The 13 636 bp mitochondrial (mt) genome sequence of the trichostrongylid nematode Cooperia oncophora was determined.Sequence data have been deposited in the EMBL/GenBank Data libraries under Accession number AY265417. The single nucleotide polymorphisms have been deposited in the dbSNP Database (http://www.ncbi.nlm.nih.gov/SNP) under ID numbers ss8485731-ss8486156. Like the mt genomes of other nematodes it is AT rich (76·75%) and cytidine is the least common nucleoside in the coding strand. There are 2 ribosomal RNA (rrn) genes, 22 transfer RNA (trn) genes and 12 protein coding genes. The relatively short AT-rich region (304 bp) and the lack of a non-coding region between two of the NADH dehydrogenase genes, nad3 and nad5, makes the mt genome of C. oncophora one of the smallest known to date, having only 525 bp of non-coding regions in total. The majority of the C. oncophora protein encoded genes are predicted to end in an abbreviated stop codon like T or TA. In total, 426 single nucleotide polymorphisms (SNP) were mapped on the mt genome of C. oncophora, which is an average of 1 polymorphism per 32 bp. The most common SNPs in the mt genome of C. oncophora were G/A (59·2%) and C/T (28·4%) transitions. Synonymous substitutions (86·4%) were favoured over non-synonymous substitutions. However, the degree of sequence conservation between individual protein genes of different parasitic nematode species did not always correspond to the relative number of non-synonymous SNPs. The mt genome sequence of C. oncophora presents the first mt genome of a member of the Trichostrongyloidea and will be of importance in refining phylogenetic relationships between nematodes. The, still limited, SNP map presented here provides a basis for obtaining insight into the genetic diversity present in the different protein coding genes, trn, rrn and non-coding regions. A more detailed study of the more variable regions will be of use in determining the population genetic structure of C. oncophora. Ultimately this knowledge will add to the understanding of the host–parasite relationship.

Karyotyping of Fasciola hepatica samples from Britain and Ireland has identified a triploid isolate which is effectively aspermic, rendering it necessarily asexually reproducing. Considering the extensive presence of asexually reproducing diploid and triploid Fasciola in Asia it is suggested that facultative gynogenesis is widespread in this parasite. This has important implications for the population genetics and evolution of Fasciola, especially in relation to the development and spread of drug resistance, and must be considered in the mathematical modelling of this process.

Sexual transmission occurs commonly in microparasites such as viruses and bacteria, but this is an unusual transmission route for macroparasites. Here we present evidence which suggests that a nematode parasite of Wood Mice (Apodemus sylvaticus) may be sexually transmitted and we have classified the nematode using molecular data. Wood Mice were collected annually in the course of work on their reproductive physiology. Larval nematodes were found in the epididymides of 19·6% of males. It seems likely that they would be transmitted to females at ejaculation. To identify these larval nematodes, which we were unable to do using morphological features, we sequenced the 18S rDNA. Sequence comparisons with the molecular phylogeny of Blaxter et al. (1998) demonstrated that they were bursate nematodes (Order Strongylida). The relationships between strongylid taxa were poorly resolved by 18S rDNA. However, both distance and parsimony analyses grouped the nematode with the superfamily Metastrongylidea in a clade containing Filaroides and Angiostrongylus sp. Importantly, the sequences were distinct from those of Heligmosomoides polygyrus and Angiostrongylus dujardini, two common strongylid nematodes of Apodemus. We were therefore unable positively to identify these worms by matching their sequences with those from morphologically identifiable adult strongylid nematodes infecting Apodemus. These results demonstrate that an as yet unidentified strongylid is quite commonly found in large numbers in the male reproductive tract of Wood Mice. Further work is required to understand the biology and transmission dynamics of this interesting system.

We found that the expression of mitochondrial apoptosis related genes (Bcl-2 associated protein X, BAX; apoptotic protease activating factor 1, Apaf-1; Caspase 9 and serine/threonine protein kinase, PKB) is elevated in Trichinella spiralis-infected muscles during encapsulation. Micro-dissection of the capsule and subsequent reverse transcription polymerase chain reaction (RT-PCR) confirmed that the expressions of these genes are restricted to the nurse cell. Immunocytochemistry revealed that pro-apoptosis factor (BAX, Apaf-1 and Caspase 9) are predominantly expressed in the basophilic cytoplasm (infected muscle cell origin) and anti-apoptosis factor (PKB) in the eosinophilic cytoplasm (satellite cell origin) of the nurse cell. Electron microscopy revealed that the pre-existing mitochondria in the muscle cells became swollen and disappeared immediately after newborn larva invasion, but new mitochondria of smaller size appeared in the cytoplasm. Nuclear fragmentation and condensation were observed in basophilic cytoplasm which is known to die. Together, the results suggest that the infected muscle cells transform but die through the process of apoptosis which is triggered by factors from the newly formed mitochondria. The anti-apoptosis factor may help the eosinophilic cytoplasm with its survival to ensure nurse cell function.

