The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

We hypothesized that expression of immunity to gastrointestinal nematodes and its relationship with dietary metabolizable protein (MP) supply in ewes depended on body protein- but not on body fat reserves. Twin-bearing ewes were trickle infected with Teladorsagia circumcincta. Three mid-pregnancy feeding treatments were used, calculated to maintain body reserves (HH), maintain body protein- and lose body fat reserves (HL), or lose body reserves (LL), and followed by 2 iso-energetic, periparturient feeding treatments, calculated to provide either scarce (LP) or adequate (HP) amounts of MP. At the end of the mid-pregnancy feeding treatment, HL- and LL-ewes had a smaller backfat depth (∼body fat reserves) than HH-ewes, and LL-ewes had a smaller muscle depth (∼ body protein reserves) and higher faecal egg counts (FEC) than HH- and HL-ewes. Overall, LL-ewes had higher periparturient FEC than HH- and HL-ewes, and LP-ewes had higher periparturient FEC than HP-ewes. However, LL-ewes offered the LP feed had higher periparturient FEC than ewes on any of the other combinations of feeding treatments. Feeding treatments affected plasma pepsinogen but not plasma IgA. The results support the view that body protein- but not body fat reserves may overcome consequences of dietary MP scarcity on periparturient breakdown of immunity to gastrointestinal nematodes.

Experiments are often carried out in the laboratory under artificial conditions. Although this can control for confounding factors, it may eliminate important factors that under natural conditions mediate the interaction under investigation. Here, we show that different results can be gained in the field and in the laboratory regarding host–parasite interaction. In the field, courting three-spined stickleback males, Gasterosteus aculeatus, were less often infected with plerocercoids of a cestode tapeworm, Schistocephalus solidus, than shoaling males. However, when a random sample of males was allowed to nest and court females in individual aquaria in the laboratory, both uninfected and infected males built nests and courted females. Moreover, while the few infected males that courted females in the field expressed less red nuptial coloration than uninfected courting males, there was no difference in redness between infected and uninfected males in the laboratory. We argue that the different results gained in the field and in the laboratory are due to differences in the cost of reproduction, due to differences in the resource pool of the males. The favourable conditions in the laboratory exclude factors such as predation risk, social interactions, and fluctuating environmental conditions that may use up resources in the field and mediate the effect of the parasite.

The statistical aggregation of parasites among hosts is often described empirically by the negative binomial (Poisson-gamma) distribution. Alternatively, the Poisson-lognormal model can be used. This has the advantage that it can be fitted as a generalized linear mixed model, thereby quantifying the sources of aggregation in terms of both fixed and random effects. We give a worked example, assigning aggregation in the distribution of sheep ticks Ixodes ricinus on red grouse Lagopus lagopus scoticus chicks to temporal (year), spatial (altitude and location), brood and individual effects. Apparent aggregation among random individuals in random broods fell 8-fold when spatial and temporal effects had been accounted for.

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.

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 community structure of monogeneans on the gills of a catfish Pimelodus albicans, in the Río de la Plata, was studied and compared with previous findings on P. maculatus. Six Monogenea species belonging to 3 genera were found: Demidospermus majusculus Kritsky & Gutiérrez, 1998; Demidospermus armostus Kritsky & Gutiérrez, 1998; Demidospermus idolus Kritsky & Gutiérrez, 1998; Demidospermus bidiverticulatum (Suriano & Incorvaia, 1995) Kritsky & Gutiérrez, 1998; Scleroductus yuncensi Jara & Cone, 1989; and Unibarra paranoplatensis Suriano & Incorvaia, 1995. The relationships between selected host and helminth variables; the interactions between the worms and the patterns obtained from the species abundance were analysed employing appropriate statistical methodology. The results obtained revealed the following common patterns within the monogenean communities of P. maculatus and P. albicans: (a) most monogenean species belonged to the genus Demidospermus; (b) S. yuncensi was a secondary species in both communities; (c) 1 of the 3 species common to both hosts was a core species (D. bidiverticulatum); (d) the selected variables of both hosts (weight and length) were not related to the number of monogeneans or species richness; (e) the dominant species in both communities showed the highest proportion of monogeneans in more than 40 % of the infracommunities; (f ) in both communities the dominant species accounted for 40–80 % of the total number of monogeneans at component community level; (g) the number of worms correlates positively with the species richness; (h) few cases of interaction among species were found; (i) a high degree of order exists among the infracommunities compared. Finally, the proportion of common species and the phylogenetic and ecological resemblances between the hosts, P. maculatus and P. albicans support a hypothesis of co-speciation among monogeneans and pimelodids in Río de la Plata. It seems improbable that these parasite communities are recent species assemblages formed by chance. The phylogenetic resemblance of the host–monogeneans system in Río de la Plata suggests that there is a single community pattern that can be observed in congeneric and sympatric hosts.

