The cloning and characterization of Ts-p41, an EF-hand calcium-binding protein of the protozoan parasite Tritrichomonas suis is describedNucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDBJ databases under the accession numbers TSU011991 and TSU011992.. A T. suis cDNA library was screened with monospecific antibodies affinity purified on an immunoreactive 41kDa antigen in a Triton X-114 membrane-protein fraction. The resulting cDNA fragments turned out to be derived from 2 different genes encoding closely related Ts-p41 variants. The deduced amino acid sequences contained 6 EF-hand domains perfectly matching the canonical consensus motif and a putative C-terminal prenylation site. Northern and Southern hybridizations revealed that Ts-p41 was highly expressed and encoded by a gene-family. A cDNA encoding Ts-p41 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant Ts-p41 proteins bind Ca2+. In immunofluorescence, epitopes recognized by anti-Ts-p41 antibodies were distributed as well on the anterior flagella as on the recurrent flagellum of the parasite. Our findings with the parabasalid T. suis suggest that multiple EF-hand bearing calcium-binding proteins might be a common phenomenon associated with flagellar motility.

Using a combination of morphological, life-cycle and molecular data, we describe a new apicomplexan parasite Eimeria telekii n.sp. from a striped grass mouse Lemniscomys striatus captured in Kenya. Oocysts are oval to spherical or ellipsoidal, 20·4×15·7 (15·5–25·0×12·0–20·0)μm with a colourless, smooth and bilayered wall. Sporocysts are ellipsoidal, 11·2×7·8 (10·0–12·0×7·0–9·0)μm with a small Stieda body and granular sporocyst residuum and contain 2 elongated, banana-shaped sporozoites with a single refractile body. Life-cycle, pathogenicity and host specificity of this parasite were studied in laboratory-bred Lemniscomys barbarus and BALB/c mice. Two asexual stages and the sexual phase took place within the enterocytes of the caecum and colon of L. barbarus but not in inoculated BALB/c mice. An infectious dose of 5000 oocysts caused severe clinical illness and mortality in 2/2 (100%) L. barbarus. Phylogenetic analysis of the small subunit rRNA gene of E. telekii and members of the genera Eimeria, Cyclospora and Isospora placed E. telekii within the eimerian rodent clade.The nucleotide sequence was deposited in the GenBankTM under the accession number AF246717.

Varroa jacobsoni is an ectoparasite of Apis mellifera which invades brood cells, on 8-day-old larvae several hours before cell capping. Reproduction of the parasite takes place in the capped brood cells during the nymphose of the bee. Cuticular hydrocarbons of unparasitized bees and of bees parasitized by Varroa jacobsoni were extracted and analysed by gas chromatography (GC) coupled with mass spectrometry (GC-MS). Three developmental stages of worker honey bees were studied: larvae, pupae and emergent adults. The comparison between unparasitized and parasitized hosts was performed with Principal Components Analysis coupled with a multivariate variance analysis. The cuticular hydrocarbon profiles of honey bees were qualitatively similar, for the 3 developmental stages and regardless of the presence of Varroa in the cells. Nevertheless, comparison of the relative proportions of hydrocarbons showed that the cuticular profiles of pupae and emergent adults parasitized by 1 mite and of larvae parasitized by 2 mites were significantly different from the corresponding unparasitized individuals. Such modifications could be regarded (i) as a cause of the multi-infestation in larvae during invasion of brood and (ii) as a consequence of stress and/or removal of proteins contained in the haemolymph of the host during its development.

Regression analysis of the relationship of serum circulating anodic and cathodic antigens (CAA and CCA), as a possible direct measure of worm burden, and fecal egg counts allows the study of phenomena like density-dependent fecundity in human Schistosoma mansoni infections. For a reliable analysis, variations in egg count measurements as well as in circulating antigen levels have to be taken into account, and an accurate estimation of these variations (represented by parameter λ in the so-called Deming regression) is of great importance. From a new, extensive data set of repeated measurements of fecal egg counts and CAA and CCA concentrations we determined the respective values for parameter λ, and (re)analysed the relationship between circulating antigens and egg counts in 3 data sets from Burundi, Senegal and Zaire by Deming regression. For comparison, ordinary linear regression was performed as well, which considerably biased the regression lines for CCA, but not for CAA. The analyses resulted in a clearly non-proportional relationship between egg counts and CAA, and, to a lesser extent, CCA. Assuming that egg counts and antigen measurements directly reflect egg production and worm burdens, respectively, our findings reinforce the indication of density-dependent fecundity in schistosomiasis mansoni, as suggested by others.

