The variability inherent in different isolates of Fasciola hepatica has been evident from reports in the literature but to date there has been no systematic examination of the relationship between these differences and the fecundity of the parasite. In this study we have attempted to remedy this situation by comparing the relative efficiencies with which 2 well-characterized isolates of the liver fluke (Oberon and Fairhurst) progress through both their definitive and intermediate hosts. We did not observe a reduction in fitness in the Oberon isolate which has been reported to be triclabendazole-resistant, compared to the triclabendazole-susceptible Fairhurst isolate, but considerable inter- and intra-isolate variability at different life-cycle stages was recorded. Thus the Oberon isolate gave 4-fold the number of cercariae when 100 snails were each challenged with a single miracidium and was more successful in establishing productive infections in rats. Fairhurst metacercariae excysted at a higher rate than those from the Oberon isolate and Fairhurst flukes produced 4-fold more eggs. The extent of the intra- and inter-isolate variability revealed in this work will provide a basis for the development of models of population dynamics aimed at predicting the response of the liver fluke to changing environmental conditions such as the use of anthelmintics or climatic change.

The fly, Philornis downsi Dodge & Aitken, was first collected in 1964 on the Galápagos Islands and is now widespread across the archipelago. Virtually nothing is known about the behaviour and ecology of the fly as well as for the genus in general. Here, we describe all larval instars for the first time, and discuss infection intensity and impacts of parasitism on nestling survival of Darwin's finches. Adult P. downsi are non-parasitic free-living flies, whereas the larvae are obligate blood-feeding parasites on nestling birds. The larvae show a marked shift in their host site specificity – a novel finding for the genus Philornis: the first and early second larval instars live as agents of myiasis in finch nostrils and other tissues, while the older second and third instar larvae reside in the nest material and feed externally on the blood of nestlings, leading to blood losses in nestlings of 18–55%. Pupation occurs in the bottom layer of the nest. The combined effects of tissue damage by the endoparasitic instar larvae and anaemia by nest-dwelling haematophagous instar larvae account for the high nestling mortality (76%) due to Philornis parasitism. This represents the highest mortality by Philornis reported in the literature and emphasizes the extremely serious threat this parasite poses for the endemic passerine fauna of the Galápagos Islands.

Transmission experiments were conducted to compare the transmissibility of genetically different Trypanosoma congolense (Savannah subgroup) strains isolated from cattle in a trypanosomiasis endemic area of eastern Zambia. A total of 17 strains were compared. Three strains were extremely virulent with a short pre-patent period, high parasitaemia and a short median survival time (between 5 and 9 days) in mice. The remainder of the strains belonged to the moderate (6 strains) or low (8 strains) virulence categories with median survival times between 10 and 30 days and >30 days, respectively. Batches of 40 teneral Glossina morsitans morsitans (Diptera: Glossinidae) were offered a single bloodmeal on mice infected with one of those strains. Flies were dissected to determine their infection status 21 days later. The proportion of flies with procyclic and metacyclic infections differed significantly between trypanosome strains and were significantly higher in flies infected with extremely virulent strains (P=0·033 and P=0·016 for the differences in the procyclic infection rate of strains with moderate and low virulence, respectively and P=0·005 and P=0·019 for the differences in the metacyclic infection rate of strains with moderate and low virulence, respectively). On the other hand, moderately virulent strains had, in general, higher procyclic and metacyclic infection rates compared to low virulent strains. But the differences were not significant (P>0·05). The outcome of those experiments shows clear differences in transmissibility of trypanosome strains associated with their virulence. This observation confirms the theory for the evolution and maintenance of virulence in a parasite population and may explain the persistence of virulent trypanosome strains in a susceptible host population.

