Vertically transmitted parasites may have positive, neutral or negative effects on host fitness, and are also predicted to exhibit sex-specific virulence to increase the proportion or fitness of the transmitting sex. We investigated these predictions in a study on the survival and sex ratio of offspring of the amphipod Gammarus roeseli from females infected by the vertically transmitted microsporidia Nosema granulosis. We found, to our knowledge, the first evidence for a positive relationship between N. granulosis infection and host survival. Infection was associated with sex ratio distortion, not by male-killing, but probably by parasite-induced feminization of putative G. roeseli males. This microsporidia also feminizes another amphipod host, Gammarus duebeni, which is phylogenetically and biogeographically distant from G. roeseli. Our study suggests that the reproductive system of gammarids is easily exploited by these vertically-transmitted parasites, although the effects of infections on host fitness may depend on specific host-parasite species interactions.

In vitro propagation followed by PCR, and a PCR-based method capable of the direct detection of Blastocystis in faeces were utilized to detect Blastocystis from various hosts in Australia, including primates and their handlers from the Perth Zoo. In addition, Blastocystis isolates from dogs and humans living in a localized endemic community in Thailand were also characterized genetically. PCR-based detection directly from faeces was shown to be more sensitive compared with in vitro culture for the detection of Blastocystis. Moreover, phylogenetic analysis of Blastocystis isolates amplified utilizing in vitro techniques prior to PCR revealed that this method favoured the preferential amplification of Blastocystis subtype 5 over subtype 1. This study is the first to provide molecular-based evidence supporting the zoonotic potential of Blastocystis in dogs, possums and primates in a natural setting.

In this paper, we describe the occurrence of a microsporidian parasite in female-biased populations of an intertidal amphipod, Corophium volutator (Pallas), at mudflat sites in the Bay of Fundy, Canada. Sequence data for the parasite's 16S rDNA indicate that it is a novel microsporidian species. This parasite was found principally in female host gonads, indicating that it might be a vertically transmitted, sex-distorting microparasite. At 4 sites each sampled in early and mid-summer, parasite prevalence varied from 0 to 21%. In the lab, infected mothers gave rise to more female-biased broods, than did uninfected mothers. Infection was not associated with size of females or with lowered survivorship of their young. Surprisingly, infected mothers actually had higher fertility controlling for body length than did uninfected mothers. Taken together, our results suggest that this novel microsporidian is likely a feminizing microparasite and is a contributing factor to local and widespread sex ratio distortion in C. volutator.

Malaria infection during pregnancy is associated with poor maternal and foetal outcomes including low birth weight. In malaria-endemic areas, low birth weight is primarily a consequence of foetal growth restriction. Little is known on the pathogenesis of foetal growth restriction and our understanding of the relationship between epidemiological observations and the pathogenesis or consequences of disease is incomplete. In this review, we describe these gaps in our knowledge and also try to identify goals for future research into malaria in pregnancy. Foetal growth restriction results from a complex four-dimensional interaction between the foetus, the mother and the malaria parasite over gestation, and research into its pathogenesis may be advanced by combining longitudinal studies with techniques and approaches new to the field of malaria in pregnancy. Such approaches would greatly increase our knowledge on the pathogenesis of this disease and may provide new avenues for intervention strategies.

Natural Cryptosporidium infections in pigs are widespread but generally apathogenic. This study was undertaken to determine the prevalence of zoonotic Cryptosporidium spp. in piggeries in Ireland, where the drinking water supply is particularly vulnerable to contamination with zoonotic species. Overall, infections were detected in 39 out of 342 animals (11·4%), with highest infection rates among weaners (15%) and sows (13·3%). Twenty-nine positive samples were genotyped based on SSU rRNA sequence analysis. Infections with Cryptosporidium parvum, the most important zoonotic species were rare and are likely to be of greater concern to animal handlers than suppliers of drinking water. In addition to C. parvum, Cryptosporidium suis, Cryptosporidium pig genotype II, Cryptosporidium muris and a previously undescribed genotype were identified. ABI-profiles indicated the presence of different alleles in at least 40% of all genotyped isolates. This was confirmed in 3 isolates by cloning of the PCR products. Since chronic mixed infections appear to be quite common in pigs they could be considered as models for mixed infections in immunocompromised humans.

Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) exhibit considerable in vitro activity against Besnoitia besnoiti tachyzoites grown in Vero cells. Real-time-PCR was used to assess B. besnoiti tachyzoite adhesion, invasion, and intracellular proliferation in vitro. A number of NTZ-derivatives, including Rm4822 and Rm4803, were generated, in which the thiazole-ring-associated nitro-group was replaced by a bromo-moiety. We here show that replacement of the nitro-group on the thiazole ring with a bromo (as it occurs in Rm4822) does not impair the efficacy of the drug, but methylation of the salicylate ring at the ortho-position in a bromo-derivative (Rm4803) results in complete abrogation of the antiparasitic activity. Treatment of extracellular B. besnoiti tachyzoites with NTZ has an inhibitory effect on host cell invasion, while treatments with TIZ, Rm4822 do not. TEM demonstrates that the effects of Rm4822 treatment upon the parasites are similar to the damage induced by NTZ. This includes increased vacuolization of the parasite cytoplasm, and loss of the structural integrity of the parasitophorous vacuole and its membrane. Thus, Rm4822, due to the absence of a potentially mutagenic nitro-group, may represent an important potential addition to the anti-parasitic arsenal for food animal production, especially in cattle.

The ‘Muguga cocktail’ live vaccine, delivered by an infection and treatment protocol, has been widely deployed in Eastern, Central and Southern Africa to protect cattle against East Coast fever, caused by Theileria parva. The vaccine contains 3 component stocks (Muguga, Serengeti-transformed and Kiambu 5). In a previous study, parasites from vaccinated and unvaccinated animals were genotyped with a panel of micro- and minisatellite markers (Oura et al.2004a) and it was shown that only the Kiambu 5 stock establishes a long-term carrier state but there was no evidence for the transmission of this stock. Also parasite genotypes different from the 3 component vaccine stocks were identified in vaccinated animals. We now report a follow-up study on the same farm, some 4 years after the initial vaccination, aimed at establishing the source of the novel parasite genotypes identified in vaccinated cattle, determining the longevity of the carrier state established by the Kiambu 5 vaccine stock and re-examining whether vaccine transmission can occur over a longer time-scale. To do this, samples were taken from vaccinated and unvaccinated cattle and the parasites were genotyped with a series of micro- and minisatellite markers. The data indicate that the vaccine stabilates contain at least 6 parasite genotypes, the Kiambu 5 stock can be detected in many but not all vaccinated cattle for up to 4 years and can be transmitted to unvaccinated cattle which share grazing and that some of the vaccinated animals become infected with local genotypes without causing overt disease.

The prevalence of Cryptosporidium in calves younger than 10 weeks was estimated in a cross-sectional epidemiological study on 100 dairy (n=499) and 50 beef (n=333) farms in East Flanders (Belgium), using a previously evaluated immunofluorescence assay (Merifluor®). The calf prevalence was 37% (95% Probability Interval (PI): 7–70%) in dairy calves and 12% (95% PI: 1–30%) in beef calves. To elucidate the genetic diversity, the Cryptosporidium 18S ribosomal DNA and the 70 kDa heat shock protein gene were targeted. In the majority of the samples C. parvum was present, although C. bovis was also identified, all but one in calves older than 1 month. The porcine-specific C. suis was identified in 1 beef calf. Subtyping of C. parvum positive isolates by sequence analysis of the 60 kDa glycoprotein gene indicated the presence of 4 allele IIa subtypes, along with 1 subtype IIdA22G1. The subtype IIaA15G2R1 was most prevalent, next to subtype IIaA13G2R1 and IIaA16G2R1, and a new subtype IIaA14G2R1. The results of the present study indicate a high prevalence of Cryptosporidium infections in calves in Belgium and confirm that these calves should be considered as a potential zoonotic reservoir for human infections.

Fitness of tick-borne pathogens may be determined by the degree to which their infection dynamics in vertebrate hosts permits transmission cycles if infective and uninfected tick stages are active at different times of the year. To investigate this hypothesis we developed a simulation model that integrates the transmission pattern imposed by seasonally asynchronous nymphal and larval Ixodes scapularis ticks in northeastern North America, with a model of infection in white-footed mice (Peromyscus leucopus) reservoir hosts, using the bacteria Borrelia burgdorferi and Anaplasma phagocytophilum as examples. In simulations, survival of microparasites, their sensitivity to reduced rodent and tick abundance, and to ‘dilution’ by a reservoir-incompetent host depended on traits that allowed (i) highly efficient transmission from acutely-infected hosts, (ii) long-lived acute or ‘carrier’ host infections, and/or (iii) transmission amongst co-feeding ticks. Minimum values for transmission efficiency to ticks, and duration of host infectivity, necessary for microparasite persistence, were always higher when nymphal and larval ticks were seasonally asynchronous than when these instars were synchronous. Thus, traits influencing duration of host infectivity, transmission efficiency to ticks and co-feeding transmission are likely to be dominant determinants of fitness in I. scapularis-borne microparasites in northeastern North America due to abiotic forcings influencing I. scapularis seasonality.

