Toxoplasma gondii is a zoonotic parasite of worldwide distribution. The consumption of infected pork meat has been suggested to be an important source for human infection in the tropical area of Yucatan, Mexico. We performed a cross-sectional study of 12 farms across the state to investigate the seroprevalence of Toxoplasma gondii infection in domestic pigs. In total, 632 samples were obtained from 2 different environmental zones (tropical deciduous low forest and tropical sub-deciduous medium forest) and 2 abattoirs. The modified agglutination test (MAT) was used to assess the seroprevalence of T. gondii in pigs and to evaluate 2 globally used serological tests, the Dye test (DT) and ID Screen® ELISA multi-species, and a commercial ELISA kit (Human Toxo IgG, Human-diagnostics), which is widely used locally in this geographical area. The overall prevalence obtained with the MAT (cut-off ≥1:25) among the 632 pigs was 1.4% (95% CI, 0.6-2.7%). The seroprevalence obtained for the different age groups was 0.6%, 0.7%, 1.8%, and 6.8% among 2-3, 3-4, 4-5, and ≥5-mo-old pigs. This increase in the seroprevalence was statistically significant for the 2 older groups (odds ratio [OR] 3.9-7.1, P < 0.05) in comparison with younger groups. DT at >4 IU dilution had a perfect agreement and 100% of sensitivity and specificity when compared with the MAT. Although ID Screen® had only a fair agreement (κ = 0.389) with the MAT, the McNemar test showed that the results of these tests were comparable (P = 0.29). The Human Toxo ELISA showed no agreement with MAT, ID Screen®, and DT (κ = 0.000-0.023, McNemar P < 0.05). This ELISA was lacking in specificity, accuracy, and precision; hence, we do not recommend its use for T. gondii diagnosis in pig serum.

Toxoplasmosis is a zoonotic disease of worldwide distribution. The parasite exhibits strong geographical patterns of strain variation with contrasting high levels of diversity across South America and restricted variation across North America. Little is known about the diversity of strains in the transitional area between the two continents. Here we present data on the prevalance and diversity of Toxoplasma gondii in the Yucatan peninsula of Mexico, through a study in commercially reared pigs. A survey of 12 farms found evidence of circulating T. gondii DNA in 125 of 632 blood samples (19.8%, CI: 16.7%-23%). In addition, 46 tongue samples were collected from culled animals and 16 of these were positive for T. gondii DNA and 3 were positive in mouse bioassay. PCR-sequencing was used to generate genotyping data from blood and tissue samples. Four loci (SAG1, 2, 3 and GRA6) were reliably amplified and revealed a high diversity among Yucatan strains with evidence of recombination and novel alleles. Sequencing data from the four loci was achieved in eight samples each of which had a different genotype. The predominant allelic type was atypical, in relation to the dominant strain types (I, II, III), the number of allelic variants being 27 (I, II-III, u-1-25), 20 (I, III, u1-18), 6 (I, III, u1-4) and 11 (I, II, u1-9) for the SAG1, SAG2, SAG3 and GRA6 loci respectively. Phylogenetic analysis showed that T. gondii strains from Yucatan shared alleles with strains originating from both North and South America. Our findings are consistent with data from other regions of Central America and suggest the genetic population structure of the parasite, with significant levels of allelic variation and recombination, constitutes a reservoir from which new strains may emerge. Positive bioassay results (7.5%) indicate that consumption of undercooked pork could be a potential T. gondii infection risk to humans.

In wild rabbits (Oryctolagus cuniculus) on an estate in Perthshire, central Scotland, the seroprevalence of Toxoplasma gondii was 18/548 (3.3%). The wild rabbit could be a T. gondii reservoir and it has potential value as a sentinel of T. gondii in environmental substrates. Toxoplasma gondii was associated with female sex (P < 0.001) and with relatively heavy infections by Eimeria stiedae (P = 0.036). It was not associated with the intensity of coccidial oocysts, the severity of myxomatosis caused by the virus Myxomatosis cuniculi, the intensity of roundworm eggs, the year or season, rabbit age or distance from farm buildings. Coinfections could have been affected by gestational down regulation of type 1 T helper cells. A sudden influx or release of T. gondii oocysts might have occurred. This is the first report of T. gondii in any wild herbivore in Scotland and also the first report of lapine T. gondii as a coinfection with E. stiedae, M. cuniculi and helminths.

