Protease expression among TCI and TCII field isolates was analysed. Gelatin-containing gels revealed hydrolysis bands with molecular masses ranging from 45 to 66 kDa. The general protease expression profile showed that TCII isolates presented higher heterogeneity compared to TCI. By utilizing protease inhibitors, we showed that all active proteases at acid pH are cysteine-proteases and all proteases active at alkaline pH are metalloproteases. However, the expression of cruzipain, the T. cruzi major cysteine-protease, did not reproduce a heterogeneous TCII cysteine zymogram profile. Dendogram analyses based on presence/absence matrices of proteases and cruzipain bands showed a TCI separation from the TCII group with 50–60% similarity. We suggest that the observed cysteine protease diversification contributes to differential host infection between TCI and II genotypes.

The benzodiazepine Ro 11-3128 (methyl-clonazepam) presents several similarities with praziquantel with regard to its anti-schistosomal mode of action, since both drugs cause spastic paralysis, calcium influx and tegumental disruption in the parasites. In order to know whether the two compounds share the same binding sites in the schistosomes, we performed in vivo and in vitro competition experiments. We took advantage of the fact that Ro 11-3128 is active against immature Schistosoma mansoni (whereas praziquantel is inactive), and praziquantel is active against S. japonicum (which is insensitive to Ro 11-3128). An excess of praziquantel did not inhibit the activity of Ro 11-3128 against immature S. mansoni and an excess of Ro 11-3128 did not inhibit the activity of praziquantel against S. japonicum, suggesting that the schistosome binding sites of the two drugs are different. On the other hand, cytochalasin D, an agent known to perturb – among other things – calcium channel function, was capable of inhibiting the schistosomicidal activity of both praziquantel and Ro 11-3128, thus adding another element of similarity between the two anti-schistosomal agents. A similar, albeit partial, inhibition of the schistosomicidal activity of the two drugs was exerted by some of the classical calcium channel blockers. Taken together, these results suggest that praziquantel and Ro 11-3128, although binding to different schistosome receptor sites, may use the same basic anti-schistosomal effector mechanisms.

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.

Carefully chosen immunological measurements, informed by recent advances in our understanding of the diversity and control of immune mechanisms, can add great interpretative value to ecological studies of infection. This is especially so for co-infection studies, where interactions between species are often mediated via the host's immune response. Here we consider how immunological measurements can strengthen inference in different types of co-infection analysis. In particular, we identify how measuring immune response variables in field studies can help reveal inter-species interactions otherwise obscured by confounding processes operating on count or prevalence data. Furthermore, we suggest that, due to the difficulty of quantifying microbial pathogen communities in field studies, innate responses against broad pathogen types (mediated by pattern response receptors) may be useful quantitative markers of exposure to bacteria and viruses. An ultimate goal of ecological co-infection studies may also be to understand how dynamics within host-parasite assemblages emerge from trade-offs involving different arms of the immune system. We reflect on the phenotypic measures that might best represent levels of responsiveness and bias in immune function. These include mediators associated with different T-helper cell subsets and innate responses controlled by pattern response receptors, such as the Toll-like receptors (TLRs).

In this study, we addressed the phylogenetic and taxonomic relationships of Trypanosoma vivax and related trypanosomes nested in the subgenus Duttonella through combined morphological and phylogeographical analyses. We previously demonstrated that the clade T. vivax harbours a homogeneous clade comprising West African/South American isolates and the heterogeneous East African isolates. Herein we characterized a trypanosome isolated from a nyala antelope (Tragelaphus angasi) wild-caught in Mozambique (East Africa) and diagnosed as T. vivax-like based on biological, morphological and molecular data. Phylogenetic relationships, phylogeographical patterns and estimates of genetic divergence were based on SSU and ITS rDNA sequences of T. vivax from Brazil and Venezuela (South America), Nigeria (West Africa), and from T. vivax-like trypanosomes from Mozambique, Kenya and Tanzania (East Africa). Despite being well-supported within the T. vivax clade, the nyala trypanosome was highly divergent from all other T. vivax and T. vivax-like trypanosomes, even those from East Africa. Considering its host origin, morphological features, behaviour in experimentally infected goats, phylogenetic placement, and genetic divergence this isolate represents a new genotype of trypanosome closely phylogenetically related to T. vivax. This study corroborated the high complexity and the existence of distinct genotypes yet undescribed within the subgenus Duttonella.

Molecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata – Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.

Given the worldwide decline of amphibian populations due to emerging infectious diseases, it is imperative that we identify and address the causative agents. Many of the pathogens recently implicated in amphibian mortality and morbidity have been fungal or members of a poorly understood group of fungus-like protists, the mesomycetozoans. One mesomycetozoan, Amphibiocystidium ranae, is known to infect several European amphibian species and was associated with a recent decline of frogs in Italy. Here we present the first report of an Amphibiocystidium sp. in a North American amphibian, the Eastern red-spotted newt (Notophthalmus viridescens), and characterize it as the new species A. viridescens in the order Dermocystida based on morphological, geographical and phylogenetic evidence. We also describe the widespread and seasonal distribution of this parasite in red-spotted newt populations and provide evidence of mortality due to infection.

Hosts are typically simultaneously co-infected by a variety of microparasites (e.g. viruses and bacteria) and macroparasites (e.g. parasitic helminths). However, the population dynamical consequences of such co-infections and the implications for the effectiveness of imposed control programmes have yet to be fully realised. Mathematical models may provide an important framework for exploring such issues and have proved invaluable in helping to understand the factors affecting the epidemiology of single parasitic infections. Here the first population dynamic model of microparasite-macroparasite co-infection is presented and used to explore how co-infection alters the predictions of the existing single-species models. It is shown that incorporating an additional parasite species into existing models can greatly stabilise them, due to the combined density-dependent impacts on the host population, but co-infection can also restrict the region of parameter space where each species could persist alone. Overall it is concluded that the dynamic feedback between host, microparasite and macroparasite means that it is difficult to appreciate the factors affecting parasite persistence and predict the effectiveness of control by just studying one component in isolation.

We investigated whether host specificity is linked to variability within species of Lamellodiscus monogeneans, which are gill ectoparasites of the Sparidae. We sampled fish parasites in the northeastern part of the Mediterranean Sea: 4 specialist species, using 1 single host species, and 3 generalist species, using 2 distinct host species. Intraspecific variability was assessed from 2 different datasets. Morphometric variability of the attachment organ, called the haptor, was estimated first from measurements of several sclerified haptoral parts on 102 individuals. Genetic variability was calculated based on comparisons of sequences derived from the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA of 62 individuals. Morphometric variances in the specialist versus generalist species were compared via principal component analysis and F-tests, and uncorrected genetic distances (p-distances) were estimated within each species. We showed that the inter-individual variance of morphometric characters, as well as p-distances, are clearly greater within generalist species than specialist ones. These findings suggest that a relative increase in morphological and molecular variability enhances the possibility to colonize new host species in monogeneans, and supports the hypothesis that intraspecific variability could be a potential determinant of host specificity.

Hookworms infect approximately 740 million humans worldwide and are an important cause of morbidity. The present study examines the role of additive genetic effects in determining the intensity of hookworm infection in humans, and whether these effects vary according to the sex of the host. Parasitological and epidemiological data for a population of 704 subjects in Papua New Guinea were used in variance components analysis. The ‘narrow-sense’ heritability of hookworm infection was estimated as 0·15±0·04 (P<0·001), and remained significant when controlling for shared environmental (household) effects. Allowing the variance components to vary between the sexes of the human host consistently revealed larger additive genetic effects in females than in males, reflected by heritabilities of 0·18 in females and 0·08 in males in a conservative model. Household effects were also higher in females than males, although the overall household effect was not significant. The results indicate that additive genetic effects are an important determinant of the intensity of human hookworm infection in this population. However, despite similar mean and variance of intensity in each sex, the factors responsible for generating variation in intensity differ markedly between males and females.

The actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cell-substrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of the Trypanosoma cruzi actin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure of T. cruzi actin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved in T. cruzi.

