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Unusual bodies have been described in the hypodermal tissues of larval Dirofilaria immitis and Brugia pahangi. Ultrastructural evidence indicates that these bodies are probably Gram-negative micro-organisms. It appears that the presence of large numbers of these bodies in an early embryo may affect development adversely. Their importance at later stages of development of filariae is not known.

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:

Schistosomes grown in mice were tested at different stages of development for susceptibility to an in vitro cytotoxic effector mechanism involving eosinophils and an antibody directed against mouse determinants. Despite the fact that 5-day lung worms and 6-week adult worms both bound the antibody to their surfaces, eosinophils attached preferentially to the adults and killed them. Complement had an enhancing effect in this system. Those eosinophils which did adhere to the lung worms degranulated onto the tegument but were unable to mediate damage or killing, even when complement was activated at the parasite surface. The resistance shown by the lung worms was shared by 2-week worms and small 3-week worms. Larger 3-week worms and older stages were, however, susceptible to cell-mediated cytotoxicity in this system. We suggest that the host antigen disguise constitutes the major protective mechanism utilized by older schistosomes to evade immunity, but that the younger stages have an additional and equally effective mechanism of resistance.

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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.

Purified eosinophil and neutrophil cationic proteins isolated from the lysosomal secretion granules of human granulocytes, evoke characteristic, dose-dependent morphological changes in young schistosomula of S. mansoni. The first sign of damage is seen with in 15-30 min of incubation and involves the formation of surface microvilli and blebs. Subsequently, tegumental evaginations of varying size are developed, but these appear to explode with rapidity, so that lengths of expanded tegumental outer membrane are deposited over the severely damaged surface of the parasite. Both types of granulocyte proteins are able to effect comparable damage at equimolar concentration. Other cationic proteins such as protamine and poly-L-arginine also damage the parasite surface but the pathological changes differ from those induced by the granulocyte proteins and they take longer to develop. In contrast, lysozyme-treated parasites are virtually similar to control schistosomula incubated in medium alone. These findings are discussed in relation to published data concerning the interaction of intact granulocytes with young schistosomula both in vitro and in vivo.

Young schistosomes collected after penetration through isolated mouse skin (3 h schistosomula) were cultured in medium containing immune rhesus monkey serum with a high titre of antibody known to kill schistosomula in the presence of complement. Morphological signs of damage in electron micrographs were confined to the surface tegument of the schistosomula. Antibodies in immune rhesus serum were shown to bind to the surface membrane of 3 h schistosomula using an antibody-enzyme bridge technique involving labelling with horseradish peroxidase and histochemical localization of the enzyme at the ultrastructural level. Schistosomula recovered from the lungs of mice 4 days after infection did not bind monkey antibodies at the surface and these 4-day schistosomula are not susceptible to damage by immune serum in vitro. Mouse erythrocyte antigens were detected on the surface of 4-day schistosomula using an appropriate antibody-enzyme bridge but these host antigens could not be found on 3 h schistosomula. This correlation between the presence of mouse host antigens on the surface of schistosomula and the inability of immune monkey antibodies to bind to the surface membrane is consistent with the hypothesis that host antigens are acquired by young schistosomes and serve to protect the surface membrane against antibody-mediated damage.