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought.

Foetal death, or abortion at term, in sheep is of major significance to the livestock industry, accounting for more than £24million lost per annum. We have investigated whether there is a genetic component to abortion within two flocks of pedigree Charollais sheep, one followed from 1989 to 2006, the other from 1992 to 2006. Abortion occurred at a rate of 5.74–8.78% per annum against a total mortality rate of 14–24%. By model covariate analysis we have shown that 15.5% aborting ewes went on to have one or more abortions and that this risk increased with parity (p=0.006). Heritability estimates were approximately 0.08 as calculated by SOLAR, pedigreemm and ASReml3, with sire and dam components of 0.046 and 0.048, respectively. Where the lamb was aborted, heritability estimates were highly variable according to the method employed, 0.046–0.378, with sex of the lamb being a significant covariate. This variability indicated one or more underlying, significant factors that were not measured in these analyses, potentially including infectious agents that may be involved. Nevertheless, the ASReml3 estimate (0.179) resolved to 0.074 variance attributable to the sire and 0.092 attributable to the dam, which, while not significant, was suggestive that genetic variants passed by the dam to the lamb may be of more weight than that from the sire in determining whether a lamb will abort.

ABSTRACT We designed fluorescence in situ hybridization probes for two distinct microsporidian clades and demonstrated their application in detecting, respectively, Nosema / Vairimorpha and Dictyoceola species. We used them to study the vertical transmission of two microsporidia infecting the amphipod Gammarus duebeni .

Many pollinator populations are declining, with large economic and ecological implications. Parasites are known to be an important factor in the some of the population declines of honey bees and bumblebees, but little is known about the parasites afflicting most other pollinators, or the extent of interspecific transmission or vectoring of parasites. Here we carry out a preliminary screening of pollinators (honey bees, five species of bumblebee, three species of wasp, four species of hoverfly and three genera of other bees) in the UK for parasites. We used molecular methods to screen for six honey bee viruses, Ascosphaera fungi, Microsporidia, and Wolbachia intracellular bacteria. We aimed simply to detect the presence of the parasites, encompassing vectoring as well as actual infections. Many pollinators of all types were positive for Ascosphaera fungi, while Microsporidia were rarer, being most frequently found in bumblebees. We also detected that most pollinators were positive for Wolbachia, most probably indicating infection with this intracellular symbiont, and raising the possibility that it may be an important factor in influencing host sex ratios or fitness in a diversity of pollinators. Importantly, we found that about a third of bumblebees (Bombus pascuorum and Bombus terrestris) and a third of wasps (Vespula vulgaris), as well as all honey bees, were positive for deformed wing virus, but that this virus was not present in other pollinators. Deformed wing virus therefore does not appear to be a general parasite of pollinators, but does interact significantly with at least three species of bumblebee and wasp. Further work is needed to establish the identity of some of the parasites, their spatiotemporal variation, and whether they are infecting the various pollinator species or being vectored. However, these results provide a first insight into the diversity, and potential exchange, of parasites in pollinator communities.

Feminizing parasites enhance their vertical transmission to the host offspring by converting genetic male hosts into phenotypic females. Crustacea are the only invertebrates where sexual differentiation is controlled by a specialised endocrine organ, the androgenic gland, rather than by the gonads. We showed that a feminizing microsporidian Microsporidium sp. inhibits androgenic gland differentiation. We investigated the effect of Microsporidium sp. and a second feminizing microsporidium, Nosema granulosis, on the masculinizing function of the androgenic gland in Gammarus duebeni. Androgenic gland implants had a masculinizing effect on the sexual characteristics and sexual behaviour of recipient female hosts, reflecting the masculinizing function of the androgenic gland. Individuals that had received androgenic glands showed changed morphology in comparison with controls; they were bigger overall, they lost their oostegite marginal setae, developed calceoli and acquired a male-like behaviour. This effect was observed in uninfected females, as well as in females infected with the Microsporidium sp. The masculinizing effect of androgenic gland implants was smaller in N. granulosis infected individuals. N. granulosis and Microsporidium sp. fall into distinct clades of the Microspora. It appears that these divergent parasites both act by inhibiting the development of the androgenic gland. However, they differ in their ability to inhibit the host’s response to the hormone that controls male sexual differentiation.

