Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37°C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

Neuroinvasive larvae of the common dog and cat roundworms, Toxocara canis and Toxocara cati, may cause severe neurological and neuropsychological disturbances in humans. Despite their pathogenic potential and high prevalence worldwide, little is known about their cell-specific influences and cerebral host–pathogen interactions in neurotoxocarosis. To address this discrepancy, a co-culture system of viable larvae with murine neuronal (CAD), oligodendrocytal (BO-1) and microglial (BV-2) cell lines has been established. Additionally, murine adult brain slices have been co-cultured with Toxocara larvae to consider complex organotypic cell–cell interplay. Cytotoxicity of larval presence was measured enzymatically and microscopically. Microscopic evaluation using trypan blue exclusion assay revealed to be less reliable and sensitive than the lactate dehydrogenase activity assay. Ultimately, even low numbers of both T. canis and T. cati larvae have impaired survival of differentiated CAD cells, which morphologically resemble primary neurons. In contrast, viability of oligodendrocytal and microglial cells as well as brain slices was not impaired by larval presence. Therefore, immune-mediated mechanisms or trauma by migrating larvae presumably induce the in vivo pathology rather than acute cytotoxic effects. Conclusively, the helminthic larvae co-culture system presented here is a valuable in vitro tool to study cell-specific effects of parasitic larvae and their products.

The helminth community in the digestive tract of the sedentary predatory fish, Sciaena umbra, near the Scandola Nature Reserve, Corsica, consists of 5 species of digeneans. Anoiktostoma coronatum (in the rectum) and Metadena pauli (in the intestine) were found in almost all the fish, and in high numbers; they are considered core species. Distomum aloysiae, Pleorchis polyorchis (both in the intestine), and Stephanostomum bicoronatum (in the rectum) were relatively uncommon, usually found in small numbers, and are considered satellite species. The two core species were very abundant in June, when the size of their populations was strongly correlated with the size of the host fish. At this time, M. pauli was found mainly in the duodenum, but in high populations was spread throughout the intestine (s. strict.). Both species were much less abundant in October, their populations were less dependent on host size, and M. pauli was more limited to the anterior intestine. Pleorchis polyorchis and Distomum aloysiae were more frequent in October, were found predominately in fish with few M. pauli, and were found posterior to M. pauli, suggesting some interactions between these species. Stephano-stomum bicoronatum showed no seasonal patterns, and no apparent interactions with A. coronatum.

Syngamid strongylids of the genus Mammomonogamus undoubtedly belong among the least known nematodes with apparent zoonotic potential and the real diversity of the genus remains hard to evaluate without extensive molecular data. Eggs of Mammomonogamus sp. are frequently found in feces of African forest elephants (Loxodonta cyclotis) and western lowland gorillas (Gorilla gorilla gorilla) in Dzanga-Sangha Protected Areas. Using sedimentation-based coproscopic techniques, we found the eggs of Mammomonogamus in 19·7% elephant and 54·1% gorilla fecal samples with 8–55 and 1–24 eggs per gram of fecal sediment for elephants and gorillas, respectively. We used a combination of light microscopy, scanning electron microscopy and analysis of cytochrome c oxidase subunit I (cox1) and a partial sequence of 18S rDNA isolated from single eggs to test the hypothesis of possible Mammomonogamus conspecificity in gorillas and elephants. Whereas 18S rDNA sequences were identical in both gorillas and elephants, we distinguished seven different haplotypes within the cox1. Two haplotypes were found in both gorillas and elephants suggesting sharing of Mammomonogamus. Assignment of the parasite to M. loxodontis is proposed. Provided sequences represent the first genomic data on Mammomonogamus spp.

