At present many laboratories throughout the world are studying the chemotherapy and immunology of Schistosoma mansoni in laboratory hosts. Many workers judge the success or failure of their attempts to cure or immunize these hosts from the ratio of the number of living adult worms recovered to the number of infecting cercariae. This ratio is affected, however, not only by the efficacy of any treatment, but also by the methods used to infect the animals and to recover the worms. If these methods result in widely varying worm recoveries amongst the animals in any experimental group, then small but significant effects of treatment might well be missed. Alternatively, such large experimental groups must be used that the work becomes tedious to perform and depends upon the availability of a great deal of technical assistance. This paper describes techniques which are rapid and do not require great skill in their performance. More important, in our hands they have given very consistent results. In this respect, particularly, we believe that these techniques have advantages over others which are currently practised.The techniques described here are those which were used in other investigations reported in this journal (Smithers & Terry, 1965a, b).The strain of S. mansoni used throughout this work was isolated in Puerto Rico and was obtained through the courtesy of Dr W. B. DeWitt of the National Institutes of Health. The parasite is maintained in an albino strain of Australorbis glabratus (Newton, 1955). Snails are exposed individually to ten miracidia overnight at 27 °C.

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Recently there has been growing evidence for the role of the eosinophil in the effector mechanism of immunity to reinfection with schistosomes. Mice immune to Schistosoma mansoni are no longer able to resist reinfection after treatment with anti-mouse eosinophil serum (1). In vitro studies using human serum and eosinophils (2) or rat serum and cells (3), have demonstrated antibody-mediated damage to schistosomula by eosinophils. These cells adhere to IgG-coated schistosomula by Fc receptors (3), and peroxidase, from the matrix of the eosinophil granule, is secreted onto the surface of the worm (4).Eosinophils have been shown to possess C3 receptors in addition to Fc (5, 6), and schistosomula are known to activate complement by the alternative pathway, binding C3 to their surface (7). It seemed appropriate, therefore, to investigate the adherence of rat eosinophils to schistosomula through the C3 receptor, and to monitor the effects of this interaction. Materials and MethodsParasite Cycle and Preparation of Schistosomula. A Puerto Rican strain of S. mansoni was maintained in laboratory bred Biomphalaria glabrata and outbred Parkes mice, as described elsewhere (8).Schistosomula were prepared in vitro from cercariae .by a mechanical method (9). Briefly, cercariae freshly shed from snails were concentrated by addition of peniciUin-stroptemycin, followed by chilling and spinning at 1,000 rpm for 15-30 s. 1 ml of deionized water was added to the pellet and the suspension was whirled in a Vortex mixer (Scientific Industries, Inc., Bohemia, N. Y.) for 1 rain. This effected the rupture of tails from bodies, which were afterwards separated by sedimentation in Hanks' balanced salt solution. The cercarial bodies were then incubated at 37°C in RPMI-1640 (Flow Labs. Ltd., Ayrshire, Scotland) and 20 mM N-2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid (Hepes) ~ for 3 h. The schistosomula were used on the day of preparation. Formalin-fixed schistosomula were prepared as previously described (10).5-Day schistosomula were recovered from the lungs of CBA mice after exposure to 1,000 * Supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Brazil), and the WeUcome Trust. Present address:

A comparison was made of the ultrastructure, development and antigenic nature of the surfaces and of the viability of three types of schistosomula of Schistosoma mansoni: schistosomula formed afrer cercariae had penetrated isolated skin (SS), schistosomula produced after mechanical separation of cercarial tails from bodies (MS), and schistosomula transformed from cercariae after incubation in fresh rat serum (RS).Within 2 h of transformation, the surface membrane of all three types of schistosomula had changed from trilaminate to heptalaminate structures and SS and MS had lost their cercarial glycocalyx. Initially a dense amorphous material was demonstrated on the surfaces of RS, which was thought to be the result of an interaction between a factor in rat serum and the glycocalyx: this material was greatly reduced within 2 h of transformation. The pre-acetabular glands of SS were emptied while those of MS and RS retained their contents. Immunofluorescent studies showed that all schistosomula bound serum from mice immune to S. mansoni, but the binding was stronger with MS and RS. The mixed agglutination reaction demonstrated the presence of human A and B blood group-like antigenic determinants on approximately 30% of 3 h old SS; these determinants were not detected on MS or RS. In vitro, the development of MS and RS was similar to SS; the first schistosomula reached the ‘gut-closed’ stage by day 10; 50–70% of SS reached this stage by day 12, in contrast to only 25–50% of MS and RS. Between 28 and 45% of all schistosomula developed to maturity when injected intravenously into mice.It was concluded that the two types of artificially prepared schistosomula fultil the main criteria of transformation from cercaria to schistosomulum. Further, it is suggested that MS are the most appropriate source of material for immunochemical and physiological studies.

A French translation of the Memorandum will be published in a future issue of the Bulletin.