Extracts of plants, such as papaya, pineapple and fig, are known to be effective at killing intestinal nematodes that inhabit anterior sites in the small intestine, such as Heligmosomoides polygyrus. In this paper, we demonstrate that similar in vitro efficacy also occurs against a rodent nematode of the large intestine, Trichuris muris, and confirm that the cysteine proteinases present in the plant extracts are the active principles. The mechanism of action of these enzymes involved an attack on the structural proteins of the nematode cuticle, which was similar to that observed with H. polygyrus. However, not all plant cysteine proteinases were equally efficacious because actinidain, from the juice of kiwi fruit, had no detrimental effect on either the motility of the worms or the nematode cuticle. Papaya latex was also shown to significantly reduce both worm burden and egg output of mice infected with adult T. muris, demonstrating that enzyme activity survived passage to the caecum and was not completely inactivated by the acidity of the host's stomach or destroyed by the gastric or pancreatic proteinases. Thus, the cysteine proteinases from plants may be a much-needed alternative to currently available anthelmintic drugs due to their efficacy and novel mode of action against different gastrointestinal nematode species.

The nature of the proteins which comprise the in vitro excretory/secretory products (ES) of the fourth-stage larva (L4) and adult Teladorsagia circumcincta are largely undefined, despite the fact that this nematode induces profound changes, in part related to parasite ES, in the cellular architecture of the glands lining the abomasal surface of infected sheep and goats. In this study, the protein components of L4 and adult ES were fractionated using 1D gel electrophoresis and the major protein bands, detected by Coomassie blue staining, excised from the gel and subjected to tryptic digest and subsequent mass spectrometric analysis. The resultant peptide mass fingerprints were used to identify 15 L4 and 13 adult ES proteins. Several proteins, such as globin and some metabolic enzymes, were present in both ES. L4 ES alone contained thioredoxin peroxidase, an enzyme that can detoxify free radicals resulting from host inflammatory responses to the parasite, a cysteine proteinase which may aid penetration of the gastric mucosa and 2 different galectins which may influence cell differentiation and morphogenesis. Adult ES contained a nucleoside diphosphate kinase homologue, an enzyme which has been linked to cellular changes and can affect liquid secretion and goblet cell degranulation.

The anthelmintic properties of tanniferous plants and of their secondary metabolites represent one possible alternative to chemotherapy that is currently being explored as a means of achieving sustainable control of gastrointestinal nematodes in ruminants. Previous in vivo and in vitro results suggest that tanniferous plants can have direct anti-parasitic effect against different stages of nematodes. However, the mode of action of the bioactive plant compounds remains obscure. The objectives of the current study were (1) to examine the hypothesis that extracts of tanniferous plants might interfere with the exsheathment of third-stage infective larvae (L3); (2) to assess the role of tannins in the process by examining the consequence of adding an inhibitor of tannins (polyethylene glycol: PEG) to extracts. The effects of 4 tanniferous plant extracts on exsheathment have been examined on L3 of Haemonchus contortus and Trichostrongylus colubriformis. Artificial exsheathment was induced in vitro by adding hypochloride solution to larval suspension. The evolution of exsheathment with time was measured by repeated observations at 10-min interval for 60 min. The selected plants were: genista (Sarothamnus scoparius), heather (Erica erigena), pine tree (Pinus sylvestris), and chestnut tree (Castanea sativa), with tannin contents ranging from 1·5 to 24·7% of DM. Extracts of a non-tanniferous plant (rye grass, tannin content: 0·3% of DM) were included in the assay as negative controls. The extracts were tested at the concentration of 600 μg/ml and the effects were compared to the rate of exsheathment of control larvae in PBS. No statistical differences in the pattern of exsheathment was observed after addition of rye grass or genista extracts for both nematode species and with heather extracts for T. colubriformis. In contrast, pine tree extracts on larvae of both species and heather extracts with H. contortus induced a significant delay in exsheathment. Last, contact with chest nut extracts led to a total inhibition of the process for both nematodes. These results suggest that extracts of tanniferous plants might affect a key process in the very early stages of larval invasion of the host. In most cases, the addition of PEG led to a total or partial restoration towards control values. This suggests that tannins are largely involved in the inhibitory process. However, other secondary metabolites may also interfere with the process that would help to explain some of the differences in response observed between the two nematode species.

