A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome.

For trophically transmitted parasites, transitional larval size is often related to fitness. Larger parasites may have higher establishment success and/or adult fecundity, but prolonged growth in the intermediate host increases the risk of failed transmission via natural host mortality. We investigated the relationship between the larval size of an acanthocephalan (Acanthocephalus lucii) and a trait presumably related to transmission, i.e. altered colouration in the isopod intermediate host. In natural collections, big isopods harboured larger worms and had more modified (darker) abdominal colouration than small hosts. Small isopods infected with a male parasite tended to have darker abdominal pigmentation than those infected with a female, but this difference was absent in larger hosts. Female size increases rapidly with host size, so females may have more to gain than males by remaining in and growing mutually with small hosts. In experimental infections, a large total parasite volume was associated with darker host respiratory operculae, especially when it was distributed among fewer worms. Our results suggest that host pigment alteration increases with parasite size, albeit differently for male and female worms. This may be an adaptive strategy if, as parasites grow, the potential for additional growth decreases and the likelihood of host mortality increases.

Anthropogenic influence on ecosystems, such as habitat fragmentation, impacts species diversity and interactions. There is growing evidence that degradation of habitats favours disease and hence affects ecosystem health. The prevalence of haemoparasites in the Common Fruit Bat (Artibeus jamaicensis) in a tropical lowland forest in Panamá was studied. We assessed the relation of haemoparasite to the general condition of the animals and tested for possible association of haemoparasite prevalence to habitat fragmentation, with special focus on trypanosomes. Overall, a total of 250 A. jamaicensis sampled from fragmented sites, here man-made, forested islands in Lake Gatùn, and sites in the adjacent, continuous forest in and around the Barro Colorado Nature Monument were examined. Using microscopy and DNA-sequencing 2 dominant types of haemoparasite infections, trypanosomes and Litomosoides (Nematoda) were identified. Trypanosome prevalence was significantly higher in bats from forest fragments, than in bats captured in continuous forest. We attribute this to the loss of species richness in forest fragments and specific characteristics of the fragments favouring trypanosome transmission, in particular changes in vegetation cover. Interestingly, the effect of habitat fragmentation on the prevalence of trypanosomes as multi-host parasites could not be observed in Litomosoides which probably has a higher host specificity and might be affected less by overall diversity loss.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered in the presence of a metabolic inhibitor. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-sensitive isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 μm). Flukes were then incubated for a further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nm); MTZ+NADPH+TCBZ (15 μg/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 μg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater surface disruption to the triclabendazole-susceptible than -resistant isolate. However, co-incubation with MTZ and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own; this was not seen for the TCBZ-susceptible Cullompton isolate. Results of this study support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Studies have been carried out to establish the relative importance of oral and trans-tegumental uptake of triclabendazole by the liver fluke, Fasciola hepatica. Experiments were designed to block either oral uptake of drug, by use of ligatures, or trans-tegumental diffusion, by allowing the drug to bind to bovine serum albumin (BSA) in the medium. Changes to the surface morphology of the tegument and gut were assessed by scanning electron microscopy. Flukes were incubated in vitro for 24 h in TCBZ.SO at a concentration of 15 μg/ml. Tegumental disruption in ligatured and non-ligatured flukes was similar, suggesting that closing the oral route did not affect drug uptake. The gut remained unaffected by drug treatment. When BSA (30 mg/ml) was present in the medium, there was a marked decline in the level of tegumental disruption. Again, the gut retained a normal morphology. Non-ligatured flukes were also incubated for 24 h in vitro in TCBZ.SO (15 μg/ml) in the presence of red blood cells. Oral ingestion of blood was demonstrated, although the gut surface retained a normal morphology. In contrast, the tegumental surface was severely affected by the drug. The findings support previous pharmacological studies which suggest that trans-tegumental uptake of triclabendazole predominates in the liver fluke.