The prevalence of Schistosoma mansoni infections of Biomphalaria pfeifferi shows seasonal variation. Field data from Zimbabwe show annual ranges from 0 to 7%. In this paper a mathematical model of B. pfeifferi population dynamics and S. mansoni epidemiology is used as a framework for analysis of these patterns. Snail fecundity is a function of snail age and of temperature, and is apparently affected by other seasonal factors. The pre-patent period is dependent on temperature. Infection affects snail fecundity and mortality. Parameter values are derived from previous field and laboratory studies. The force-of-infection is estimated from the analysis of size-prevalence data. Using observed temperatures, model output agrees well with field data on snail abundance and prevalence of infection over a 14-month period. Seasonal variation in prevalence largely reflects variation in the pre-patent period and in snail population age structure. The possible role of seasonality in the force-of-infection is discussed. Prevalence patterns are not greatly affected by year-to-year differences in temperature. Significant seasonal variation in snail–man transmission rates is expected.

Information relating to the characterization of cestode surface macromolecules is limited. This is especially the case with Taenia crassiceps, a well-recognized model for the study of larval cestodiasis. Here, the protein and glycoprotein composition of the tegumental surface and cyst fluid of the metacestode have been investigated using radio-isotope labelling, immunoprecipitation, SDS–PAGE and lectin affinity chromatography. A restricted number of surface proteins was labelled with the 125I/Iodogen method although the majority were immunogenic; in contrast an array of cyst fluid antigens were labelled. Host serum proteins, including immunoglobulins, were identified on the surface and in the cyst fluid. Some of the 125I-labelled surface proteins, including a 37 kDa molecule, have been shown to be glycoproteins and probably contain-D-mannose and/or D-glucose; there is limited or no N-acetylglucosamine and no terminal galactose present on these components. A 37 kDa surface molecule, possibly the same glycoprotein, was also precipitated by infection sera and this may endorse the theory that highly immunogenic carbohydrates are continuously shed by T. crassiceps as a mechanism for diverting the immune response of the host. Radio-iodinated and biosynthetically labelled T. crassiceps antigens were highly cross-reactive with antibody raised to other cestodes and not one antigen was identified as a possible candidate for use in specific immunodiagnosis of any of the important taeniid infections.

Insecticide resistance is a serious problem in the control of many vector-borne diseases, particularly in developing countries where the possibility of changing to an alternative compound may be restricted. Consequently genes giving susceptibility to insecticides in vector species can be viewed as constituting a valuable resource to be used up with discretion. The exchange of information and thought taking place in the Workshop was centred upon possible ways to conserve this resource, i.e. to slow down the evolution of resistance while maintaining adequate control, so preserving the effective life of available insecticides as long as possible. The subject was treated mainly in relation to insect vectors of disease although niost of the discussion would also be relevant to other insect pests.

The effects of dietary urea supplementation and of a 10-week trickle infection regime, simulating chronic exposure to Haemonchus contortus, on the zymogenic population of the abomasa of Hampshire Down lambs was examined. At necropsy a variety of parameters including plasma pepsinogen concentrations, the wet weights of abomasal fundic mucosal pieces and the amounts of pepsinogen contained in them, were assessed. Tissue pepsinogen concentration was measured as the total, acid-stable proteolytic activity present in mucosal homogenates, as well as immunohistochemically. The immunohistochemical findings were quantified using computer-aided image analysis. Elevation of plasma pepsinogen concentrations in infected animals was of borderline significance (P=0·06). The fundic mucosae of infected animals were heavier (P<0·02) than those of control animals, but there was no overall change in the pepsinogen content of tissues. Immunohistochemistry revealed that infected animals had increased numbers of zymogenic cells, due to mucous cell hyperplasia and the adaptation of cells to produce both mucins and pepsinogen. The pepsinogen content of chief cells, the major source of pepsinogen in uninfected animals, was reduced in infected lambs. Image analysis confirmed that at a mid-point of the mucosa of infected animals there was increased pepsinogen-specific immunoreactivity that corresponded with areas of mucosal hyperplasia. Mucous cell hyperplasia might therefore allow the maintenance of pepsinogen secretion in infected animals even if chief cell output is reduced.

