Several approaches were adopted for the disruption and removal of the tegumental surface from protoscoleces of the horse strain of the hydatid organism, Echinococcus granulosus. The effectiveness of each method and the purity of subsequent microthrix-enriched fractions obtained by differential centrifugation were evaluated by electron microscopy, by the amount of protein released and by the degree of enrichment of surface plasma membrane marker enzymes. Incubation in saponin for 10 min produced the purest microtriche preparation, but in low yield; freeze/thawing, incubation in Triton X-100 for 10 mm or in saponin for 20 min produced fractions containing significant amounts of relatively pure microtriches, but mild homogenization was a poor method for surface disruption and subsequent isolation of microtriches. Phosphodiesterase, adenosine triphosphatase (total and ouabain-inhibited), leucine aminopeptidase and glutamyltransferase were active in the protoscoleces but none were enriched in any of the microthrix fractions. In contrast, alkaline phosphatase, acid phosphatase, 5′ nucleotidase and maltase were enriched significantly in all of the isolated microtriche preparations, which suggests that these enzymes are predominantly surface membrane bound. The protein profiles of the microthrix-enriched fractions, following SDS—PAGE, were basically similar, although there were some qualitative and quantitative differences in the proteins released by each isolation procedure. Three major PAS-staining components were present in all the preparations and these probably originated from the glycocalyx. One of these PAS-positive components, with an approximate molecular weight of 110 kDa, may be a glycoprotein specific to the horse strain of E. granulosus.

A single dose of Ro 11–3128 was found to be 98–100% effective against Schistosoma mansoni infections at intervals of 3 h to 15 days following infection, and apparently killed the schistosomula stages soon after administration, thus allowing an assessment of the immunizing potential of progressive larval stages. Following infection with 500 unirradiated cercariae, optimum resistance was manifest by groups drug-treated at 48–96 h (60–75%). Significantly lower levels of resistance occurred with early (3 h) or later (6–15 day) treatments. Superimposition of an infection treated at 15 days on a prior infection which had been treated at 2 days did not reduce the level of resistance caused by the latter, indicating that the infection plus delayed treatment had not induced suppression. Thus the peak resistance manifest during the 48–96 h period may reflect optimum expression of protective antigens. Comparison of irradiated (20 krad.) with unirradiated infections showed that, when drug-terminated 24, 48 or 96 h post-infection, irradiated cercariae induced significantly less resistance than unirradiated cercariae, perhaps indicating a delayed appearance of protective antigens following radiation treatment.

Starting with the work showing activity in the research programme (Table 1), compounds will usually have been entered for various reasons, first into the in vitro testing systems, or more rarely into the in vivo system unless this is the only testing system available for that particular organism.

The participation of products released from Schistosoma mansoni schistosomula (SRP-A) in the IgG antibody response of infected Brown-Norway rats and infected humans has been studied using immunoprecipitation with various antigenic preparations and in in vitro cytotoxicity assays. A large number of SRP-A molecules with a wide range of molecular weights was recognized by infected rat and human sera. Anti-SRP-A antibodies appeared in rat sera from day 28 after infection. In infected humans, a variable pattern of SRP-A recognition was observed between individuals. IgG antibodies obtained by immunization of rats with SRP-A without addition of adjuvants reacted with 3 major schistosomula surface proteins with molecular weights of 38, 32 and 21 kDa. These latter molecules were also revealed strongly by infected rat sera. Moreover, these antibodies were able to kill schistosomula in vitro in the presence of complement or eosinophils.

The fluorescent probe 1-anilino-8-naphthalene suiphonic acid (ANS) was used to investigate the effect of temperature on the physical state of the mitochondrial membranes of adult and larval schistocephalus solzdus together with that of their hosts Gasterosteus aculeatus and Gallus domesticus. Arrhenius plots of ANS/membrane fluorescence for S. solidus plerocercoids was linear over the temperature range 15 to 58 °C, while that for the adult was biphasic with a discontinuity at 39·9 °C. This was interpreted as a physical change which occurred in the adult membrane but not in the plerocercoid membrane and pointed to an alteration in membrane composition during infection. Gasierosteus aculeatus showed a linear Arrhenius plot for membrane fluorescence, irrespective of acclimation temperature. Gallus domesticus showed a discontinuity in the Arrhenius plot for membrane fluorescence at 46·9 °C, outside the normal physiological temperature range.