Robinia aurata n. g., n. sp. is described from Liza aurata (Mugilidae), the golden grey mullet, from the Ebro Delta, Spanish Mediterranean. The new genus differs from all other hemiurid genera in the combined possession of muscular flanges and a vestigial ecsoma. Within the Bunocotylinae, which currently accommodates 2 genera, Bunocotyle and Saturnius, the new genus exhibits a unique combination of blind caeca, Juel's organ, post-ovarian bulk of the uterus in the hind-body, and tegumental papillae surrounding the oral and ventral sucker apertures. Furthermore, Robinia n. g. differs from both Bunocotyle and Saturnius in the nature of the muscular extensions around the oral sucker, with the shape of a muscular belt in the latter and numerous muscular papillae in the former. The phylogenetic hypothesis for the Bunocotylinae developed from sequence data analyses based on partial lsrDNA and complete ssrDNA combined (22 species) and V4 domain of the ssrRNA gene (37 species) supports the erection of the new genus and confirms its position within the Hemiuroidea. Both molecular analyses confirmed the monophyly of the Hemiuroidea, its division into 2 major clades and the polyphyly of the Derogenidae, as in previous studies, and suggest that the Gonocercinae (with 2 genera, Gonocerca and Hemipera), may require a distinct familial status. Finally, there was poor support for the distinct status of the Lecithasteridae and Hemiuridae, following previous suggestions based on different sequence data sets. A key to genera of the Bunocotylinae is presented.

Every few years a large proportion of the feral sheep on Hirta, St Kilda die due to food shortage. The effects of malnutrition are exacerbated by gastrointestinal nematodes. As found in sheep flocks in mainland Britain, Teladorsagia circumcincta has long been considered the predominant and most pathogenic nematode species in all age classes of Soay sheep. Previous research indicated that intensity of this species showed a negative association with host age and comprised 75% of the entire gastrointestinal burden. Here we present new data that show Trichostrongylus axei and Trichostrongylus vitrinus to be the predominant worm pathogens in young Soay sheep. In the present study, Trichostrongylus spp. burdens declined with host age whereas T. circumcincta actually increased in burden over the first few age classes. Also, male hosts had significantly higher burdens of Trichostrongylus spp. than females, with this genus making up a higher proportion of the strongyle egg producing community in male hosts than female hosts. These new findings raise questions concerning our previous interpretation of the main nematode species contributing to strongyle egg count in the population, and the contrasting infection patterns of these nematode species in unmanaged St Kilda Soay sheep compared with domestic sheep in mainland Britain.

The understanding of the patterns and processes underlying the structure of parasite communities has progressed significantly in the last years; however, much of the evidence available to date on parasite community comes from unconfirmed studies. In this study the ectoparasite communities of 2 related rockfishes, Sebastes capensis and Helicolenus lengerichi, from the southern Chilean coast (ca. 40 °S) were examined to determine whether their specific compositions are similar, structured in non-random ways, and repeatable among years. From 2001 to 2004, 189 specimens of S. capensis and 101 of H. lengerichi were examined, 10 and 9 parasite species were recorded in S. capensis and H. lengerichi, respectively. Component ectoparasite communities of these hosts were taxonomically similar (60%), but the abundances and prevalences of their parasites differed significantly among years and between hosts. The most prevalent species were Lepeophtheirus chilensis, and Microcotyle sp. for S. capensis, and Microcotyle sp., Juanetia continentalis, and Interniloculus chilensis for H. lengerichi. Infracommunities of S. capensis showed higher species richness and parasite numbers than those of H. lengerichi. The similarity among infracommunities (measured as parasite abundance) varied significantly among years both within and between hosts. Despite this, temporal nestedness patterns were observed in infracommunities of these hosts, suggesting that their ectoparasite communities are structured and might be predictable in time, at least in the southern Chilean coast where they share the same habitat.

The success of a pathogen depends not only on its transmission to new hosts, but also on its ability to colonize and persist within its current host. Studies of within-host dynamics have focused on only a few diseases of humans, whereas little is known about the factors that influence pathogen populations as they develop inside non-human hosts. Here, we investigate pathogen dynamics occurring within bumble bees (Bombus impatiens) infected by the gut trypanosome Crithidia bombi. Infection by C. bombi showed several features characteristic of vertebrate diseases, including a rapid initial increase in infection intensity, marked oscillations in parasitaemia, and the stimulation of a systemic immune response in infected bees. Within-host dynamics generated substantial variation in the infectiousness and flower-visiting behaviour of bumble bees. Changes in bee foraging that arise from infection may influence the probability of C. bombi transmission between bees at flowers.

