws (1968, Exp. Parasitol. 23: 1-10) reported on the hatching in vitro of Taenia hydatigena, T. pisiformis, and Echinococcus granulosus. Hatching is here defined as the liberation of the oncosphere from all enclosing coats and membranes, with the activation of the embryo. There is no evidence from published papers that by our definition, eggs of T. solium have been hatched successfully in vitro. We report here the hatching in vitro of oncospheres of Taenia solium. Ten terminal gravid proglottids were obtained from a patient treated with niclosamide. The segments were passed by the patient 8 hr after treatment. They were washed in 0.85% NaCI and the uterine branches were counted for appropriate identification (Brown and Neva, 1983. Appleton-Century-Crofts, pp. 186187). The segments were placed in Petri dishes lined with filter paper moistened in saline, and were covered with moist cotton and filter paper. The dishes were sealed with Parafilm to prevent drying and kept for 1 wk at room temperature until experiments began. The following procedures were used as precautionary measures. Containers sufficiently large to accommodate Petri dishes, Pasteur pipettes, slides and other small glassware were filled with 95% ethyl alcohol. All material possibly contaminated with eggs was discarded into the container, taking care that the items were fully submerged in the liquid. The work area was swabbed periodically with 70% alcohol, and, at the end of the experiment, hands were rinsed in alcohol before washing with soap and water. Observations on eggs subjected to 50% ethyl alcohol (final concentration) or higher showed that, on a slide, embryophores fell apart within 5 min of addition of the alcohol and within 1 min when preparation was covered with a coverslip. The embryos appeared black and shrivelled. However, if the final alcohol concentration was 35%, the eggs remained intact during a 15min observation period under the microscope. Eggs were dissected from proglottids using a dissecting microscope and were transferred into Solution 1 consisting of: (a) 120 mg pepsin, 1: 10,000 (DIFCO Laboratories, Detroit, Michigan); (b) 2 ml HC1, 1 N; and (c) 20 ml distilled H20. The pH of this solution was 2.0. Ten ml of his solution was added to eggs in a Petri dish, and placed in an incubator at 41 C for 15 min. The eggs were then washed three times in 0.85% NaCl to prevent transfer of ingredients from the first solution into the second. Eggs were then pipetted into a 10-mm-diameter screw cap tube, containing Solution 2, consisting of: (a) 10 ml 0.85% NaCI; (b) 35 mg trypsin, crystalline (Type XII, Sigma Chemical Company, St. Louis, Missouri); (c) 0.5 ml whole bile from pig (kept frozen until use); and (d) 1 ml NaHCO3, 10% solution in distilled water. The pH of this solution was 7.2 to 7.4. The tube was then tightly capped and placed in an incubator for 15 min at 41 C, removed to room temperature (21-22 C) for observation and kept for another 15 min.