In the present study, we constructed gender-enriched cDNA libraries for the adult stage of the parasitic nematode Oesophagostomum dentatum (order Strongylida) using suppressive-subtractive hybridization (SSH), sequenced clones from the female-library and male-library (480 from each) and conducted bioinformatic and microarray analyses of the expressed sequence tags (ESTs). In total, 873 ESTs (440 male and 433 female) were obtained, achieving a sequencing success of 91%The nucleotide sequences reported in this article (Tables 1–5) have been deposited in the EMBL, GenBank and DDJB databases under the Accession nos. AM157797–AM158083. Microarray analyses of 516 unique ESTs representing both gender-enriched libraries revealed differential hybridization for 391 of them (75·8%). Of these, 220 (56·3%) had significantly greater signal intensities in the female than in the male, and 154 (70%) of these were predicted to have homologues in C. elegans. These homologues were predicted to be involved in key biological processes, including embryonic nutrition, gametogenesis, molecular binding/transport or metabolism, nucleic acid synthesis and function, and signal transduction. Of the 171 ESTs with statistically higher signal intensities in male O. dentatum, 43·8% had homologues in C. elegans. These homologues included major sperm proteins (MSPs) or MSP-like molecules, keratin-like molecules, molecules involved in metabolism, PDZ domain-containing proteins, sugar binding proteins, protein kinases, serine proteases or protease inhibitors, molecules involved in proteolysis and other proteins, such as enzymes and various putative proteins. Of the 287 ESTs (from both gender-enriched cDNA libraries) with no known homologues in C. elegans, 50 (17·4%) had homologues in other nematodes, 8 had homologues in various other organisms and 104 (36·2%) had no homology to any sequence in current gene databases. The present study lays a foundation for the isolation and molecular, biochemical and functional characterization of selected genes from the gender-enriched cDNA archives established for O. dentatum.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

We describe the epidemiology of the protostrongylid parasites Parelaphostrongylus odocoilei and Protostrongylus stilesi in Dall's sheep (Ovis dalli dalli) from the Mackenzie Mountains, Northwest Territories, Canada (65 °N; 128 °W). Peak numbers of 1st-stage larvae of both parasites were shed by Dall's sheep on their winter range from March until May. In larval development experiments in the Mackenzie Mountains, peak numbers of infective 3rd-stage larvae of P. odocoilei were available in gastropod intermediate hosts in August–September. For both protostrongylids, the majority of transmission likely occurs on the winter range, with infection of gastropods when they emerge from hibernation in spring, and infection of Dall's sheep upon their return in fall. We validated a degree-day model for temperature-dependent development of larval P. odocoilei in gastropods, and applied degree-day models to describe and predict spatial and temporal patterns in development of P. odocoilei and P. stilesi in northern North America. Temperature-dependent larval development may currently limit northward range expansion of P. odocoilei into naïve populations of Dall's sheep in the Arctic, but climate warming may soon eliminate such constraints. In Subarctic regions where both P. odocoilei and P. stilesi are endemic, the length of the parasite ‘growing season’ (when temperatures were above the threshold for larval development) and amount of warming available for parasite development has increased over the last 50 years. Further climate warming and extension of the seasonal window for transmission may lead to amplification of parasite populations and disease outbreaks in host populations.

SSU ribosomal sequences of trypanosomes from Brazilian cattle and water buffalo were used to infer phylogenetic relationships between non-pathogenic T. theileri and allied species parasitic in artiodactyls. T. theileri trypanosomes from distinct geographical regions in Brazil and from other countries were tightly clustered into the ‘clade T. theileri’ distant from the ‘T. brucei clade’ of pathogenic parasites of artiodactyls, and also distinct from trypanosomes of other mammals. The existence of this monophyletic assemblage (T. theileri clade) composed only by isolates from artiodactyl species justifies the continued recognition of the subgenus T. (Megatrypanum) with T. theileri as its type species. Phylogenies based on SSU and ITS1 ribosomal sequences produced the same branching pattern with isolates from different mammalian hosts clustered in 5 lineages: A, related to water buffalo; B, C and D, to cattle; E, to fallow deer. The pattern of host specificity allied to some congruence between host and parasite phylogenies suggested association of these trypanosomes with their respective hosts. Segregation of cattle isolates into three lineages revealed an overall geographical structure. Moreover, positioning of trypanosomes infecting tabanids in the T. theileri clade is consistent with the role of these flies as important vectors of these trypanosomes.

