Although the folate metabolic pathway in malaria parasites is a major chemotherapeutic target, resistance to currently available antifolate drugs is an increasing problem. This pathway, however, includes a number of enzymes that, to date, have not been characterized despite their potential for clinical exploitation. As a step towards evaluation of additional targets in this pathway, we report the isolation and characterization of 3 new genes that encode homologues of GTP cyclohydrolase I (GTP-CH), dihydrofolate synthase/folylpolyglutamate synthase (DHFS/FPGS) and serine hydroxymethyltransferase (SHMT). The genes encoding GTP-CH and SHMT are unambiguously assigned to chromosome 12, while that for DHFS/FPGS is tentatively assigned to chromosome 13. All 3 genes are expressed in blood-stage parasites, yielding transcripts of which only ca 60–70% is accounted for by coding sequence. All 3 of the proteins predicted to be encoded by these genes display sequence differences compared to the human host homologues that may be of functional significance. These data bring the complement of cloned genes that encode activities in the pathway to seven, leaving only the gene encoding dihydroneopterin aldolase (DHNA) to be identified in the route from GTP to folate synthesis and folate turnover in the thymidylate cycle.

Toxoplasma gondii has a unique enzyme, a NTPase, which has a wide specificity toward NTP. In the present study, we produced a monoclonal antibody (IgG1, 6C6) against the enzyme which could recognize NTPase isozymes among several strains of T. gondii. Three avirulent strains of T. gondii, ME49, Beverley and Nakayama, were found to have 1 NTPase (63kDa, pI 6·0), while a virulent strain RH and an avirulent strain Fukaya had 2 isozymes (63kDa) with different pIs (pIs 6·0 and 6·5 for the former, and pIs 6·2 and 6·4 for the latter, respectively), suggesting that this monoclonal antibody recognizes a common epitope of NTPase among T. gondii strains. Furthermore, 6C6 could inhibit NTPase activity in the presence of dithiothreitol in a dose-dependent manner, and immuno-EM study of NTPase revealed that this molecule is located on the surface membrane of T. gondii tachyzoites. When Vero cells were co-cultured with tachyzoites pre-treated with 6C6, the number of infected cells significantly decreased, suggesting that 6C6 inhibits invasion of the parasites to host cells. These data suggest that the molecule recognized by 6C6 might be considered a potential candidate antigen for vaccines against T. gondii tachyzoites.

The gp63 encoding genes were characterized by PCR–RFLP in 35 isolates representative of the Leishmania donovani complex (L. infantum, L. donovani, L. archibaldi and L. chagasi), with special attention to Mediterranean L. infantum from different geographical origins, and in separate groups from Old World Leishmania (L. major, L. tropica and L. aethiopica). The aim was to evaluate how the possible selective pressure by the host on these important surface proteins would influence structuring of our sample. Comparison was carried out with the structure obtained (i) from reported isoenzyme data, characters supposed to vary neutrally, and (ii) from PCR–RFLP analysis of gp63 inter-genic regions, containing non-translated spacers and regulatory genes. Polymorphism within the gp63-encoding region, was much higher than in gp63 inter-genic regions. In the gp63 intra-genic dendrogram, the 4 species of L. donovani complex were discriminated and quite distinct from outgroups. Within L. infantum, geographical structuring was observed and did not overlap with the structure built-up from isoenzymes and inter-genic data. These results support the idea of a strong host-selection on gp63, at vector level but most of all at vertebrate (human or dog) immunological level. Furthermore, they illustrate how the nature of genetic characters may influence the perception of population structuring.

