Researchers have learned much concerning the population biology of Toxoplasma gondii over the past 2 decades. It is now apparent that many atypical genotypes exist besides the typical 3 genotypes (type I, type II and type III) first described from samples from Europe and the United States. These genotypes can differ in pathogenicity and transmissibility from the typical genotypes that have been used in the majority of scientific research over the past 70 years. These differences impact much of what we used to believe as facts about congenital toxoplasmosis (CT) and will be important in developing new recommendations for prevention of CT and the monitoring of women at risk for developing CT. The present review highlights new information on T. gondii genotypes and how this information will change the way we convey information about CT to pregnant women, physicians and students.

Here we present the detection of a gene cluster for Neospora caninum surface genes, similar to the Toxoplasma gondii SRS9 locus, and the cloning and characterization of the NcSRS9 gene. PCR genome walking, using NcBSR4 gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to the SRS5 and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtained in vitro and RT-PCR analysis showed no significant variations of NcSRS9 transcripts during in vitro tachyzoite-bradyzoite switch, probably due to incomplete maturity of in vitro bradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding of N. caninum persistence.

Understanding the origin of invasive parasites and ecological transmission barriers on the distribution of mosquito-borne pathogens is enriched by molecular phylogenetic approaches now that large databases are becoming available. Here we assess the biogeographical relationships among haemosporidian blood parasites and an avian host, the New Zealand bellbird (Meliphagidae, Anthornis melanura). Four Plasmodium haplotypes were identified among 93 infected bellbirds (693 screened) using nested PCR of a mitochondrial DNA cytochrome b gene fragment. The most common lineage, LIN1 (11%), is confined to northern New Zealand and falls within a known clade of Plasmodium (subgenus Novyella) sp. infecting Australian meliphagids. LIN1 differs within that clade by 4 9% sequence divergence suggestive of an endemic lineage to New Zealand. The most widespread lineage, LIN2 (2%), is an exact match with a global cosmopolitan (P. elongatum GRW06). Two rare lineages, LIN3 and LIN4 are less abundant, geographically restricted within New Zealand and have <1% sequence divergence with P. (Novyella) sp. (AFTRU08) and P. relictum (LINOLI01) documented from Africa. For the first time, we provide invaluable information on possible rates of entry of invading parasites in New Zealand and their distribution from temperate to cold environments.

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.

The present study aimed to determine the in vitro biological efficacy and selectivity of 7 novel AIAs upon bloodstream trypomastigotes and intracellular amastigotes of Trypanosoma cruzi. The biological activity of these aromatic compounds was assayed for 48 and 24 h against intracellular parasites and bloodstream forms of T. cruzi (Y strain), respectively. Additional assays were also performed to determine their potential use in blood banks by treating the bloodstream parasites with the compounds diluted in mouse blood for 24 h at 4°C. Toxicity against mammalian cells was evaluated using primary cultures of cardiac cells incubated for 24 and 48 h with the AIAs and then cellular death rates were determined by MTT colorimetric assays. Our data demonstrated the outstanding trypanocidal effect of AIAs against T. cruzi, especially DB1853, DB1862, DB1867 and DB1868, giving IC50 values ranging between 16 and 70 nanomolar against both parasite forms. All AIAs presented superior efficacy to benznidazole and some, such as DB1868, also demonstrated promising activity as a candidate agent for blood prophylaxis. The excellent anti-trypanosomal efficacy of these novel AIAs against T. cruzi stimulates further in vivo studies and justifies the screening of new analogues with the goal of establishing a useful alternative therapy for Chagas disease.