Our preliminary observation, that sera from schistosomiasis patients react with carcinomas, raised the possibility of antigenic cross-reactivity. We here extend this observation to show that mice experimentally infected with Schistosoma mansoni react with human urothelial and transitional bladder carcinomas and also with a gastric carcinoma cell line, AGS. To identify cross-reacting epitopes, we looked for the expression of carcinoma markers in schistosome worms and eggs using monoclonal antibodies against tumour antigens MUC1, Tn and TF (also known as the oncofetal Thomsen-Friedenreich antigen or T antigen). Immunohistochemical staining showed that the TF-epitope is present in adult intravascular S. mansoni worms and eggs deposited in tissues of infected animals. The localization of TF-immuno-reactive material in schistosomes was seen at the parasite surface between male and female worms and around trapped eggs in the liver. This localization is consistent with the antigen being secreted. Mice experimentally infected with S. mansoni, developed circulating antibodies against the TF-epitope (identified as Gal(beta1-3) GalNAc-O-R) as seen in ELISA using TF-expressing asialoglycophorin (AGP) as antigen. The observed anti-TF response in S. mansoni-infected mice reflects the complexity of host-parasite interactions in this infection.

The effects of cold storage of infective third-stage larvae (L3) of different isolates of the parasitic nematode Haemonchus contortus were studied with respect to infectivity, pre-patent period and propensity for larval arrestment. Two complementary experiments were conducted with 2 groups of lambs, each animal being inoculated with 2000 L3 of either Swedish or Kenyan origin. In a first experiment, L3s were cold treated at 5 °C for 9 months prior to infection, whereas in a second experiment larvae were newly hatched. Individual faecal egg counts (FECs), and worm burdens were determined for each experiment. The results showed that the greatest differences were associated with the pre-treatment of larvae. The pre-patent period and the FECs differed significantly between the experiments but not between the isolates used in each experiment. However, the extent of hypobiosis was significantly different between the two isolates when fresh larvae were used (36% Kenyan isolate and 70% Swedish). The storage of H. contortus at 5 °C had no apparent effect on the infectivity of L3s, as high establishment ranging from 43 to 74% were observed, irrespective of isolates used. This study showed that H. contortus exhibited similar phenotypic traits regardless of geographical origin. Thus, there was limited evidence for adaptations to temperate climatic conditions.

The comparative development of Echinococcus multilocularis was studied in its definitive hosts, the fox, dog, cat and raccoon dog, beyond the pre-patent period to 90 days post-infection. All host species, apart from cats were susceptible to infection and capable of supporting substantial worm burdens. Although worms in cats matured and produced thick-shelled eggs, their overall development was retarded compared to that in other species in which the parasite matured rapidly producing large populations of gravid worms. E. multilocularis matured rapidly in foxes and raccoon dogs and this was sustained in raccoon dogs but not in foxes in which maturation of worms declined during the later stages of infection, in contrast to that in both raccoon dogs and dogs. These populations were sustained for longer in raccoon dogs and dogs compared to foxes. Cats would appear to have only a minor role in the maintenance of E. multilocularis in endemic areas, and infections in cats may be of minimal public health significance. In contrast, foxes, dogs and the recently recognized definitive host the raccoon dog, are all capable of playing significant roles in the epidemiology of alveolar echinococcosis. This study also demonstrated that the developmental processes of growth, segmentation, proglottization and maturation in adult Echinococcus are independent and can be influenced by environmental factors thus confirming earlier in vitro observations.

Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.

