Toxoplasma gondii is largely transmitted to definitive felid hosts through predation. Not all prey species represent identical risks of infection for cats because of differences in prey susceptibility, exposure and/or lifespan. Previously published studies have shown that prevalence in rodent and lagomorph species is positively correlated with body mass. We tested the hypothesis that different prey species have different infection risks by comparing infection dynamics of feral cats at 4 sites in the sub-Antarctic Kerguelen archipelago which differed in prey availability. Cats were trapped from 1994 to 2004 and anti-T. gondii IgG antibodies were detected using the modified agglutination test (⩾1:40). Overall seroprevalence was 51·09%. Antibody prevalence differed between sites, depending on diet and also on sex, after taking into account the effect of age. Males were more often infected than females and the difference between the sexes tended to be more pronounced in the site where more prey species were available. A difference in predation efficiency between male and female cats may explain this result. Overall, our results suggest that the composition of prey items in cat diet influences the risk of T. gondii infection. Prey compositon should therefore be considered important in any understanding of infection dynamics of T. gondii.

During the acute phase of Trypanosoma cruzi infection, strong haematological and immune system alterations are observed. The parasite expresses trans-sialidase, a virulence factor responsible for the sialylation of its surface glycoconjugates. This enzyme is also shed to the bloodstream where it is associated with immune system alterations triggered during the infection. During experimental and human infections, the host elicits antibodies able to neutralize the enzyme activity that would be responsible for restricting systemic trans-sialidase to the early steps of the infection, when major immune alterations are induced. The actual relevance of these antibodies was tested by passive transference of monoclonal neutralizing antibodies in acute infection models displaying extreme sensitivity to the infection. Mice were inoculated with virulent parasite strains that induce high parasitaemia, early mortality and strong immune tissue abnormalities. The trans-sialidase-neutralizing antibodies were able to preserve B cell areas both in ganglia and spleen as well as the thymus architecture even in these extreme models. Although no differences between control and treated mice regarding animal survival were found, a major role for the humoral response in controlling the damage of the immune system induced by a systemically distributed virulence factor was defined in an infection with a eukaryotic pathogen.

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 76 free-range chickens from Guyana, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 50 (65·8%) of 76 chickens with titres of 1:5 in four, 1:10 in one, 1:20 in five, 1:40 in seven, 1:80 in six, 1:160 in eight, 1:320 in four, 1:640 or higher in 15. Hearts and brains of 26 chickens with titres of <1:5 were pooled in 5 batches and bioassayed in mice. Hearts and brains of 50 chickens with titres of 1:5 or higher were bioassayed in mice. Toxoplasma gondii was isolated by bioassay in mice from 35 chickens with MAT titres of 1:20 or higher. All mice inoculated with tissues of 30 infected chickens remained asymptomatic. Toxoplasma gondii isolates from 35 chickens were genotyped using 11 PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, a new SAG2, and Apico. A total of 9 genotypes were identified, with 5 genotypes (nos 1, 4, 5, 6 and 7) unique to Guyana, 2 genotypes (nos 2 and 3) previously identified in chickens from Brazil, 1 genotype (no. 8) previously identified in chickens from Brazil, Costa Rica and Nicaragua, and 1 genotype (no. 9) belonging to the clonal type III lineage that exists globally. Infection with 2 genotypes was found from 1 chicken. This is the first report of genetic characterization of T. gondii isolates from any host from Guyana.

We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.

A Neospora caninum 17–19 kDa antigenic protein fraction (p17) in one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) is the immunodominant antigen recognized by sera from bovines naturally infected by N. caninum. To identify the proteins making up the p17 fraction, we screened a new N. caninum tachyzoite cDNA library with an affinity-purified antibody against p17 (APA17). We isolated several cDNA clones with 100% sequence identity to the NcGRA7 gene. This previously described gene encodes a dense granule protein with an apparent molecular mass of 33 kDa. A second line of evidence emerged through a combined proteomic approach associating two-dimensional PAGE (2D-PAGE) to Western blotting and to mass spectrometry to characterize the p17 fraction. Two acidic immunodominant but minority protein spots were recognized by APA17 and by bovine sera. These antigens of 17 and 33 kDa are respectively composed of 4 and 2 isoforms. Furthermore, p17 isolation by 2D-PAGE and peptide sequencing by tandem mass spectrometry yielded a partial sequence of 17 amino acids, which allowed the putative amino terminal region of the NcGRA7 protein to be identified unambiguously.The NcGRA7 protein, without the putative signal peptide at the NH2-terminus, was cloned and expressed in Escherichia coli and when the purified recombinant protein (rNcGRA7) was analysed by SDS-PAGE and mass spectrometry, 2 bands of 24 and 33 kDa were resolved and identified as NcGRA7. These results demonstrate that the immunodominant 17 kDa antigen of N. caninum is encoded by the NcGRA7 gene.

A cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism)-based strategy has been used to identify levamisole (LEV) resistance markers in the nematode Haemonchus contortus. Transcript profiles of adult nematodes from two LEV-resistant and two susceptible isolates were compared. Among the 17 280 transcript-derived fragments (TDFs) amplified, 26 presented a polymorphic pattern between resistant and susceptible nematodes: 11 TDFs were present in both resistant isolates and absent from both susceptible isolates whereas 15 TDFs were present in both susceptible isolates and absent from both resistant isolates. 8 TDFs specifically present in resistant isolates were cloned and sequenced. Some of these TDFs could represent novel genes, as their sequences presented no homologies in databases. Interestingly, specific expression of one candidate (HA17) in resistant nematodes from different isolates was confirmed by RT-PCR experiments. The finding that HA17 expression correlates with LEV resistance in three H. contortus isolates vs five susceptible isolates strongly suggest that we identified a new potential marker of LEV resistance. This differential approach at the transcriptome level could be of great interest for the identification of the molecular mechanism involved in this phenotype.

Parasitic infection is often associated with changes in host life-history traits, such as host development. Many of these life-history changes are ultimately thought to be the result of a depletion or reallocation of the host's resources driven either by the host (to minimize the effects of infection) or by the parasite (to maximize its growth rate). In this paper we investigate the energetic budget of Aedes aegypti mosquito larvae infected by Vavraia culicis, a microsporidian parasite that transmits horizontally between larvae, and which has been previously shown to reduce the probability of pupation of its host. Our results show that infected larvae have significantly less lipids, sugars and glycogen than uninfected larvae. These differences in resources were not due to differences in larval energy intake (feeding rate) or expenditure (metabolic rate). We conclude that the lower energetic resources of infected mosquitoes are the result of the high metabolic demands that microsporidian parasites impose on their hosts. Given the fitness advantages for the parasite of maintaining the host in a larval stage, we discuss whether resource depletion may also be a parasite mechanism to prevent the pupation of the larvae and thus maximize its own transmission.

The Bd37gene encoding for a glycosyl-phosphatidyl-inositol anchored protein of Babesia divergens displays genetic polymorphisms among isolates. Five major polymorphic groups (clades) were shown by PCR-RFLP among different B. divergens isolates. Each group has been characterized according to a reference Bd37 gene (Rouen87, W8843, Y5, 6303E and 1705B). Recombinant (GST fusion) protein (Bd37r) expressed from the Bd37 gene, was used as antigen in a saponin-based formulation and was able to protect gerbils, after 2 injections at low dose vaccine concentration (1 μg per dose), against a virulent challenge with the B. divergens Rouen87 isolate. In spite of polymorphism of Bd37 gene, Bd37r induced complete immunoprotection against challenges with each of the 5 reference isolate groups defined by PCR-RFLP.

A nutritional characteristic of trypanosomatid protozoa is that in vitro they need a haem-compound as a growth factor, which is supplied as haemoglobin, haematin or haemin. Because haemin and related porphyrins are an important source of oxidative stress in biological systems, the effect of haemin on growth, protein content and the antioxidant defence system in Trypanosoma cruzi was evaluated. We have observed that, in epimastigotes grown under different haemin concentrations in the culture medium (0–30 mg/l), 5 mg/l was the haemin concentration yielding optimum growth. Above 15 mg/l there was a clear decrease in growth rate, producing the epimastigote to amastigote transformation. Such morphological change was observed together with a marked injury of the enzymatic machinery of the parasite, leading to diminished protein synthesis as well as lower activity of the antioxidant enzymes (superoxide dismutase, ascorbate peroxidase and trypanothione reductase), reduced total thiol content and a marked increase in the HaemOx-1 activity and expression. The current work demonstrates that there is a correlation between higher haemin concentrations in the culture medium and oxidative damage in the cells. Under these conditions induction of HaemOx-1 would indicate the important role of this enzyme as an antioxidant defence response in Trypanosoma cruzi.

The epidemiology of pregnancy malaria infection and disease is complex but reflects underlying interactions between the Plasmodium falciparum parasite, the mother, and the foetus. Parasites sequester in the human placenta by binding to chondroitin sulfate A (CSA), a novel receptor that does not commonly support binding of other parasites. Women become resistant to P. falciparum malaria over successive pregnancies as they acquire antibodies against the CSA-binding placental parasite forms. Due to acquired immunity, placental malaria is briefer and less inflammatory in multigravid women than primigravid women, and these parity differences may account for the different outcomes these women and their offspring experience. Commonly recognized sequelae of malaria-like maternal anaemia and low birth weight primarily occur in first and second pregnancies. Hypertension may result from maternal-foetal conflict over the inflammatory response to placental malaria, and occurs in young, first-time mothers. Placental malaria can either increase or decrease parasitaemia risk in the offspring, depending on the mother's parity. The burden of disease due to pregnancy malaria, and the benefits of an effective vaccine, may be much greater than is currently appreciated.