The induction of schistosomulicidal activity of peritoneal macrophages by concanavalin A-stimulated supernatants from long-term T-cell clones and by interferon-gamma (IFN-γ) was investigated in detail. Optimal conditions of in vitro macrophage activation by T-cell clone supernatants were established. Macrophages from 13-week S. mansoni-infected mice responded to lymphokine activation as well as resident mnacrophages from uninfecteci mice. IFN-γ was shown to play an essential role in induction of schistosomulicidal macrophage activity: recombinant IFN-γ at high concentration could induce schistosomula killing, and an anti-IFN-γ antiserum inhibited the induction ofschistosomulicidal activity by T-cell clone supernatants. Our data also indicate that macrophage activation could be obtained by IFN-γ in synergy with other lymphokines in the supernatant of long-term T-cell clones. Macrophages from mice injected with T-cell clone supernatants were primed in vivo and triggered to kill schistosomula in vitro in the presence of lipopolysaccharide (LPS). The data demonstrate that lymphokines produced by T-cell clones and, in particular, IFN-γ can participate in the activation of schistosomulicidal macrophages.

Monoclonal antibody 2A6 binds to the surface of the Dictyocaulus viviparus L3 sheath and identifies a molecule which is highly antigenic in both infected and vaccinated cattle. Immunogold electron microscopy reveals that the antibody binds to a highly stable surface coat overlying the epicuticle of the L3 sheath. The binding to the cuticular surface is stage specific, being restricted to the surface of the L3 sheath, although the antigen can be detected on Western blots of parasite homogenates from the L1 stage. Immunofluorescence on live parasites demonstrates that the antibodies also bind to the L3 sheath surface of many other strongylid nematodes. The antigen detected by 2A6 differs in molecular weight between the species; in D. viviparus the antigen is poorly resolved between 29 and 40 kDa by SDS-PAGE whereas in nematodes from the family Trichostrongyloidea a single band or doublet of approximately 20 kDa is detected, suggesting that a related molecule is present in these species. Although infection and vaccination with D. viviparus elicits a strong immune response to the L3 sheath antigen this does not appear to be the case following infection with other strongylid nematodes.

Detergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl p-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z–Phe–Arg–MCA. Two of these were optimally active at alkaline pH, the other at pH 4·0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz–Arg–MCA readily, whilst the other cleaved Bz–Arg–MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukayrotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc–Ala–Ala–pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz–Arg–MCA hydrolase, the aminopeptidases and the Boc–Ala–Ala–pNA serine peptidase are cytosolic.

Schistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

Laboratory reared Culex quinquefasciatus Say, 1823 originating from the vicinity of Belem, in northern Brazil, were allowed to engorge on caimans (Caiman c. crocodilus) infected with Hepatozoon caimani (Carini, 1909) and boas (Boa constrictor) infected with H. cf. terzii (Sambon and Seligmann, 1907) both from Para State. Engorged mosquitoes killed on successive days post-feeding (p.f.) were studied by transmission electron microscopy. Images of oocysts from 13 days p.f., caiman-fed mosquitoes were also examined by scanning electron microscopy. The Hepatozoon spp. from the respective hosts differed in their ability to develop in C. quinquefasciatus. Most female mosquitoes fed on caimans, became fully engorged, and survived beyond 22 days p.f., whereas those engorged on boa became debilitated and did not survive beyond 9 days p.f. In boa-fed mosquitoes oocysts were found on the 6th day p.f. The few mosquitoes surviving to the 9th day p.f. contained either undivided oocysts or those that had already commenced sporogenic division. By 8–10 days p.f. caiman-fed mosquitoes contained uninucleate oocysts. Sporogonic divisions were observed from day 12 p.f. onwards. Although sporogenic development conformed in general with the previously reported accounts, the study allowed a more detailed examination of the plasmalemmal endocytotic system, and the consolidation of the crystalline body in specialized ‘factories’ of cystalline material. Sporozoite differentiation occasionally started on the 18th day p.f., but otherwise was observed only on day 22 p.f.