In the present study, we investigated the involvement of sulfated glycosaminoglycans in both the in vivo development and adhesion of T. cruzi epimastigotes to the luminal surface of the digestive tract of the insect vector, Rhodnius prolixus. Pre-incubation of T. cruzi, Dm 28c epimastigotes with heparin, chondroitin 4-sulfate, chondroitin 6-sulfate or protamine chloridrate inhibited in vitro attachment of parasites to the insect midgut. Enzymatic removal of heparan sulfate moieties by heparinase I or of chondroitin sulfate moieties by chondroitinase AC from the insect posterior midgut abolished epimastigote attachment in vitro. These treatments also reduced the labelling of anionic sites exposed at the luminal surface of the perimicrovillar membranes in the triatomine midgut epithelial cells. Inclusion of chondroitin 4-sulfate or chondroitin 6-sulfate and to a lesser extent, heparin, in the T. cruzi-infected bloodmeal inhibited the establishment of parasites in R. prolixus. These observations indicate that sulfated glycosaminoglycans are one of the determinants for both adhesion of the T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine and the parasite infection in the insect vector, R. prolixus.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971–2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5–20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5–20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.

LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in protozoans of the genus Leishmania which is associated with the immunopathogenesis and susceptibility of BALB/c mice to L. major infection. Previously, we demonstrated that intranasal immunization with a plasmid carrying the LACK gene of Leishmania infantum (LACK-DNA) promotes protective immunity in BALB/c mice against Leishmania amazonensis and Leishmania chagasi. In the present study, we investigated the protective immunity achieved in hamsters intranasally vaccinated with 2 doses of LACK-DNA (30 μg). Compared with controls (PBS and pCI-neo plasmid), animals vaccinated with LACK-DNA showed significant reduction in parasite loads in the spleen and liver, increased lymphoproliferative response and increased nitric oxide (NO) production by parasite antigen-stimulated splenocytes. Furthermore, hamsters vaccinated with LACK-DNA presented high IgG and IgG2a serum levels when compared to control animals. Our results showed that intranasal vaccination with LACK-DNA promotes protective immune responses in hamsters and demonstrated the broad spectrum of intranasal LACK-DNA efficacy in different host species, confirming previous results in murine cutaneous and visceral leishmaniasis.

NK cells represent one of the first lines of defence in the immune reaction after invasion of Leishmania parasites. Depletion of mouse natural killer (NK) cells dramatically enhances susceptibility of normally resistant mice. In this study we evaluated the fate of NK cells and parasites after contact formation. The hydrophilic fluorescent dye CMFDA (chloro-methylfluorescin diacetate) that allows analysis of cytotoxicity in flow cytometry and microscopy was used. Furthermore, these findings were confirmed with scanning and transmission electron microscopy. Direct contact points were found between Leishmania promastigotes and naïve human NK cells. These contacts were associated with transfer of cytosol by membrane bridges and cytotoxicity of NK cells against Leishmania. However, in contrast to other target cells which allow repeated exocytosis of lytic granules, contact with Leishmania causes immediate destruction of NK cells in a non-apoptotic way. Our results give a reasonable explanation for ex vivo observations of reduced NK cell numbers and impaired NK response in patients with acute cutaneous leishmaniasis. Animal models have clearly shown that NK cells play a key role in the induction and direction of the immune response. Thus inhibition of NK cells at the onset of infection would be advantageous for the survival of the parasite.

We investigated whether parasites or hosts benefit from reduced reproduction in infected hosts. When parasites castrate their hosts, the regain of host reproduction is necessary for castration to be a host adaptation. When infecting Daphnia magna with Pasteuria ramosa, in a lake water based medium, 49 2% of the castrated females regained reproduction. We investigated the relationship between castration level, and parasite and host fitness proxies to determine the adaptive value of host castration. Hosts which regained reproduction contained less spores and had a higher lifetime reproduction than permanently castrated hosts. We also found a negative correlation between parasite and host lifetime reproduction. For hosts which regained reproduction we found no optimal level of castration associated with lifetime reproduction. These results support the view that host castration only is adaptive to the parasite in this system. In addition, we suggest that permanent castration might not be the norm under natural conditions in this system. Finally, we argue that a reduction in host reproduction is more likely to evolve as a property favouring parasites rather than hosts. To our knowledge this is the only experimental study to investigate the adaptive value of reduced host reproduction when castrated hosts can regain reproduction.