It is generally accepted that free-living stages of parasitic helminths are dependent on aerobic degradation of endogenous energy sources for their energy generation. This concept, however, is not the result of extensive experimental evidence, but originated mainly intuitively as oxygen is widely available in their habitat and these stages generally have a small size. Schistosoma mansoni, the sole parasitic helminth whose energy metabolism has been studied throughout its life-cycle indeed has aerobically functioning free-living stages. However, large differences exist in energy metabolism between adult stages of distinct parasitic helminths, and caution should be taken in predicting that all free-living stages of all parasitic helminths have the same, aerobic energy metabolism. Hence, this report studied the energy metabolism of Fasciola hepatica miracidia and demonstrated that F. hepatica miracidia are also dependent on aerobic degradation of their endogenous glycogen stores by glycolysis and on Krebs cycle activity for energy generation. However, in contrast to S. mansoni, F. hepatica miracidia cannot function anaerobically, as inhibition of the respiratory chain blocked motility and carbohydrate degradation, and finally resulted in death of the miracidia. Therefore, this report demonstrated that differences exist between miracidia of distinct species, in pre-adaptation of their energy metabolism to the occasional hypoxic conditions within their next host.

Opisthorchis viverrini infection is an endemic disease that causes a serious public health problem in southeast Asia, especially in northeast Thailand. We have developed a PCR method using a pair of primers named OV-6F/OV-6R for detecting O. viverrini eggs in stool samples and compared it with Stoll's egg-count method. The primers were designed based on the pOV-A6 specific DNA probe sequence which gave a 330 base pair product. The PCR method can detect a single egg in artificially inoculated faeces or as little as 2×10-17ng of O. viverrini genomic DNA. The method gave 100% sensitivity in all hamster groups except in animals exposed to the lowest intensity of infection (1 metacercaria/hamster). In the first month of infection, the PCR method was more sensitive than using the egg-count method in all infected groups especially in the light infections. The PCR method was also successfully used in monitoring a therapeutic study. Since the PCR method showed no cross-reaction with Heterophyid flukes, it can be useful for specific identification of O. viverrini eggs in stool samples without the risk of false positives. It also has great potential for application in clinical epidemiological studies.

More than 20 species of the entomopathogenic nematode Steinernema have been described; to date, all reproduce exclusively by cross-fertilization of male and female individuals. Steinernema sp. strain T87 from Indonesia was found to consist largely of self-fertile hermaphrodites. Progeny were produced by morphological females both in insects (Galleria mellonella) and in hanging drops of insect haemolymph inoculated with a single infective juvenile. Sperm were present in the oviduct of unmated morphological females. Approximately 1% of infective juveniles developed into males, and males were also present in the second generation where they constituted 1–6% of the population. Under the same conditions the related species Steinernema longicaudum strain CB2B displayed typical steinernematid reproduction: cross-fertilization and a 1:1 sex ratio. It is argued that the development of hermaphroditism in Steinernema sp. T87 represents convergent evolution with Heterorhabditis, the other major genus of entomopathogenic nematode.

If the deleterious effects of non-specific parasites are greater on vulnerable host species than on reservoir host species then exclusion of the vulnerable host through apparent competition is more likely. Evidence suggests that such a mechanism occurs in interactions between the ring-necked pheasant (Phasianus colchicus), the grey partridge (Perdix perdix), and their shared caecal nematode Heterakis gallinarum. Modelling of the system predicts that the reduced parasite impact on the pheasant compared to the partridge results in the force of infection transmitted from pheasants to partridges being sufficient to cause partridge exclusion. Since the parasite impacts are currently estimated from correlational work, controlled infections were conducted to experimentally compare the impact of H. gallinarum on the two hosts and verify cause and effect. While challenged partridges showed reduced mass gain, decreased food consumption, and impaired caecal activity, in comparison to controls, the only detectable effect of parasite challenge on the pheasant was impaired caecal activity. The impact of H. gallinarum on challenged partridges conforms with previous correlational data, supporting the prediction that parasite-mediated apparent competition with the ring-necked pheasant may result in grey partridge exclusion. However, the observed decrease in the caecal activity of challenged pheasants could imply that H. gallinarum may also have an impact on the fecundity and survival of pheasants in the wild, particularly if food is limiting. If this is the case, the associated decrease in the force of infection to which the partridge is exposed may be sufficient to change the model prediction from partridge exclusion to pheasant and partridge coexistence.