The fate of 75Se-labelled schistosomula in mice treated at 24 h post-infection with either Ro 11–3128 or oxamniquine, compared to untreated controls, was followed by compressed organ autoradiography. No difference in the total percentage of schistosomula detected as foci was found between the three groups at each sampling time. However, the distribution of schistosomula was altered. In oxamniquine-treated mice there was a delayed migration from the skin relative to controls and fewer parasites in total appeared to reach the lungs. In Ro 11–3128 treated mice very few parasites left the skin. Ro 11–3128 treatment induces resistance to challenge whereas oxamniquine does not. The fate of challenge schistosomula in previously infected Ro 11–3128 treated mice was also compared to that in control mice. There was evidence for delayed or reduced migration from the skin of Ro 11–3128 treated mice. Significant death of challenge parasites occurred in the lungs, or post-lung sites in both control and treated mice. It was not possible to conclude that any of the late attrition was immune-dependent in the treated mice and the results appeared to indicate that a significant fraction of parasite death occurred at the skin stage.

In Medium 199 supplemented with calf serum (+ 10%), adult Schistosoma mansoni produced a mean total of 400 eggs/worm pair during maintenance for 10 days in vitro. During the period of egg deposition in vitro, the dry weight of worm pairs decreased by 35%. Biomass decreases were proportionally greater in female worms than in males. Declines in the dry weight of female worms were mainly due to decreases in protein content, while biomass decreases in males resulted from losses of both protein and glycogen. Glucose was depleted from the maintenance medium at a rate of approximately 200 μg/worm pair/day during each of the first 3 days in vitro, while concentrations of glucose in the medium of above 1 g/litre did not affect the degree of glycogen depletion observed in adult S. mansoni during culture for 24 h. The contribution of the loss of schistosome gut contents in vitro to the observed changes is discussed. The work described represents part of an attempt at logical design of in vitro culture.

The cuticle of parasitic nematodes, the main contact site with the host, plays an important role in host-parasite interaction and thus also in immunological control. We compared different surface-iodinated life-stages of the filarial worm Dipetalonema viteae (microfilariae, infective 3rd-stage larvae (L3), adult males and females) with respect to changes in their surface composition. Autoradiographs of peptide maps show that all stages present an identical set of peptide spots reflecting common surface protein(s). Spots specific for larvae L3 show that the composition of the iodinated surface differs in microfilariae and adults i.e. it changes during development. Adults show a spot typical for males or females. Identical spots are found in L3. This suggests that a surface component is also sex specific.

Restriction endonuclease digestion of genomic DNA generates DNA fragments of unique size, dependent upon the particular base sequence. Following fractionation by agarose gel electrophoresis, repetitive DNA can be visualized as distinct bands in stained gels and the restriction fragment length of such bands used as diagnostic characters. Restriction fragment length differences were detected between species within the genera Trichinella, Caenorhabditis, Romanomermis, Steinernema (syn. Neoaplectana) and Meloidogyne. This technique provides a new tool for the taxonomist, which is independent of phenotypic variation and it enables the rapid and reliable separation of closely related species.