Non-availability of adult worms from living hosts remains a key problem in population genetic studies of schistosomes. Indirect sampling involving passage through laboratory animals presents significant ethical and practical drawbacks, and may result in sampling biases such as bottlenecking processes and/or host-induced selection pressures. The novel techniques reported here for sampling, storage and multi-locus microsatellite analysis of larval Schistosoma mansoni, allowing genotyping of up to 7 microsatellite loci from a single larva, circumvent these problems. The utility of these assays and the potential problems of laboratory passage, were evaluated using 7 S. mansoni population isolates collected from school-children in the Hoima district of Uganda, by comparing the associated field-collected miracidia with adult worms and miracidia obtained from a single generation in laboratory mice. Analyses of laboratory-passaged material erroneously indicated the presence of geographical structuring in the population, emphasizing the dangers of indirect sampling for population genetic studies. Bottlenecking and/or other sampling effects were demonstrated by reduced variability of adult worms compared to their parent field-collected larval samples. Patterns of heterozygote deficiency were apparent in the field-collected samples, which were not evident in laboratory-derived samples, potentially indicative of heterozygote advantage in establishment within laboratory hosts. Genetic distance between life-cycle stages in the majority of isolates revealed that adult worms and laboratory-passaged miracidia clustered together whilst segregating from field miracidia, thereby further highlighting the utility of this assay.

Much evolutionary theory assumes that parasite virulence (i.e. parasite-induced host mortality) is determined by within-host parasite reproduction and by the specific parasite genotypes causing infection. However, many other factors could influence the level of virulence experienced by hosts. We studied the protozoan parasite Ophryocystis elektroscirrha in its host, the monarch butterfly, Danaus plexippus. We exposed monarch larvae to wild-isolated parasites and assessed the effects of within-host replication and parasite genotype on host fitness measures, including pre-adult development time and adult weight and longevity. Per capita replication rates of parasites were high, and infection resulted in high parasite loads. Of all host fitness traits, adult longevity showed the clearest relationship with infection status, and decreased continuously with increasing parasite loads. Parasite genotypes differed in their virulence, and these differences were maintained across ecologically relevant variables, including inoculation dose, host sex and host age at infection. Thus, virulence appears to be a robust genetic parasite trait in this system. Although parasite loads and genotypes had strong effects on virulence, inoculation dose, host sex and age at infection were also important. These results have implications for virulence evolution and emphasize the need for a detailed understanding of specific host-parasite systems for addressing theory.

Toxoplasma gondii is one of the few pathogens that can cross the placenta. Frequency and severity of transmission vary with gestational age. While the control of acquired toxoplasmosis is already well explored, the control of materno-foetal transmission of the parasite remains almost unknown. This is partly due to the lack of an animal model to study this process. This review summarises the studies which have been undertaken and shows that the mouse is a valuable model despite obvious differences to the human case. The paramount role of the cellular immune response has been shown by several experiments. However, IFN-γ has a dual role in this process. While its beneficial effects in the control of toxoplasmosis are well known, it also seems to have transmission-enhancing effects and can also directly harm the developing foetus. The ultimate goal of these studies is to develop a vaccine which protects both mother and foetus. Therefore, it is useful to study the mechanisms of natural resistance against transmission during a secondary infection. In this setting, the process is more complicated, involving both cellular and also humoral components of the immune system. In summary, even if the whole process is far from being elucidated, important insights have been gained so far which will help us to undertake rational vaccine research.

Heat shock is believed to be a developmental inductor of differentiation in Leishmania. Furthermore, heat shock genes are extensively studied as gene models to decipher mechanisms of gene regulation in kinetoplastids. Here, we describe the organization and expression of the HSP70 loci in representative Leishmania species (L. infantum, L. major, L. tropica, L. mexicana, L. amazonensis and L. braziliensis). With the exception of L. braziliensis, the organization of the HSP70 loci was found to be well conserved among the other Leishmania species. Two types of genes, HSP70-I and HSP70-II, were found to be present in these Leishmania species except for L. braziliensis that lacks HSP70-II gene. Polymorphisms in the HSP70 locus allow the differentiation of the Old and New World species within the subgenus Leishmania. A notable discrepancy between our data and those of the L. major genome database in relation to the gene copy number composing the L. major HSP70 locus was revealed. The temperature-dependent accumulation of the HSP70-I mRNAs is also conserved among the different Leishmania species with the exception of L. braziliensis. In spite of these differences, analysis of the HSP70 synthesis indicated that the HSP70 mRNAs are also preferentially translated during heat shock in L. braziliensis.

