C57BL6 mice were protected against Plasmodium berghei sporozoite challenge by immunization with live 12 krad dose-irradiated sporozoites, but not by 20 krad dose-irradiated sporozoites. Immunization with 12 krad irradiated sporozoites generated low levels of antibody reactive to liver-stage parasites (titres of 1/100). Inoculation of as few as 100 live P. berghei sporozoites induced complete host protection accompanied by a very quick and high boost of antibody titres up to 1/4000. This sporozoite challenge-drive antibody boost was absent in mice immunized by 20 krad sporozoites and in non-protected, and non-immunized mice. Antibody was mainly liver-stage (LS) specific and due to an increase of IgG2a and IgG2b. The in vitro effect of pre- and post-challenge sera upon either sporozoite invasion or LS development was assessed in Hep-G2 cultures. Both were found to have a strong effect upon LS development even at 1/2500 dilution, and conversely a low effect upon invasion. These results suggest that sporozoites irradiated at doses that induce protection are able to prime T-cells which, upon challenge by non-irradiated sporozoites, provide help to B-lymphocytes to trigger the production of high titres of anti-LS antibodies that can inhibit LS development in vitro.

The development of Sarcocystis falcatula merozoites in bovine turbinate (BT) cell cultures is described and compared with development of Sarcocystis neurona merozoites. Merozoites of S. falcatula entered BT cell cultures and increased in size until 3 days post-inoculation when the nucleus of some merozoites developed lobes. Developing schizonts present at 4 days contained a lobed nucleus or appeared multinucleate. A single mature schizont was observed 4 days p.i. Schizonts were numerous 5 and 6 days p.i. Merozoites were produced from blastophores on the schizont. S. neurona merozoites developed to mature schizonts by 3 days p.i. in BT cells and a residual body was often present. Transmission electron microscopy revealed that S. falcatula merozoites possessed more micronemes than did S. neurona merozoites. Our study demonstrates that S. falcatula and S. neurona are not the same parasite.

The affinity of merozoites of Sarcocystis singaporensis obtained from the lungs of acutely infected rats to muscle cells and other cell lines grown in vitro was examined. Two distinct types of mature schizonts developed in the lungs 11–13 days p.i. with sporocysts: those containing PAS− merozoites (type 1) which mainly reacted with antibodies prepared against sporozoites, and others containing PAS+ merozoites (type 2) which were antigenically close to bradyzoites. When inoculated onto cell cultures, type 1-merozoites induced schizogonic development in brain capillary endothelial cells of the rat. In contrast, type 2-merozoites invaded L6 myoblasts. In long-term cultures (50 days) of L6 cells, zoites transformed to a 8–15 μm long uninucleate stage which, tentatively, could be unizoite sarcocysts. Although the observed dichotomy in merozoite development is unprecedented in this form, evidence from previous work suggests that these observations are relevant to other Sarcocystis species. The presented cell culture system could be a first step towards successful growth of sarcocysts in vitro.

Polysporoplasma sparis infection was studied in gilthead sea bream from different mariculture systems of the Spanish coasts. Culture conditions influenced the infection dynamics, as the parasite appeared only in semi-intensive cultures and was not found in intensive closed systems nor in open ones. No clear seasonal pattern was observed. No fish weighing less than 51 g was found parasitized in any group. A statistically significant dependence between infection prevalence and host weight was observed in some growing stocks. Light and transmission electron microscope observations revealed serious damage in the trunk kidney. Glomerular disease was provoked by the progressive occupation of the glomerular capillaries by P. sparis spores. Tubular epithelial cells were also affected. Inflammatory responses appeared towards the end of the infection, and consisted mainly of melanomacrophages and eosinophils. Rodlet cells were common close to infected capillaries and debris of rodlet sacs formed a belt encircling capillary vessels. Cytochemistry demonstrated the lipidic nature of these sacs and the glycogen and glycoprotein composition of the cytoplasmic granules of rodlet cells.