Competition between parasite species has been predicted to be an important force shaping parasite and host ecology and evolution, although empirical data are often lacking. Using the Mus musculus-Schistosoma mansoni and Schistosoma rodhaini host-parasite systems we characterized mate choice and inter-specific competition between these two schistosome species. Simultaneous infections revealed species-specific mate preferences for both species as well as suggesting mating competition, with male S. rodhaini appearing dominant over male S. mansoni. S. rodhaini homologous pairs were also shown to have increased reproduction per paired female in the presence of a competitor in simultaneous infections. Overall total reproductive success was, however, similar between the two species under conditions of direct competition due to the greater initial infectivity of S. mansoni in comparison to S. rodhaini. Inter-specific competition was also implicated as increased parasite virulence to the host. The potential effects of such interactions on parasite and host ecology and evolution in nature are discussed.

Trypanosomatid parasites possess 2 distinct iron-containing superoxide dismutases (Fe-SODs) designated SODA and SODC, both of which are targeted to their mitochondria. In contrast to SODAs that carry typical mitochondrial transit peptides, SODCs have highly unusual mitochondrial targeting signals. Our analyses clearly show that these pre-sequences are bipartite possessing a signal peptide-like domain followed by a transit peptide-like domain. Consequently, they resemble N-terminal extensions of proteins targeted to multi-membrane plastids, suggesting that trypanosomatids once contained a eukaryotic alga-derived plastid. Further support for this hypothesis comes from striking similarities in length, hydropathy profile, and amino acid composition of SODC pre-sequences to those of Euglena and dinoflagellate plastid proteins. To account for these data, we propose that the Trypanosomatidae initially possessed a gene encoding a mitochondrial Fe-SOD with a classical mitochondrial transit peptide. Before or after plastid acquisition, a gene duplication event gave rise to SODA and SODC. In a subsequent evolutionary step a signal peptide was linked to SODC, enabling its import into the plastid. When the trypanosomatid plastid subsequently was lost, natural selection favoured adaptation of the SODC N-terminal signal as a mitochondrial transit peptide and re-targeting to the mitochondrion.

Although co-infection is the norm in most human and animal populations, clinicians currently have no practical tool to assist them in choosing the best treatment strategy for such patients. Given the vast range of potential pathogens which may co-infect the host, obtaining such a practical tool may seem an intractable problem. In ecology the joint concepts of functional groups and guilds have been used to conceptually simplify complex ecosystems, in order to understand how their component parts interact and may be manipulated. Here we propose a mechanism by which to apply these concepts to pathogen co-infection systems. Further, we describe how these groups could be incorporated into a mathematical modelling framework which, after validation, could be used as a clinical tool to predict the outcome of any particular combination of pathogens co-infecting a host.

Juvenile female Litomosoides sigmodontis secrete a protein (Juv-p120) highly modified with dimethylethanolamine (DMAE). In an attempt to establish the source of this decoration worms were pulsed with [3H]-choline and [3H]-ethanolamine and the radio-isotope labelled products analysed. Both isotope labels were successfully taken up by the worms, as demonstrated by labelling of phospholipids with [3H]-choline, being predominantly incorporated into phosphatidylcholine and [3H]-ethanolamine into phosphatidylethanolamine. Isotope labelling of phosphatidylethanolamine was particularly striking with the worms taking up ~30 times as much labelled ethanolamine as choline. It was possible to detect faint labelling of Juv-p120 with [3H]-ethanolamine after prolonged exposure periods but, unlike the situation with the phospholipids, it was much more readily labelled with [3H]-choline. When pulsing with [3H]-ethanolamine it was also possible to detect isotope-labelled phosphatidylcholine, which may ultimately account for the low levels of labelling of Juv-p120. Overall our results raise the previously unconsidered but intriguing possibility that in L. sigmodontis, choline may be the precursor of DMAE.