Almost nothing is known about the natural ecology of the nematode Caenorhabditis elegans, including its interactions with parasites. To help rectify this discrepancy, we assessed natural variation in the response of C. elegans towards a potential parasite, the soil bacterium Bacillus thuringiensis. Our results show that 10 isolates from across the world differ significantly in survival rate and infection level when confronted with a parasitic strain of B. thuringiensis. Furthermore, behavioural responses are identified as an important component of C. elegans defence, including evasion and possibly reduced ingestion of parasites. Again, the natural isolates show significant differences in these traits. In conclusion, worm defence is indicated to be complex and variable across space, implying that parasites play an important role in the ecology of this species. Based on these results, we expect C. elegans to be a promising model host for future analysis of the evolutionary dynamics of parasite–host interactions.

The work reported here describes the comparative ability of albendazole (ABZ), fenbendazole (FBZ) and triclabendazole (TCBZ) to penetrate through the tegument of mature Fasciola hepatica, and the influence of the physicochemical composition of the incubation medium on the drug diffusion process. The data obtained from the trans-tegumental diffusion kinetic studies were complemented with the determination of lipid-to-water partition coefficients (octanol-water) for the benzimidazole (BZD) anthelmintic drugs assayed. Sixteen-week-old F. hepatica were obtained from untreated artificially infected sheep. The flukes were incubated (37 °C) over 60 and 90 min in incubation media (pH 7·4) prepared with different proportions of ovine bile and Krebs' Ringer Tris (KRT) buffer (100, 75, 50, 25 and 0% of bile) containing either ABZ, FBZ or TCBZ at a final concentration of 5 nmol/ml. After the incubation time expired, the liver fluke material was chemically processed and analysed by high performance liquid chromatography (HPLC) to measure drug concentrations within the parasite. Additionally, the octanol-water partition coefficients (PC) for each molecule were calculated (as an indicator of drug lipophilicity) using reversed phase HPLC. The 3 BZD molecules were recovered from F. hepatica at both incubation times in all incubation media assayed. The trans-tegumental diffusion of the most lipophilic molecules ABZ and FBZ (higher PC values) tended to be greater than that observed for TCBZ. Interestingly, the uptake of ABZ by the liver flukes was significantly greater than that measured for TCBZ, the most widely used flukicidal BZD compound. This differential uptake pattern may be a relevant issue to be considered to deal with TCBZ-resistant flukes. Drug concentrations measured within the parasite were lower in the incubations containing the highest bile proportions. The highest total availabilities of the 3 compounds were obtained in liver flukes incubated in the absence of bile. Altogether, these findings demonstrated that the entry of the drug into a target parasite may not only depend on a concentration gradient, the lipophilicity of the molecule and absorption surface, but also on the physicochemical composition of the parasite's surrounding environment.

Substrate preferences, spatial aggregation patterns and seasonal dynamics in the egg laying of ectoparasitic Argulus coregoni were studied at a commercial fish farm in Finland. Pilot experiments showed that A. coregoni females selected specific types of substrates for egg laying. Significantly more A. coregoni eggs were laid on dark substrates than on light ones suggesting the use of visual cues. Therefore, egg-laying plates of dark colour were constructed for further experiments. Most A. coregoni eggs were deposited in locations in shadow and in the deepest water in a 2 m deep farming canal. Relatively more eggs were laid on bottom stones situated near each egg-laying trap than on artificial egg-laying plates indicating a preference for irregular stones in the deeper locations in the canal. The plates were located 20 cm above the bottom. However, a total of 5863 A. coregoni egg clutches, corresponding approximately to 1·5 million unhatched metanauplii, were successfully destroyed with the plates indicating that egg-laying traps can be used as an ecological control method against argulids in certain situations. For traps to be effective, ponds should not contain stones or any other hard substrata attracting female lice. The egg laying of A. coregoni in this study started on 5 July in 2001 and extended over 3·5 months up to mid-October. The egg-laying pattern of A. coregoni population was unimodal, supporting the view that only a single A. coregoni generation occurred annually in Central Finland.

A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6·5 and 7·5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52–76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.