Current models of the regulation of host populations by parasite infection assume that death of a host results in death of all the parasites contained within or on the host. This assumption acts as a density-dependent constraint on parasite population growth and contributes to the stability of the interaction between host and parasite populations. The protozoan Ichthyophthirius multifiliis, a parasite of freshwater fish, is one of a number of pathogens in which the reproductive capacity of an individual is not entirely destroyed by the death of its host. The effect of host death on the reproductive potential of I. multifiliis was investigated with a series of short-term experiments examining (i) the relationship between parasite burden and host mortality, (ii) parasite survival on the host and (iii) the relationship between parasite age and the number of infective stages released/parasite. Results of a series of experimental epidemics in which host numbers were maintained constant showed large oscillations in parasite numbers. Deterministic models ignoring the age structure of the parasite population were unable to reproduce the behaviour observed in the experimental epidemics. Using the results of the short-term experiments, a difference equation model was developed. This model was able to qualitatively reproduce the observed epidemic behaviour.

Myxobolus cyprini has, until now, been considered an ‘organo-cosmopolitan’ parasite of the common carp (Cyprinus carpio) and less frequently of other carps, producing spores in various organs in small plasmodia and possible in cysts. The present observations of naturally infected common carp fry and two-summer carp have revealed that M. cyprini is a specific muscle parasite, developing intracellularly in the muscle fibres of the skeletal muscle. The sarcoplasm of the infected muscle fibres is filled with developmental stages, followed by spores of M. cyprini, which are held together in a 1–1·5 mm long pseudocyst by the sarcolemma of the muscle fibre. After maturation of the spores and disintegration of the pseudocysts the spores are transported in the bloodstream to different parts of the body where they are retained in the capillaries.

Cytochemical staining with diaminobenzidine (DAB) has been used to locate cytochrome oxidase activity in mitochondria from major tissues of three intestinal parasitic nematodes, Nippostrongylus brasiliensis, Haemonchus contortus and Ascaridia galli. Deposition of DAB reaction product was uniform and relatively intense in mitochondria from all tissues studied in N. brasiliensis. However, mitochondria from different tissues of H. contortus and A. galli varied considerably in their cytochemical staining, decreasing in the order hypodermis < muscle < gut < reproductive tissue. Mitochondria in gut and reproductive tissue of A. galli possessed little or no cytochrome oxidase activity. These results are compatible with respect to oxygen availability and the extent and localization of the mammalian-type aerobic mitochondrial electron transport pathway in intestinal parasitic nematodes.

The abomasum of living sheep infected with 50000 larvae of Haemonchus contortus was examined before and during infection, by means of fibre optic endoscopy. Biopsy specimens were removed from the abomasum of the living sheep and were examined by means of scanning electron microscopy. Changes were noted in the surface structure of the abomasum 2 days after infection and larvae were seen on, and burrowing into, the mucosa. These changes became more pronounced as the infection proceeded, especially after day 10 of the infection when the adult worms had appeared. The behaviour of adult nematodes was observed within the abomasum and on occasions they were seen to move from surrounding areas into an area of haemorrhage, caused by removal of a biopsy specimen. Individual nematodes were removed from the abomasum by means of the biopsy forceps. Fibre optic endoscopy was shown to be a useful tool in the study of parasitic nematodes in vivo.

The ability of Eimeria vermiformis, a coccidium which normally parasitizes the mouse, to develop in rats was investigated. The Rowett strain (Lac: RNU) rats were euthymic (rnu/+), treated or untreated with cortisone acetate, and athymic (rnu/rnu). E. vermiformis completed its development only in rnu/rnu rats, which passed small numbers of oocysts capable of sporulating and infecting C57BL/6 mice. In the rnu/+ rats, irrespective of cortisone treatment, development appeared to terminate with the completion of the asexual (schizogonic) stages since no gametocytes were recognized in the tissues and no oocysts were detected in the faeces. The findings are discussed with reference to the factors which govern host specificity.

A limited number of metacestodes of Hymenolepis diminuta from Tenebrio molitor had host plasmatocytes and cell debris of coagulocyte origin associated with the microvillar surface. There was evidence of phagocytosis of microvilli by plasmatocytes, involving clathrin-like endocytotic vesicles. In contrast to these otherwise ‘normal’ cysticercoids, occasional batches were ‘sticky’ and, on flushing into saline, rapidly developed large bladders possibly associated with osmotic stress. In addition, numerous small surface blebs were present. These ‘surface-stressed’ cysticercoids also had host plasmatocytes and cell debris associated with the bladder bases and the blebs, and phagocytosis of microvilli was observed. In no case had precipitation of haemolymph, melanization or encapsulation (features typical of the insect immune response) occurred. The possibility that the limited host response indicates a spatially restricted impairment of the surface compatibility of the parasite, and thus insufficient to elicit the full immune response, is discussed.

