A number of authors have demonstrated that the schistosomicidal compound, Praziquantel (Pzq), depends for its action upon the immune status of the host (Sabah et al. 1985; Brindley & Sher, 1987; Doenhoff et al. 1987). We have attempted to define the synergistic interaction between immuno- and chemotherapy further, using the murine irradiated vaccine model of schistosomiasis mansoni. In vaccinated mice, resistance operates in the skin but not the lungs; drug targeted towards lung-stage worms exacerbates lung-phase immunity, however, as depicted by the increased number and size of inflammatory reactions in the pulmonary tissues. Parasites are often found trapped within such foci. In the present investigation, light and ultrastructural studies have been utilized to examine the nature and extent of damage inflicted upon lung-stage larvae recovered from day 6 Pzq-treated vaccinated mice. Such studies have revealed that damage involves muscle disorganization, internal disruption and occasionally, loss of the tegument; in the latter case, cells are often seen attached to the denuded lung worms. To identify the crucial cellular effector cell(s) involved in the synergy between immuno- and chemotherapy, cell depletion studies have been performed in vivo. It would appear from these experiments that eosinophils or lymphocytes rather than neutrophils or macrophages are important effector cells in this synergy. Histological studies argue in favour of eosinophils being the key effector cells.

The major glycoprotein of the sheath of Litomosoides carinii microfilariae (gp22) was analysed for its amino acid and amino sugar composition. It is rich in proline, glutamine/glutamic acid and glycine and contains (N-acetyl)galactosamine. The N-terminal amino acid sequence was determined up to position 37. It consists of a group of 6 repeats of the pentapeptide sequence methionine-glycine-proline-glutamine-proline with two minor modifications in repeats 3–6, while the first two repeats follow the general pattern more loosely. Identical N-terminal amino acid sequences were found in at least two other sheath polypeptides (33 kDa, 39 kDa). Antisera prepared against 3 overlapping synthetic peptides corresponding to the amino terminus of gp22 recognized different epitopes. They all reacted with identical patterns of sheath polypeptides. The antisera failed to recognize antigens of 4th-stage larvae of L. carinii. In contrast, cross-reacting epitopes were detected in other parasite stages. Antisera reacted with material surrounding embryos and microfilariae in the uterus of females, and caused patchy fluorescence on the sheath of blood-derived and in vitro-released microfilariae.

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.

T cell-deprived mice acutely infected with S. mansoni suffer microvesicular hepatocyte damage which is not seen in infected, immunological intact animals. A cationic fraction (CEF6) of the PBS-soluble portion of S. mansoni eggs (SEA) induces antibodies which, on passive transfer, prevent hepatocyte damage. CEF6 contains 2 antigens, ω1 and α1, and has also been shown to be a useful serodiagnostic reagent. This paper describes the purification and characterization of the 2 antigens present in CEF6. ω1 is a monomeric glycoprotein with a pI > 9·0 and a molecular weight of 31 kDa. α1 consists of two immunologically cross-reactive dimers, 41 and 36 kDa in non-reducing conditions, each of which consists of one unique and one common glycoprotein subcomponent. In ELISA with mouse and human infection sera ω1 is shown to be S. mansoni specific and is better able to distinguish S. mansoni infections from other schistosome infections than are unfractionated SEA, CEF6 or α1. Passive transfer of monospecific anti-ω1 sera into S. mansoni infected, T cell-deprived mice completely prevented the occurrence of microvesicular hepatocyte damage in these animals. Monospecific anti-α1 serum had no hepatoprotective capacity.

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.

Culture characteristics of two species of piscine trypanosomes were compared. Trypanosoma catostomi from Catostomus commersoni was isolated in hypo-osmotic blood agar media but not in hyper-osmotic media. However, T. phaleri from Amia calva was isolated in both media. Only T. catostomi was stimulated to divide by diluting infected blood with water. The maximum number of T. phaleri and percentage of trypomastigotes were not affected by NaCl concentration. The maximum number of T. catostomi was inversely related to osmotic pressure and the percentage of trypomastigotes was directly related to osmotic pressure. This was confirmed by using equi-osmotic concentrations of NaCl, NaBr, Na acetate, KCl, choline-Cl or sucrose. Supplementation with urea had no effect on the number or morphology of trypanosomes. The survival of T. catostomi following cryopreservation was enhanced by prior incubation in hyper-osmotic media. Differential culture characteristics of these species may reflect adaptation to different host–vector systems.

