The occurrence of multiple-clone Plasmodium falciparum haploid blood-stage infections is a pre-requisite for cross-fertilization and genetic exchange at the diploid stage in the mosquito. Using monoclonal antibodies against 3 polymorphic blood-stage antigens, a method of two-colour differential immunofluorescence allowed the resolution of between 1 and 4 clones/isolate. A mean of 2 P. falciparum clones was detected in the blood of malaria patients in The Gambia. The mean number of clones/patient showed no correlation with age, parasitaemia, or disease severity. There was a slight difference in mean number between sample periods, probably reflecting temporal differences in transmission intensity. A statistical analysis of 2-locus genetic diversity of clones within isolates concludes that not all multiple-clone infections result from superinfection, but that some are due to single multiple-clone inoculations.

Serotyping with monoclonal antibodies was used to estimate the number and frequencies of allelic variants of two merozoite surface proteins, MSP1 and MSP2, and an exported protein Exp-1, in a sample of 344 clinical isolates of Plasmodium falciparum from an urban region of The Gambia. Represented among the isolates were 36, 8 and 2 alleles of the MSP1, MSP2 and Exp-1 loci respectively. Relative frequencies of these alleles remained stable in the parasite population over the 2 years of the study. A computer program was used to calculate from the frequencies of individual alleles at the three loci, the probable number of different genotypes in samples from the population, assuming random assortment among the loci. No significant difference was found between the expected and the observed genotype diversity. It is concluded that recombination among unlinked loci is a common consequence of sexual reproduction of P. falciparum in The Gambia. Slightly lower genotype diversity was observed in each of two villages, which may be a consequence of smaller population size compared with the urban region.

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages of Plasmodium berghei in cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in ‘mature’ cultures. These did not represent a distinct population, but the ‘tail’ of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigens de novo in the invaded hepatoma cell. A comparison of the reactivity of text-abstract EE parasites with anti-circumsporozoite antibody and with anti-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate.

Human neutrophils were infected with tachyzoites of Toxoplasma gondii in vitro. Infection rates after 1 h of incubation were 38·6–52·0% and 16·0–25·8% in the presence or absence of specific antibody respectively. Apparently, neutrophils killed tachyzoites 18 h after infection in the presence of antibody, whereas tachyzoites proliferated in 25·0–35·0% of infected neutrophils in the absence of antibody. Total tachyzoite counts 18 h after infection were approximately 4 times as high as those 1 h after infection in the absence of antibody and complement. Complement also enhanced phagocytosis and killing of tachyzoites by neutrophils, but the effects were less than that of antibody. Superoxide anion was produced intensively within 1 h after infection, depending on the presence of antibody or complement. Neutrophils seem to be responsible for dissemination of tachyzoites in the host during the initial phase of Toxoplasma infection until antibody is produced.

Neutrophil chemotactic activity was detected in the fluid of Toxoplasma gondii cultures by the agarose plate and the Boyden chamber methods. Toxoplasma culture fluid was obtained by incubating the tachyzoites at 37 °C in a 5% CO2 atmosphere for 6 h in Dulbecco's modified Eagle's minimum essential medium containing 10% heat-inactivated foetal calf serum. Soluble extracts from tachyzoites had negligible activity, indicating that the chemotactic factors were metabolites secreted from tachyzoites. The chemotactic activity in Toxoplasma culture fluid depended on the number of tachyzoites and the period of incubation. The substances responsible for neutrophil chemotaxis were two heat-labile peptides with estimated Mr 4·5 and 14 kDa with N-terminal groups free.

