Schistosoma Mansoni: Characterization and Comparison of The Surfaces of Developing Schistosomula and Adult Worms

An animal Infected with schistosome worms is stimulated by antigen associated with the living, adult parasite to mount an Immunological reaction against the surface of invading schlstosomula. The adult worms, however, remain unaffected by the Immune response of the host and the. young schistosomula develop resistance to the host response during the 5 days in which they migrate from the skin to the lungs. The aim of the experiments described In this thesis was to compare the biochemical composition and antigenicity of the surface of the parasite at different stages of development from the cercarla to the adult, in order to understand more about how resistance to the host is acquired and maintained. The initial step was to establish a method for producing sufficient quantities of schlstosomula which were uniformly developed and uncontaminated by host material. Comparative studies were made of several types of artificially transformed schlstosomula and the prototype organisms, namely schistososaila resulting from cercarial penetration of isolated skin (SS). An organism (MS) produced by mechanically separating cercarial tails from bodies with subsequent incubation of the bodies in a defined medium was shown to undergo surface changes similar to those observed during cercarlae-SS transformation. Surface microvilli were produced in the 1st hour, the glycocalyx was lost within 2 hours, there was a change from a trilaminate to a heptalamlnata surface membrane by 2-3 hours and the inclusions sesn in the tegument were similar to those previously described in the SS tegument. Only one morphological difference was noted between MS and SS: the pre-acetabular glands of MS retain their contents for up to 48-72 hours while SS secrete the granular contents within 3 hours of skin penetration. MS were therefore used as the reference organisms In all further studies. Comparisons were made with cercarlae, the in vivo stages of in vitro schlstosomula (CS) and adult worms. Immunological techniques and reagents, i.e. immunofluorescence,125 I-Protein A binding, and radio-labelled myeloma proteins and antiglobulin were adapted to the study of living schistosomes. The use of triple-layer antibody techniques and the use of radioimmunoassays are considered to be significant Improvements over previous studies. A combined study of antibody class and subclass development and the binding capacity of the surfaces of the various organisms revealed differences both in the host response of different mice strains and in the quantity and distribution of antibody bound by the various schistosome stages. With identical cercarlal infections, Parkes mice produced da tec table levels of schistosome surface specific antibody by 10 days while antibody was not detected until 2-3 weeks after infection in CHA mice. IgM levels were shown to appear late and remained high throughout the course of the infection. IgG1 IgG2a, IgG2b, IgG3 and IgD levels were only slightly Increased while IgA levels were moderately Increased over that observed with normal mouse sera. Furthermore, the various schistosome stages differed in the binding of antlbody: cercariae, MS and SS bound significantly more antibody than did the later stages, namely, CS, LS and the adult schlatosomea. Only one morphological difference was noted between MS and SS: the pre-acetabular glands of MS retain their contents for up to 48-72 hours while SS secrete the granular contents within 3 hours of skin penetration. MS were therefore used as the reference organisms in all further studies. Comparisons were made with cercarlae, the In vivo stages of schistosomula collected from the skin (SS) and from the lungs (LS), the in vitro schlstosomula (CS) and adult worms. Immunological techniques and reagents, i.e. immunofluorescence, 125 I-Proteln A binding, and radio-labelled myeloma proteins and antiglobulin were adapted to the study of living schistosomes. The use of radioimmunoassays are considered to be significant Improvements over previous studies. A combined study of antibody class and subclass development, and the binding capacity of the surfaces of the various organisms revealed differences both In the host response of different mice strains and In the quantity and distribution of antibody bound by the various schistosome stages. With identical cercarlal infections, Parkes mice produced de tec table levels of schistosome surface specific antibody by 10 days while antibody was not detected until 2-3 weeks after Infection In CBA mice. IgM levels were shown to appear late and remained high throughout the course of Ota Infection. IgCj, IgG2a, IgC2b, IgG3 and IgD levels were only slightly Increased while IgA levels were moderately Increased over that observed with normal mouse sera. Furthermore, the various schistosome stages differed In the binding of antibody:cercarlae, MS and SS bound significantly store antibody than did the later stages, namely, CS, LS and the adult schistosomes. A phenomena observed during the inmunofluorescence studies was the sloughing of the fluorescent surface coat of MS as the organism moved across the microscopic field. Sloughing could be prevented at A°C; electron microscopy revealed no damage to the tegument, but microvilli were observed In the sloughing surface coat. Attempts to restaln "sloughed organisms" with Immune serum were unsuccessful. Sloughing may represent a mechanism for eliminating host antibodies but further studies are necessary to prove this. The use of radioimmunoassays revealed the adsorption of host Immunoglobulins onto the surface of all the schlstosomula stages. The use of lodlnated myeloma proteins and F(ab)2 fragments indicated that this adsorption was non-specific and did not require the presence of an Fc receptor. Schistosome synthesis of a mouse a2- macroglobulin-like determinant was confirmed using the artificially prepared MS. However,adsorption was also observed In studies using 125 I-Mo a2-macroglobulin. Correlations between parasite antigens and the presence of host antigens on the schistosome surfaces have been observed. The expression of human blood group-llke antigens and mouse erythrocytes antigens was studied by mixed agglutination, Immunofluorescence and immunoradloassay. With all schistosome stages the binding of specific antibody is relative to the amount of host material detected on the surface. A variety of biochemical techniques were used to compare the tegumental surfaces of cercarlae, schlstosomula and adults. These techniques Included lactoperoxldasa lodlnatlon and galactose oxldase- tritlated borohydrlde labelling of surface proteins and glycoproteins, followed by detergent solubilization and separation of components by polyacrylamide gel electrophoresis. Similarities and differences in the total proteins and surface components of the various schistosome stages were noted. The number of proteins obtained by 1° SDS-PAGE was between 45-55, which was considered minimal. The majority of these proteins were common to all stages, two of these proteins migrated at rates similar to actln and myosin. The actln-llke component was labelled by the lactoperoxldase. technique, Indicating that it is exposed to the external surface. The proteins unique to a particular developmental stage included a 160,000-180,000 molecular weight component of cercarlae, three components (58,000-60,000 daltons, 23,000 daltons and 11,000 daltons in adult schistosomes and a protein of about 29,000 daltons in adult females. Carearlas had 8 surface components, which were iodinated by the lactoperoxldase technique, 3 of these components were also labelled by the galactose oxidase- 3H borohydrlde procedure. MS and SS shared 8 lactoperoxldase labelled surface components, 3 of these were similar to cercarlal surface components. Two surface components were also labelled by the galactose oxidase- 3H borohydrlda procedure. LS had 7 components which wars iodinated by the lactoperoxldase procedure; 3 components are also labelled by tritium. CS possess 9 lactoperoxldase labelled components, three of which were tritiated. Adult schistosomes had 8 iodinated surface components, 3 of which were labelled by tritium. It Is concluded that the schistosomes maintain a complex Interaction of acquisition, elimination and mimicry of host antigenic material and thus evade the action of the host Immune response. It is suggested that the development of these evasive mechanisms occurs during the period between skin penetration and migration through the lungs.