Although the relationship between intestinal parasitism, the ingestion and use of energy, and host survival is expected, little work has been done to outline the effect of such organisms upon their host's nutritional requirements in an ecological context. This study is the first to demonstrate that an intestinal helminth previously reported to be of little or no histopathological consequence, Plagiorhynchus cylindraceus, has a significant detrimental impact upon the flow of food energy through a definitive host, the European starling, Sturnus vulgaris. Within both male and female adult European starlings reductions in standard metabolic rates occurred as the result of initial infection, indicating that the host's basal metabolism/thermal regulatory abilities were altered. Moreover, initially infected male starlings, but not females, had an increased consumption and excretion of energy and maintained lower average daily body weights versus controls when temperature stressed. These results appear to be due to either a parasite-mediated alteration in host activity and/or to the disruption of host-digestive abilities. Additionally, these data indicate that, overall, male and female S. vulgaris respond differently to infection and that intestinal helminths normally thought to be of little or no pathological consequence to the host are factors that should be addressed in future studies regarding animal energetics, ecology, and behaviour.

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 relationship between host dietary protein and intestinal helminth population biology during primary infection was studied using a mouse–T. muris experimental model. Uninfected CBA/Ca mice fed either a 4% (by mass) or 16% protein diet grew at a similar rate, but both suffered significant growth retardation when infected with T. muris. T. muris initial establishment and fecundity were unaffected by dietary protein content, but were significantly influenced by infection dose, declining at the higher level of infection. Dietary protein deficiency increased parasite survival at high infection levels. Heavily infected hosts fed the 16% protein diet were found to expel almost the whole of their infections around day 21 p.i. By contrast, this response appeared defective in hosts fed the lower 4% protein diet, resulting in the establishment and survival of large, chronic and patent infections. Mean faecal egg output per mouse increased with infection dose in animals fed a low protein diet, but was low and unrelated to infection exposure in hosts fed the 16% protein diet. It is concluded that low protein levels in diet, via alterations in host resistance, may have an important influence on the transmission of T. muris.

The accumulation of the trypanocide isometamidium chloride (Samorin®, RMB Animal Health Ltd, UK) by a range of clones of Trypanosoma congolense with varying sensitivity to the drug, was measured by methods based on the fluorescence of isometamidium. Fluorescence microscopy and flow cytometry showed a reduction in drug accumulation by resistant clones. Fluorescence spectrophotometry demonstrated an inverse correlation between the intensity of cell-associated fluorescence and the level of resistance of the clones expressed in vivo. The addition of the metabolic inhibitor SHAM/glycerol to the incubation medium resulted in a reduction of this apparent difference in drug accumulation between the clones; those clones which were sensitive to isometamidium showed a reduction in fluorescence while a percentage increase in fluorescence was observed as clones became more resistant to the trypanocide. These observations may be of value for the in vitro detection of resistant T. congolense populations and may also be used to estimate the mean level of resistance in a given sample. The results also imply that decreased accumulation of isometamidium by drug-resistant clones of the parasite may be responsible for the reduction in sensitivity.

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.

Larvae of the monogeneans Pseudodiplorchis americanus and Neodiplorchis scaphiopodis develop within egg capsules retained in the uterus of the parent parasite. This study reveals adaptations for the storage and nutrition of infective stages which have no known precedent amongst other platyhelminths. The vitelline system is greatly reduced and appears to contribute little to embryo nutrition. An electron-dense ‘shell’ which encloses the newly formed embryos is subsequently replaced by a thin flexible sac composed of concentric layers of membranes which are derived from the uterus wall. Further membranes are added to this multi-laminate structure as the encapsulated embryos pass along the uterus, and this enables the sac to expand continuously as the larva inside it grows. The capsule lining extends into fine cytoplasmic processes which ‘plumb in’ to the larval tegument between ciliated cells. These connexions are packed with glycogen and appear to perform a placenta-like function. The unique adaptations correlate with the demands of the life-cycle. Large numbers of oncomiracidia (up to 300) can be packed, in membranous sacs, within the storage capacity of the uterus; direct transfer of nutrients from parent to offspring enables resources to be supplied over an extended period; and infective larvae can be maintained in readiness for an unpredictable opportunity for transmission.

Ultrastructural changes associated with exsheathment of the infective juveniles of Haemonchus contortus are described. Hyaline ring formation occurs associated with annulations 10 and 20 and consists of the dissolution of the basal zone and the inner electron-dense layer resulting in the rupture of the sheath. These changes are consistent with the action of a protease-exsheathing enzyme. There is a significant reduction associated with exsheathment in the size of the excretory cells and the number of excretory granules contained within them. No changes in the oesophagus were observed associated with exsheathment. These, and the observations of earlier workers, suggest that it is the excretory cells and not the oesophagus which are the source of exsheathing fluid during exsheathment.

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.

