We have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytostomes and the resulting food vacuoles in the parasite cytoplasm.

As stated by Jennie Blackwell in her introduction to this symposium, the surface of protozoan parasites has long been the province of immunologists, driven by interest in the diagnostic and vaccine potential of surface molecules. This preoccupation with vaccines led to the generation of a great deal of sequence data for surface molecules but gave few clues as to their function.

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4–5·5, 37 °C) human red blood cell lysates were used to simulate this process, measuring the denaturation of haemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, their rates decreasing with increasing pH, and increasing in the presence of protease. Both processes were inhibited by the quinoline-containing anti-malarial drugs (QCDs) chloroquine, quinine, mefloquine and amodiaquine at concentrations well below those expected in the acidic food vacuole of the parasite. Spectrophotometric analysis indicated that chloroquine complexes with heme in acid-denatured haemoglobin. Other weak bases as well as verapamil and diltiazem, known to reverse the resistance of malarial parasites to chloroquine, were without efifect indicating that the action of QCDs is specific. Based on our previous results and the present report, we suggest that iron release in acidified lysates is mediated through the formation of ferryl (Fe(IV)) radicals. QCDs possibly complex with this radical, as they do with heme, and prevent its contact with an adjacent heme molecule which is required for ring opening and iron release. These results may suggest that one of the anti-malarial effects of QCDs is to deprive the parasite of an adequate iron supply. Addition of iron to cultures of Plasmodium falciparum was expected to circumvent the deprivation of iron and reduce the anti-malarial eflfect of QCDs. However, adding iron as penetrating fructose or nitrilotriacetate complexes did not alter the parasite's susceptibility to chloroquine. Ascorbate markedly increased the release of iron in acidified lysates, and this effect was not reduced by chloroquine. Ascorbate was found to decrease parasite susceptibility to chloroquine, suggesting that iron deprivation may be an important factor in the anti-malarial action of QCDs.

Associations between social rank, immunodepression and resistance to Babesia microti infection within single-sex groups of male house mice suggest rank-dependent suites of response involving different hormonal and immune changes in relation to aggressive behaviour and group size prior to infection. Reduced resistance among high-ranking males was associated with increased serum testosterone and corticosterone concentration and reduced serum immunoglobulin, but was independent of group size. Among low-ranking males, hormonal changes were not associated with resistance to B. microti but changes in corticosterone concentration and measures of immunodepression increased with group size and aggressive behaviour. The results concur with earlier findings suggesting differences between high- and low-ranking mice in their physiological responses to social experience and consequently reduced resistance to B. microti infection among high-ranking individuals.

A genomic library of Entamoeba histolytica (pathogenic strain HM-1: IMSS) was screened to detect repetitive DNA clones other than those from the highly abundant ribosomal DNA (rDNA). One such clone (HMc) had a 2·3 kb insert which hybridized with the main genome and not the rDNA circle. Southern hybridization of E. histolytica genomic DNA, digested with EcoR I and probed with HMc, showed multiple bands. The banding pattern was identical in all axenic pathogenic strains tested. Differences, however, existed when the banding pattern of a pathogenic strain was compared with that of a non-pathogenic strain. HMc was present in about 25–30 copies per genome in strain HM-1: IMSS. Nucleotide sequence analysis of HMc revealed a partial open reading frame which hybridized with a 1·35 kb poly A+ transcript in Northern blots. The deduced amino acid sequence did not, however, show significant homology with known proteins. The HMc sequence was found only in E. histolytica as it hybridized with 5 different axenic strains of E. histolytica but did not recognize other closely related species of Entamoeba. It has thus the potential to be used as a species-specific DNA probe.

A complete developmental sequence of Leishmania mexicana has been produced in axenic culture for the first time. This was achieved by manipulation of media, pH and temperature conditions over a period of 16 days. All experiments were initiated with lesion amastigotes that were transformed to multiplicative promastigotes by culture in HOMEM, 10% foetal calf serum, pH 7·5, at 25 °C. Metacyclogenesis was induced by subpassage in Schneider's Drosophila medium, 20% foetal calf serum, pH 5·5, and the resulting forms transformed to axenically growing amastigotes by subpassage in the same medium and raising the temperature to 32 °C. Parasites from each day were characterized with respect to their general morphology using light microscopy of Giemsa-stained smears, and biochemically by analysis of total protein content, proteinases, nucleases and secretory acid phosphatase. The results demonstrated that the three main stages identified - amastigotes, multiplicative promastigotes and metacyclic promastigotes - each exhibited the expected suite of biochemical properties. Further, the changes in morphology observed as the developmental sequence proceeded from stage to stage were accompanied by appropriate changes in biochemical properties. These results provide both useful biochemical markers and a culture system in which to examine the regulation of differentiation and transformation during the Leishmania life-cycle.

