Objective. The aim of this work was to study the prevalence and genetic diversity of Babesia in Ixodes persulcatus ticks and small mammals from Ural and Siberia in Russia. Methods. In total, 481 small mammals and 922 questing adult I. persulcatus from North Ural (Sverdlovsk region) and West Siberia (Novosibirsk region) were examined for the presence of Babesia by nested PCR based on the 18S rRNA gene. Results. Babesia microti of the ‘Munich’-type was found in 36·2% of blood samples of the small mammals from the Sverdlovsk region and B. microti of the ‘US’-type in 5·3% of the animals from the Novosibirsk region. Babesia DNA was not detected in 133 analysed I. persulcatus from the Sverdlovsk region; however, it was found in 24 of 789 ticks from the Novosibirsk region. Three distinct Babesia species were detected in I. persulcatus. B. microti ‘US’-type was identified in 10 ticks, Babesia closely related to B. divergens/B. capreoli in 2 ticks, and Babesia closely related to B. venatorum (EU1) in 12 ticks. Conclusion. To our knowledge, this is the first detection of Babesia sensu stricto in I. persulcatus ticks and of B. microti in I. persulcatus in the Asian part of Russia.

Systematics involves resolving both the taxonomy and phylogenetic placement of organisms. We review the advantages and disadvantages of the two kinds of information commonly used for such inferences – morphological and molecular data – as applied to the systematics of metazoan parasites generally, with special attention to the malaria parasites. The problems that potentially confound the use of morphology in parasites include challenges to consistent specimen preservation, plasticity of features depending on hosts or other environmental factors, and morphological convergence. Molecular characters such as DNA sequences present an alternative data source and are particularly useful when not all the parasite's life stages are present or when parasitaemia is low. Nonetheless, molecular data can bring challenges that include troublesome DNA isolation, paralogous gene copies, difficulty in developing molecular markers, and preferential amplification in mixed species infections. Given the differential benefits and shortcomings of both molecular and morphological characters, both should be implemented in parasite taxonomy and phylogenetics.

The recent implementation of mass drug administration (MDA) for control of uro-genital schistosomiasis has identified an urgent need for molecular markers to both directly monitor the impact of MDA, for example to distinguish re-infections from uncleared infections, as well as understand aspects of parasite reproduction and gene flow which might predict evolutionary change, such as the development and spread of drug resistance. We report the development of a novel microsatellite tool-kit allowing, for the first time, robust genetic analysis of individual S. haematobium larvae collected directly from infected human hosts. We genotyped the parasite populations of 47 children from 2 schools in the Ségou region of Mali, the first microsatellite study of this highly neglected parasite. There was only limited evidence of population subdivision between individual children or between the two schools, suggesting that few barriers to gene flow exist in this population. Complex relationships between parasite reproductive success, infection intensity and host age and gender were identified. Older children and boys harboured more diverse infections, as measured by the number of unique adult genotypes present. Individual parasite genotypes had variable reproductive success both across hosts, a pre-requisite for evolutionary selection, and, phenotypically, in hosts of different ages and genders. These data serve as a baseline against which to measure the effect of treatment on parasite population genetics in this region of Mali, and the tools developed are suitable to further investigate this important pathogen, and its close relatives, throughout their range.

Selfing has been considered the most common mode of reproduction in Echinococcus flatworms. However, population genetic studies on the asexual larval stage involving nuclear co-dominant markers have not always revealed significant heterozygote deficiencies – the expected outcome of a regularly and highly inbred population. In this study, we analysed the genetic structure of Echinococcus granulosus sensu lato populations from Southern Brazil during their adult (sexual) stage using 1 mitochondrial and 1 nuclear marker (cox 1 and mdh, respectively). We show that parasite genetic differentiation is largest among definitive hosts (domestic dogs) from different farms, suggesting that transmission is mostly maintained within a farm. Moreover, we show that heterozygote deficiencies are not significant, and we suggest that outbreeding is the most common mode of reproduction of the parasite in that region.