Community data on the prevalence of helminth infections is important for guiding health policy, but expensive to collect. As a result most surveys focus on school-aged children, frequently using schools as a sentinel population. Since there already exists a vast amount of data on infection levels in school-aged children, but limited community-based data, we undertook a literature search on age-stratified infection data for intestinal nematode infections and schistosomiasis in Africa, to investigate whether estimates of the prevalence of infection in school-aged children could provide an index for determining community prevalence. The observed data on prevalence of infection in infants, school-aged children and adults were fitted using linear and logistic regression models which take into account variation in sample prevalences. Despite the wide variation in study sites, the observed relationship between community prevalence and school-aged prevalence was remarkably consistent for each parasite species. The prevalence of infection in school-aged children alone was shown to be higher than the predicted prevalence in the community, but the degree of overestimation was dependent on the parasite species and the level of infection. The results suggest that the prevalence of infection in school-aged children could provide a cost-effective predictive tool which can be used at a district/national level to identify target areas for control and to evaluate the numbers at risk of infection.

Although eosinophilic bar- or droplet-like inclusions are frequently detectable inside eggs deposited in the livers of Schistosoma japonicum-infected animals, little is known of their exact nature. In the livers of mice implanted with freshly laid eggs, inclusion-positive eggs were found in 28·7 and 46·2% of deposited eggs at 2 and 4 weeks, respectively, after implantation, but in 4·3% at 5 weeks when most of the eggs had already degenerated. When the extent of granuloma formation was investigated, granulomas around inclusion-positive eggs were smaller than those around negative eggs. Host factors associated with the formation of inclusion were sought using in vivo and in vitro studies. Following the administration of anti-egg antigen serum into egg-implanted mice, no increase in occurrence of inclusion-positive eggs was seen. In a co-culture of mature eggs with infected rabbit or mouse serum, inclusions were rarely found. In contrast, they were found in 17·9% of eggs in the presence of splenic cells. The present study is the first to show that there is decreased granuloma formation in the presence of eosinophilic inclusions inside eggs and our in vitro study suggests that host cell–egg interaction is responsible for the formation of inclusions.

Soluble extracts of adult Schistosoma japonicum and S. mansoni were examined for the presence of proteolytic activities ascribable to dipeptidyl peptidases (DPPs) at a range of pH from 4 to 11 using synthetic peptidyl substrates diagnostic of DPPs I, II, III and IV. Activity capable of cleaving the DPP I-specific substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec which exhibited a pH optimum of 5·5 was observed in extracts of schistosomes. Female schistosomes exhibited greater DPP I activity than male schistosomes, while female S. japonicum showed substantially more activity than female S. mansoni. The specific activities against H-Gly-Arg-NHMec were 21·5 and 1·9 nmoles NHMec/min/mg protein for female and male S. japonicum and 8·5 and 1·9 nmoles NHMec/min/mg for female and male S. mansoni. The biochemical properties of schistosome DPP I were similar to mammalian DPP I (=cathepsin C) in that schistosome DPP I was only slowly inhibited by the cysteine protease inhibitor trans-epoxysuccinyl-1-leucylamido (4-guanidino)- butane, partly inhibited by the blocked diazomethyl ketones Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2, but enhanced by halide ions. At pH 8·5, activity against the DPP III-specific substrate H-Arg-Arg-NHMec was evident in schistosome extracts, and this activity appeared to be due to a zinc metallo-exopeptidase because it was inhibited by 1,10-phenathroline and by EDTA. DPP II or DPP IV activity was not detected in the schistosome extracts.

Oesophagostomum bifurcum, as well as hookworm infections are hyperendemic among humans in northern Togo and Ghana. For parasite-specific diagnosis a coproculture is obligatory, because only the infective larvae, and not the eggs, can be distinguished morphologically. The sensitivity of duplicate coprocultures from a single stool sample was found to be above 90% in comparison to a gold standard of 10 coprocultures made from a single stool specimen. Prevalence of infection with O. bifurcum and hookworm further increased with the number of coprocultures made from each individual stool. Notwithstanding the high sensitivity, intensity of infection per individual varied considerably from day-to-day and the number of larvae found in different samples out of 1 stool also varied highly, both showing a heterogeneous distribution. Surprisingly, daily fluctuation and within-specimen variation could not be differentiated from each other, probably because of the variation created by the coproculture technique. To estimate the intensity of infection, it is sufficient to make repeated coprocultures from only 1 individual stool sample. Laborious collection of stool samples on subsequent days does not give better estimates of the individual infection status.

