Nucleoside triphosphate diphosphohydrolases (NTPDases, GDA1_CD39 protein superfamily) play a diverse range of roles in a number of eukaryotic organisms. In humans NTPDases function in regulating the inflammatory and immune responses, control of vascular haemostasis and purine salvage. In yeast NTPDases are thought to function primarily in the Golgi, crucially involved in nucleotide sugar transport into the Golgi apparatus and subsequent protein glycosylation. Although rare in bacteria, in Legionella pneumophila secreted NTPDases function as virulence factors. In the last 2 decades it has become clear that a large number of parasites encode putative NTPDases, and the functions of a number of these have been investigated. In this review, the available evidence for NTPDases in parasites and the role of these NTPDases is summarized and discussed. Furthermore, the processes by which NTPDases could function in pathogenesis, purine salvage, thromboregulation, inflammation and glycoconjugate formation are considered, and the data supporting such putative roles reviewed. Potential future research directions to further clarify the role and importance of NTPDases in parasites are proposed. An attempt is also made to clarify the nomenclature used in the parasite field for the GDA1_CD39 protein superfamily, and a uniform system suggested.

When every individual has an equal chance of mating with other individuals, the population is classified as panmictic. Amongst metazoan parasites of animals, local-scale panmixia can be disrupted due to not only non-random mating, but also non-random transmission among individual hosts of a single host population or non-random transmission among sympatric host species. Population genetics theory and analyses can be used to test the null hypothesis of panmixia and thus, allow one to draw inferences about parasite population dynamics that are difficult to observe directly. We provide an outline that addresses 3 tiered questions when testing parasite panmixia on local scales: is there greater than 1 parasite population/species, is there genetic subdivision amongst infrapopulations within a host population, and is there asexual reproduction or a non-random mating system? In this review, we highlight the evolutionary significance of non-panmixia on local scales and the genetic patterns that have been used to identify the different factors that may cause or explain deviations from panmixia on a local scale. We also discuss how tests of local-scale panmixia can provide a means to infer parasite population dynamics and epidemiology of medically relevant parasites.

Immune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

Humans are infected by 2 genetic assemblages (A and B) of Giardia duodenalis, a protozoan parasite that causes gastro-intestinal disease. Sub-assemblages AI, AII, BIII and BIV are commonly identified in human cases. Detection requires amplification of G. duodenalis loci. Subsequent DNA sequencing or restriction fragment length polymorphism (RFLP) identifies sub-assemblages but is expensive (DNA sequencing) or insensitive (RFLP). This study investigated a fluorescence-based detection method, using terminal-restriction fragment length polymorphism (T-RFLP) of the glutamate dehydrogenase gene to characterize human infections. Clinical samples (n=73), positive for Giardia were collected in New South Wales, Australia, and were used to evaluate T-RFLP detection. The accuracy and sensitivity of T-RFLP detection was established by comparison to DNA sequencing and RFLP. Sub-assemblage assignment by T-RFLP identified BIV as the common subtype in N.S.W cases, whilst AI, AII and BIII were also detected. When compared to DNA sequencing and RFLP, analysis by T-RFLP was a reliable and reproducible method. Automated fluorescent detection enabled accurate sizing of restriction fragments and provided a sensitive alternative to RFLP. Discrimination of sub-assemblages by T-RFLP was comparable to DNA sequencing, but was efficient and inexpensive. The protocol described here provides a rapid and sensitive diagnostic tool for routine sample screenings in epidemiological research.

Blastocystis infection has a worldwide distribution especially among the disadvantaged population and immunocompromised subjects. This study was carried out to determine the prevalence and the association of Blastocystis infection with the socio-economic characteristics among 300 primary schoolchildren, living in rural communities in Lipis and Raub districts of Pahang state, Malaysia. Stool samples were collected and examined for the presence of Blastocystis using direct smear microscopy after in vitro cultivation in Jones' medium. The overall prevalence of Blastocystis infection was found to be as high as 25·7%. The prevalence was significantly higher among children with gastrointestinal symptoms as compared to asymptomatic children (x2=4·246; P=0·039). Univariate and multivariate analyses showed that absence of a piped water supply (OR=3·13; 95% CI=1·78, 5·46; P<0·001) and low levels of mothers’ education (OR=3·41; 95% CI=1·62, 7·18; P<0·01) were the significant predictors of Blastocystis infection. In conclusion, Blastocystis is prevalent among rural children and the important factors that determine the infection were the sources of drinking water and mothers' educational level. Interventions with provision of clean water supply and health education especially to mothers are required.