Host–parasite interactions were studied for the microsporidium Pleistophora intestinalis and its host, Daphnia magna. Two host clones were established from the same population from which the parasites were taken (home-1 and 2), and two clones from two other ponds (neighbour and Munich clone). With increasing clutch number infected females from home-1 clone produced relatively smaller clutches than uninfected females. Age and body length at maturity were not affected by the infection, but body length of the sixth adult instar was reduced. In an experiment including all four host clones, the parasite reproduced well in the two home clones and in the neighbour clone, but poorly in the Munich clone. Juvenile growth and age at maturity was not affected in the two home clones, but for the neighbour and the Munich clone age was delayed by 2·2 days and 4·1 days, and juvenile growth reduced by 16 and 23%, respectively. Significant host-clone x parasite-treatment interactions were also found for size at maturity and clutch size. This pattern of host-parasite interactions suggests that there is no general positive relation between disease severity and parasite multiplication rate.

An experiment was conducted to determine the rate and maximum percentage of sporulation of Eimeria acervulina oocysts at various environmental conditions relating to temperature (21 versus 33 °C) and relative humidity (RH) (40 versus 80%). Measurements were made during 44 h after excretion of oocysts in 3 substrates: dry litter, clammy litter and pure faeces respectively. Maximum sporulation percentage in both dry (22·6%) and clammy litter (19·5%) was higher (P < 0·005) than in pure faeces (11·6%). Neither temperature nor RH had a significant influence on percentage of oocysts that sporulated. Under these simulated practical conditions approximately 25% of all oocysts sporulated, whereas sporulation under optimal conditions (29 °C, aeration, 2% K2Cr2O7) showed a higher (68%) sporulation ability of oocysts. At 33 °C sporulation proceeded at a faster pace than at 21 °C (P < 0·005). With respect to RH and substrate, once sporulation started, the rate of increase to maximum percentage was not different. Time of onset of sporulation was influenced by temperature (P < 0·0001) and RH (P < 0·001). Time of onset occurred 15 h later at 21 °C compared with 33 °C and 5 h later at 40% RH compared with 80%. Also, an interaction effect (P < 0·01) was found with effect of RH being stronger at 21 °C compared with 33 °C. It was concluded that the most important aspect in the epidemiology of E. acervulina during a flock cycle is the time of onset of sporulation with temperature being the most important factor.

Mice were fed diets containing 960 mg (control), 100 mg (moderately Mg deficient) and 30 mg (severely Mg deficient) of Mg/kg. After 20 days, mice were inoculated with Babesia hylomysci (from Dr Wery, Anvers, Belgium). Significant increases in RBC Mg levels were observed following infection. All the control and moderately deficient mice died from infection, whereas the severely Mg-deficient diet protected mice against infection, as shown by a decrease in parasitaemia and mortality. The decrease in RBC Mg, modifications in membrane properties and increased oxidant stress are possible explanations for the protective effect of severe Mg deficiency. When mice were maintained for 2 months after inoculation on a severely Mg-deficient diet and were then switched to a control diet, all survived and had low parasitaemias. After 1 month, these mice were rechallenged with B. hylomysci and 89% survived.

The activity of atovaquone against Plasmodium berghei ANKA during sporogonic development has been examined. Anopheles stephensi mosquitoes were fed on gametocyte infected mice which had been treated 8 h previously with atovaquone or diluent alone. Mosquito midguts were examined for oocysts, and salivary gland infections were estimated using an ELISA for the circumsporozoite protein (CSP). The number of oocysts per midgut fell by at least 97% when mosquitoes were fed on mice dosed with 0·1–10 mg atovaquone/kg body weight. This was paralleled by a decrease in the prevalence of oocyst-infected mosquitoes from 70–90% in controls to 40% or 10% respectively. No oocysts were observed at a dose of 100 mg/kg. CSP ELISA results indicated that mosquitoes fed on atovaquone failed to produce sporozoites. Mosquitoes which fed on gametocytaemic, atovaquone-treated mice (0·1–100 mg/kg) did not transmit malaria to naive mice. These results demonstrate that atovaquone has a highly potent inhibitory activity against the mosquito stages of P. berghei.

A striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4–5 kDa. Two different monoclonal antibodies directed against the 4–5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the low Mr antigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4–5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability between Toxoplasma gondii tachyzoites grown in vivo and in vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

Production of elevated haemolymph agglutination titres by Biomphalaria glabrata following exposure to Echinostoma paraensei miracidia was investigated, to characterize this parasite-induced response and to understand its functional relevance. Both the dose of infection (1, 10 or 100 miracidia per snail) or the number of separate exposures to infection (between one and three, over a 4 or 8 day interval) were varied, and assuming a threshold dosage (10 miracidia per snail or higher) was exceeded, titres of juvenile snails peaked at 8–16 times the values for unexposed control snails, regardless of the exposure regimen. Adult snails, which are relatively refractory to infection, have slightly higher resting titres than juveniles, but exhibit only a 2- to 4-fold increase in titre following exposure. Juveniles exposed to infection but lacking demonstrable infection had lower titres than snails with confirmed infections. Exposure to infection increased heterogeneity of plasma agglutinins and provoked production of unique specificities not found in unexposed snails. However, the overall pattern of agglutination responses for snails with successfully developed parasites did not differ from those in which parasite development was unsuccessful. Agglutinating activity was inhibitable by several different monosaccharides, although plasma from infected snails was relatively unaffected by N-acetyl-glucosamine or N-acetyl-galactosamine. Wounding of snails provoked no change in plasma agglutination activity. As the highest agglutination titres were produced in snails with successfully developing parasites and agglutinin composition did not differ between snails with successful or unsuccessful parasites, the functional relevance of the response remains enigmatic. The production of unique agglutinins following exposure deserves additional study.

A simulation model for the population dynamics of Eimeria acervulina infection in broilers is presented. The model describes the development of the numbers of parasites in the various life-stages during the growing period of broilers and the empty house period between grow-outs. The model includes assumptions with respect to development of immunity to E. acervulina infection and effects of application of anticoccidial drugs. The model consists of a set of difference equations that are solved numerically at 1 h intervals. Under constant conditions, an equilibrium level was reached after a few grow-outs during which infection always peaked around the 21st day in the growing period. Within a growing period, infection peaked earlier (later) than the 21st day in case initial numbers of sporulated oocysts were higher (lower) than the equilibrium number.

The malaria parasite exists in an extracellular form at several stages in its life cycle. Within the vertebrate host, sporozoites and merozoites have to invade specific cell types. Proteins on the surface of the parasite or externalized from specialized organelles have been implicated as ligands for receptors on the host cell surface. Direct binding studies have identified parasite proteins that interact with the target cell surface. Examination of the deduced amino acid sequences has allowed the identification of primary structural motifs which may have roles in this process. On the sporozoite, the circum-sporozoite protein and sporozoite surface protein-2, a protein initially located within micronemes, have been found to contain an amino acid sequence thought to be involved in mediating recognition of sulphated polysaccharides on the surface of a liver cell. On the merozoite, merozoite surface protein-1 may be involved in the initial recognition of red blood cells; this protein undergoes a complex series of modifications in the time between its synthesis as a precursor molecule and successful erythrocyte invasion. Other merozoite proteins located at the apical end of the parasite have been identified as erythrocyte or reticulocyte binding proteins.

The activity of lysophospholipase of human erythrocytes increased by about 3 orders of magnitude upon infection with Plasmodium falciparum. The apparent Km for hydrolysis of lysophosphatidylcholine by this enzyme was 50 ± 7μM and the apparent Vmax 6·8±0·6 nmol/h × 106 cells. The activity was Ca2+ independent and had a broad pH maximum at pH 8. The enzyme was insensitive to such anti-malarials as mefloquine and arteether and was only weakly inhibited by chloroquine, with a 50% inhibition concentration (IC50) of 70 mM. The anti-malarials quinine and quinacrine were more efficient inhibitors, with IC50s of 2·6 mM and 0·7 mM, respectively. The sulphydryl agents p–hydroxymercuribenzoate (pHMB) and thimerosal were considerably more potent, inhibiting the plasmodial lysophospholipase with IC50s of 18 μM and 10 μM, respectively. When present at 10 μM prior to invasion, both pHMB and thimerosal arrested the growth and reinvasion capacity of P. falciparum in culture. In a synchronized P. falciparum culture the continuous presence of 5 μM thimerosal dramatically decreased total parasitaemia and, within 4 days, totally abolished the capacity of the surviving parasites to reinvade. Thus the plasmodial lysophospholipase may represent a potential new target for anti-malarial chemotherapy.