The discovery of Trichinella zimbabwensis in farm crocodiles of Zimbabwe has opened up a new frontier in the epidemiology of the Trichinella genus. The objective of the present study was to investigate the infectivity of encapsulated species (T. spiralis, T. nativa, T. britovi, T. murrelli and T. nelsoni) and non-encapsulated species (T. pseudospiralis, T. papuae and T. zimbabwensis) in caimans (Caiman crocodilus), varans (Varanus exanthematicus), pythons (Python molurus bivittatus) and turtles (Pelomedusa subrufa) raised at their natural temperature range (26–32 °C). Mice and chickens were used as controls. At 6 days post-infection (p.i.), adult worms were detected in the small intestine of reptiles infected with T. papuae and T. zimbabwensis, of chickens infected with T. pseudospiralis and of mice infected with all encapsulated and non-encapsulated species. At 60 days p.i., T. papuae and T. zimbabwensis adult worms were collected from the intestine of varans and caimans and larvae from muscles of the four reptile species, T. pseudospiralis larvae from muscles of chickens, and larvae of all Trichinella species from mouse muscles. The highest reproductive capacity index of both T. papuae and T. zimbabwensis was observed in varans. The results show that T. papuae and T. zimbabwensis are able to complete their entire life-cycle in both poikilothermic and homoiothermic animals.

The individual and combined effects of cadmium (Cd) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) on the antibody response of fish against metazoan parasites were tested. Eels experimentally infected with the swim bladder nematode Anguillicola crassus were exposed to sublethal concentrations of Cd and PCB 126. Cd was added to the water resulting in an effective concentration of 21·7±12·8 μg/l (mean±S.D.). PCB 126 was applied orally at a dose of approximately 100 ng PCB 126 per g body weight. At the end of the experiment, 76 days post-infection (p.i.), eels were found to be infected with 2–3 worms. Immunoblot analyses revealed that the body wall of adult worms was the most suitable crude antigen, and was subsequently used for an ELISA to evaluate the immune response of A. anguilla under various conditions. A significant increase of Anguillicola-specific antibodies in the peripheral blood was first detected 61 days p.i., indicating that it was not the invasive larvae but the adult worms which elicit the antibody response. The presence of Cd in the concentrations applied did not appear to modulate the production of antibodies. In contrast, the exposure to PCB 126 resulted in a complete suppression of the antibody response. A similar effect was also found for the combined exposure of the infected eels to Cd and PCB 126. A suppressed immune response, as demonstrated here, may be the reason why hosts exposed to environmental pollution became often much more easily infected than unexposed conspecifics.

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequencedNucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the Accession number AF162691., allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35 582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Kmapp values describing interactions with dUMP (28·8 μMvs. 3·9 μM) and (6RS, αS)-N5,10-methylenetetrahydrofolate (383 μMvs. 54·7 μM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Ion channels are important target sites of anthelmintics, but little is known about those in Fasciola hepatica. In this work, we applied a planar lipid bilayer technique to characterize the properties of single ion channels in F. hepatica. Under a 200/40 mM KCl gradient, a large conductance channel of 251 pS was observed in 18% of the membranes studied. The channel was selective to K+ over Cl− with a permeability ratio of K+ to Cl− (PK/PCl) of 4·9. Open state probability (Po) of the channel was less than 0·5 and dependent on voltage (−60~+40 mV) and Ca2+ (~100 μM). The other two types of single channels observed in 11 and 5% of membranes, respectively, were a K+-permeable channel of 80 pS (PK/PCl=4·6) and a Cl−-permeable channel of 64 pS (PK/PCl=0·058). Open state probability of both channels showed little voltage dependence. The results indicate that distinct single channels of 60~251 pS are present in relative abundance and, in addition, that the planar lipid bilayer technique can be a useful tool for the study of single ion channels in F. hepatica.

The host immune response has profound effects on parasitic nematode infections. Here we have investigated how a range of infection parameters are affected by host immune responses and by their suppression and enhancement. The infection parameters considered were the number of parasitic females, their size, per capita fecundity and intestinal position. We found that in immunosuppressive treatments worms persist in the gut, sometimes with a greater per capita fecundity, maintain their size and have a more anterior gut position, compared with worms from control animals. In immunization treatments there are fewer worms in the gut, sometimes with a lower per capita fecundity and they are shorter and have a more posterior gut position, compared with worms from control animals. Worms from animals immunosuppressed by corticosteroid treatment reverse their changes in size and gut position. This description of these phenomena pave the way for a molecular biological analysis of how these changes in infection parameters are brought about by the host immune response.