Density-dependent effects on adult and larval stages of the introduced nematode parasite Anguillicola crassus in the European eel definitive host were examined in the laboratory in naturally infected fish, and in field samples. The effect of adult nematode subpopulations on larval development over time and the effect of increasing adult intensity on the gravid female subpopulations were investigated. At high adult subpopulations the movement of larvae from the swimbladder wall into the swimbladder lumen appears to be inhibited, and A. crassus larvae to be arrested in development in a density-dependent manner. The number of gravid females per host reaches a constant level, and so the proportion of gravid female nematodes per adult subpopulation decreases relative to further increasing total adult nematode numbers. Both these mechanisms have the potential to regulate infrapopulations of A. crassus within the eel definitive host and thus the parasite suprapopulation.

Vaccination of lambs with the membrane-bound (S3) thiol-Sepharose binding protein (TSBP) fraction derived from the gut of Haemonchus contortus confers significant protection against homologous challenge. The S3 TSBP peptide profile is dominated by a major protein of ca. 60 kDa which is strongly recognized by antisera from sheep demonstrably protected following immunization with S3 TSBP. In an attempt to identify this protein, sera from protected lambs were employed to screen a λgt11 cDNA library of the adult parasite and resulted in the isolation of numerous clones encoding a homologue of the mitochondrial enzyme, glutamate dehydrogenase (GDH). GDH enzyme activity was readily demonstrable in S3 TSBP material and immunolocalization studies showed that the enzyme was localized to the cytoplasm of the parasite's gut. Furthermore, the enzyme appeared to be developmentally regulated, with both GDH mRNA and protein expressed almost exclusively during the blood-feeding parasitic stages.

A recombinant cysteine protease inhibitor, onchocystatin of the parasitic nematode Onchocerca volvulus, was tested for its role in microfilarial development in the simuliid vector. Onchocystatin was found to be present in female adults and skin microfilariae of the bovine parasite O. ochengi, the closest relative of O. volvulus. In addition the inhibitor could be detected as an excretory–secretory (E–S) product of the microfilariae. Co-injection of onchocystatin and the O. ochengi microfilariae into the surrogate vector Simulium ornatum s.l. significantly enhanced the recovery rates of the parasite within 24 h into the infection (P>0·001). The findings suggest a possible role of onchocystatin in the evasion by the parasite of the immune response of its vector.

Phosphorylcholine (PC) is a common constituent of proteins secreted by filarial nematodes. As this substance has been shown to interfere with immune responses, we are interested in designing strategies for blocking its attachment. Towards this end, we are investigating the mechanism of incorporation of PC into filarial molecules and in the present manuscript we describe experiments relating to elucidating the source of PC for attachment. Synthesis of phosphatidylcholine in eukaryotic organisms can occur by a mechanism involving the transfer of PC from CDP-choline to diacylglycerol (the Kennedy pathway). By (i) measuring transfer of radio-isotope labelled PC from CDP-choline to parasite molecules and (ii) employing inhibitors of CDP-choline synthesis, we have investigated whether CDP-choline can act as a source of PC for transfer to ES–62, a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae. Although we can find no evidence of this, we show that attachment of PC is blocked by hemicholinium-3, an inhibitor of choline kinase, the first enzyme in the Kennedy pathway. Thus, at least the first step in this pathway – phosphorylation of choline, would appear to be necessary for attachment of PC to ES-62.

Murine antibodies, raised against a purified recombinant 30 kDa laminin-binding protein from Echinococcus granulosus, were used to investigate the tissue distribution of the native protein in protoscoleces and brood capsules. Immunofluorescence, in combination with confocal microscopy, revealed that the protein was distributed in small annular foci near the peripheral regions of the protoscoleces. Immunoelectron microscopy of thawed cryosections demonstrated that the laminin-binding protein was present in the cytoplasm of tegumentary cytons and myocytons, but not in cells of the excretory system. The protein was associated with amorphous regions of the cytoplasm, and was not expressed at the surfaces of cells. This distribution resembles those of other invertebrate laminin-binding proteins, which are thought to act in the cell cycle and cell proliferation events. A low degree of label was consistently detected in extracellular matrices of the protoscolex.