Cysteine peptidases of protozoa have been implicated in a variety of biological events, and the expression of these enzymes is modulated in response to distinct stimuli, including environmental changes and differentiation. In the present work, we have examined the expression of cysteine peptidases from Herpetomonas samuelpessoai grown at distinct temperatures and during dimethylsulfoxide (DMSO)-elicited differentiation. We demonstrated that a 45 kDa cysteine peptidase had its activity reduced during the parasite growth at 37°C in comparison to 26°C, and when cultured up to 72 h in the presence of DMSO. The modulation in the 45 kDa cysteine peptidase expression is connected to the differentiation process, since both temperature and DMSO are able to trigger the promastigote to paramastigote transformation in H. samuelpessoai. The possible immunological similarity of H. samuelpessoai proteins with well-known cysteine peptidases produced by trypanosomatid pathogens, including cruzipain (Trypanosoma cruzi) and cysteine peptidase b (cpb) from Leishmania mexicana, was also investigated, as well as with calpain molecules. The protein cellular lysate of H. samuelpessoai reacted with antibodies raised against cpb of L. mexicana and calpain of Drosophila melanogaster; however, no reaction was observed against cruzipain. The 35 kDa cpb-like protein had its expression diminished in DMSO-treated parasites, while the 80 kDa calpain-like molecule was enhanced and an additional 30 kDa calpain-related polypeptide was exclusively observed in these cells. Fluorescence microscopy and flow cytometry analyses corroborated these data. The results described above add H. samuelpessoai to the list of parasites whose differentiation seems to be correlated with cysteine peptidase expression.

Little is known regarding the diversity, distribution or host-parasite associations of Trypanosoma spp. in Australian wildlife. Here we report on an investigation based on divergence of the 18S rRNA gene of trypanosomes isolated from a range of hosts and varied geographical locations. A total of 371 individuals representing 19 species of native animals from 14 different locations were screened. In total, 32 individuals from 9 different species tested positive for the parasite. Phylogenetic analysis revealed considerable parasite diversity with no clear geographical distribution and no evidence of host specificity. In general, it appears that Australian Trypanosoma spp. are widespread, with several genotypes appearing in multiple host species and in varied locations including both mainland areas and offshore islands. Some host species were found to be susceptible to multiple genotypes, but no individuals were infected with more than a single isolate.

The phylum Microspora is ancient and diverse and affects a wide range of hosts. There is unusually high use of vertical transmission and this has significant consequences for transmission and pathogenicity. Vertical transmission is associated with low pathogenesis but nevertheless can have significant impact through associated traits such as sex ratio distortion. The majority of microsporidia have mixed transmission cycles and it is not clear whether they are able to modify their phenotype according to environmental circumstances. There is a great need to understand the mechanisms controlling transmission and one of the first challenges for the genomics era is to find genes associated with life cycle stages. Similarly we cannot currently predict the ease with which these parasites might switch between host groups. Phylogenetic analysis suggests that there are strong relationships between Microsporidia and their hosts. However closer typing of parasite isolates, in relation to host range and disease phenotype, is required to assess future environmental risk from these pathogens.

Free-living animals are usually inhabited by a community of parasitic species that can interact with each other and alter both host susceptibility and parasite transmission. In this study we tested the prediction that an increase in the gastrointestinal nematode Heligmosomoides polygyrus would increase the infestation of the tick Ixodes ricinus, in free-living yellow-necked mice, Apodemus flavicollis. An extensive cross-sectional trapping survey identified a negative relationship between H. polygyrus and I. ricinus counter to the prediction. An experimental reduction of the nematode infection through anthelmintic treatment resulted in an increase in tick infestation, suggesting that this negative association was one of cause and effect. Host characteristics (breeding condition and age) and habitat variables also contributed to affect tick infestation. While these results were counter to the prediction, they still support the hypothesis that interactions between parasite species can shape parasite community dynamics in natural systems. Laboratory models may act differently from natural populations and the mechanism generating the negative association is discussed.

Our present study is the first attempt to characterize Leishmania parasites from foci in Uzbekistan and Tajikistan endemic for visceral leishmaniasis (VL). PCR-sequencing of the ribosomal internal transcribed spacer 1 and multilocus microsatellite typing (MLMT) were applied to DNA extracted from preparations of Giemsa-stained bone marrow aspirates from 13 cases of VL. L. infantum was shown to cause VL currently occurring in this area. MLMT applying 14 microsatellite markers, previously shown to be polymorphic for strains of the L. donovani complex, revealed that microsatellite profiles of parasites causing human VL in the Namangan and Jizzakh regions in Uzbekistan, and Penjikent region in Tajikistan, basically coincide with those of strains of L. infantum MON-1. Furthermore, these parasites were assigned to a distinct cluster genetically clearly separated from the populations of L. infantum MON-1 from Europe, the Middle East and North Africa. The existence of a genetically homogeneous but distinct group of L. infantum MON-1 indicates that the parasites circulating in the Uzbeki and Tajiki foci studied have been restricted there for a long time rather than having been recently introduced from elsewhere by human or animal reservoir migration.

