With a view to developing putatively neutral markers based on Single Nucleotide Polymorphisms (SNPs) in the human malaria parasite, Plasmodium vivax, we utilized the published whole genome sequence information of P. falciparum and P. vivax to find a ~200 kb conserved syntenic region between these two species. We have selected 27 non-coding DNA fragments (in introns and intergenic regions) of variable length (300–750 bp) in P. vivax in this syntenic region. PCR of P. vivax isolates of a population sample from India could successfully amplify 17 fragments. Subsequently, DNA sequencing and sequence analysis confirmed the polymorphic status of only 11 fragments. Altogether, 18 SNPs were detected and 2 different measures of nucleotide diversity showed variable patterns across different fragments; in general, introns were less variable than the intergenic regions. All 11 polymorphic fragments were found to be evolving according to a neutral equilibrium model and thus could be utilized as putatively neutral markers for population genetic studies in P. vivax. Different molecular population genetics parameters were also estimated, providing initial insight into the population genetics of Indian P. vivax.

Certain species of the protozoan genus Besnoitia cause clinical disease in livestock and wildlife. In the present paper a new species, Besnoitia neotomofelis is described from the southern planes woodrat (Neotoma micropus). The parasite was detected by bioassay of woodrat tissues in outbred Swiss Webster mice in an attempt to isolate Toxoplasma gondii. Initially, the organism was misdiagnosed as T. gondii because it was highly pathogenic for mice and its tachyzoites resembled T. gondii tachyzoites. Further studies revealed that it differed structurally and biologically from T. gondii. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey kidney cells, and in vivo in mice. Non-dividing, uninucleate tachyzoites were approximately 1×5 μm in size. Longitudinally-cut bradyzoites in tissue sections measured 1·5–1·6×7·7–9·3 μm. Tissue cysts were microscopic, up to 210 μm long, and were infective orally to mice. Cats fed tissue cysts shed unsporulated 13×14 μm sized oocysts. All mice inoculated with B. neotomofelis died of acute besnoitiosis, irrespective of the dose, and Norwegian rats became infected but remained asymptomatic. Entero-epithelial stages (schizonts, gamonts) were found in cats fed tissue cysts. Large (up to 40×50 μm) first-generation schizonts developed in the lamina propria of the small intestine of cats. A second generation of small sized (8 μm) schizonts containing 4–8 merozoites was detected in enterocytes of the small intestine. Gamonts and oocysts were seen in goblet cells of the small intestinal epithelium. Tachyzoites were present in mesenteric lymph nodes of cats. Phylogenetic analysis indicated that B. neotomofelis was related to other Besnoitia species from rodents, rabbits, and opossums. Besnoitia neotomofelis is distinct from the 3 other species of Besnoitia, B. wallacei, B. darlingi and B. oryctofelisi that utilize cats as a definitive host.

Encephalitozoon cuniculi is an obligate intracellular pathogen that has a wide host distribution, but primarily affects rabbits. The aim of this study was to characterize both the cell-mediated and the antibody response in rabbits after experimental infection using 2 different infection routes: oral and ocular. SPF rabbits were infected with low (103 spores) and high (107 spores) infection doses. Monitored parameters included clinical signs, detection of spores in urine, antibody response detected with ELISA, and cell-mediated immunity detected by antigen-driven lymphocyte proliferation. At week 13 post-infection, half of the rabbits in each group were suppressed by intramuscular administration of dexamethasone. At week 18 post-infection, animals were euthanized. Clinical signs were mild with exacerbation after immunosuppression. Spores in urine and antigen-specific cell-mediated immunity were detected from weeks 5 and 4 post-infection, respectively. Specific IgM was detected 1 week after infection, and IgG antibodies followed 1 week later in rabbits infected with the high dose. Immunological responses were dose dependent. The authors can conclude that both oral and ocular experimental infection with E. cuniculi resulted in an immune response of the infected animals. Rabbits could be used as an experimental model for the study of ocular microsporidiosis.

Some Kudoa species display variations in the number of polar capsules in spores within an individual pseudocyst. Nonetheless, there is usually a dominant morphotype which forms a significant element of diagnosis. In 2007, a Kudoa isolate from whiting (spores with 5 (dominant) or 6 (minor) polar capsules) was characterized by Burger et al. (2007) as being 100% identical in SSU rDNA to Kudoa yasunagai (spores with 7 polar capsules) from a halibut, despite its obvious morphological differences. The authors hypothesized that either SSU rDNA had reached its level of resolution or that the genetic identity revealed conspecificity. To further investigate these hypotheses, SSU and LSU rDNA sequence data were coupled with principal components, correlation, and regression analyses of morphometric data from different kudoid isolates that infect brain tissue to determine the relationships between spore morphotypes and different kudoid isolates. The trends in morphometrics between the spores of particular isolates were so similar that it was concluded that the molecular results did indicate conspecificity rather than SSU reaching its level of resolution. This phenotypic influence on a significant diagnostic character within the Kudoidae has a major impact on the diagnosis of this, and potentially other, pathogenic species.

Objective. RhD-positive subjects are protected against toxoplasmosis-associated impairment of psychomotor performance. Here we searched for RhD-positivity-associated maternal protection against the effects of toxoplasmosis. Methods. In the present retrospective cohort study, we analysed data from 785 (139 RhD-negative) Toxoplasma-free and 194 (27 RhD-negative) Toxoplasma-infected pregnant women. We searched for effects of toxoplasmosis and Rhd-phenotype on maternal weight before pregnancy, pregnancy weight gain, fetal ultrasound data (biparietal diameter, abdominal circumference, femur length) and on birth length and weight. Results. At pregnancy week 16, the RhD-negative mothers with toxoplasmosis gained more weight than others (P<0·001). The difference of about 1600 g remained approximately constant from pregnancy week 16 until the end of pregnancy. Neither toxoplasmosis nor RhD phenotype had any effect on fetal bioparameter data or birth length and weight. Conclusion. The most parsimonious explanation for the observed data is that the RhD-positive phenotype might protect infected subjects against a broad spectrum of detrimental effects of latent toxoplasmosis, including excessive gestational weight gain.