Preparations of isolated brush border plasma membrane of Hymenolepis diminuta and H. microstoma possess the following enzymatic activities: alkaline phosphohydrolase (E.C. 3.1.3.1); Type I phosphodiesterase (E.C. 3.1.4.1); ribonuclease (E.C. 3.1.4.22); adenosine triphosphatase (E.C. 3.6.1.3); and 5′-nucleotidase (E.C. 3.1.3.5). The following enzymatic activities could not be demonstrated in either membrane preparation: Type II phosphodiesterase (E.C. 3.1.4.18); cyclic adenosine-3′, 5′-monophosphate phosphodiesterase (E.C. 3.1.4.17); leucine aminopeptidase (E.C. 3.4.11.1); maltase (α-glucosidase; E.C. 3.2.1.20); and lactase (β-galactosidase; E.C. 3.2.1.23). These data generally agree with those of previous studies in which similar membrane-bound enzymes were demonstrated in intact (living) worms.

Manipulative parasites with complex life cycles are known to induce behavioural and physiological changes in their intermediate hosts. Cyathocephalus truncatus is a manipulative parasite which infects Gammarus pulex as intermediate host. G. pulex males display pre-copulatory mate guarding as a response to male-male competition for access to receptive females. In this paper, we tested the influence that C. truncatus-infection might have on male G. pulex sperm number and pairing success. We considered 3 classes of G. pulex males in our experiments: (i) uninfected males found paired in the field, (ii) uninfected males found unpaired in the field, or (iii) infected males found unpaired in the field. Both infected males and uninfected unpaired males paired less with a new female than uninfected paired males did. Furthermore, infected males appear to be at a strong disadvantage when directly competing for females with a healthy rival male, and had fewer sperm in their testes. We discuss the potential effect of male and female mating strategies on such male host mating alteration.

This memoir presents the results of the study of a number of specimens of Filariid worms from Cattle and other Ruminants which had accumulated during the last few years from various collections of Nematodes, sent to me for identification and laid aside for further investigation.

Trypanocide resistance remains a huge challenge in the management of animal African trypanosomiasis. Paucity of data on the prevalence of multi-drug resistant trypanosomes has greatly hindered optimal veterinary management practices. We use mathematical model predictions to highlight appropriate drug regimens that impede trypanocide resistance development in cattle. We demonstrate that using drugs in decreasing resistance order results in a negligible increase in number of cattle with resistant infection, in contrast to a more pronounced increase from trypanocide use in increasing resistance order. We demonstrate that the lowest levels of trypanocide resistance are achieved with combination therapy. We also show that increasing the number of cattle treated leads to a progressive reduction in the number of cattle with drug resistant infections for treatments of up to 80% of the cattle population for the combination treatment strategy. Our findings provide an initial evidence-based framework on some essential practices that promote optimal use of the handful of trypanocides. We anticipate that our modest forecasts will improve therapeutic outcomes by appropriately informing on the best choice, and combination of drugs that minimize treatment failure rates.

Human African trypanosomiasis (HAT) or sleeping sickness is caused by protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. Despite the enormous technological progress in molecular parasitology in recent years, the diagnosis of HAT is still problematic due to the lack of specific tools. To date, there are two realities when it comes to HAT; the first one being the world of modern experimental laboratories, equipped with the latest state-of-the-art technology, and the second being the world of HAT diagnosis, where the latest semi-commercial test was introduced 30 years ago (Magnus et al.1978). Hence, it appears that the lack of progress in HAT diagnosis is not primarily due to a lack of scientific interest or a lack of research funds, but mainly results from the many obstacles encountered in the translation of basic research into field-applicable diagnostics. This review will provide an overview of current diagnostic methods and highlight specific difficulties in solving the shortcomings of these methods. Future perspectives for accurate, robust, affordable diagnostics will be discussed as well.

The development of Paramphistomum sukari Dinnik in a snail host is described with the emphasis laid on the succession of redial generation.

The sporocyst gives birth to about twenty to thirty rediae. These rediae of the first generation commence with the production of daughter rediae then enter the second phase of their productivity during which they produce cercariae. The daughter rediae, or the rediae of the second generation, repeat these two phases during their lives, commencing with redial production and after that changing to the production to cercariae. Both the first- and second-generation rediae are able to produce a few daughter rediae at the end of their life. There is evidence that the subsequent generations of rediae are also able to give birth to daughter rediae and cercariae.

As a result the successive generations of rediae maintain the infection in an intermediate host for a long time, probably as long as the infected snail can survive.