The tick-borne protozoan parasites Theileria parva and Theileria annulata cause economically important diseases of cattle in tropical and sub-tropical regions. Because of shortcomings in disease control measures based on therapy and tick control, there is a demand for effective vaccines against these diseases. Vaccines using live parasites have been available for over two decades, but despite their undoubted efficacy they have not been used on a large scale. Lack of infrastructure for vaccine production and distribution, as well as concerns about the introduction of vaccine parasite strains into local tick populations have curtailed the use of these vaccines. More recently, research has focused on the development of subunit vaccines. Studies of immune responses to different stages of the parasites have yielded immunological probes that have been used to identify candidate vaccine antigens. Immunisation of cattle with antigens expressed in the sporozoite, schizont or merozoite stages has resulted in varying degrees of protection against challenge. Although the levels of protection achieved have not been sufficient to allow exploitation for vaccination, there are clearly further lines of investigation, relating to both the choice of antigens and the antigen delivery systems employed, that need to be pursued to fully explore the potential of the candidate vaccines. Improved knowledge of the molecular biology and immunology of the parasites gained during the course of these studies has also opened up opportunities to refine and improve the quality of live vaccines.

Mitochondrial DNA (mtDNA) sequence data and amplified fragment length polymorphism (AFLP) patterns were compared for the lungworm Dictyocaulus viviparus, a nematode parasite of cattle. Eight individual D. viviparus samples from each of 8 herds in Sweden and 1 laboratory isolate were analysed, with the aim of describing the diversity and genetic structure in populations using different genetic markers on exactly the same DNA samplesNucleotide sequence data reported in this paper have been submitted to GenBank under the Accession numbers DQ299539-DQ299826.. There was qualitative agreement between the whole-genome AFLP data and the mtDNA sequence data, both indicating relatively strong genetic differentiation among the Swedish farms. However, the AFLP data detected much more genetic variation than did the mtDNA data, even after allowing for the different inheritance patterns of the markers, and indicated that there was much less differentiation among the populations. The mtDNA data therefore seemed to be more informative about the most recent history of the parasite populations, as the general patterns were less obscured by detailed inter-relationships among individual worms. The 4 mtDNA genes sequenced (1542 bp) showed consistent patterns, although there was more genetic variation in the protein-coding genes than in the structural RNA genes. Furthermore, there appeared to be at least 3 distinct genetic groups of D. viviparus infecting Swedish cattle, 1 of which was predominant and showed considerable differentiation between farms, but not necessarily within farms. Second, the 2 smaller genetic groups occurred on farms where the predominant group also occurred, suggesting that these farms have had multiple introductions of D. viviparus.

In Trypanosoma brucei brucei, an invariant surface glycoprotein of molecular weight 75 kDa (ISG75) is uniformly distributed over the surface of a trypanosome and is specific for bloodstream-form parasites. For the other taxa of the Trypanozoon subgenus no data about this surface molecule are available. Therefore, we investigated the ISG75 in the genomes of several pathogenic Trypanozoon by Southern blot, PCR and RT-PCR and sequence analysisNucleotide sequence data reported in this paper are available in the GeneBank™, EMBL and DDBJ databases under the Accession numbers DQ200175-DQ200256.. This study reveals that (i) all members of the Trypanozoon subgenus, i.e. T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. evansi and T. equiperdum, harbour ISG75 as multiple gene copies with at least 4–16 copies per genome; (ii) ISG75 gDNA and cDNA sequences are distributed in 2 groups that share at least 75% and 77% identity respectively; (iii) sequences from both groups are transcribed in all species and subspecies of the Trypanozoon subgenus; (iv) the main differences between group I and group II are located in the variable region at the amino-terminus of the putative proteins; (v) however, all the sequences in both groups have some well-conserved features, such as the cysteine residues, an amino-terminal cleavable signal peptide, a single α-helix transmembrane domain and a cytoplasmic domain at the carboxy-terminus.

A cDNA encoding Fg14-3-3 protein 1 was cloned by immunoscreening of an adult-stage Fasciola gigantica cDNA library using a rabbit antiserum against tegumental antigens of the parasite. The protein has a deduced amino acid sequence of 252 residues and a calculated molecular weight of 28·7 kDa. It shows sequence identity values between 57·6 and 58·1% to the human 14-3-3 beta, zeta, theta, and eta proteins and is in a phylogenetic cluster with the 14-3-3 protein 1 of Schistosoma spp. Nucleic acid analyses indicate that the Fg14-3-3 protein 1 is encoded by a single copy gene and that this gene is expressed as a transcript of 1250 nucleotides. In adult and 4-week-old parasites the gene's transcriptional and translational products were localized in the gut epithelium, parenchyma, tegument cells, and in the reproductive organs. An antiserum against recombinant Fg14-3-3 protein 1 detected a slightly smaller 14-3-3 protein in the parasite's excretion/secretion material and showed cross-reactivity with 14-3-3 proteins in extracts of other trematodes and mouse. Antibodies against Fg14-3-3 protein were detected in the sera of rabbits as early as 2 weeks after infection with metacercariae of F. gigantica and the antibody titre increased continuously over a 10-week observation period.