Chagas' disease, caused by Trypanosoma cruzi, affects 16–18 million people in Central and South America. Patient treatment is based on drugs that have toxic effects and limited efficacy. Therefore, new chemotherapeutic agents need to be developed. Snake venoms are sources of natural compounds used in various medical treatments. We observed that Crotalus viridis viridis venom was effective against all developmental forms of T. cruzi. Ultrastructural analysis revealed swelling of mitochondria, blebbing and disruption of the plasma membrane, loss of cytoplasm components and morphological changes of the cell. Staining with propidium iodide and rhodamine 123 confirmed the observed alterations in the plasma and mitochondrial membranes, respectively. The effects of the venom on the parasite intracellular cycle were also analysed. Pre-infected LLC-MK2 cells incubated with Cvv venom showed a 76–93% reduction in the number of parasites per infected cell and a 94–97·4% reduction in the number of parasites per 100 cells after 96 h of infection. Free trypomastigotes harvested from the supernatants of Cvv venom-treated cells were incapable of initiating a new infection cycle. Our data demonstrate that Cvv venom can access the host cell cytoplasm at concentrations that cause toxicity only to the amastigote forms of T. cruzi, and yields altered parasites with limited infective capacity, suggesting the potential use of Cvv venom in Chagas' disease chemotherapy.

Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features. The localization and the aggregation state of L. braziliensis H1 has been determined by immunolocalization, mass spectrometry, cross-linking and electrophoretic mobility shift assays. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. braziliensis. An antibody raised against recombinant L. braziliensis H1 recognized specifically that protein by immunoblot in L. braziliensis extracts, but not in other Leishmania species, a consequence of the sequence divergences observed among Leishmania species. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. braziliensis H1 is monomeric in solution, but oligomerizes upon binding to DNA. Finally, despite the lack of a globular domain, L. braziliensis H1 is able to form complexes with DNA in vitro, with higher affinity for supercoiled compared to linear DNA.

This work reports a new type of actinospore, Unicapsulactinomyxon, which exhibits a unique morphological characteristic in that it has a single and large polar capsule (9·3×4·1 μm) (which possesses a longitudinally-folded polar filament) instead of the 3 polar capsules previously described for actinosporeans. The spore has a binucleated sporoplasm and 3 valves, each of which forms a long process. The spore has a total length of 241·3 μm. This parasite develops in groups of 8 inside pansporocysts in the coelomic cavity of the polychaete host. Molecular investigations on the SSU rDNA show that the new actinospore type is most closely related to Enteromyxum species (81–84% similarity). A survey of actinospore infections of the marine polychaete Diopatra neapolitana in 2007 and 2009, in the Aveiro Estuary (Portugal), showed an annual prevalence of 1·0% and 0·3%, respectively.

Plasmodium falciparum has for some time been developing resistance against known anti-malarial drugs, and therefore a new drug is urgently needed. Selenium (Se), an essential trace element, in the form of inorganic Se, selenite (SeO32−), has been reported to have an anti-plasmodial effect, but its mechanism is still unclear. In the present study, we evaluated the anti-plasmodial effect of several Se compounds against P. falciparum in vitro. The anti-plasmodial effect of several Se compounds was analysed and their apoptosis-inducing activity was evaluated by morphological observation, DNA fragmentation assay and mitochondrial function analysis. SeO32−, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial effects with 50% inhibition concentration at 9, 10, 45, and 65 μm, respectively, while selenate and methylselenocysteine up to 100 μm have no effect on parasite growth. The effective Se compounds caused the parasites to become shrunken and pyknotic and significantly increased mitochondrial damage against P. falciparum compared to the untreated control. In conclusion, SeO32−, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial activities that induce apoptosis-like cell death in P. falciparum, and the anti-plasmodial effects of Se seem to be based on its chemical forms. The apoptosis-like cell-death mechanism in P. falciparum can be beneficial to respond to the growing problem of drug resistance.