The aim of this study was to develop, perfect and validate the PCR (polymerase chain reaction) technique using mitochondrial (mt) and ribosomal (ITS-2) DNA for the accurate identification of Dicrocoelium dendriticum in molluscs and ants, the first and second intermediate hosts, and their early detection. The first primers that were designed amplified a 169 pb mtDNA fragment of D. dendriticum permitted the detection of a single D. dendriticum metacercaria from the Formica rufibarbis and Formica pratensis abdomen, as well as the detection of the brainworm in the head of the ants collected in tetania. Although these primers did not amplify Dicrocoelium chinensis DNA and permitted detected D. dendriticum in the molluscs, they did not discriminate Brachylaimidae metacercariae found in the same mollusc. The PCR that was designed to amplify a 93 bp fragment of the ITS-2 is D. dendriticum specific as it did not amplify D. chinensis, Brachylaimidae and other trematodes. This technique is very sensitive since it permitted the detection of D. dendriticum in the molluscs from the first day post-infection, the brainworm in the head of the ants and only 1 D. dendriticum metacercaria from the abdomen of the ants. Both techniques are important, mainly the latter.

Hematodinium is a genus of parasitic dinoflagellates whose species have caused significant mortalities in marine crustacean fisheries worldwide. A species of Hematodinium infects the blue crab, Callinectes sapidus on the eastern seaboard of the USA. The mode of transmission of the parasite in blue crabs is unknown. We established several continuous in vitro cultures of Hematodinium sp. isolated from the haemolymph of infected blue crabs. One isolate has been continuously maintained in our laboratory through serial subcultivation for over 12 months, and is capable of infecting new hosts when inoculated into healthy crabs. Cells of the parasite undergo characteristic developmental changes in vitro consistent with the identifiable stages of Hematodinium sp.: filamentous trophonts, amoeboid trophonts, arachnoid trophonts and sporonts, sporoblasts, prespores and dinospores (macrospores and microspores). Additionally, we describe an unusual shunt in the life cycle wherein presumptive schizonts derived from arachnoid sporonts developed into filamentous and arachnoid trophonts that can then initiate arachnoid sporonts in new cultures. This may explain the rapid proliferation of the parasite in blue crab hosts. We also found that temperature and light intensity affected the growth and development of the parasite in vitro.

Corridor disease is an acute, fatal disease of cattle caused by buffalo-adapted Theileria parva. This is a nationally controlled disease in South Africa and strict control measures apply for the movement of buffalo, which includes mandatory testing for the presence of T. parva and other controlled diseases. Accurate diagnosis of the T. parva carrier state in buffalo using the official real-time hybridization PCR assay (Sibeko et al. 2008), has been shown to be affected by concurrent infection with T. sp. (buffalo)-like parasites. We describe the Hybrid II assay, a real-time hybridization PCR method, which compares well with the official hybridization assay in terms of specificity and sensitivity. It is, however, not influenced by mixed infections of T. sp. (buffalo)-like parasites and is as such a significant improvement on the current hybridization assay.

Properties of ecological networks facilitate the understanding of interaction patterns in host-parasite systems as well as the importance of each species in the interaction structure of a community. The present study evaluates the network structure, functional role of all species and patterns of parasite co-occurrence in a host-parasite network to determine the organization level of a host-parasite system consisting of 170 taxa of gastrointestinal metazoans of 39 marine fish species on the coast of Brazil. The network proved to be nested and modular, with a low degree of connectance. Host-parasite interactions were influenced by host phylogeny. Randomness in parasite co-occurrence was observed in most modules and component communities, although species segregation patterns were also observed. The low degree of connectance in the network may be the cause of properties such as nestedness and modularity, which indicate the presence of a high number of peripheral species. Segregation patterns among parasite species in modules underscore the role of host specificity. Knowledge of ecological networks allows detection of keystone species for the maintenance of biodiversity and the conduction of further studies on the stability of networks in relation to frequent environmental changes.