Although immunisation protocols for a wide variety of parasitic diseases have been developed, it is often questioned why these do not always reach the market. In this review information about the regulations and procedures that apply to licensing the production and marketing of medicinal preparations, especially parasite vaccines, is presented. These general regulations specify issues on product (quality, safety, efficacy and potency) and production (facilities and consistency). Vaccine developers and manufacturers have to comply with these regulations, which may involve years of research and development. Moreover, where the manufacturer claims specific features of the product, these claims have to be corroborated by (experimental) data. A series of principles has been used to develop vaccines against parasite infections varying from the use of (attenuated) live vaccines to killed vaccines and subunit vaccines. The implications of some specific regulatory issues associated with these approaches are discussed.

Burrowing depth may affect predation rate, feeding ability and reproduction in bivalve clams. We studied the effect of burrowing depth on the abundance of the ergasilid Paraergasilus rylovi in the freshwater bivalve clam Anodonta piscinalis. We transplanted uninfected clams to a lake where they were allowed to choose their preferred burrowing depth, and were exposed naturally to copepodids of the parasite. There was a significant positive correlation between proportionate burrowing depth (PBD) and the abundance of P. rylovi at the end of the 17-day experiment, the deeper-burrowed clams harbouring more P. rylovi. Original PBD (0%, 50%, 100%) did not influence the final PBD or parasite abundance. Clam length affected PBD, smaller clams burrowing deeper, but it did not affect parasite abundance. Infected experimental clams and naturally-burrowed uninfected clams, both originating from the same lake, did not differ in their mean PBD. This indicated that burrowing of the experimental clams affected parasitism rather than the parasites altering burrowing of the clams. In line with the experimental result, we observed a significant positive correlation between PBD and the abundance of P. rylovi also among clams collected from 2 natural A. piscinalis populations.

Parasite life-cycles present intriguing model systems to study divergence in resource use and ecology between parasite taxa. In ecologically similar taxa, consistent selective forces may lead to convergence of life-history traits, but resource overlap and similarity of life-cycles may also promote divergence between the taxa in (1) use of host species or (2) specific niche within a host. We studied the life-history characteristics of 2 sympatric species of Diplostomum parasites, D. spathaceum and D. gasterostei, concentrating particularly on differences in intermediate host use and characteristics of the infective stages between the species. This group of trematodes is a notoriously difficult challenge for morphological taxonomy and therefore any information on the ecology of these species can also be helpful in resolving their taxonomy. We observed that these species indeed had diverged as they used mainly different snail and fish species as intermediate hosts and in controlled experiments infected different regions of the eye (lens and vitreous body) of a novel fish host. Interestingly, cercarial characteristics (activity and life-span) were similar between the species and the species were difficult to separate at the cercarial stage unless one observes their swimming behaviour. The release of cercaria from the snail hosts was higher in D. spathaceum, but when cercarial numbers were proportioned to the volume of the snail host, the production was higher in D. gasterostei suggesting differences in the rate of snail host exploitation between the parasite species. These results corroborate the prediction that closely related parasite taxa which are competing for the same resources should have diverged in their life-history characteristics and host use.

Severe adverse events (SAEs) following ivermectin treatment may occur in people harbouring high Loa loa microfilarial (mf) densities. In the context of mass ivermectin distribution for onchocerciasis control in Africa, it is crucial to define precisely the geographical distribution of L. loa in relation to that of Onchocerca volvulus and predict the prevalence of heavy infections. To this end, we analysed the distribution of mf loads in 4183 individuals living in 36 villages of central Cameroon. Mf loads were assessed quantitatively by calibrated blood smears, collected prior to ivermectin distribution. We explored the pattern of L. loa mf aggregation by fitting the (zero-truncated) negative binomial distribution and estimating its overdispersion parameter k by maximum likelihood. The value of k varied around 0·3 independently of mf intensity, host age, village and endemicity level. Based on these results, we developed a semi-empirical model to predict the prevalence of heavy L. loa mf loads in a community given its overall mf prevalence. If validated at the continental scale and linked to predictive spatial models of loiasis distribution, this approach would be particularly useful for optimizing the identification of areas at risk of SAEs and providing estimates of populations at risk in localities where L. loa and O. volvulus are co-endemic.