Infection with Giardia lamblia often causes only minor mucosal changes to the small intestine yet frank fat malabsorption may still occur. Some evidence suggests abtext-abstract pancreatic exocrine function in subjects with giardiasis although the mechanism and significance of this is unclear. Studies were conducted in vitro to determine the effect of G. lamblia trophozoites or culture filtrates from the organism on lipolysis of triglyceride by porcine pancreatic lipase. Live trophozoites significantly inhibited lipolysis. The degree of inhibition increased with longer duration of lipase exposure to trophozoites. Total amounts of enzyme inhibited were proportional to enzyme concentration, while the percentage inhibition was greatest at lowest concentration. At a lipase concentration of 1·7 i.u./ml, enzyme activity was reduced by 89·7% compared to controls after incubation for 4 h with trophozoites. The effect was abolished using killed, intact trophozoites. Culture filtrates of G. lamblia did not inhibit lipolysis. Specificity of the effect was suggested by the failure of another flagellate protozoan, Trichomonas vaginalis, to inhibit lipase. In this assay system the inhibition of lipolysis was not dependent on the bile salt concentration present. The impact of this effect in vivo remains to be determined but it may contribute to fat malabsorption in giardiasis.

A monoclonal antibody (MAb G11) species-specific to the Em2 antigen of Echinococcus multilocularis was generated for (i) further biological characterization of the Em2 antigen, (ii) easy affinity-purification of Em2 antigen for immunodiagnostic and immunological investigations and (iii) development of a sandwich-ELISA for the detection of Em2 antigen in diagnostic samples and thus species-specific identification of E. multilocularis metacestode material. The MAb G11 was used in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E. multilocularis antigen/ml of solution. MAb G11 specifically detected Em2 antigen in all of 15 E. multilocularis-isolates originating from various geographical areas and in none of other helminth isolates (e.g. Echinococcus granulosus, E. vogeli, and others). Further biological analysis by FITC-labelled MAb G11 demonstrated unique binding activity to the laminated layer of the metacestode. Also, oncospheres were binding FITC-labelled MAb G11 on an outer layer synthesized during cultivation in vitro for 13 days after hatching. Application of the MAb G11 antibody sandwich-ELISA for investigation of solubilized oncospheres confirmed the in vitro synthesis of Em2 antigen by oncospheres on day 13 p.i. Adult stages (somatic antigens) and freshly hatched oncospheres were always MAb G11 negative. Solid-phase MAb G11 was used for purification of the corresponding Em2 antigen by affinity chromatography. A preliminary serological evaluation of the Em2(G11) antigen by ELISA revealed identical immunodiagnostic characteristics, compared to Em2 obtained by classical means, thus suggesting the presented method for future isolation of large-scale Em2 antigen.

Miracidia and in vitro-derived primary sporocysts of the avian schistosome Trichobilharzia ocellata were studied for the expression and the characteristics of glycoconjugate moieties comprising the surface coat. Using a panel of 9 peroxidase labelled lectins, several different lectin binding sites were demonstrated on the larvae. Fixed miracidia have binding sites for 7 of the lectins; wheat-germ agglutinin binds to both the ciliated plates and the tegumental ridges between them; the other 6 lectins bind to the plates only. Three of the miracidia-binding lectins, wheat-germ agglutinin, concanavalin A and peanut agglutinin, also bind to fixed sporocysts. Since the miracidial ridges are devoid of binding sites for concanavalin A and peanut agglutinin, whereas the sporocyst tegument binds these lectins, it appears that these sites become exposed during or shortly after transformation. In saturation experiments, low concentrations of peanut agglutinin and concanavalin A are bound more avidly by sporocysts than by miracidia, indicating a higher binding affinity of the former. The two larval forms do not differ in affinity for wheat-germ agglutinin but they have different binding capacities; when offered in high concentrations, more of this lectin is bound by sporocysts than by miracidia. Lectin binding was competitively inhibited by adding the appropriate free saccharides. Live larvae showed the same lectin binding pattern as did fixed specimens. Proteinase treatment reduced lectin binding to living and, to a lesser extent, to fixed larvae, suggesting that binding sites are constituents of proteoglycoconjugates. After SDS–PAGE of extracts from miracidia and sporocysts and subsequent Western blotting, some of the lectins failed to bind glycoproteins, others reacted with an array of bands. The patterns differed among the lectins and each lectin gave different patterns for miracidia and sporocysts. The results obtained with these two lectin-binding techniques support the conclusion that stage-specific proteoglycoconjugates occur at the surface of T. ocellata larvae.