The sheep has worldwide agricultural importance, yet the genetic control of the immune responses underlying susceptibility or resistance to ovine disease is little understood. Here, we identify six novel polymorphisms in the ovine immune response genes interferon-γ (IFNG), tumour necrosis factor-α (TNF), interleukin-1β (IL1B) and interleukin-4 (IL4) in pedigree Charollais flocks. We confirm the presence of previously reported polymorphisms in IFNG and IL1B in Charollais. Restriction fragment length polymorphism (RFLP) genotyping assays have been developed for four polymorphisms, IFNGg.168C>T, IFNGg.285A>G, IL1Bg.689C>T and TNFg.3UTRA>G, and a Taqman genotyping assay has been developed for IL4g.485C>T. The previously described IL2g.647C>T polymorphism is adapted for RFLP analysis. Allele frequencies are described in Charollais, Lleyn and Suffolk cross sheep. Polymorphisms are typed in both Charollais ewes and lambs and analysed against abortion phenotypes. A subset of animals have also been analysed for the presence of Toxoplasma gondii, an abortion-causing protozoan. The IFNGg.168T allele is shown to be associated with increased risk of a ewe having an abortion, while the IFNGg.285G allele is associated with increased risk of a lamb being aborted. These assays provide tools for the investigation of the genetic basis of other phenotypes in sheep, including infectious disease susceptibility.

BACKGROUND:Sex-ratio distorting parasites are of interest due to their effects upon host population dynamics and their potential to influence the evolution of host sex determination systems. In theory, the ability to distort host sex-ratios allows a parasite with efficient vertical (hereditary) transmission to dispense completely with horizontal (infectious) transmission. However, recent empirical studies indicate that some sex-ratio distorting parasites have retained the capability for horizontal transmission.RESULTS:Numerical simulations using biologically realistic parameters suggest that a feminising parasite is only likely to lose the capability for horizontal transmission if its host occurs at low density and/or has a male-biased primary sex ratio. It is also demonstrated that even a small amount of horizontal transmission can allow multiple feminising parasites to coexist within a single host population. Finally it is shown that, by boosting its host's rate of population growth, a feminising parasite can increase its own horizontal transmission and allow the invasion of other, more virulent parasites.CONCLUSIONS:The prediction that sex-ratio distorting parasites are likely to retain a degree of horizontal transmission has important implications for the epidemiology and host-parasite interactions of these organisms. It may also explain the frequent co-occurrence of several sex-ratio distorting parasite species in nature.

Although the intracellular bacterium Wolbachia is ubiquitous in insects, it has a unique relationship with New World ants on which particular bacterial strains have specialized. However, data are from distantly related hosts and detailed phylogenetic information which could reveal transmission dynamics are lacking. Here, we investigate host-Wolbachia relationships in the monophyletic fungus-growing ant tribe Attini, screening 23 species and using multilocus sequence typing to reliably identify Wolbachia strains. This technique reduces the significant problem of recombination seen using traditional single gene techniques. The relationship between Wolbachia and the fungus-growing ants appears complex and dynamic. There is evidence of co-cladogenesis, supporting vertical transmission; however, this is incomplete, demonstrating that horizontal transmission has also occurred. Importantly, the infection prevalence is frequently different between closely related taxa, with the Acromyrmex leaf-cutting ants appearing particularly prone to infection and there being no consistent relationship with any of the major life history transitions. We suggest that infection loss and horizontal transmission have driven epidemics or selective sweeps of Wolbachia, resulting in multiple gains and losses of infection across the fungus-growing ants.