The effect of waterborne zinc (Zn) on Gyrodactylus population dynamics was studied on isolated guppies maintained at concentrations ranging from 0 to 240 μg Zn/l. After 1 week pre-exposure to Zn, each fish was experimentally infected with 3 gyrodactylids and parasite numbers were recorded daily on each fish until the fish either died or recovered from infection. Parasite establishment was most successful at 0 and 240 μg Zn/l (97%) compared with the intermediate Zn concentrations. Low to moderate concentrations of Zn were beneficial to the parasite, as evidenced by the concentration-dependent increase in peak parasite burden on recovered fish up to 120 μg Zn/l. In contrast, 240 μg Zn/l may have been toxic to the parasite, as both peak parasite burden (in fish that recovered from infection), and maximum rate of increase of the parasite population (in fish that died) declined at this concentration. The combined effect of infection and Zn is harmful to the fish, because mortality of infected fish (but not uninfected fish) increased with increasing Zn concentrations. We suggest that the observed mortality occurs because of the inability of fish to continuously produce mucous that is a key factor for protecting fish from both waterborne Zn and ectoparasites.

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica on the East Coast of the United States. Transmission dynamics of this parasite were investigated in situ for 2 consecutive years (May through October) at 2 lower Chesapeake Bay sites. Compared to previous studies where seasonal infection patterns in oysters were measured, this study also provided parasite water column abundance data measured using real-time PCR. As previously observed, salinity and temperature modulated parasite transmission dynamics. Using regression analysis, parasite prevalence, oyster mortalities and parasite water column abundance were significantly positively related to salinity. Perkinsus marinus weighted prevalence in wild oysters and parasite water column abundance both were significantly related to temperature, but the responses lagged 1 month behind temperature. Parasite water column abundance was the highest during August (up to 1200 cells/l) and was significantly related to P. marinus weighted prevalence in wild oysters, and to wild oyster mortality suggesting that parasites are released in the environment via both moribund and live hosts (i.e. through feces). Incidence was not significantly related to parasite water column abundance, which seems to indicate the absence of a linear relationship or that infection acquisition is controlled by a more complex set of parameters.

Infective larvae of an intestinal nematode, Strongyloides venezuelensis, enter rodent hosts percutaneously, and migrate through connective tissues and lungs. Then they arrive at the small intestine, where they reach maturity. It is not known how S. venezuelensis larvae develop during tissue migration. Here we demonstrate that tissue invasion ability of S. venezuelensis larvae changes drastically during tissue migration, and that the changes are associated with stage-specific protein expression. Infective larvae, connective tissue larvae, lung larvae, and mucosal larvae were used to infect mice by various infection methods, including percutaneous, subcutaneous, oral, and intraduodenal inoculation. Among different migration stages, only infective larvae penetrated mouse skin. Larvae, once inside the host, quickly lost skin penetration ability, which was associated with the disappearance of an infective larva-specific metalloprotease. Migrating larvae had connective tissue migration ability until in the lungs, where larvae became able to settle down in the intestinal mucosa. Lung larvae and mucosal larvae were capable of producing and secreting adhesion molecules.

Niche apportionment models have only been applied once to parasite communities. Only the random assortment model (RA), which indicates that species abundances are independent from each other and that interspecific competition is unimportant, provided a good fit to 3 out of 6 parasite communities investigated. The generality of this result needs to be validated, however. In this study we apply 5 niche apportionment models to the parasite communities of 14 fish species from the Great Barrier Reef. We determined which model fitted the data when using either numerical abundance or biomass as an estimate of parasite abundance, and whether the fit of niche apportionment models depends on how the parasite community is defined (e.g. ecto, endoparasites or all parasites considered together). The RA model provided a good fit for the whole community of parasites in 7 fish species when using biovolume (as a surrogate of biomass) as a measure of species abundance. The RA model also fitted observed data when ecto- and endoparasites were considered separately, using abundance or biovolume, but less frequently. Variation in fish sizes among species was not associated with the probability of a model fitting the data. Total numerical abundance and biovolume of parasites were not related across host species, suggesting that they capture different aspects of abundance. Biovolume is not only a better measurement to use with niche-orientated models, it should also be the preferred descriptor to analyse parasite community structure in other contexts. Most of the biological assumptions behind the RA model, i.e. randomness in apportioning niche space, lack of interspecific competition, independence of abundance among different species, and species with variable niches in changeable environments, are in accordance with some previous findings on parasite communities. Thus, parasite communities may generally be unsaturated with species, with empty niches, and interspecific interactions may generally be unimportant in determining parasite community structure.