Sodium fluoride (NaF) is an anion that has been previously shown to block the moulting process of Ascaris suum larvae. This study describes moulting and development-specific protein expression profiles of A. suum lung-stage L3 (AsLL3) following NaF exposure. AsLL3s cultured in the presence or absence of NaF were prepared for protein analysis using two-dimensional (2D) electrophoresis. NaF exposure inhibited at least 22 proteins in AsLL3 compared with moulted larvae (i.e. AsLL4). A further comparison of AsLL4 with those of pre-cultured AsLL3 and NaF-exposed AsLL3 revealed 8 stage-specifically and 4 over-expressed proteins. Immunoblot analysis revealed an inhibition by NaF of 19 immunoreactive proteins. Enzyme assay and immunochemical data showed an inhibition of the moulting-specific inorganic pyrophosphatase activity by 41% and a decreased expression in NaF-treated larvae, indicating its significance in the moulting process. A protein spot associated with NaF inhibition was isolated and identified by peptide mass spectrometry and bioinformatics approaches to be a member of 3–hydroxyacyl–CoA dehydrogenase/short-chain dehydrogenase enzyme families. These results have implications for the identification of proteins specific to the moulting process as potential chemotherapeutic targets.

We have developed an individual-based model to reflect the complexity of the early phase of drug resistance selection in a nematode/sheep model. The infection process consists of the stochastic ingestion of infective larvae spatially aggregated in clumps. Each clump corresponds to infective larvae, which are the offspring of the mature nematodes from a given sheep. We studied the dynamics of the parasitic population and the frequency of the recessive resistance alleles during selection by anthelmintic treatments. The interaction between genetic and demographic processes illustrated the trade-off between the control of the infection and the delay of resistance selection. We confirmed the importance of the number of treatments and their timing. The same treatment frequency may result in different outcomes on resistance selection in relation to the size of the refuge (infective larvae on pasture). Treatment applied during the summer (when the mortality of infective larvae on pasture was high), may lead to a rapid selection of drug resistance and a lack of control of sheep and pasture contamination. We showed that higher stocking rates were also a force in promoting the resistance allele selection.

The gastro-intestinal (GI) nematode Heligmosomoides polygyrus is an important experimental model in laboratory mice and a well-studied parasite of wood mice in the field. Despite an extensive literature, the taxonomy of this parasite in different hosts is confused, and it is unclear whether laboratory and field systems represent the same or different Operational Taxonomic Units (OTUs). Molecular analyses reveal high sequence divergence between H. p. bakeri (laboratory) and H. p. polygyrus (field); 3% difference in the ribosomal DNA Internal Transcribed Spacers (ITS) and 8·6% variation in the more rapidly evolving mitochondrial cytochrome c oxidase I (COI) gene. The COI sequence of U.K. H. p. polygyrus is more similar to H. glareoli from voles than to H. p. bakeri, while a single isolate of H. p. polygyrus from Guernsey confirms the extent of genetic variation between H. p. polygyrus populations. Analysis of molecular variance demonstrated that mtCOI sequence variation is associated primarily with groups with distinct ITS2 sequences, and with host identity, but is not partitioned significantly with a single combined taxon H. polygyrus incorporating European and North American isolates. We conclude therefore that the laboratory OTU should be raised to the level of a distinct species, as H. bakeri from the laboratory mouse Mus musculus, and we reject the hypothesis that H. bakeri has diverged from H. polygyrus in the recent past following introduction into America. However, we are unable to reject the hypothesis that H. polygyrus and H. bakeri are sister taxa, and it may be that H. polygyrus is polyphyletic or paraphyletic.

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, β-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.

Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus datasetNucleotide sequence data described in this paper have been deposited in the EMBL database under the Accession number AM177177.. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34–40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.