Lysozyme secretion from macrophages of Schistosoma mansoni-infected mice was time dependent, rising significantly from the 8th week post-infection, the macrophages thereafter exhibiting very high levels (> 90%) of schistosomulicidal activity. Despite the ability of lysozyme to kill schistosomula in vitro, the concentrations required for such killing were several hundred-fold to several thousand-fold higher than those detected in the supernatants from infected-mice macrophages cultured with or without schistosomula. An in vitro lysozyme inhibitor, N, N, N-triacetyl chitobiose, did not abrogate the cytotoxic ability of macrophages from schistosome-infected mice, but an inhibitor of arginine-dependent cytotoxicity, NG-monomethyl arginine, markedly inhibited schistosomulicidal activity. Evidently, concentrations of ambient lysozyme from macrophage cultures are too low to affect schistosomula in culture, while the main schisto-somulicidal pathway in vitro seems to be arginine dependent.

A footpad assay was used to measure the DTH of mice to soluble worm antigens (SWAP), and to living day 7 lung schistosomula, following vaccination and challenge infections with Schistosoma mansoni. DTH to SWAP was first observed on day 10, and reached its maximum on day 17 post-vaccination. Treatment of mice with anti-CD4 antibody on the 3 days prior to footpad challenge completely abrogated this response. Reactivity to living parasites was of a lower order than that to SWAP; it also peaked earlier, on day 10 post-vaccination. By day 35, responsiveness to both sets of antigens had declined almost to control levels. There was no correlation between the level of DTH to living schistosomula, at any time, and the degree of resistance subsequently developed. Percutaneous challenge of vaccinated mice was followed by a resurgence of reactivity to SWAP. This secondary response occurred more rapidly than the primary response, peaking on day 7 post-challenge, and was of a similar magnitude. We were unable to detect a similar recall of DTH to living schistosomula, possibly because the assay was insufficiently sensitive. We conclude that the intensity and kinetics of DTH responsiveness are crucial features of the irradiated vaccine model, and suggest that further investigation of cell-mediated immune reactions, particularly those occurring in the lungs, is vital to a better understanding of events underlying the development and expression of immunity.

The routes of penetration and the strategies of invasion of Schistosoma japonicum miracidia in the snail vector Oncomelania hupensis were observed in a histological study. In all species of the genus Schistosoma, it is usually assumed that the miracidia achieve penetration through the tegument. Our results showed that at least 57% of S. japonicum miracidia penetrated the snail by natural openings (branchial cavity, mouth and rectum). Throughout the invasion phase, the larvae were observed in all the tissues and organs with the exception of the genital gland. The spatial distribution of parasites in the snail revealed that the migration towards the visceral organs such as the kidney, heart and sinuses (which are the most usual microhabitats of the mother sporocysts of S. japonicum) appeared to take place via the circulatory system. Using natural openings as routes for penetration probably provides a selective advantage in a host–parasite system in which the target mollusc is amphibious: we presume that the miracidia inside these natural openings are protected against desiccation when the snail leaves the water, and that they can subsequently invade the tissues.

Adult Litomosoides carinii, recovered from cotton rats (Sigmodon hispidus) 4–5 months post-infection (p.i.), were cultivated in vitro with emphasis on investigations into the development of intra-uterine embryonic stages. Baseline values for the embryonic status of female worms were established immediately after recovery from the hosts. In such females, on average, 16% of the intra-uterine stages were fully formed microfilariae while the remainder belonged to the early embryonic classes that were characterized. For the evaluation of culture success, apart from survival of worms in vitro, the rate of microfilariae development (mf rate) served as a major parameter. Of the five basic culture media RPMI 1640, F12, L15, NCTC 135, and IMDM, tested without supplementation, RPMI 1640 yielded by far the best results (survival = 14 days; mf rate = 41%), and was therefore chosen as the routine medium. In comparison with 5% CO2 in nitrogen, a gas phase of 5% CO2 in air was superior, although the resulting oxygen tension of 138 mmHg in the medium was not physiological. Addition of 10% newborn or foetal bovine serum to the medium in some cases distinctly influenced results. Effects of different batches of sera varied from ‘filaricidal’ to ‘very promoting’. Co-cultivation of worms and Sigmodon cells, or rhesus monkey LLCMK2 cells, only marginally improved results. Of the serum substitutes Ultroser G, BMS, and Clex, the latter had a moderately promoting effect. The protein supplements bovine serum albumin, transferrin and haemoglobin significantly improved results, and could replace certain batches of serum. Supplementation with the haemin moiety alone was less effective than with haemoglobin. The anti-oxidants glutathione plus ascorbic acid proved beneficial in combination with a serum supplement only. Results from other experiments with multiple supplementation also suggest that various supplements may act only in a synergistic manner. The longest average time that adult L. carinii survived in vitro was 3–4 weeks. The highest mf rate was 78%, which indicated that all text-abstract embryonic stages present in the uteri of a female at the start of culture completed their development to microfilariae, however, oogenesis ceased in vitro. The parameters for embryonic development employed proved to be highly sensitive for the judgement of various culture conditions.