Infection of small macropodids with the larval stage of Echinococcus granulosus can cause fatalities as well as significant pulmonary impairment and other adverse sequelae. The brush-tailed rock-wallaby (Petrogale penicillata) is a small macropodid listed as vulnerable on the IUCN's Red List of Threatened Species. This study used radiographic techniques to determine the prevalence and severity of pulmonary hydatid infection and growth rates of hydatid cysts in a wild population of this macropodid. The overall prevalence was 15·3% (9/59 animals) with 20·0% (8/40 animals) of adults infected. During the study period, the death of at least 1 infected animal was directly attributed to pulmonary hydatidosis. Rapid cyst growth occurred in some animals (up to 43% increase in cyst volume in 3 months). Cyst volume reduced lung capacity by up to 17%. Secondary pulmonary changes were uncommon but, in 1 animal, resulted in reduction in lung capacity by approximately 50%. Infection was associated with a higher blood urea concentration, but no significant differences in other blood variables were detected. These results indicate that hydatid infection may be a significant risk to threatened populations of small macropodids and should be addressed in conservation management plans for these animals.

The route of transmission and host finding behaviour are fundamental components of a parasite's fitness. Riccardoella limacum, a haematophagous mite, lives in the mantle cavity of helicid land snails. To date it has been assumed that this parasitic mite is transmitted during courtship and mating of the host. Here we present experimental evidence for a new transmission route in the host snail Arianta arbustorum. Parasite-free snails were kept on soil on which previously infected host snails had been maintained for 6 weeks. R. limacum was successfully transmitted via soil without physical contact among hosts in 10 out of 22 (45·5%) cases. In a series of experiments we also examined the off-host locomotion of R. limacum on snail mucus and control substrates using an automated camera system. Parasitic mites showed a preference to move on fresh mucus. Our results support the hypothesis that R. limacum uses mucus trails to locate new hosts. These findings should be considered in commercial snail farming and when examining the epidemiology of wild populations.

Nycteribiids (Diptera: Nycteribiidae) are specific haematophagous ectoparasites of bats, which spend nearly all their adult lives on hosts. However, females have to leave bats to deposit their larva on the walls of the roosts, where they later emerge as adult flies. Nycteribiids had thus to evolve efficient sensorial mechanisms to locate hosts from a distance. We studied the sensory cues involved in this process, experimentally testing the role of specific host odours, and general cues such as carbon dioxide, body heat, and vibrations. As models we used two nycteribiids (Penicillidia conspicua and Penicillidia dufourii) and their primary bat hosts (Miniopterus schreibersii and Myotis myotis, respectively). Carbon dioxide was the most effective cue activating and orientating the responses of nycteribiids, followed by body heat and body odours. They also responded to vibration, but did not orientate to its source. In addition, sensory cues combined (carbon dioxide and body heat) were more effective in orientating nycteribiids than either cue delivered alone. Results suggest that nycteribids have some capacity to distinguish specific hosts from a distance, probably through their specific body odours. However, the strong reliance of nycteribiids on cues combined indicates that they follow these to orientate to nearby multispecies bat clusters, where the chances of finding their primary hosts are high. The combination of sensory cues seems therefore an effective strategy used by nycteribiids to locate bat hosts at a distance.

It has been shown that the bovine abomasal parasite, Ostertagia ostertagi, drastically modulates its microenvironment, causing epithelial cell damage, accumulation of inflammatory cells and pH changes in the stomach. The mechanisms used by the parasite to change the abomasal environment are largely unknown, but an important role has been attributed to excretory-secretory (ES) products from the parasite. In this study we have identified proteins representing a novel ES protein family, characterized by the SCP/Tpx-1/Ag5/PR-1/Sc7 protein motif. These proteins were named Oo-AL1 and Oo-AL2 (O. ostertagi ASP-like protein). Both proteins contain a signal peptide and 1 predicted N-glycosylation site. The transcript for Oo-AL1 was present from the L4 stage onwards in both male and female adult worms, whereas the Oo-AL2 transcript was hardly detectable. Western blots of somatic extracts and ES products from different developmental stages of O. ostertagi, probed with anti-Oo-AL1 antibodies, revealed Oo-AL proteins in the ES products of adult worms. An analysis of the nematode genome and EST databases indicated that these novel ES proteins are unique to O. ostertagi and its relative, Teladorsagia circumcincta, suggesting a key function in these abomasal parasites.

In human Toxocara canis infection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through a T. canis infection in BALB/c mice. Infection with T. canis embryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies against T. canis (IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40–80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDa T. canis proteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.