Scuticociliates are histophagous marine parasites that cause mortality in fish. Acid phosphatases (AcPs) are considered virulence factors and they are used by different parasites to dephosphorylate host molecules. The aim of this work was to characterize the AcPs from 3 scuticociliate species, Uronema marinum, Miamiensis avidus and Parauronema virginianum, which parasitize marine finfish species. We identified AcP activity (pH 5·2) with differential cellular distribution in the 3 parasite species. Native gel electrophoresis of ciliate lysates revealed the presence of 1 high molecular weight AcP activity band in M. avidus (tartrate-sensitive), several low molecular weight AcPs in U. marinum and 1 low molecular weight band only in P. virginianum (tartrate-resistant). Scuticociliate AcP was inhibited by specific inhibitors of tyrosine protein phosphatases. AcP decreased upon starvation but rapid reactivation occurred following exposure to skin mucus. Groper (Polyprion oxygeneios) peripheral blood leucocytes (PBLs) and, to a lesser extent, red blood cells, also increased AcP activity. Protein tyrosine phosphatase PTP1b was primarily detected in the plasma membrane of M. avidus and ingestion of groper PBLs upregulated its expression. M. avidus recovered from experimentally infected groper had greater levels of PTP1b expression than the injected suspension. The present results highlight the importance of PTPs in histophagous parasites and their interaction with fish host's factors.

Descriptions of Babesia occultans have previously been restricted to sub-Saharan Africa. Here, we report the finding, for the first time, of this low or non-pathogenic bovine Babesia species in Tunisia, northern Africa. B. occultans DNA was detected by molecular methods in Hyalomma marginatum unfed ticks collected in 3 bioclimatic regions of Tunisia. The near-full-length 18S rRNA gene was sequenced and compared with related sequences retrieved from GenBank. Phylogenetic analysis indicated that other sequences deposited as Babesia sp. could also correspond to B. occultans, suggesting that this species may have a wide distribution in Mediterranean and Asiatic regions, and not only in sub-Saharan Africa as previously described. A B. occultans-specific Reverse Line Blot (RLB) oligonucleotide probe was designed for future epidemiological studies that would help to clarify this possibility.

Introduction. Evaluation of Leishmania drug susceptibility depends on in vitro SbV susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of SbV-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as SbV-resistant and -sensitive, in order to identify potential resistance markers. Methods. The differential expression of 13 genes involved in SbV metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. Results. Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of SbV-resistant compared to the group of SbV-sensitive parasites (P<0·01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. Discussion. Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro SbV resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro SbV susceptibility assays, but interfering with the gene expression patterns.

The synergism facilitated by a series of recent developments has conspired to catalyze rapid advancements in the taxonomy and systematics of elasmobranch tapeworms. These developments are: (1) increased interest in global biodiversity; (2) globalization-facilitated communication; (3) enhanced microscopic and digital technologies; (4) availability of web-based taxonomic resources; (5) ease of use and low cost of molecular techniques and (6) the impressive repertoire of available phylogenetic methods. As a consequence, an estimation of global elasmobranch tapeworm diversity is now within our grasp, as is a basic understanding of the effort and resources required to complete the discovery and description of this fauna globally. The generation of robust hypotheses of the phylogenetic relationships for most elasmobranch-parasitizing cestode orders is also well underway. An international community of cestodologists has emerged and through their sharing of knowledge and specimens is making great strides towards expanding knowledge of the cestode fauna of vertebrates worldwide. It is important that these efforts continue to move forward in a collaborative fashion, integrating morphological and molecular data, but also fully engaging elasmobranch taxonomists and systematists. It is equally important that efforts to characterize and describe global biodiversity are not derailed by such seductive, but ultimately unrewarding impracticable initiatives as molecular taxonomy. Integrated taxonomy is certainly not for the feint of heart, but those with the courage to pursue this strategy will be responsible for maintaining and enhancing the biologically relevant context required for effective species recognition well into the future.