Saliva inoculated by sandfly females during feeding stimulated production of high levels of anti-saliva antibodies. To determine whether 3 species of the genus Phlebotomus have species-specific salivary antigens we performed dot-blots and immunoblots using sera from mice, hamsters and rabbits repeatedly bitten by sandflies. Important differences were found in the antigen components of the salivary gland lysates (SGL) of Phlebotomus papatasi, P. perniciosus and P. halepensis. In total 4–9 species-specific antigens were detected in each species by immunoblotting. Cross-reactivity was not detected between P. papatasi and the other species tested; in the SGL of P. papatasi sera from animals bitten by this species recognized 5–7 major antigens while sera from animals bitten by other species did not react. A weak cross-reaction was observed between P. perniciosus and P. halepensis; in SGL from P. perniciosus, the sera from rabbits and hamsters bitten by this species recognized about 8 intense bands while sera from animals bitten by P. halepensis reacted weakly with up to 4 saliva polypeptides.

The prevalence and abundance of infections with haemoparasites were studied over a 3 year period in Clethrionomys glareolus (bank vole, n=420) sampled from forests in the NE of Poland. Total species richness was 5 (Prevalence=Haemobartonella sp. 63·1%, Bartonella grahamii 27·4%, Hepatozoon erhardovae 31·4%, Trypanosoma evotomys 15% and Babesia microti 1·0%) with 81·9% of the voles carrying at least 1 species and a mean infracommunity species richness of 1·4. Variation in species richness was determined primarily by season and year, and the interaction of these factors. The observed frequency distribution of infracommunity species richness did not differ from that predicted by a null model, suggesting that there were no marked associations between the species. Analyses of prevalence and abundance of infection with each species in turn, revealed that overall the principal causes of variation were temporal and seasonal and their interaction, intrinsic factors such as age and sex playing only a minor role. However, the relative importance of specific extrinsic, and rarely intrinsic, factors varied and was distinct for each of the species in the study. Prevalence data revealed 4 sets of 2-way associations between species, mostly varyingly dependent on combinations of intrinsic and extrinsic factors. Analysis of quantitative associations suggested 4 sets of positive 2-way interactions, 3 of which remained after controlling for the effect of extrinsic and intrinsic factors on the abundance of each species, but only one could be unequivocally accepted (Haemobartonella sp.+B. grahamii) after correction for multiple comparisons. These data are discussed in the context of the changing ecological profiles in this region of Eastern Europe and, in a wider context, in relation to current understanding of the factors which shape component community structures of haemoparasites in wild rodents.

Six myxosporidian species were found in chub (Leuciscus cephalus) originating from Lower Austrian rivers. The frequency of the parasites and their localization was recorded. In all chub, independent of size and origin, Myxobolus cyprini occurred predominantly in the macrophage centres (MCs) of the haematopoietic organs, spleen and kidney. Exclusively in the head kidney of young fish not yet described vermicular plasmodia containing spores of M. cyprini were found. In muscle tissue the prevalence of M. cyprini was comparatively low. Other species of Myxobolus characterized by plasmodial cysts frequently occurred in gills and swimbladder but were rarely detected, and only in small numbers, in the haematopoietic organs. The number of M. cyprini spores and the relative volume of MCs in the haematopoietic organs were estimated in order to examine possible correlations. Significant interrelated changes were found only in juvenile fish up to a size of 15cm. In bigger fish, the number and size of macrophage aggregates were highly variable and independent of infection intensity and fish size, but the number of spores never exceeded that of the aggregated macrophages. The data suggest that due to an early date of infection M. cyprini is the only species which is closely associated with macrophage aggregation.

The aggressive use of praziquantel to combat schistosomiasis in Egpyt raises concern about the possible emergence of resistance. Eggs from Egyptian patients with praziquantel-resistant infections (not cured by 3 doses of praziquantel) have been used to establish infection-specific schistosome isolates in mice. The response of these worms to the drug was observed in vitro, in order to determine if the isolates obtained from these resistant infections were, in fact, less responsive to praziquantel. One of the hallmark effects of praziquantel on schistosomes in vitro is a disruption of the worm's outer surface, the tegument. Here, praziquantel-induced tegumental damage is observed in 3 distinct isolates, 2 derived from resistant infections and 1 from an infection cured by a single dose. The isolates from the resistant infections were less susceptible to praziquantel-induced tegumental damage in vitro, suggesting that the worms are in some way less responsive to the drug.