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.

Schistosoma kisumuensis n. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species, Pelomys isseli, Mastomys natalensis, and Dasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of the S. haematobium group, currently represented by 8 species parasitizing artiodactyls and primates, including humans. Schistosoma kisumuensis differs from these species by producing relatively small Schistosoma intercalatum-like eggs (135·2×52·9 μm) with a relatively small length to width ratio (2·55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partial cox1, nad6, 12S) strongly supports the status of S. kisumuensis as a new species and as a sister species of S. intercalatum. The cox1 genetic distance between these two species (6·3%) is comparable to other pairwise comparisons within the S. haematobium group. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33·7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.

The ability of pathogens to neutralize host defence mechanisms represents a fundamental requisite in the successful establishment of an infection. Host-pathogen interactions between quahog parasite unknown (QPX) and its hard clam host are poorly understood. Our prior in vivo investigations have shown that different QPX isolates display varying levels of pathogenicity toward clams. Similarly, field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. An in vitro approach was developed in this study to evaluate the toxicity of QPX cells and extracellular products toward haemocytes using a neutral red uptake assay. Results demonstrated that QPX produces virulence factors that are cytotoxic to M. mercenaria haemocytes. This cytotoxicity appears to be induced by clam factors, suggesting that it may play an important role in supporting QPX infection and proliferation within the host. Moreover, application of this technique to different QPX isolates and clam broodstocks indicates variations of QPX cytotoxicity in agreement with previous in vivo experiments, strengthening the existence of different QPX strains.

The polymerase chain reaction was used to detect trypanosomes in samples collected from cattle, wild animals and tsetse flies in KwaZulu-Natal Province, South Africa. A total of 673 samples from cattle and 266 from tsetse flies in the study area located near the Hluhluwe-Umfolozi Game Reserve were analysed. Both Trypanosoma congolense and T. vivax were found as single or mixed infections in cattle and tsetse flies. Moreover, the T. congolense in the infections were found to comprise 2 genotypic groups: the Savannah-type and the Kilifi-type, which were present either as single or mixed infections in cattle and in tsetse flies.

We studied the prevalence and intensity of the parasitic copepod Myicola ostreae in 2 closely related oysters Crassostrea angulata and C. gigas and their F1 hybrids. The effects on host and host reaction were also analysed to better understand host-parasite relationships between copepods and bivalve molluscs. Full reciprocal crosses were carried out between C. angulata and C. gigas and the progenies were reared in the wild in Ria Formosa Lagoon (Portugal), allowing natural infestation by M. ostreae. Prevalence and intensity were significantly higher in C. angulata than in C. gigas. The parasite level of F1 hybrids was similar to C. angulata and significantly higher than in C. gigas. The results of our study support a hypothesis of dominantly inherited susceptibility to M. ostreae infestation. Moreover, copepods were observed on the gill surface of C. gigas engulfed by a capsule-like structure. Histological analyses revealed that the copepods were surrounded by a massive agglomerate of haemocyte-like cells encircled by a thin layer of fibroblast-like cells. This encapsulation response was not observed in C. angulata or in F1 hybrids. These results suggest that the differential susceptibility to M. ostreae between C. angulata and C. gigas may be ascribed to host defence factors.