The rostellar distal cytoplasm of Echinococcus granulosus protoscoleces is characterized by extensive basal membrane infoldings, prominent hemidesmosomes and is subtended by a lamina reticularis with microfibrils of approximately 10 nm diameter that occasionally show a 55 nm banding periodicity. The rostellar hooks, in 2 rows, each have a blade, guard and handle region and possess a central amorphous pulp, a middle microfibrillar medulla with microfibrils of approximately 4 nm diameter, and a complex outer cortex in all but the proximal region of the guard and the base of the handle. In these regions additional material, of similar electron density to the medulla, but lacking the fibrillar substructure, occurs and gives the areas a lobed appearance. Energy-dispersive X-ray microanalysis of whole hooks demonstrated the presence of sulphur and trace quantities of phosphorus. X-ray near-edge absorption spectra resembled those of cystine, feather and hair and showed the sulphur to be predominantly in the form of disulphide linkages. X-ray diffraction patterns of whole hook preparations revealed 2 diffuse rings with equatorial spacings of 7·99 Å and 15·22 Å, thus differing from vertebrate keratins.

Clavibacter sp. (syn. Corynebacterium rathayi) adhered to both Anguina funesta (syn. Anguina agrostis) and Anguina tritici, but differences in the nature of adhesion were noted. Similar patterns of binding of the bacteria and of anti-wheat germ agglutinin antibody initially led us to believe that the mechanism of bacterial adhesion was related to the presence of wheat-germ agglutinin (WGA) on the outer cuticle of both species of nematodes and its complementary carbohydrate on the bacterial capsule. However, treatment of either species of nematode with sodium metaperiodate inhibited bacterial adhesion but not the binding of anti-WGA antibody. Bacterial adhesion, therefore, is not mediated by WGA on the nematodes' surface. Moreover, differences in patterns of bacterial adhesion to Anguina species, both before and after treatments with NaCl and detergents, suggest basic interspecific differences in the nature of adhesion. Electron microscopy confirmed the contribution of the nematodes' cuticular surface coat (SC) to the process of adhesion, but it is still not clear how the SC interacts with the bacterial capsule or which of its components are involved. While complete removal of the SC with periodate prevented bacterial adhesion, juveniles that naturally resisted bacterial adhesion did not lack a SC. One explanation could be that the SC of individuals, to which bacteria do not adhere naturally, lacks crucial components that cannot be defined by conventional EM.

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.

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PH1-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

BALB/c mice were characterized as more resistant to infection with Heligmosomoides polygyrus (Nematoda) than C57BL/6 mice based on lower establishment and survival during a primary infection and stronger protection induced by an immunizing regime. It was hypothesized, therefore, that C57BL/6 mice would be more heavily infected than BALB/c mice when they lived together as a single population in a large indoor arena where transmission occurred through contact between the mice and damp peat trays where parasite eggs developed into larvae. Fifty female mice (including 5 infected mice) of each strain were placed in a 3.2 x 0.8 m arena. Net egg production and numbers of larvae acquired by sentinel mice of each strain were monitored every two weeks. The experiment was replicated twice. The results did not support the hypothesis. No difference was detected between strains of mice in the number of larvae acquired by sentinel mice during 24 h exposure periods, or in the numbers of worms present after 12 or 23 weeks. Net egg production was also comparable between strains. A hypothesis that the unexpected similarity of infection in the two strains was related to differences in rates of contact with the peat trays was not supported by preliminary data on mouse behaviour that revealed equal frequency of contact with peat trays between strains. A second hypothesis that continuous exposure to larvae led to similar infection levels in the two strains (in contrast to the controlled characterization experiments) was also unsupported. Mice were infected weekly with 10, 50 or 100 larvae for 5 or 10 weeks. Net egg production and numbers of worms were consistently higher in C57BL/6 than BALB/c mice. At this time it is not clear why infection in the two strains was virtually identical in the large arenas but clearly distinct in all controlled infection experiments.

A study to compare effects of mass, targeted and selective chemotherapy with levamisole (Ketrax) as an action for the control of Ascaris lumbricoides was carried out in three communities in rural Oyo State, Nigeria. Selective treatment was applied in one village by treating the most heavily infected 20% of the inhabitants, targeted treatment in the second village involved children aged 2–15 years, while mass treatment was offered to all inhabitants excluding infants under 1 year and pregnant women in the third village. Recommended doses of levamisole were given in the villages, as described above, at 3-monthly intervals during a period of 1 year. Prevalence and intensity (epg) of A. lumbricoides infection were determined immediately before and 3 months after the period of intervention using a modified Kato-Katz technique. In the selective treatment village, no significant differences were found between the pre- and post-treatment egg counts (mean(±S.D.) epg 6776±10791 versus 4259±10909 respectively) of A. lumbricoides in the total population. In the targeted treatment village, significant differences were recorded in pre- and post-treatment egg counts for the total population (9057 ± 15 797 versus 2579±6529) among the children alone (10935±20094 versus 992±3175) and among the untreated adults (7742±9782 versus 4561±8798). In the mass treatment village, significant differences in pre- and post-treatment egg count values were also recorded (11 907±17220 versus 1489±5165). The intensity of Trichuris trichiura and hookworm infections among the villagers before and after intervention were not observed to have changed significantly regardless of selective, targeted or mass treatment.