This paper describes patterns of infection with Opisthorchis viverrini within a human community assessed by egg count, parasite-specific antibody levels and worm burden determined by expulsion chemotherapy. All three measurements increased significantly with age, reaching a plateau in young adults. There were no significant sex-associated differences in the prevalence or intensity of infection, antibody levels, or in the frequency of heavy worm burdens. A striking 81·5% of the total Opisthorchis population and 74% of the total egg output were expelled by the most heavily infected 10% of the humans sampled. This extreme over-dispersion, together with recent findings that hepatobiliary disease frequently accompanies heavy infections, argues that the priority for community-based parasite control programmes should be to maximize treatment and health promotion efforts toward heavily infected individuals.

The mature thread-like spermatozoon of Paragonimus ohirai was shown to be composed of 7 distinct segments which have morphologically different characteristics, following staining with silver nitrate. The first segment (1S) is a needle-shaped structure with a perforatorium. The 2S is composed of two filaments loosely paired; 3S consists of a somewhat coarse thread (vestige of the cytoplasmic projection); 4S is composed of two flagella tightly paired; in 5S, two silver-positive filaments are independently seen as in 2S; 6S has a ‘spiral’ structure composed of two flagella; in 7S, two filaments are seen dissociated from each other. The sperm nucleus exists within the ‘spiral’ structure of 6S and this structure may serve to hold and/or protect the nucleus. The technique presented here is useful for gross morphological observations of the long sperm of flatworms.

Pseudodiplorchis americanus has an extremely brief opportunity for transmission, restricted to less than 24 h in the year when the host (Scaphiopus couchii) enters water to breed. This strict annual cycle means that invading worms have 1 year to complete reproductive development. Despite this, a large proportion of the parasite suprapopulation is not prepared for transmission at the time of host breeding. The present study correlates detailed soil temperatures for one field site in S.E. Arizona, spanning 3·5 years, with laboratory data on parasite growth and reproductive development at a series of controlled temperatures. Development is totally inhibited at 16°C. Optimal growth and development occurs at 25°C and is slower at both 20°C and a diurnal cycle fluctuating between 20 and 34°C (mean 27°C) (simulating summer temperatures experienced by host and parasite). The effective period for accumulation of infective stages by P. americanus in any one season varies according to the timing of the summer rains in consecutive years. Between 1985 and 1988, the interval between host breedings varied from 11 to 13 months and the period when temperatures were above 20°C varied from 4 to 5·5 months per year. Since the first month post-infection (p.i.) consists of pre-reproductive development, first-year worms have only 3–4·5 months to produce infective larvae. In the shortest seasons, these worms may not be able to complete reproductive preparation in time for the first opportunity for transmission. In different parts of the geographical distribution of S. couchii, different cycles of temperature, rainfall and host breeding occur; varying temperature regimes represent an important abiotic control of P. americanus reproductive biology and transmission.