We investigated population differences in immunological adaptation of three-spined sticklebacks (Gasterosteus aculeatus) to one of their most abundant macroparasites, the eye fluke Diplostomum pseudospathaceum. We compared infection success in lab-bred fish of 2 populations in northern Germany, from a lake, where eye flukes are prevalent, and a river, where these parasites do not occur. In order to discriminate between protection through innate and acquired immunity, we exposed fish either only once or repeatedly. Lake fish were significantly less susceptible than river sticklebacks already after a single exposure, indicating that in sympatric hosts innate immunity plays the major role in the defence against this helminth infection. In both habitat types, previous exposures only marginally decreased infection rates within 12 weeks. Lake fish showed higher immunocompentence by means of respiratory burst activity and spleen size, regardless of the infection status. Furthermore, they were in a better energy status than river fish, as indicated by a higher hepatosomatic index and haematocrit value. Interestingly, F1 hybrid fish of both populations ranged between the pure habitat types in parasite susceptibility as well as in immunological and condition parameters. Our results suggest that sticklebacks from lakes are better adapted to cope with higher parasite abundance in this habitat.

Great Island Virus (GIV) is an arbovirus present in the tick Ixodes uriae, a common ectoparasite of nesting seabirds. Common guillemot (Uria aalge) and black-legged kittiwake (Rissa tridactyla) are the preferred and most abundant hosts of I. uriae on the Isle of May, Scotland. As part of a study to understand the epidemiology of GIV, the ability of guillemot and kittiwake to support tick-borne transmission of GIV was examined. GIV was present in ticks feeding in isolated guillemot colonies and guillemots had virus-specific neutralizing antibodies demonstrating previous GIV infection. By contrast, only uninfected ticks were found in colonies inhabited solely by kittiwakes. GIV was isolated from kittiwake ticks in colonies which also contained breeding guillemots but no virus-specific neutralizing antibodies were present in blood samples of kittiwake on which infected ticks were feeding. Thus guillemots are the main vertebrate hosts of GIV on the Isle of May whereas kittiwakes do not appear to be susceptible to infection. Virus infection of adult ticks feeding on guillemots was highly efficient and may involve both viraemic transmission and transmission from infected to uninfected ticks feeding together on birds that do not develop a patent viraemia.

Ivermectin treatment may induce severe adverse reactions in some individuals heavily infected with Loa loa. This hampers the implementation of mass ivermectin treatment against onchocerciasis in areas where Onchocerca volvulus and L. loa are co-endemic. In order to identify factors, including co-infections, which may explain the presence of high L. loa microfilaraemia in some individuals, we analysed data collected in 19 villages of central Cameroon. Two standardized skin snips and 30 μl of blood were obtained from each of 3190 participants and the microfilarial (mf) loads of both O. volvulus and L. loa were quantified. The data were analysed using multivariate hierarchical models. Individual-level variables were: age, sex, mf presence, and mf load; village-related variables included the endemicity levels for each infection. The two species show a certain degree of ecological separation in the study area. However, for a given individual host, the presence of microfilariae of one species was positively associated with the presence of microfilariae of the other (OR=1·79, 95% CI [1·43–2·24]). Among individuals harbouring Loa microfilariae, there was a slight positive relationship between the L. loa and O. volvulus mf loads which corresponded to an 11% increase in L. loa mf load per 100 O. volvulus microfilariae. Co-infection with O. volvulus is not sufficient to explain the very high L. loa mf loads harboured by some individuals.

Post-mortem examinations of harbour porpoises, Phocoena phocoena, regularly reveal heavy parasitic worm burdens. These same post-mortem records show varying levels of polychlorinated biphenyls (PCBs) accumulating in the blubber of porpoises. Although a number of papers have documented geospatial and temporal changes of PCBs and their detrimental effects on marine mammal health, as yet none have examined their role in determining nematode burdens in wild marine mammal populations. Using a data set consisting of harbour porpoises stranded in the UK between 1989 and 2002, we found a significant, positive association between PCB levels and nematode burdens, although the nature of the relationship was confounded with porpoise sex, age and cause of death. It was also apparent that individuals with the heaviest infestations of nematodes did not have the highest PCB level: while PCBs are important, they are clearly not the sole determinants of nematode burdens in wild populations of the harbour porpoise around the UK.