Biological Activities of Various Lipid Fractions from Echinococcus granulosus Scolices on In vitro Cultures of Hymenolepis diminuta

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Thin-layer chromatography of lipid extracts of Echinococcus revealed substances with stimulatory and depressive effects on growth of excysted cysticercoids of Hymenolepis diminuta in in vitro cultures. Cholesterol depressed growth and increased mortality, unidentified steroids increased mortality and a substance with Rfs similar to farnesal stimulated growth of these forms by as much as 100%. Similar substances, untested as yet for biological activity, were found in lipid extracts of infective larvae of Trichinella spiralis and miracidia of Fasciola hepatica. The complexity of the life cycles of parasitic helminths dictates reasonably sophisticated control mechanisms. The molts of larvae and maturation to adulthood are not unlike those of insects. The bulk of literature during the last 10 years on in vitro cultivation of parasitic helminths illustrates the rather primitive state of our knowledge regarding these phenomena. A finer understanding of the involved mechanisms might well open up new avenues for control of these parasites. A chance observation that lipoidal materials were being leached from scolices of Echinococcus granulosus during preparation of ES (excretion and secretion) antigens, and the fact that scolices of this organism live in hydatid cysts in an excellent culture medium, yet do not mature, prompted these studies. MATERIALS AND METHODS The procedure followed for the collection of scolices was essentially that of Agosin et al. (1957). Livers collected from sheep and cattle at the Beirut abattoir were brought to the laboratory within 2 hr after slaughter of the host animals. The fluid was withdrawn aseptically from hydatid cysts with a syringe and needle. The settling scolices were transferred to centrifuge tubes with a Pasteur pipette, washed 3 or 4 times with distilled water Received for publication 16 February 1968. * These studies were supported in part by research grants from the Medical Research Committee, American University of Beirut and from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Public Health Service (AI-02205-08 and AI-07218-01). t Present address: Department of Biology, University of Notre Dame, Notre Dame, Indiana. + Present address: School of Pharmacy, University of Kentucky, Lexington, Kentucky. and separated by low speed centrifugation. The thoroughly washed scolices were used alive for one experiment and lyophilized to a dry powder for the remainder. Total lipids from scolices were extracted by a procedure similar to that of Folch et al. (1957). The lyophilized scolices (0.495 g) were mixed with 100 volumes (w/v) of a mixture of chloroform: methanol (2:1, v/v) and agitated on a shaker for 6 hr. The insoluble residue was then separated from the solvent by low speed centrifugation and the supernatant fluid filtered through paper. This extraction was repeated 3 more times. The combined extracts were reduced to 10 ml under reduced pressure at 40 C and were subsequently washed with an equal volume of distilled water (Beames, 1965). After standing 1 hr, the separated chloroform layer was washed with 1/2 volume of 1% KC1 solution. To the aqueous phase, an equal volume of a mixture of chloroform: methanol (2:1) was added, and in a few minutes the chloroform layer (lower phase) was easily separated. The combined chloroform extracts were kept overnight at -20 C and quickly filtered in the cold. The filtrate was dried over Na2SO4 and evaporated to dryness under reduced pressure at 40 C. The residue was weighed (0.0672 g; 13.6% of dry weight) and immediately picked up in a minimum volume of chloroform and spotted on thin layer chromatography (TLC) plates for the fractionation of its lipid content. Preliminary qualitative extractions of infective larvae of Trichinella spiralis and miracidia of Fasciola hepatica were done in a similar way. Unless otherwise specified, thin layer plates 0.25 mm thick were prepared from Silica Gel G (Merck according to Stahl). The plates were oven-dried at 110 C for 30 min and stored in a dessicator. They were reactivated at 110 C for 10 min immediately before use. Eastman Chromagrams (0.1 mm thick) were also used. Solvent development was allowed to proceed to 12 to 13 cm from the point of sample application (1.5 cm from the lower edge of the plate) in preequilibrated chambers away from light.