Arid zones of northern Venezuela are represented by isolated areas, important from an ornithological and ecological perspective due to the occurrence of restricted-range species of birds. We analysed the prevalence and molecular diversity of haemosporidian parasites of wild birds in this region by screening 527 individuals (11 families and 20 species) for parasite mitochondrial DNA. The overall prevalence of parasites was 41%, representing 17 mitochondrial lineages: 7 of Plasmodium and 10 of Haemoproteus. Two parasite lineages occurred in both the eastern and western regions infecting a single host species, Mimus gilvus. These lineages are also present throughout northern and central Venezuela in a variety of arid and mesic habitats. Some lineages found in this study in northern Venezuela have also been observed in different localities in the Americas, including the West Indies. In spite of the widespread distributions of some of the parasite lineages found in northern Venezuela, several, including some that are relatively common (e.g. Ven05 and Ven06), have not been reported from elsewhere. Additional studies are needed to characterize the host and geographical distribution of avian malaria parasite lineages, which will provide a better understanding of the influence of landscape, vector abundance and diversity, and host identity on haemosporidian parasite diversity and prevalence.

Cysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with Ki's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.

Temperature can be a limiting factor on parasite development. Riccardoella limacum, a haematophagous mite, lives in the mantle cavity of helicid land snails. The prevalence of infection by R. limacum in populations of the land snail Arianta arbustorum is highly variable (0–78%) in Switzerland. However, parasitic mites do not occur in host populations at altitudes of 1290 m or higher. It has been hypothesized that the host's hibernation period might be too long at high elevations for mites and their eggs to survive. To test this hypothesis, we experimentally infected snails and allowed them to hibernate at 4°C for periods of 4–7 months. Winter survival of host snails was negatively affected by R. limacum. The intensity of mite infection decreased with increasing hibernation duration. Another experiment with shorter recording intervals revealed that mites do not leave the host when it buries in the soil at the beginning of hibernation. The number of mites decreased after 24 days of hibernation, whereas the number of eggs attached to the lung tissue remained constant throughout hibernation. Thus, R. limacum survives the winter in the egg stage in the host. Low temperature at high altitudes may limit the occurrence of R. limacum.

A major issue for the proper understanding of the evolution of life-cycle histories is the regulation of voltinism and its variation. Diapause characteristics are known to regulate voltinism, but the underlying mechanisms are poorly understood. This paper studies diapause duration and voltinism variation in a haematophagous diptera parasitizing 2 sympatric hosts with very different breeding phenologies. We hypothesize that bivoltinism will be more frequent in carnid flies parasitizing an early breeding, multi-brooded species than in flies parasitizing a late breeder, single-brooded species. We obtained evidence of the co-occurrence of uni- and bivoltinism in both clutches of the multi-brooded Spotless starling (Sturnus unicolor) as well as in clutches of the single-brooded European roller (Coracias garrulus). Unexpectedly, the proportion of bivoltine flies was similar in both host species. A remarkable degree of host-parasite synchronization at the population level was found for bivoltine flies. Our findings reveal the facultative nature of diapause in Carnus. We discuss the influence of abiotic conditions and host availability on polymorphism in life-history cycles and the consequences both for the parasite and the host.

In disturbed environments, infected organisms have to face both parasitic and chemical stresses. Although this situation is common, few studies have been devoted to the effects of infection on hosts' energy reserves and antitoxic defence capacities, while parasite survival depends on host survival. In this study, we tested the consequences of an infection by Polymorphus minutus on the energy reserves (protein, lipid and glycogen) and antioxidant defence capacities (reduced glutathione, γ-glutamylcysteine ligase activity) of Gammarus roeseli males and females, in the absence of chemical stress. Moreover, malondialdehyde concentration was used as a toxicity biomarker. The results revealed that in infected G. roeseli, whatever their gender and the sampling month, protein and lipid contents were lower, but glycogen contents were higher. This could be explained by the fact that the parasite diverts part of the host's energy for its own development. Moreover, glutathione concentrations and γ-glutamylcysteine ligase activity were both lower, which could lead to lower antitoxic defence in the host. These results suggest negative effects on individuals in the case of additional stress (e.g. pollutant exposure). In the absence of chemical stress, the lower malondialdehyde level in infected gammarids could imply a probable protective effect of the parasite.

Cyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, additional IGS DNA sequences were generated from individual worms of 8 species already studied. Comparative analysis of these sequences revealed a greater range of intra-specific variation than previously reported (up to 23%); whilst the level of inter-specific variation (3–62%) was similar to that identified in earlier studies. The reverse line blot (RLB) method has been used to exploit the cyathostomin IGS DNA region for species identification. Here, we report validation of novel and existing DNA probes for identification of cyathostomins using this method and highlight their application in differentiating life-cycle stages such as third-stage larvae that cannot be identified to species by morphological means†.

In vitro tests were performed to evaluate the ability of 6 isolates of the nematophagous fungus Pochonia chlamydosporia to infect immature and mature Toxocara canis eggs on cellulose dialysis membrane. There was a direct relationship between the number of eggs colonized and the increase in the days of interaction, as well as between the number of eggs colonized and the increase in the concentration of chlamydospores (P<0·05). Immature eggs were more susceptible to infection than mature eggs. The isolate Pc-04 was the most efficient egg parasite until the 7th day, and showed no difference in capacity to infect mature and immature eggs in comparison to Pc-07 at 14 and 21 days of interaction, respectively. Isolate Pc-04 was the most infective on the two evolutionary phases of the eggs at most concentrations, but its ability to infect immature eggs did not differ from that presented by the isolates Pc-07 and Pc-10 at the inoculum level of 5000 chlamydospores. Colonization of infective larvae inside or outside the egg was observed in treatments with the isolates Pc-03, Pc-04, Pc-07 and Pc-10. The isolate Pc-04 of P. chlamydosporia has great biological capacity to destroy immature and mature T. canis eggs in laboratory conditions.

In order to quantify the impact of parasites on host population dynamics, experimental manipulations that perturb the parasite-host relationship are needed but, logistically, this is difficult for wild hosts. Here, we describe the use of a delayed-release anthelmintic delivery system that can be administered when the hosts can be captured and its activity delayed until a more appropriate period in the host-parasite cycle. Our model system is Svalbard reindeer infected with a nematode parasite, Marshallagia marshalli, which appears to accumulate during the Arctic winter. To determine the extent to which this occurs and the effect on host fitness, reindeer need to be treated with anthelmintics in late autumn but they can only be caught and handled in April. To solve this problem, we devised an intra-ruminal capsule that releases the anthelmintic from up to 6 months after being administered. The capsule was trialed in cannulated sheep and red deer to determine optimum capsule orifice size and release rates. Capsules were estimated to release placebo for 100–153 days followed by abamectin for 22–34 days. To test the efficacy of treatment in reindeer, capsules were administered in April and retrieved in October. All capsules had fully released the anthelmintic and treated reindeer had significantly lower worm burdens than controls. Thus, success of this system allows repeated treatment over several years to test the effect of winter parasitism on host fitness.

Climate change in the Arctic is anticipated to alter the ecology of northern ecosystems, including the transmission dynamics of many parasite species. One parasite of concern is Ostertagia gruehneri, an abomasal nematode of Rangifer ssp. that causes reduced food intake, weight loss, and decreased pregnancy rates in reindeer. We investigated the development, availability, and overwinter survival of the free-living stages of O. gruehneri on the tundra. Fecal plots containing O. gruehneri eggs were established in the Northwest Territories, Canada under natural and artificially warmed conditions and sampled throughout the growing season of 2008 and the spring of 2009. Infective L3 were present 3–4 weeks post-establishment from all trials under both treatments, except for the trial established 4 July 2008 under warmed conditions wherein the first L3 was recovered 7 weeks post-establishment. These plots were exposed to significantly more time above 30°C than the natural plots established on the same date, suggesting a maximum temperature threshold for development. There was high overwinter survival of L2 and L3 across treatments and overwintering L2 appeared to develop to L3 the following spring. The impact of climate change on O. gruehneri is expected to be dynamic throughout the year with extreme maximum temperatures negatively impacting development rates.