A histopathological study was performed to clarify the characteristics of granuloma formation and liver fibrosis in Schistosoma mekongi infection in comparison with S. japonicum infection. Mice were exposed to S. mekongi (Laotian strain) and S. japonicum (Japanese strain) cercariae, and were dissected at 6, 8, 12, 16, and 20 weeks post-exposure. In the liver, granulomas in S. mekongi infection were cellular, initially organized with foam cells, and continuously appeared in the intralobular area, while granulomas in S. japonicum infection were fibrous and did not continuously appear in the intralobular area. Portal fibrosis was not seen in S. mekongi infection, but was commonly seen in S. japonicum infection in the later weeks. Granulomas in the small intestine were seen mainly in the submucosa with foam cells in S. mekongi infection and without foam cells in S. japonicum infection. The lung granulomas contained mainly histiocytes in both S. mekongi and S. japonicum infection. The absence of portal fibrosis in S. mekongi infection allows schistosome eggs to infiltrate into the intralobular area continuously, which can be what lies behind the ultrasonographic differences; the echogenic network pattern as was seen in S. japonicum infection, has not been noted in S. mekongi infection.

The use of chemotherapy on a mass scale in endemic areas may lead to the appearance of resistant isolates through the mechanism of selective drug pressure. Studies have demonstrated that praziquantel (PZQ) is able to inhibit the excretory activity and to cause tegumental damage in Schistosoma mansoni adult worms. The use of the probe resorufin to evaluate excretory activity, as well as the probe Hoechst 33258 to detect tegumental damage in adult worms, may represent a method to identify resistant (or less susceptible) isolates. The purpose of the present work was to compare the changes caused by PZQ in the function of the excretory system and in the integrity of the tegument of adult worms from the LE isolate (susceptible to PZQ) and the LE-PZQ isolate (less susceptible to PZQ). Worms from the isolate LE-PZQ showed less severe tegumental lesions, in both in vitro and in vivo experiments, detected by labelling with Hoechst 33258 and continued to have a functional excretory system as shown by labelling with resorufin in vitro.

The sheep ked has been largely eradicated in the UK but persists in the feral Soay sheep of St Kilda in the Outer Hebrides. Sheep keds transmit Trypanosoma melophagium, but parasitaemias are typically cryptic and this trypanosome has not been recorded in the St Kilda sheep. Trypanosomes were detected by PCR in preserved keds and were also found in gut smears from live keds; one infected gut was used to establish the trypanosome in vitro. Examination of the morphology of bloodstream forms from culture confirmed its identity as T. melophagium. Most keds were found to harbour the trypanosome, particularly those collected from lambs. DNA was extracted from preserved keds and from trypanosomes grown in vitro. Sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene and the spliced leader transcript showed the T. melophagium sequences to be very similar to those from T. theileri. A partial sequence of the ked SSU rRNA gene was also obtained. The close genetic relationship of T. melophagium and T. theileri suggests that T. melophagium represents a lineage of T. theileri that adapted to transmission by sheep keds and hence became a specific parasite of sheep.

Fatty acid binding proteins are considered to be promising vaccine candidates against trematodiasis. In order to provide additional information about their function in Fasciola gigantica we performed a comparative analysis of FgFABP1 and FgFABP3, two isoforms with quite different isoelectric points of 4·9 and 9·9 and 67% sequence identity. Both are expressed in the juvenile and adult parasite but differ in their tissue-specific distribution. In addition, the sequence of FABP3 is identical in F. hepatica and F. gigantica indicating the protein's functional importance in this genus. Immune sera produced against soluble recombinant FgFABPs reacted with 14 kDa antigens in crude worm, soluble egg, cirrus sac extracts, and excretion/secretion product. Both FgFABPs were located in the parenchyma of the parasite but in addition, FgFABP1 was abundant in testes and spermatozoa while FgFABP3 was abundant in vitelline cells, eggs, and caecal epithelium. Mass spectrometry identified FgFABP1 and FgFABP3 in the ES product whereas only FgFABP3 was identified in egg extract. Serum samples of an experimentally infected rabbit reacted from week 6 post-infection with FgFABP3 and from week 12 with FgFABP1 while sera of infected sheep were not reactive. The results suggest differences in the biological functions of these 2 isoforms and differences in the host/parasite interaction that should be considered for their potential as vaccines against fascioliasis.

The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.

We address the effect of spatial scale and temporal variation on model generality when forming predictive models for fish assignment using a new data mining approach, Random Forests (RF), to variable biological markers (parasite community data). Models were implemented for a fish host-parasite system sampled along the Mediterranean and Atlantic coasts of Spain and were validated using independent datasets. We considered 2 basic classification problems in evaluating the importance of variations in parasite infracommunities for assignment of individual fish to their populations of origin: multiclass (2–5 population models, using 2 seasonal replicates from each of the populations) and 2-class task (using 4 seasonal replicates from 1 Atlantic and 1 Mediterranean population each). The main results are that (i) RF are well suited for multiclass population assignment using parasite communities in non-migratory fish; (ii) RF provide an efficient means for model cross-validation on the baseline data and this allows sample size limitations in parasite tag studies to be tackled effectively; (iii) the performance of RF is dependent on the complexity and spatial extent/configuration of the problem; and (iv) the development of predictive models is strongly influenced by seasonal change and this stresses the importance of both temporal replication and model validation in parasite tagging studies.