The ability of the larval forms of Schistosoma mansoni to invade and parasitize their molluscan host, Biomphalaria glabrata, is determined by a multitude of factors. In this study we sought to elucidate the possible mechanisms by which the invading larvae are able to counteract the potentially harmful oxidative environment presented by the host upon initial miracidial infection. This was attempted by examining the gene expression profile of parasite antioxidant enzymes of the linked glutathione-(GSH) thioredoxin (Trx) redox pathway during early intramolluscan larval development. Three such enzymes, the peroxiredoxins (Prx1, Prx2 and Prx3) were examined as to their activity and sites of expression within S. mansoni miracidia and in vitro-cultured mother sporocysts. Results of these studies demonstrated that the H2O2-reducing enzymes Prx1 and 2 are upregulated during early mother sporocyst development compared to miracidia. Immunolocalization studies further indicated that Prx1 and Prx2 proteins are expressed within the apical papillae of miracidia and tegumental syncytium of sporocysts, and are released with parasite excretory-secretory proteins (ESP) during in vitro larval transformation. Removal of Prx1 and Prx2 from larval ESP by immunoabsorption significantly reduced the ability of ESP to breakdown exogenous H2O2, thereby directly linking ESP Prx proteins with H2O2-scavenging activity. Moreover, exposure of live sporocysts to exogenous H2O2 stimulated an upregulation of Prx1 and 2 gene expression suggesting the involvement of H2O2–responsive elements in regulating larval Prx gene expression. These data provide evidence that Prx1 and Prx2 may function in the protection of S. mansoni sporocysts during the early stages of infection.

Macrocyclic lactones (MLs) are highly lipophilic anthelmintics which are known to bind to and open ligand-gated ion channels. However, these anthelmintics, and particularly the avermectin members of the ML class of endectocides, are potent substrates for ABC transporters and these transporters may regulate drug concentration in both the host and the parasite. There is accumulating evidence that ivermectin (IVM), and to a lesser extent moxidectin (MOX), selects for certain alleles of P-glycoprotein and other ABC transporter genes, selects for constitutive overexpression of some of these gene products, and induces overexpression of some P-glycoproteins in nematodes. However, such mechanisms of ML resistance do not easily lend themselves to the identification of SNP markers for resistance because of the diversity of ABC transporters in nematodes, the apparent diversity of effects of different MLs, and because regulatory elements for ABC transporter gene expression are not well understood in nematodes. Another non ligand-gated ion channel gene which appears to be under IVM selection, at least in Onchocerca volvulus and Haemonchus contortus, is β-tubulin, and a simple genetic test for this selection has been described in O. volvulus. However, further work is required to elucidate a reliable marker associated with this gene in H. contortus or other parasitic nematodes of livestock. The possible involvement of ABC transporter genes and β-tubulin in ML resistance provides a start in developing our understanding of this phenotype and markers for its detection in field populations of parasitic nematodes. However, more work is required before these leads can provide practical SNP markers for ML resistance.

Efficient, cyclical transmission of trypanosomes through tsetse flies is central to maintenance of human sleeping sickness and nagana across sub-Saharan Africa. Infection rates in tsetse are normally very low as most parasites ingested with the fly bloodmeal die in the fly gut, displaying the characteristics of apoptotic cells. Here we show that a range of antioxidants (glutathione, cysteine, N-acetyl-cysteine, ascorbic acid and uric acid), when added to the insect bloodmeal, can dramatically inhibit cell death of Trypanosoma brucei brucei in tsetse. Both L- and D-cysteine invoked similar effects suggesting that inhibition of trypanosome death is not dependent on protein synthesis. The present work suggests that antioxidants reduce the midgut environment protecting trypanosomes from cell death induced by reactive oxygen species.