An experiment was designed to investigate aspects of the population dynamics of acquired immunity to Heligmosomoides polygyrus in laboratory mice. The influence of host strain (CBA or NIH), rate of exposure (5 or 40 L3/mouse/2 weeks) and diet (3 or 16% protein w/w) on the population dynamics of repeated infection and the response to a standard challenge infection were investigated. The time delay between the end of the period of repeated infection and the subsequent challenge (between 1 and 24 weeks) had no effect on worm recovery. The effects of both exposure and diet were significant and similar whether assessed on the basis of the dynamics of repeated infection or response to challenge: low rates of exposure and low dietary protein were both associated with low levels of acquired immunity. Mouse strain was the most important determinant of worm recovery after challenge, but had no significant effect on the degree to which parasite population growth was constrained by acquired immunity during repeated infection. It is suggested that both CBA and NIH mice raise immune responses which act on parasite survival, but that only NIH mice raise responses operative against larval establishment.

Twelve populations of bats were examined to determine the extent of interspecific associations in determining the species richness of intestinal helminth infracommunities. The pool of helminth species which was available to individual bats ranged from 2 to 21. The ‘summed binomial’ distribution was determined to underlie the host frequency distribution of the number of helminth species per host. Overall covariation in occurrences of species in replicated communities can be detected by testing for the equality of the observed variance of the host frequency distribution to the variance expected when species are allocated to hosts at random. Where statistically significant the covariance was indicative of a majority of positive rather than negative interspecific associations. As the mean number of species per host in a host population increases not only does the number of positive associations increase but so does the proportion of species pairs which exhibit positive associations. Although there is an increase in the proportion of species pairs which exhibit positive associations as the number of species increases, the magnitude of the associations (as indicated by the mean positive or the mean negative pairwise covariances) does not. Therefore, we concluded that positive interactions are more common than negative interactions in determining the species richness of helminth infracommunities of bats. Further, positive associations become even more important as the community becomes more complex. However, the increased importance is derived from the number rather than the strength of the associations.

Taenia multiceps coenurus fluid was analysed by fast protein liquid chromatography in order to separate the factors responsible for previously reported modification of immunological activity in macrophages and T-cells. One factor, F7, was found to be mitogenic for murine L3T4+ T-cells, to be macrophage dependent, to require macrophage compatibility at the I region of the H2 complex, to increase the sensitivity of T-cells to regulatory signals from macrophages and to increase the rate of generation of splenic rosette-forming cells (RFC) against sheep red cells. A second factor, F24, was found to alter macrophages so as to render them suppressive, rather than stimulatory, for parasite-activated and Con A-activated lymphocyte transformation, to depress the rate of generation of RFC and to antagonize the mitogenic effect of F7. The combined actions of these two factors are, therefore, sufficient to explain the known immunomodulatory effects of the metacestode.

The behaviour of Heterodera schachtii second-stage juveniles in response to mustard (Sinapis alba) rooxudates was observed and analysed under aseptic conditions in a standardized bioassay. Aggregation of juveniles on an agarose layer occurred within less than 30 min in the area where root exudates had been applied and persisted for several hours. Analysis of time-lapse video recordings showed that the aggregation did not result from a directed orientation of the juvenile towards the root exudate. This was supported by an orientation assay using single juveniles. Aggregated juveniles showed pre-infection exploratory behaviour, including stylet thrusting and head-end bending, while staying at rest for several minutes.

The course of primary infection was studied in BALB and B10 H-2 congenic mouse strains. The duration of infection, as assessed with regular faecal egg counts and worm burdens, was shorter in mice carrying the H-2s, H-2d or H-2q haplotypes when compared to mice with H-2b. Strains with H-2k were intermediate. An experiment was carried out to test the hypothesis proposed by Wassom, Krco & David (1987) predicting that the progeny of I–E+ve mouse strains crossed with I–E-ve strains, would show susceptibility rather than resistance to infection. This hypothesis was not substantiated by our data and we conclude that it does not apply to primary infections with Heligmosomoides polygyrus. It is proposed that the gene products of at least two loci within the H-2 (associated with the H-2b and H-2k haplotypes) are crucial in determining the response phenotype of mice to primary infection with H. polygyrus. One allele, (associated with the H-2b haplotype) may be preferentially affected by parasite-mediated immunosuppression.