Food intake, body weight changes, serum protein and amino acid concentrations were measured during the course of primary infections of Nippostrongylus brasiliensis in rats fed on either a 2% protein (casein) diet or a 16% protein diet (Oxoid 41 B). Total serum protein concentration declined from 77·84±5·35 mg/ml in uninfected well-nourished rats to 54±3·29 mg/ml in rats at 9 days post-infection (p.i.). A reduction from 47·80±2·78 to 40·38±5·62 mg/ml had occurred in protein-malnourished rats by day 6 p.i. The hypoproteinaemia was accompanied by significant hypoalbuminaemia in the protein-malnourished rats and concentrations fell from approximately 33 to 19 mg/ml at the time of peak infection. Six days after inoculation, a significant increase was detected in the concentration of serum amino acids in both well-nourished and protein-malnourished rats; the effect was more prolonged for non-essential than for essential amino acids. The results are discussed briefly in relation to recent work on the control of protein metabolism during malnutrition.

The activities of glycolytic and related enzymes and the tricarboxylic acid cycle enzymes were measured in freshly isolated 1st- (Li), 2nd- (L2) and 3rd-stage (L3) larvae of both Ancylostoma tubaeforme and Haemonchus contortus. All enzymes of the glycolytic pathway were present in all developmental stages of both strongylid nematodes although higher levels of activities were obtained in the pre-infective 1st- and 2nd-stage larvae than in the infective 3rd stage. However, the pre-infective larvae contained lower levels of pyruvate kinase (PK) than the infective larvae. Consequently, the pyruvate kinase to phosphoenolpyruvate carboxykinase (PEPCK) ratios were 0·23 and 0·26 for the L1s and L2s for A. tubaeforme and 0·36 and 0·21 for those of H. contortus respectively. High levels of activity of glucose-6-phosphate dehydrogenase obtained in the bacteriophagous pre-infective larvae were consistent with high rates of morphogenesis and substrate synthesis characteristic of the pre-infective stages. All the tricarboxylic acid cycle enzymes were present in the infective larvae of both nematodes while in the pre-infective Li and L2 stages, the enzymes at the beginning of the cycle, namely aconitate hydratase and NAD-linked isocitrate dehydrogenase, were not detected. A scheme was proposed for the energy metabolism of these developing larvae. In this scheme, the pre-infective larvae were shown to operate an anaerobic metabolic pathway involving the carboxylation of phosphoenolpyruvate (PEP) by phosphoenolpyru vate carboxykinase (PEPCK) to form oxaloacetate (OAA), whereas in the infective larvae the metabolic pathway favouring the direct dephosphorylation of PEP, as in vertebrate tissues, was followed.

Evidence exists that lethal activity is associated with extracts from Eimeria tenella-infected caecal tissues. We examined tissues of the small intestine infected with other species of Eimeria for a similar lethal activity. Extracts were prepared from intestinal tissues of chickens infected with either E. acervulina, E. brunetti, E. maxima, E. mitis or E. necatrix. Appropriate control extracts from tissues of non-infected chickens were also prepared. Intravenous injection of the extracts into healthy chickens showed that lethality of infected tissue extracts was higher than control extracts and was independent of the site of infection within the intestine. Comparison of the lethality of caecal extracts from chickens infected with embryo-adapted or parent lines of E. tenella showed that lethality was independent of the recognized pathogenic characteristics of the lines. Also, it was demonstrated that lethal activity is associated with extracts of tissues containing massive numbers of arrested and non-arrested sporozoites and early trophozoites.