Stability of trichostrogylid populations indicates that some form of density-dependent regulation occurs which could act through fecundity. We present evidence for intraspecific density-dependent effects in 1 of 2, dominant, abomasal nematodes species (Ostertagia gruehneri) of Svalbard reindeer (Rangifer tarandus platyrhynchus). We found evidence in O. gruehneri, for density-dependent regulation of female worm length in April, July and October 1999. However, it is only in July that female worm length explains the variation in the number of eggs in utero which is also related to egg production per female worm only in this month and not at other times of the year. The seasonal pattern in faecal egg output in this species focuses egg production in the summer months when conditions are favourable to transmission. In contrast, we found no evidence in the other common species (Marshallagia marshalli) for density-dependent regulation of female worm length during or the number of eggs in utero. Faecal egg output in M. marshalli was positively related to worm burden but not to the mean number of eggs in utero. Neither inter-specific interactions nor host body condition appeared to influence worm fecundity. The contrasting patterns of density-dependent regulation of fecundity provides further evidence for divergent life-histories in this nematode community.

Electrophysiological techniques were used to obtain recordings of extracellular electrical activity from the anterior end of live adult females of Brugia pahangi. Stimulation with 100mM, 10mM and 1mM acetylcholine resulted in an increase in spike activity which was concentration dependent, whereas stimulation with phosphate-buffered saline and 0·1mM acetylcholine gave no increase in activity. The delay in response was not concentration dependent. The action of possible host cues was investigated. Stimulation with heat-inactivated foetal calf serum (IFCS) and 10mM glutathione gave an increase in spike activity but exposure to 5mg/dl haemoglobin elicited no response. The response to IFCS was found to be suppressed completely by pre-incubation for 30min in ivermectin.

The glycolipids of Ascaris suum represent either neutral, zwitterionic or acidic structures. The acidic fraction comprises a sulphatide and an unusual phosphoinositolglycosphingolipid (Lochnit et al. 1998b). The sulphatide was previously localized to the hypodermis, contractile zone of somatic muscle cells and the external musculature of the female uterus, whereas the presence of the phosphoinositolglycosphingolipid species was restricted to the intestine. The neutral and zwitterionic components belong to the arthro-carbohydrate series, which are substituted in their zwitterionic structures by phosphocholine (PC) and in one glycolipid by an additional phosphoethanolamine residue. In previous immunohistochemical localization studies, however, the chemical nature of the PC-substituted biomolecules has not been investigated in detail. Here, we report on the immunohistochemical localization and differentiation of phosphocholine-containing structures into lipid- and protein-bound species in adult A. suum. The patterns of immunostaining, obtained with a PC-specific monoclonal antibody and anti-zwitterionic glycolipid hyperimmune serum in the female worm, indicated a parallel organ distribution for glycolipid- and protein-bound PC-epitopes. Immunoreactivity was localized to specific tissues of the body wall, intestine and reproductive tract. This is the first report of surface-located PC-epitopes for ascarids. The patterns of immunolabelling obtained with antibodies directed against the unsubstituted arthro-carbohydrate series backbone suggested that the glycolipid-bound epitope was restricted to the hypodermis, whilst the protein-bound antigenic determinant resembled that for PC.