Trypanosoma brucei gambiense sleeping sickness follows a long asymptomatic phase and persists in ancient foci from which epidemic clinical disease arises. A putative focus of T. b. gambiense infections has been identified, initially in mothers and young children, on the Lake Albert shoreline of Western Uganda leading to mass screening of 6207 individuals in September 2008. T. b. gambiense infections were identified by Card Agglutination Test for Trypanosomiasis (CATT) and sub-species-specific PCR although parasitological methods failed to confirm any patent trypanosome infections. In April 2009, CATT positives were re-visited; diagnosis of individuals by CATT and PCR was unstable over the two time points and parasites remained undetected, even using mini Anion Exchange Centrifugation Technique (mAECT). These observations suggest the possibility of a silent focus of disease, where all infected individuals are in a latent stage, and highlight our limited understanding of the local natural history and disease progression of T. b. gambiense in children and adults.

Toxoplasmosis is a significant public health threat for Inuit in the Canadian Arctic. This study aimed to investigate arctic seals as a possible food-borne source of infection. Blood samples collected from 828 seals in 7 Canadian Arctic communities from 1999 to 2006 were tested for Toxoplasma gondii antibodies using a direct agglutination test. Polymerase chain reaction (PCR) was used to detect T. gondii DNA in tissues of a subsample of seals. Associations between seal age, sex, species, diet, community and year of capture, and serological test results were investigated by logistic regression. Overall seroprevalence was 10·4% (86/828). All tissues tested were negative by PCR. In ringed seals, seroprevalence was significantly higher in juveniles than in adults (odds ratio=2·44). Overall, seroprevalence varied amongst communities (P=0·0119) and by capture year (P=0·0001). Our study supports the hypothesis that consumption of raw seal meat is a significant source of infection for Inuit. This work raises many questions about the mechanism of transfer of this terrestrial parasite to the marine environment, the preponderance of infection in younger animals and the natural course of infection in seals. Further studies to address these questions are essential to fully understand the health risks for Inuit communities.

We describe the new microsporidium Hamiltosporidium tvaerminnensis gen. et sp. nov. with an emphasis on its ultrastructural characteristics and phylogenetic position as inferred from the sequence data of SSU rDNA, alpha- and beta-tubulin. This parasite was previously identified as Octosporea bayeri Jírovec, 1936 and has become a model system to study the ecology, epidemiology, evolution and genomics of microsporidia - host interactions. Here, we present evidence that shows its differences from O. bayeri. Hamiltosporidium tvaerminnensis exclusively infects the adipose tissue, the ovaries and the hypodermis of Daphnia magna and is found only in host populations located in coastal rock pool populations in Finland and Sweden. Merogonial stages of H. tvaerminnensis have isolated nuclei; merozoites are formed by binary fission or by the cleaving of a plasmodium with a small number of nuclei. A sporogonial plasmodium with isolated nuclei yields 8 sporoblasts. Elongated spores are generated by the most finger-like plasmodia. The mature spores are polymorphic in shape and size. Most spores are pyriform (4·9–5·6×2·2–2·3 μm) and have their polar filament arranged in 12–13 coils. A second, elongated spore type (6·8–12·0×1·6–2·1 μm) is rod-shaped with blunt ends and measures 6·8–12·0×1·6–2·1 μm. The envelope of the sporophorous vesicle is thin and fragile, formed at the beginning of the sporogony. Cytological and molecular comparisons with Flabelliforma magnivora, a parasite infecting the same tissues in the same host species, reveal that these two species are very closely related, yet distinct. Moreover, both cytological and molecular data indicate that these species are quite distant from F. montana, the type species of the genus Flabelliforma. We therefore propose that F. magnivora also be placed in Hamiltosporidium gen. nov.