This study investigated sequence variability in the second internal transcribed spacer of ribosomal DNA for 3 species of Metastrongylus (porcine lungworms). The ITS-2 region was amplified by PCR from individuals of M. elongatus, M. pudendotectus and M. salmi, and then subjected directly to single-strand conformation polymorphism analysis (SSCP), which allowed the direct display of sequence variation within and among individuals representing each speciesNucleotide sequences data reported in this paper are available in the EMBL, GenBankTM accession numbers: AJ305373 to AJ305404.. There were marked differences in SSCP profiles among species, making this approach useful for species identification. For individual species, representative bands were excised from electrophoretic gels, reamplified by PCR and subjected to direct sequencing. For all 3 taxa, variability in the ITS-2 was related chiefly to the presence of microsatellites. Eight different microsatellites were identified, namely (A)n, (TG)n, (TCG)n, (TA)n, (TATG)n, (G)n, (TACA)n and (T)n. Considerable variability in microsatellite repeat number (ranging from 1 to 23) was found among individual nematodes of a species and between species. The microsatellites were located to specific stem or loop regions in the predicted ITS-2 precursor rRNA secondary structure. The results may suggest that slipped-strand mispairing in microsatellite regions contributes to sequence variability in the ITS-2 of Metastrongylus species under structural constraint as a consequence of microsatellite location in the precursor rRNA. Similar studies of the ITS-2 for a wide range of parasitic nematodes may lead to a better understanding of concerted evolution in these organisms.

The pharmacological profiles of the 5-hydroxytryptamine (5-HT) receptors on Ascaris suum pharyngeal and somatic body wall muscles were investigated. The mechanisms involved following activation of these receptors were also studied. 5-HT activated and maintained pumping in isolated pharynxes with an EC-50 value of 44±1·7μM. The 5-HT agonists, tryptamine, sumatriptan 8-OH-DPAT and 5-carboxyamidotryptamine all failed to stimulate pumping. The 5-HT2 antagonist, ketanserin, initially excited and then inhibited pumping while the 5-HT3 antagonist, ondansetron, had no effect. 5-HT and 5-HT agonists, 8-OH-DPAT, 5-carboxyamidotryptamine, α-methyl-5-HT and tryptamine all inhibited ACh-induced contractions of a somatic body wall muscle strip. Ketanserin partially blocked the inhibitory effect of α-methyl-5-HT and ACh-induced contractions while the 5-HT uptake blocker, fluoxetine, potentiated the effect of 5-HT on ACh-induced contractions. Basal levels of cAMP, 1540±232pmol/mg, in pharyngeal muscle and 1721±134pmol/mg, somatic body wall muscle, were both increased by forskolin. 5-HT had no effect on pharyngeal muscle cAMP levels but raised cAMP levels in somatic body wall muscle, e.g. 100μM 5-HT, raised the level to 2851±212pmol/mg and 1000μM raised levels to 4578±1234pmol/mg., 1000μM, increased inositol phosphate levels in pharyngeal muscle. These results provide some evidence for a 5-HT2-like receptor on pharyngeal muscle. In contrast, the situation on somatic body wall muscle is more confusing since the pharmacological profile partly indicates a 5-HT2-like receptor but this receptor is linked to a rise in cAMP levels. Further studies are required to resolve the position but they must be based on the rational design of ligands specifically for nematode 5-HT receptors and not simply using ligands developed for the classification of mammalian 5-HT receptors. Such a design must take into account data from molecular biology studies of nematode 5-HT receptors.

In this paper we report on the biochemical presence of nitric oxide synthase (NOS)-like activity in Ascaris suum tissue and examine the pharmacological effect of NO donors on A. suum muscle strip preparation. NOS activity was determined by monitoring the formation of [3H]citrulline from [3H]L-arginine and NO formation via the oxyhaemoglobin assay. Neuromuscular tissue from A. suum which stained positively for NADPH diaphorase, contained NOS activity. Neither NOS activity nor NADPH diaphorase staining was detected in intestinal tissue. The absence of Ca2+, NADPH and other co-factors normally required for mammalian neuronal NOS activity only partially reduced the formation of both citrulline and NO by A. suum neuromuscular homogenate. The results of the biochemical assays indicate the presence of an enzyme capable of producing NO and citrulline, but with a different profile from that of rat neuronal NOS. We also present preliminary evidence for the action of NO (NO donors) in the neuromuscular system of A. suum.

Human populations are often infected with more than one species of parasite, especially in developing countries where overall rates of parasitism are high. Infections with multiple parasite species may not necessarily be independent within an individual as physiological, immunological or ecological factors may result in positive or negative associations between infections with different parasite species. A general framework for estimation of these associations is presented. Data from over 215000 individuals are analysed and the associations between geohelminth (Ascaris lumbricoides, Trichuris trichiura and hookworm) and malaria species are investigated. A method is presented for analysing data from multiple communities and testing whether the associations in different communities are equal. Overall estimates of the associations between species are obtained for each country and continent where data were available. Associations between geohelminth species were, in general, found to be positive whilst both positive and negative associations were found between the different Plasmodium species. There was evidence for significant geographical heterogeneity between the associations. A method for using these parameter estimates to predict the distribution of multiple infections when only marginal prevalence data are available is described and demonstrated.