Small subunit ribosomal RNA (srRNA) genes of three Theileria species, one Cytauxzoon and four Babesia species were amplified using the polymerase chain reaction (PCR), cloned and sequenced. Our sequences were aligned with srRNA sequences previously published for eight species of Apicomplexa, one ciliate and one dinoflagellate, the last two being included as free-living outgroup species. Phylogenetic relationships between the organisms were inferred by four in-dependent methods of phylogenetic tree construction using the ciliate Oxytricha nova to root the trees. Our trees fail to show a consensus branching order. They do, however, clearly indicate that the theilerias form a monophyletic taxon derived from a paraphyletic group which includes the species B. equi, C. felis and B. rodhaini. The distance trees indicate that the babesias sensu stricto (B. canis, B. caballi, B. bigemina and B. bovis) form another monophyletic taxon which diverged before the theilerias separated from the above-mentioned paraphyletic group. The parsimony and maximum likelihood trees suggest that the babesias and theilerias are sister taxa, both of which were derived from the paraphyletic group.

Rabbit polyclonal and mouse monoclonal antibodies (Mabs) prepared against synthetic peptides patterned on exofacialloops 3 (amino acids 546–555) and 7 (821–834) of the human anion transport protein band 3 inhibited the cytoadherence of Plasmodium falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Mabs directed against exofacial loop4 (amino acids 628–642) did not inhibit adherence to a significant degree. The murine Mabs recognized only P. falciparum- infected erythrocytes suggesting that the epitopes of loops 3, 4 and 7 are normally cryptic in uninfected erythrocytes.

A simulation model for effects of Eimeria acervulina infection on technical and economic characteristics in broiler production is presented. The model describes development over time of the growth depression, feed intake reduction, and decrease in feed efficiency associated with infection. The model also shows a phase of compensatory growth during which earlier negative effects are counterbalanced, at least partly. Major assumptions made were: infection with E. acervulina occurs in each flock; production is affected in each flock; compensatory growth takes place because immunity develops and cell regeneration occurs. The results show that the pattern of development of the production characteristics during a flock cycle depends on the initial contamination level. Both a high and low initial contamination level results in a lower average daily gain, a worse feed to gain ratio, and a reduced net revenue compared to an intermediate contamination level.

The schistosome intermediate snail host, Bulinus truncatus (Mollusca: Planorbidae), has two reproductive (phally) morphs. Both aphallics and euphallics can self-fertilize, but aphallics cannot donate sperm because they do not develop a functional penis and prostate. This study investigated the interactions between phally and fitness consequences of Schistosoma haematobium infection in B. truncatus. Snails which developed patent infections produced 26% fewer eggs than controls and 35% fewer eggs than exposed snails which did not develop infections. This reduction was due to a lower lifetime production of egg masses and a smaller mean number of eggs/mass in infected snails relative to control or exposed snails. However, there was no evidence of increased mortality in infected snails. Contrary to reports of fecundity compensation in other intermediate host snails, egg production post-exposure during the pre-patent period did not increase relative to that of controls in either infected or exposed snails. Phally did not influence susceptibility to infection or length of the prepatent period. Furthermore, lifetime egg, egg mass and hatchling production, as well as mean eggs/mass and number of hatchlings reaching maturity, did not differ significantly between aphallics and euphallics within control or exposed experimental groups. However, within the infected group euphallics produced 38% fewer eggs, smaller egg masses, and fewer hatchlings reaching maturity than aphallics, supporting the prediction of a cost to the growth and maintenance of a full male tract. This cost was detectable only when snails were under the stress of infection. The proportion of euphallic offspring produced did not differ across experimental groups. We discuss these results in light of alternative host strategies to minimize fitness costs of infection.