Toxoplasma gondii is an obligate intracellular parasite that interacts with calcium storage organelles and induces calcium-dependent signalling in macrophages. This study was performed to determine whether Toxoplasma induces changes in intracellular calcium in these cells. Ratiometric imaging of live, Fura-2 loaded macrophages challenged with T. gondii revealed robust elevations in intracellular calcium. These elevations were late in onset, beginning 15–20 min after addition of parasites and occurred in up to 20% of macrophages in an imaging field. Further characterization of these events revealed that they follow from challenge with live T. gondii, but not heat-killed parasites or soluble Toxoplasma antigen (STAg). Parasite-induced calcium elevations derived from extracellular sources, and were independent of host recognition factors MyD88 and CCR5. These findings indicate that Toxoplasma gondii alters calcium homeostasis in macrophages and this activity is independent of known pathways involved in the innate recognition of this organism.

Balamuthia mandrillaris, a soil amoeba, is the causative agent of Balamuthia granulomatous amoebic encephalitis, a life-threatening brain infection. This amoeba is acquired from contaminated soil and may enter the host through cutaneous lesions or through nasal passages, migrating to the lungs or brain. During invasion, B. mandrillaris has access to components of the extracellular matrix (ECM) of the host. Therefore, we investigated the interaction of B. mandrillaris with 3 ECM glycoproteins (collagen-I, fibronectin and laminin-1) that are encountered in host connective tissues and at the basal lamina. Using optical microscopy, amoeba association on ECM-coated surfaces was examined. Binding of amoebae on laminin was greater than that on collagen or fibronectin. Laminin-adhered B. mandrillaris exhibited elongated and spread forms, distinctive from those observed for amoebae on a plastic surface. Collagen and fibronectin-adhered B. mandrillaris presented elongated shapes with cellular expansions. Binding to collagen, fibronectin, or laminin was inhibited when amoebae were pre-treated with sialic acid. Treatment with galactose resulted in diminished binding of amoebae on laminin, while mannose increased binding in all coating conditions tested. Dependence of divalent cations on amoeba binding was demonstrated for laminin-amoeba interaction. Collectively, the results indicate that B. mandrillaris recognizes specific glycoproteins of the mammalian extracellular matrix.

The presence of haemoparasites belonging to the taxa Anaplasma, Bartonella and Trypanosoma was determined among 76 common shrews (Sorex araneus) from Northwest England. Anaplasma phagocytophilum DNA was recovered from the blood of 1 shrew (1·3%), with the amplified 16S rRNA sequence identical to one previously reported from a bank vole (Clethrionomys glareolus). Trypanosoma spp. DNA was detected in 9 shrews (11·8%), the amplified 18S rDNA fragments being indistinguishable from one another, and distinct from previously published data. This represents the first report of trypanosome infection in S. araneus and suggests they are susceptible to an uncharacterized Trypanosoma species. Blood from 11 shrews (14·5%) yielded Bartonella spp., with characterization of isolates using comparative sequence analysis of partial gltA and 16S-23S rRNA intergenic spacer regions revealing 2 different genotypes. Phylogenetic inference from alignment of partial gltA sequences found that both UK S. araneus types formed a well-supported cluster with Bartonella sp. isolated from S. araneus in Sweden. No significant effect of host age, sex, or year of collection was found on prevalence of Bartonella or trypanosome infections. The results of this survey demonstrate that common shrews in the UK are susceptible to haemoparasitic infections, at prevalences similar to those reported from sympatric rodents.

The 70 kDa heat-shock protein (Hsp70) sequences are considered one of the most conserved proteins in all domains of life from Archaea to eukaryotes. Hammondia heydorni, H. hammondi, Toxoplasma gondii, Neospora hughesi and N. caninum (Hammondia-like organisms) are closely related tissue cyst-forming coccidians that belong to the subfamily Toxoplasmatinae. The phylogenetic reconstruction using cytoplasmic Hsp70 coding genes of Hammondia-like organisms revealed the genetic sequences of T. gondii, Neospora spp. and H. heydorni to possess similar levels of evolutionary distance. In addition, at least 2 distinct genetic groups could be recognized among the H. heydorni isolates. Such results are in agreement with those obtained with internal transcribed spacer-1 rDNA (ITS-1) sequences. In order to compare the nucleotide diversity among different taxonomic levels within Apicomplexa, Hsp70 coding sequences of the following apicomplexan organisms were included in this study: Cryptosporidium, Theileria, Babesia, Plasmodium and Cyclospora. Such analysis revealed the Hammondia-like organism to be the lowest divergent group when compared to other groups within the phylum Apicomplexa. In conclusion, the Hsp70 coding sequences proved to be a valuable genetic marker for phylogenetic reconstruction and may constitute a good candidate to be used with other genes for species phylogeny within this group of organisms.