Toxoplasma gondii in sheep is important as a cause of lambing losses and as a food hazard. We aimed to assess the prevalence of infection in lambs via development of a standardised PCR technique applied to neonates together with follow-up serology at age 4 months. We measured the sensitivity of PCR targeting the T. gondii sequences B1, SAG1, 5′SAG2, 3′SAG2 and SAG3 in the presence of abundant sheep DNA. B1-PCR was the most sensitive protocol, achieving 50% positivity when 0.02 parasite genome copies were present in an assay testing 10 ng of template. Standardised B1-PCR, and serological follow-up using the modified agglutination test (MAT), were used to estimate infection prevalence in lambs from two flocks in Northern England. Neonatal prevalence detected by PCR on umbilical cord did not differ significantly between viable Charollais (16/243 (6.6%)) and viable Swaledale (30/264 (11.4%)). In contrast, at age 4 months seroprevalence was higher (P < 0.001, OR = 4.42) in Charollais (50/411 (12.2%)) than in Swaledale (10/335 (3.0%)). There was no evidence of a relationship between the results of PCR and those of serology. In addition, prenatal exposure was not associated with mortality: among non-viable lambs, 3/54 Charollais but 0/16 Swaledale were PCR positive, and 1/26 Charollais and 1/14 Swaledale were seropositive. These results indicate that both standardised B1-PCR, and serology, can be used to detect T. gondii in lambs. Frequent prenatal exposure was detected without mortality and sometimes without an IgG response. Some lambs, without PCR evidence of prenatal exposure, seroconverted early.

Parasites can strongly influence the success of biological invasions. However, as invading hosts and parasites may be derived from a small subset of genotypes in the native range, it is important to examine the distribution and invasion of parasites in the context of host population genetics. We demonstrate that invasive European populations of the North American Crangonyx pseudogracilis have experienced a reduction in post-invasion genetic diversity. We predict that vertically transmitted parasites may evade the stochastic processes and selective pressures leading to enemy release. As microsporidia may be vertically or horizontally transmitted, we compared the diversity of these microparasites in the native and invasive ranges of the host. In contrast to the reduction in host genetic diversity, we find no evidence for enemy release from microsporidian parasites in the invasive populations. Indeed, a single, vertically transmitted, microsporidian sex ratio distorter dominates the microsporidian parasite assemblage in the invasive range and appears to have invaded with the host. We propose that overproduction of female offspring as a result of parasitic sex ratio distortion may facilitate host invasion success. We also propose that a selective sweep resulting from the increase in infected individuals during the establishment may have contributed to the reduction in genetic diversity in invasive Crangonyx pseudogracilis populations.

We introduce a statistical method that explores host-parasite coevolution by testing the null hypothesis that hosts and their associated parasites evolved independently. This test is simple and intuitive and involves only suitable randomization of the observed data. It is not even necessary to construct host and parasite phylogenetic trees, as the test can be performed directly on distance matrices. Statistical power of the test was evaluated using simulated data consistent with the alternative hypothesis of cospeciation. Results were compared with the method of Mantel (1967) and the ParaFit method of Legendre et al. (2002). We observed that our method has greater power overall and thus a higher ability to detect cospeciation in closely related host-parasite systems. Our test was also successful when applied to the pocket gopher and chewing lice system.

The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s) of these bi-functional enzymes during host infection are discussed.

BACKGROUND:Toxoplasma gondii is a zoonotic parasite of global importance. In common with many protozoan parasites it has the capacity for sexual recombination, but current evidence suggests this is rarely employed. The global population structure is dominated by a small number of clonal genotypes, which exhibit biallelic variation and limited intralineage divergence. Little is known of the genotypes present in Africa despite the importance of AIDS-associated toxoplasmosis.RESULTS:We here present extensive sequence analysis of eight isolates from Uganda, including the whole genome sequencing of a type II/III recombinant isolate, TgCkUg2. 454 sequencing gave 84% coverage across the approximate 61 Mb genome and over 70,000 single nucleotide polymorphisms (SNPs) were mapped against reference strains. TgCkUg2 was shown to contain entire chromosomes of either type II or type III origin, demonstrating chromosome sorting rather than intrachromosomal recombination. We mapped 1,252 novel polymorphisms and clusters of new SNPs within coding sequence implied selective pressure on a number of genes, including surface antigens and rhoptry proteins. Further sequencing of the remaining isolates, six type II and one type III strain, confirmed the presence of novel SNPs, suggesting these are local allelic variants within Ugandan type II strains. In mice, the type III isolate had parasite burdens at least 30-fold higher than type II isolates, while the recombinant strain had an intermediate burden.CONCLUSIONS:Our data demonstrate that recombination between clonal lineages does occur in nature but there is nevertheless close homology between African and North American isolates. The quantity of high confidence SNP data generated in this study and the availability of the putative parental strains to this natural recombinant provide an excellent basis for future studies of the genetic divergence and of genotype-phenotype relationships.