We used nestedness analysis to seek non-random patterns in the structure of component communities of metazoan parasites collected from 31 sympatric fish species from the northeastern Bothnian Bay, the most oligohaline area of the Baltic Sea. Only 8 marine parasite species were found among the 63 species recorded, although some marine fish species reproduce in the bay and others occasionally visit the area. Marine parasite species can utilize both freshwater and marine fish species as intermediate or final hosts, and marine fish can harbour freshwater parasite species. This exchange of parasite species between marine and freshwater fish has probably resulted from ecological factors acting over short time scales rather than from evolutionary processes acting over longer time; the key factor probably being the immediate presence of suitable intermediate and definitive hosts. Marine fish were expected to harbour species-poor parasite communities consisting mainly of generalists acquired from the sympatric freshwater fish species, which would result in a nested pattern among the different component communities. However, an anti-nested pattern was found in the component communities of metazoan parasites of fishes from the Bothnian Bay. A likely explanation for the observed pattern is that there are specialist parasite species, the majority of which are cestodes, in some of the freshwater fish species which otherwise have depauperate parasite communities.

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.

Free-living Soay sheep (Ovis aries) on the island of Hirta, St Kilda, Scotland, are naturally parasitized by gastrointestinal nematodes, predominantly Teladorsagia circumcincta. In this paper we show that reduced faecal egg counts (FEC) are associated with an allele at a microsatellite locus located in the first intron of the interferon gamma gene (o(IFN)-γ) in Soay sheep lambs and yearlings, measured at approximately 4 and 16 months of age, respectively. The same allele was also associated with increased T. circumcincta-specific antibody (IgA) in lambs, but not associated significantly in yearlings. Flanking control markers failed to show a significant association with either FEC or IgA. These results suggest that a polymorphic gene conferring increased resistance to gastrointestinal nematode parasites is located at or near the interferon gamma gene, and support previous reports which have mapped a quantitative trait locus (QTL) for resistance to this region in domestic sheep. Our data are consistent with the idea that a functional polymorphism leading to reduced expression or efficacy of (IFN)-γ could enhance the immune response to gastrointestinal nematodes by favouring the activity of the Th2 cell subset and antibody associated immune mechanisms.

One α- and 2 β-tubulin isotypes (isotypes 1 and 2) from the parasitic nematode Haemonchus contortus were artificially expressed in E. coli and purified to obtain tubulin that was capable of polymerizing into microtubules. Binding of [14C] mebendazole (MBZ), a benzimidazole compound, to each individual unpolymerized isotype and to microtubules polymerized from recombinant α- and β-tubulin was assessed and Kd and Bmax values determined. Mebendazole bound to the individual tubulin isotypes with a stoichiometry of 1:1. Binding occurred with highest affinity to α-tubulin followed by β-tubulin isotype 2 and β-tubulin isotype 1 indicating that α-tubulin may play a role in benzimidazole binding to microtubules. Upon polymerization of α- and β-tubulin isotype 2 into microtubules the stoichiometry of binding increased to 2:1 (mebendazole:tubulin) while binding affinity remained the same. Mebendazole binding to α/β-isotype 1 microtubules remained unchanged following polymerization. The increase in the number of benzimidazole receptors on α/β-isotype 2 microtubules suggests the formation of a new benzimidazole receptor upon polymerization.

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.

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.

Heat shock and stress responses are documented for the first time in larval stages of the cestodes Taenia solium and Taenia crassiceps. Radioactive metabolic labelling after in vitro incubation of cysts at 43 °C, revealed the induction of heat shock proteins. In T. crassiceps, the major heat shock proteins were 80, 70 and 60 kDa. After prolonged incubation, a set of low molecular weight heat shock proteins (27, 31, 33 and 38 kDa), were also induced. In vitro incubation of cysts at 4 °C, induced the synthesis of stress proteins ranging from 31 to 80 kDa, indicating the parasite is also able to respond to cold shock. T. solium cysts exposure to temperature stress also resulted in an increased synthesis of 2 major heat shock proteins of 80 and 70 kDa. Western blots using the excretory–secretory products of T. solium showed that 2 heat shock proteins were recognized by antibodies in the sera of cysticercotic patients: one of 66 kDa and another migrating close to the run front. The T. solium 66 kDa protein was also recognized by specific antibodies directed to a 60 kDa bacterial heat shock protein, suggesting that it belongs to this family of proteins.