Genetic analyses of Echinococcus granulosus isolates from different intermediate host species have demonstrated substantial levels of variation for some genotype (strain) clusters. To determine the range of genetic variability within and between genotypes we amplified and cloned partial cox1 and nadh1 genes from 16 isolates of E. granulosus from 4 continents. Furthermore, we sequenced different clones from a PCR product to analyse the intra-individual genetic variance. The findings showed a moderate degree of variance within single isolates and a significant degree of variance between the cluster of genotypes G1–G3 (sheep, Tasmanian sheep and buffalo strain), genotypes G4 (horse strain) and G5 (cattle strain) and the cluster of the genotypes G6 (camel strain) and G7 (pig strain). The variance of up to 2·2% within genotypes was relatively low compared with that of 4·3–15·7% among genotypes. The present results indicate that a re-examination of the classification of 5 genotypes of Echinococcus is warranted. Hence, our data highly support a re-evaluation of the taxonomy of the clades G1–G3, G4, G5, G6/7 and G8 (cervid strain) within the genus Echinococcus.

Recent studies of the forces behind the diversification of parasite assemblages have shed light on many aspects of parasite biodiversity. By using only parasite species richness as their measure of diversity, however, previous investigations have ignored the relatedness among parasite species and the taxonomic structure of the assemblages, which contain much information about their evolutionary origins. Here, we performed a comparative analysis across 50 species of fish from the coast of Brazil; we evaluated the effects of several host traits (body size, social behaviour, feeding habits, preference for benthic vs. pelagic habitats, depth range, and ability to enter brackish waters) on the diversity of their assemblages of metazoan parasites. As measures of diversity, we used parasite species richness, as well as the average taxonomic distinctness of the assemblage and its variance; the latter measures are based on the average taxonomic distance between any two parasite species in an assemblage. Unlike parasite species richness, taxonomic distinctness was unaffected by the number of host individuals examined per species. Fish body length proved to be the main predictor of parasite species richness, even when controlling for the confounding influences of host phylogeny and sampling effort, although it did not correlate with measures of parasite taxonomic distinctness. Predatory fish also had higher parasite species richness than planktivores, but this trend could not be confirmed using phylogenetically independent contrasts between host taxa. The main host feature associated with the taxonomic diversity of parasites was schooling behaviour, with schooling fish having more taxonomically diverse parasite assemblages than those of their non-schooling relatives. When focusing on endoparasite species only, both predatory feeding habits and a broad depth range were associated with the taxonomic distinctness of parasites. Our results suggest that certain host traits (i.e. body size) determine how many parasite species a host can accumulate over evolutionary time, whereas different host features influence the processes causing the taxonomic diversification of parasite assemblages.

Experimental intraperitoneal Taenia crassiceps cysticercosis in mice exhibits distinct genetical, immunological and endocrinological features possibly resulting from the complex interactive network of their physiological systems. Very notable is the tendency of parasites to grow faster in hosts of the female sex. It is also remarkable in the feminization process that the infection induces in chronically infected male mice, characterized by their estrogenization, deandrogenization and loss of sexual and aggressive patterns of behaviour. The proto-oncogene c-fos is a sex steroid-regulated transcription factor gene, expressed basally and upon stimulation by many organisms. In the CNS of rodents, c-fos is found expressed in association to sexual stimulation and to various immunological and stressful events. Hence, we suspected that changes in c-fos expression in the brain could be involved in the feminization of the infected male mice. Indeed, it was found that c-fos expression increased at different times during infection in the hypothalamus, hippocampus, less so in the preoptic area and cortex, and not in several other organs. The significant and distinctive regional changes of c-fos in the CNS of infected mice indicate that the brain of the host senses intraperitoneal cysticercosis and may also announce its active participation in the regulation of the host–parasite relationship. Possibly, the host's CNS activity is involved in the network that regulates the estrogenization and deandrogenization observed in the chronically infected male mice, as well as in the behavioural and immunological peculiarities observed in this parasitic infection.

Parasites have been found to be more infective to sympatric hosts (local adaptation) in some systems but not in others. The variable nature of results might arise due to differences in host and/or parasite migration rates, parasite virulence, specificity of infection, and to differences in the dose–response functions. We tested this latter possibility by manipulating the dose of trematode (Microphallus sp.) eggs on sympatric and allopatric host populations (Potamopyrgus antipodarum). We found that infection rapidly increased to a high asymptote (0·88±0·02, 1 S.E.) in the sympatric host population, but infections were low and surprisingly unrelated to dose in the allopatric host. We also found that host survival and growth rate were not negatively affected by increasing parasite dose in either population. These results suggest that defences in the allopatric host were not overwhelmed at high parasite doses, and that any life-history costs of defence are not plastic responses to parasite dose.