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.

The Regent Honeyeater (Xanthomyza phyrigia) is an endangered Australian bird species. Breeding populations have been established at Australian zoos in support of re-introduction programmes. This species is the host of a new species of Isospora (Apicomplexa). Oocysts are spherical, 25·8 (22·5–28·75) by 23·8 (20–26·25) μm with a colourless to pale yellow smooth wall undergoing rapid exogenous sporulation, 90% sporulated oocysts in 8 h at 20°C. Each oocyst contains 1 polar granule. Sporocysts are ovoid, 18·67 (17–19) by 9·49 (9–10) μm with a flat Stieda body and spherical substieda body devoid of a hyaline body. The asexual stages and sexual phase is within the enterocytes of the duodenum and jejunum. Faeces collected in the morning (AM, n=84) and in the afternoon (PM, n=90) revealed significant diurnal periodicity in oocyst shedding; 21% (18 of 84) of the AM were positive with the mean of 499 oocysts.g−1 compared to the PM with 91% (82 of 90) bird faeces positive with the mean of 129 723 oocysts.g−1. Therefore, parasite checks for these birds should be carried out in the afternoon to obtain an accurate result. The ecological significance of the high parasite burden in captive birds requires further investigation and comparison to the wild counterparts.

The in vitro effects of 4 arylimidamides (DB811, DB786, DB750 and DB766) against the proliferative tachyzoite stage of the apicomplexan parasite Besnoitia besnoiti were investigated. These four compounds had been shown earlier to exhibit in vitro activities in the nanomolar range against the related apicomplexans Neospora caninum and Toxoplasma gondii. Real-time-PCR was used to assess B. besnoiti intracellular proliferation in vitro. Preliminary assessment by light microscopy identified DB811 and DB750 as the most promising compounds, while DB786 and DB766 were much less effective. Three-day-growth assays and quantitative real-time PCR was used for IC50 determination of DB811 (0·079 μM) and DB750 (0·56 μM). Complete growth inhibition was observed at 1·6 μM for DB 811 and 1·7 μM for DB750. However, when infected cultures were treated for 14 days, proliferation of parasites occurred again in cultures treated with DB750 from day 4 onwards, while the proliferation of DB811-treated tachyzoites remained inhibited. Electron microscopy of B. besnoiti-infected fibroblast cultures fixed and processed at different time-points following the initiation of drug treatments revealed that DB811 exerted a much higher degree of ultrastructural alterations compared to DB750. These results show that arylimidamides such as DB811 could potentially become an important addition to the anti-parasitic arsenal for food animal production, especially in cattle.

Genomic DNAs from 3 oocyst isolates of Hammondia sp. from foxes (Vulpes vulpes and V. lagopus) and 1 oocyst isolate of Hammondia heydorni from a dog, were examined by PCR and sequence analysis of 6 loci in order to determine whether the isolates were conspecific. Consistent genetic differences were found between the fox and dog isolates, respectively, at the ITS-2 region, the lsu rRNA gene, the alpha tubulin gene and the HSP70 gene, but not at the ssu rRNA gene or ITS-1 locus. Infection experiments established that dogs were unsuitable as definitive hosts for Hammondia sp. of foxes; hence this species is regarded as separate from H. heydorni of wolf-like canids, but probably identical with Isospora triffittae (syn. Isospora triffitti) previously reported from foxes. This species has therefore been named Hammondia triffittae n. comb. Reindeer, moose, sheep, goats, foxes and rabbits may act as intermediate hosts for H. triffittae. Muscle tissues of inoculated intermediate hosts were infectious for foxes from 16 days post-infection. Oocysts of H. triffittae were subspherical, averaging 12·5×10·9 μm in size. The lsu rRNA gene and the alpha tubulin gene seem to be suitable genetic markers for differentiating between H. triffittae and H. heydorni.

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20 000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.