During trans-dermal invasion of the vertebrate host, larval schistosomes (cercariae) transform into schistosomula and become enveloped by a double lipid bilayered, tegumental membrane. The glucose transporter protein SGTP4 is found exclusively in these host-interactive tegumental membranes and in membranous vesicles proposed to be their precursor. In this study, we monitored the appearance and migration of this tegumental marker protein during larval transformation to test the current model of tegumental membrane biosynthesis in parasitic blood flukes. Only minutes after transformation was initiated, SGTP4 began accumulating in a previously unrecognized, bilaterally symmetrical, ‘cyton network’ beneath the peripheral muscle. Approximately 30min after the initiation of transformation the marker protein was seen in tubules connecting the network to the surface and erupting onto the surface in discrete patches. After 1h the patches were regularly arrayed over the schistosomula body and began to cover the anterior organ. By 3h the staining has largely resolved into a contiguous layer of fluorescence covering most of the worm surface. These findings confirm earlier suggestions, based on electron microscopy, that the parasite's surface tegumental membranes are derived from the migration of membranous vesicles produced within cytons and reveal a new subtegumental architecture interconnecting the cytons.

The toxicity of cadmium, zinc and cadmium/zinc mixtures at concentrations ranging from 1000 to 50000μg/l were investigated against cercariae and metacercariae of Parorchis acanthus obtained from the dog whelk Nucella lapillus. Cercarial encystment at concentrations of 25000μg/l or higher was significantly impaired by all test metals; however, at lower concentrations only zinc demonstrated toxicity. Mixtures of cadmium and zinc had a synergistic effect compared with single metal toxicity but only at 50000μg/l. Excystment in vitro was only significantly affected by cercariae exposed to cadmium/zinc mixtures whilst encysting. Twenty-four h exposures of fully formed cysts had no effect on excystment in vitro. Effects on in vitro excystment rates over a 2h period demonstrated widespread effects for cercariae-exposed P. acanthus. No effects were evident on excystment rates of cyst-exposed parasites.

The specific objectives of this study were to elucidate metal toxicity to hatching, survival and avoidance behaviour of Schistosoma mansoni miracidia. The toxicity of cadmium, zinc, and cadmium/zinc mixtures at concentrations ranging from 10000 to 10μg/l was investigated. Metal mixture toxicity investigation was undertaken with equal concentrations of the metals. The hatching of miracidia from eggs was inhibited by concentrations of 1000–10000μg/l of single metals. Metal mixtures had no effect on egg hatching. Survival of miracidia was reduced by increasing metal concentration except at concentrations of 10μg/l for single metal toxicity where survival was increased above the control. Miracidia demonstrated a rapid avoidance behaviour when briefly exposed to heavy metals. The mechanisms of metal toxicity to miracidia are briefly discussed.

The distribution of nitric oxide synthase (NOS) immunoreactivity and putative NOS activity in adult Schistosoma mansoni was analysed using 3 different types of NOS antibodies and NADPH-diaphorase histochemistry. Although potential involvement of the gaseous radical nitric oxide (NO) in host response to infection by schistosomes has been suggested, there is little or no information available regarding the role, or even the presence, of the NO pathway in schistosomes themselves. Here, we demonstrate that antibodies against neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms stain adult worms with distinctive patterns; anti-endothelial NOS (eNOS) shows no selective labelling. nNOS-like immunoreactivity is found in the main nerve cords and the peripheral nervous system. Putative sensory neurons with apical neuronal processes leading to the tegument of male worms are also immunoreactive for nNOS. Anti-iNOS labels a variety of predominantly non-neuronal tissues, showing intense labelling at or near the surface of the worm and in components of the gastrointestinal tract. The distribution of NADPH-diaphorase reactivity (a histochemical marker of NOS), is generally similar to the pattern of NOS immunoreactivity, including labelling of neuronal-like cells as well as developing eggs. These results suggest that an NOS-like enzyme is present in S. mansoni, and indicate potential roles for the different NOS isoforms in neuronal signalling, reproduction and development.