We tested whether the prevalence of ticks can be predicted reliably from a simple epidemiological model that takes into account only mean abundance and its variance. We used data on the abundance and distribution of larvae and nymphs of 2 ixodid ticks parasitic on small mammals (Apodemus agrarius, Apodemus flavicollis, Apodemus uralensis, Clethrionomys glareolus and Microtus arvalis) in central Europe. Ixodes trianguliceps is active all year round, occurs in the study area in the mountain and sub-mountain habitats only and inhabits mainly host burrows and nests, whereas Ixodes ricinus occurs mainly during the warmer seasons, occupies a large variety of habitats and quests for hosts outside their shelters. In I. ricinus, the models with k values calculated from Taylor's power law overestimated prevalences. However, if moment estimates of k corrected for host number were used instead, expected prevalences of both larvae and nymphs I. ricinus in either host did not differ significantly from observed prevalences. In contrast, prevalences of larvae and nymphs of I. trianguliceps predicted by models using parameters of Taylor's power law did not differ significantly from observed prevalences, whereas the models with moment estimates of k corrected for host number in some cases under-estimated relatively lower larval prevalences and over-estimated relatively higher larval prevalences, but predicted nymphal prevalences well.

Parasite transmission dynamics are fundamental to explaining the evolutionary epidemiology of disease because transmission and virulence are tightly linked. Horizontal transmission of microsporidian parasites, e.g. Nosema bombi, may be influenced by numerous factors, including inoculation dose, host susceptibility and host population heterogeneity. Despite previous studies of N. bombi and its bumble bee hosts, neither the epidemiology nor impact of the parasite are as yet understood. Here we investigate the influence N. bombi spore dosage (1000 to 500 000 spores), spore source (Bombus terrestris and B. lucorum isolates) and host age (2- and 10-day-old bees) have on disease establishment and the presence of patent infections in adult bumble bees. Two-day-old bees were twice as susceptible as their 10-day-old sisters, and a 5-fold increase in dosage from 100 000 to 500 000 spores resulted in a 20-fold increase in the prevalence of patent infections. While intraspecific inoculations were 3 times more likely to result in non-patent infections there was no such effect on the development of patent infections. These results suggest that host-age and dose are likely to play a role in N. bombi's evolutionary epidemiology. The relatively low levels of horizontal transmission success are suggestive of low virulence in this system.

Using a combination of morphological, life-history and molecular data, we redescribe Sphaerospora ranae (Morelle, 1929) n. comb. (previously Leptotheca ranae) and emend its taxonomic status. Renal infection was recorded in 2 spp. of frogs (out of 5 amphibian spp. examined), Rana dalmatina (proposed type host) and Rana temporaria, suggesting restricted host specifity of S. ranae. We provide a description of sporogonic stages of S. ranae for the first time and suggest possible modes of its developmental cycle. Phylogenetic analysis inferred from the small-subunit ribosomal DNA revealed a close relationship of S. ranae with piscine Sphaerospora elegans (type species of the genus) and Sphaerospora truttae, forming together with distantly related Leptotheca fugu a ‘Sphaerosporid clade’, the basal branch to all myxosporean species. The close relationship of the 3 Sphaerospora spp. is further supported by the presence of 2 areas with extensive nucleotide insertions in the V4 region of the SSU rDNA (absent in L. fugu), morphology and life-history features. We conclude, that the spore morphology of Sphaerospora s.l., is very simple and probably represents a ‘primitive’, basal morphotype retained in most myxosporean lineages. Based on presented data, we propose emendation of the genus Sphaerospora using morpological, life-history and molecular features.

A key aim of anthelmintic resistance research is to identify molecular markers that could form the basis of sensitive and accurate diagnostic tests. These would provide powerful tools to study the origin and spread of anthelmintic resistance in the field and to monitor strategies aimed at preventing and managing resistance. Molecular markers could also form the basis of routine diagnostic tests for use in surveillance and clinical veterinary practice. Much of the research conducted to date has focused on the investigation of possible associations of particular candidate genes with the resistance phenotype. In the future, as full parasite genome sequences become available, there will be an opportunity to apply genome-wide approaches to identify the genetic loci that underlie anthelmintic resistance. Both the interpretation of candidate gene studies and the application of genome-wide approaches require a good understanding of the genetics and population biology of the relevant parasites as well as knowledge of how resistance mutations arise and are selected in populations. Unfortunately, much of this information is lacking for parasitic nematodes. This review deals with a number of aspects of genetics and population biology that are pertinent to these issues. We discuss the possible origins of resistance mutations and the likely effects of subsequent selection on the genetic variation at the resistance-conferring locus. We also review some of the experimental approaches that have been used to test associations between candidate genes and anthelmintic resistance phenotypes and highlight implications for future genome-wide studies.