Previous studies have shown that the secreted phosphorylcholine-containing glycoprotein of filarial nematodes, ES-62, is only present in the post-infective life-cycle stages, but that the mRNA is transcribed throughout the worm's life-cycle. The aim of this current study was to investigate whether the presence or absence of protein expression simply reflects differences in mRNA abundance. To this end, we investigated the relative abundance of ES-62 using TaqMan real time RT-PCR, in different life-cycle stages of 2 model filarial nematode parasites, Acanthocheilonema viteae and Brugia pahangi. For B. pahangi, microfilariae, infective larvae and adult worms were each found to have approximately similar levels of ES-62 mRNA. However, the corresponding stages of A. viteae differed greatly from each other with a pattern of increased mRNA production with maturation. As a rule A. viteae had higher levels of ES-62 mRNA than B. pahangi, and this was particularly noticeable in the adult stage where the difference was approximately 3500-fold higher. However, this significant difference in mRNA abundance was not reflected in the quantity of ES-62 protein secreted by the adult worms of each species, as A. viteae only secreted ~3 times as much ES-62 as B. pahangi. Thus, overall, the results obtained from this study indicate that ES-62 protein production does not solely reflect mRNA levels, and also suggest that the 2 nematodes may employ different mechanisms for regulating protein production.

Dogs infected with adult tapeworms of Echinococcus granulosus release antigens (coproantigens) in faeces which can be detected by a capture ELISA. Supernatants prepared from E. granulosus-infected dog faecal samples were fractionated by size-exclusion fast protein liquid chromatography (FPLC) on a Superose-6 column. Coproantigen ELISA and Western blotting were used to demonstrate the immunoreactivity of eluted fractions. Two main FPLC peaks of antigenic activity were detected and designated as fraction F1 and fraction F2 with approximate relative molecular weights >670 kDa, and in the range of 146 to 440 kDa respectively. These two antigenic fractions (F1 and F2) fractionated from infected dog faeces were heat stable and largely protease-insensitive, but were highly sensitive to sodium periodate treatment, which strongly suggested the involvement of carbohydrates. Capture IgG antibodies against E. granulosus proglottis somatic extracts, detected a molecule with an approximate molecular weight of 155 kDa in fraction F2 after immunoblotting. The 155 kDa antigen could be completely ablated by sodium periodate treatment, but not after protease or lipase treatment. A surface tegument preparation of adult E. granulosus tapeworms contained large amounts of antigen that corresponded in size range and antigenicity to that observed in the FPLC fraction F2. There was also a peak of antigenic activity at >670 kDa corresponding to fraction F1 from a culture derived excretory–secretory (E–S) adult tapeworm preparation. The involvement of carbohydrate moieties in coproantigen activity present in the FPLC fractions F1 and F2 from faecal supernatants of E. granulosus-infected dogs was confirmed by lectin-binding assays and exoglycosidase treatment, which showed that α-D-mannose and/or α-D-glucose, β-galactose and N-acetyl-β-glucosamine residues were the most important carbohydrate components in putative coproantigens present in both fractions. N-acetyl-β-glucosamine and sialic acid residues were also contained in coproantigen molecules present in fraction F2. These results suggested that coproantigens detected in faeces of E. granulosus-infected dogs are large molecular weight molecules that may be derived from the carbohydrate-rich surface glycocalyx of adult worms, and are shed, released or secreted during the life-span of the tapeworm.

Recent studies of Echinococcus granulosus molecular strain typing have enabled a better understanding of the transmission cycle of cystic echinococcosis. There have been many publications in this area but there is a need for the evaluation of these tools. We have attempted to respond to this need in our study, which assessed 8 DNA fragments of 40 E. granulosus cysts from North Africa. Parasitological material was collected from 5 types of intermediate hosts, in 5 different countries. The primers chosen to amplify DNA targets were defined either in nuclear DNA, or in mitochondrial DNA. After amplification, PCR products were sequenced. The sequences obtained were aligned and comparisons were made within the group and with GenBank sequences. Whether the target was nuclear or mitochondrial, the same 2 main groups of genotypes were found. The first one, the ‘sheep’ strain, was found in the human, sheep and cattle samples collected in North Africa. The second one, the ‘camel’ strain, was found in the camel cysts and cattle and human cysts from Mauritania. These findings further confirm the congruence of the data given by the nuclear and the mitochondrial genome.