Infection with the endosymbiotic bacteria Wolbachia is widespread in filarial nematodes. Previous studies have suggested concordance between the phylogeny of Wolbachia with that of their nematode hosts. However, there is only one published molecular phylogenetic study of filarial species, based on the 5S rRNA gene spacer. The phylogeny proposed by this study is partially incongruent with previous classifications of filarial nematodes, based on morphological characters. Furthermore, both traditional classifications and molecular phylogenies are, in part, inconsistent with the phylogeny of Wolbachia. Here we report mitochondrial cytochrome oxidase I (COI) gene sequences for 11 species of filaria and for another spirurid nematode which was included as an outgroup. In addition, 16S rRNA, wsp and ftsZ gene sequences were generated for the Wolbachia of several filarial species, in order to complete the available data sets and further resolve the phylogeny of Wolbachia in nematodes. We used these data to evaluate whether nematode and Wolbachia phylogenies are concordant. Some of the possible phylogenetic reconstructions based on COI gene were congruent with the phylogeny of Wolbachia and supported the grouping of the rodent filaria Litomosoides sigmodontis with the lymphatic filariae (i.e. Brugia spp. and Wuchereria spp.) and the sister group relationship of Dirofilaria spp. and Onchocerca spp. However, the placement of the Wolbachia-free filaria Acanthocheilonema viteae is ambiguous and dependent on the phylogenetic methods used.

A 24-week randomized double blind intervention trial was conducted on adult female tea pluckers from an estate in Bangladesh to investigate the impact of iron supplementation and anthelmintic treatment on changes in ferritin and haemoglobin levels as well as on prevalence and intensity of helminth infections. A total of 553 women were randomly assigned to 1 of 4 intervention groups: group 1 received iron supplementation on a weekly basis, group 2 received anthelmintic treatment at the beginning and half way through the trial, group 3 received both iron supplementation as group 1 and anthelmintic treatment as group 2, and group 4 was a control group and received placebos for both iron supplementation and anthelmintic treatment. Prevalence and intensity of helminth infections (egg counts/g stool) of Ascaris lumbricoides, Trichuris trichiura and hookworms significantly fell in the 2 groups receiving anthelmintic treatment and there were some reductions in the 2 groups not receiving anthelminthic treatment. Haemoglobin and haematocrit concentrations increased significantly in the iron supplemented groups with smaller increases in the anthelmintic only group. All women showed a decrease in serum ferritin levels post-trial with greater losses in the 2 dewormed groups. Significant negative associations were found between hookworm egg counts and ferritin levels and Trichuris trichiura egg counts and haemoglobin concentration.

The cuticle is a major barrier prohibiting the infection of nematodes against micro-organisms. The attachment of bacterial spores of the nematode hyperparasite Pasteuria penetrans (PP1) to field populations of root-knot nematodes (RKN, Meloidogyne spp.) from Burkino Faso, Ecuador, Greece, Malawi, Senegal and Trinidad and Tobago were assayed in standard attachment tests. The attachment of spore population PP1 to different field populations of root-knot nematode showed that the rates of attachment differed between countries. Similar tests were also undertaken on P. penetrans spores from these countries against 2 species of RKN, M. incognita and M. arenaria. The results showed a high degree of variability in spore attachment with no clear distinction between the 2 species of nematode. It has been hypothesized that Pasteuria spore attachment is linked to nematode species designations and this study clearly shows that this is not the case. Further tests showed that variation in spore attachment was not linked to nematode phylogeny. The results therefore beg the question of how do parthenogenetic root-knot nematodes maintain cuticle variability in the face of such an aggressive hyperparasite.