Neospora caninum infection persists throughout the life of its intermediate host due to the conversion of tachyzoites to slowly dividing bradyzoites that encyst in the brain. This event results in persistent N. caninum infection in bovine herds and partially explains the poor efficacy of many chemotherapeutic agents and vaccine formulations. Thus, there is a need for greater understanding of the tachyzoite-to-bradyzoite conversion mechanisms. Here we studied for the first time the transcription kinetics of the N. caninum bradyzoite-specific gene NcSAG4 in brain samples from chronically infected mice by means of real-time RT-PCR. NcSAG4-messenger RNA (mRNA) levels increased significantly during the chronic phase but followed 2 different expression patterns depending on the isolate used for murine inoculation. NcSAG4-mRNA levels in brains from Nc-1-inoculated mice peaked during late chronic infection (on day 64 post-infection, p.i.), whereas those from Nc-Liv-inoculated mice peaked earlier during the chronic infection (on day 32 p.i.). This difference could be a reflection of the different abilities of these isolates to replicate and form cysts in parasitized brains. These results are consistent with our observations of anti-rNcSAG4 antibody production; low levels were present at seroconversion and slowly increased during the chronic phase. In contrast, NcSAG1 transcription levels, which mark the tachyzoite stage, were maintained without variation in both groups of mice. This suggests the presence of a significant amount of tachyzoites or intermediate zoites expressing NcSAG1 in the brain, even during the late chronic infection.

Schistosomiasis remains one of the most prevalent parasitic diseases in developing countries. After malaria, schistosomiasis is the most important tropical disease in terms of human morbidity with significant economic and public health consequences. Although schistosomiasis has recently attracted increased focus and funding for control, it has been estimated that less than 20% of the funding needed to control the disease in Africa is currently available. In this article the following issues are discussed: the rationale, development and objectives of the Schistosomiasis Control Initiative (SCI)-supported programmes; the management approaches followed to achieve implementation by each country; mapping, monitoring and evaluation activities with quantifiable impact of control programmes; monitoring for any potential drug resistance; and finally exit strategies within each country. The results have demonstrated that morbidity due to schistosomiasis has been reduced by the control programmes. While challenges remain, the case for the control of schistosomiasis has been strengthened by research by SCI teams and the principle that a national programme using ‘preventive chemotherapy’ can be successfully implemented in sub-Saharan Africa, whenever the resources are available. SCI and partners are now actively striving to raise further funds to expand the coverage of integrated control of neglected tropical diseases (NTDs) in sub-Saharan Africa.

The taxonomic status of Echinococcus, an important zoonotic cestode genus, has remained controversial, despite numerous attempts to revise it. Although mitochondrial DNA (mtDNA) has been the source of markers of choice for reconstructing the phylogeny of the genus, results derived from mtDNA have led to significant inconsistencies with earlier species classifications based on phenotypic analysis. Here, we used nuclear DNA markers to test the phylogenic relationships of members of the genus Echinococcus. The analysis of sequence data for 5 nuclear genes revealed a significantly different phylogeny for Echinococcus from that proposed on the basis of mitochondrial DNA sequence data, but was in agreement with earlier species classifications. The most notable results from the nuclear phylogeny were (1) E. multilocularis was placed as basal taxon, (2) all genotypes of Echinococcus granulosus grouped as a monophyletic entity, and (3) genotypes G8 and G10 clustered together. We conclude that the analysis of nuclear DNA data provides a more reliable means of inferring phylogenetic relationships within Echinococcus than mtDNA and suggest that mtDNA should not be used as the sole source of markers in future studies where the goal is to reconstruct a phylogeny that does not only reflect a maternal lineage, but aims to describe the evolutionary history at species level or higher.

Zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum is an important disease of humans and dogs. Here we review aspects of the transmission and control of ZVL. Whilst there is clear evidence that ZVL is maintained by sandfly transmission, transmission may also occur by non-sandfly routes, such as congenital and sexual transmission. Dogs are the only confirmed primary reservoir of infection. Meta-analysis of dog studies confirms that infectiousness is higher in symptomatic infection; infectiousness is also higher in European than South American studies. A high prevalence of infection has been reported from an increasing number of domestic and wild mammals; updated host ranges are provided. The crab-eating fox Cerdocyon thous, opossums Didelphis spp., domestic cat Felis cattus, black rat Rattus rattus and humans can infect sandflies, but confirmation of these hosts as primary or secondary reservoirs requires further xenodiagnosis studies at the population level. Thus the putative sylvatic reservoir(s) of ZVL remains unknown. Review of intervention studies examining the effectiveness of current control methods highlights the lack of randomized controlled trials of both dog culling and residual insecticide spraying. Topical insecticides (deltamethrin-impregnated collars and pour-ons) have been shown to provide a high level of individual protection to treated dogs, but further community-level studies are needed.