Leishmania mutants have contributed greatly to extend our knowledge of this parasite's biology. Here we report the use of the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding a DNA-directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite. Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its chromosomal location but was targeted into supernumerary episomal molecules.

Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence of B. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. Gorging B. divergens infected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.

Multiple invasions (MIs) or infections, i.e. those by more than 1 parasite in the same erythrocyte, could be the result of parasite density or, alternatively, to parasite-related factors or host-related factors. According to the last possibility, to our knowledge, only 3 laboratory studies of malaria have found an increase in the occurrence of MIs when antibodies to parasite antigens were present. Therefore, we tested the possibility that MIs were influenced by the host immune status, using as model the malaria-like parasite Haemoproteus infecting blue tits (Cyanistes caeruleus). Avian hosts infected with Haemoproteus were medicated with primaquine or injected with saline solution and the density of infection and the presence of MIs counted. Medication treatment reduced significantly the density of infection by Haemoproteus in females but not in males. For females, the presence of MIs was positively associated with both the density of infection and the immunoglobulin levels on each capture, but no association was found between the treatment and the presence of MIs. For males, the density of infection but not the immunoglobulin levels was positively associated with the presence of MIs. In addition, medicated males supported more MIs than controls. Our results represent the first line of evidence in the wild for a possible role of the host immune system promoting MIs.

The sex ratio may be influenced by many factors, such as stress and immunosuppression, age of parents, parity and sex of preceding siblings. In animal systems, parasitism often changes the sex ratio of infected hosts, which can increase the probability of their transmission. The most common human protozoan parasite in developed countries, Toxoplasma gondii (prevalence 20%−80%), is known to change the behaviour of its intermediate hosts, thereby increasing the probability of transmission to its definitive host (the cat) by predation. The intermediate hosts, which under natural conditions are rodents, serve as the vector for Toxoplasma. Therefore, we speculate that Toxoplasma can alter the secondary sex ratio (i.e. male to female ratio in the offspring) of infected females to increase the proportion of (congenitally infected) male offspring, which are the more migratory sex in most rodent species. Here we studied the sex ratio of experimentally infected laboratory mice, expressed here as the proportion of males in the litter. In accordance with our hypothesis and results of previous retrospective cohort studies on human subjects, mice with toxoplasmosis produced a higher sex ratio than controls, in the early phase of latent infection. In the later phase of infection, mice with congenital toxoplasmosis had a lower sex ratio than controls, which is in accord with the Trivers-Willard hypothesis of sex ratio manipulation, suggesting that females in poor physical condition give birth to more female offspring.

Common arthropod vectors for trypanosomes are flies, fleas and bugs. This study reports on an unknown trypanosome species isolated from naturally infected Haemaphysalis hystricis ticks, hereby, referred to as Trypanosoma KG1 isolate. The parasite has been successfully cultured in vitro with L929 or HEK 293T cell line as feeder cells. This trypanosome cannot survive in vitro without feeder cells. Following experimental infections of ticks, the trypomastigote-like and the epimastigote-like forms of this trypanosome could be detected by Giemsa-stained smears of the midgut and salivary glands of Ornithodoros moubata ticks which were made to feed on a culturing medium containing Trypanosoma KG1 isolate through an artificial membrane. Trypanosoma KG1 isolate could also be detected from Giemsa-stained smears of the haemolymph up to 30 days post-inoculation into the O. moubata haemocoel. Trypanosoma KG1 isolate cannot be propagated in laboratory animals including mice, rats, rabbits and sheep. A phylogenetic tree constructed with the 18S rRNA gene indicates that Trypanosoma KG1 is a member of the stercorarian trypanosomes.

Ligand-gated chloride channels, including the glutamate-(GluCl) and GABA-gated channels, are the targets of the macrocyclic lactone (ML) family of anthelmintics. Changes in the sequence and expression of these channels can cause resistance to the ML in laboratory models, such as Caenorhabditis elegans and Drosophila melanogaster. Mutations in multiple GluCl subunit genes are required for high-level ML resistance in C. elegans, and this can be influenced by additional mutations in gap junction and amphid genes. Parasitic nematodes have a different complement of channel subunit genes from C. elegans, but a few genes, including avr-14, are widely present. A polymorphism in an avr-14 orthologue, which makes the subunit less sensitive to ivermectin and glutamate, has been identified in Cooperia oncophora, and polymorphisms in several subunits have been reported from resistant isolates of Haemonchus contortus. This has led to suggestions that ML resistance may be polygenic. Possible reasons for this, and its consequences for the development of molecular tests for resistance, are explored.