The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.

The impact of di-cationic pentamidine-analogues against Toxoplama gondii (Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites of T. gondii Rh and T. gondii Me49 with an IC50 of 0·11 and 0·13 μm, respectively. Pre-incubation of fibroblast monolayers with 1 μm DB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5–6 days of drug exposure, T. gondii tachyzoites adapted to the compound and resumed proliferation up to a concentration of 1·2 μm. Out of a set of 32 di-cationic compounds screened for in vitro activity against T. gondii, the arylimidamide DB745, exhibiting an IC50 of 0·03 μm and favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adapted T. gondii (IC50=0·07 μm). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adapted T. gondii tachyzoites, but not on DB750-adapted T. gondii. Adaptation of T. gondii to DB745 (up to a concentration of 0·46 μm) was much more difficult to achieve and feasible only over a period of 110 days. In cultures infected with DB750-adapted T. gondii seemingly intact parasites could occasionally be detected by TEM. This illustrates the astonishing capacity of T. gondii tachyzoites to adapt to environmental changes, at least under in vitro conditions, and suggests that DB745 could be an interesting drug candidate for further assessments in appropriate in vivo models.

Eimeria tenella is recognized worldwide as a significant pathogen in the poultry industry. However, a lack of methods for isolating developing schizonts has hindered the use of transcriptome analyses to discover novel and developmentally regulated genes. In the present study, we characterized the long-term successive development of E. tenella in infected chicken caeca and assessed the utility of laser microdissection (LMD) for the isolation of schizont RNA. Developmental stages, including those of the first, second, and third-generation schizonts and gametocytes, were synchronous. Using LMD, only the mature second-generation schizonts were successfully excised from the lamina propria, and non-degraded RNA was purified from the schizonts. E. tenella-specific genes were amplified by reverse transcription polymerase chain reaction (RT-PCR). These results augment our understanding of the E. tenella life cycle, and reveal LMD as a potentially useful tool for gene expression analyses of the intracellular stages of E. tenella.

The mainstream forestry policy in many European countries is to convert coniferous plantations into (semi-natural) deciduous woodlands. However, woodlands are the main habitat for Ixodes ricinus ticks. Therefore, assessing to what extent tick abundance and infection with Borrelia spirochetes are affected by forest composition and structure is a prerequisite for effective prevention of Lyme borreliosis. We selected a total of 25 pine and oak stands, both with and without an abundant shrub layer, in northern Belgium and estimated tick abundance between April and October 2008–2010. Additionally, the presence of deer beds was used as an indicator of relative deer habitat use. Borrelia infections in questing nymphs were determined by polymerase chain reactions. The abundance of larvae, nymphs, and adults was higher in oak stands compared to pine stands and increased with increasing shrub cover, most likely due to differences in habitat use by the ticks' main hosts. Whereas tick abundance was markedly higher in structure-rich oak stands compared to homogeneous pine stands, the Borrelia infection rates in nymphs did not differ significantly. Our results indicate that conversion towards structure-rich deciduous forests might create more suitable tick habitats, but we were unable to detect an effect on the infection rate.

Chagas disease chemotherapy is based on drugs that exhibit toxic effects and have limited efficacy, such as Benznidazole. Therefore, research into new chemotherapeutic agents from natural sources needs to be exploited. Apis mellifera venom consists of many biologically active molecules and has been reported to exhibit remarkable anti-cancer effects, often promoting an apoptosis-like death phenotype. This study demonstrates that A. mellifera venom can affect the growth, viability and ultrastructure of all Trypanosoma cruzi developmental forms, including intracellular amastigotes, at concentrations 15- to 100-fold lower than those required to cause toxic effects in mammalian cells. The ultrastructural changes induced by the venom in the different developmental forms led us to hypothesize the occurrence of different programmed cell death pathways. Autophagic cell death, characterized by the presence of autophagosomes-like organelles and a strong monodansyl cadaverine labelling, appears to be the main death mechanism in epimastigotes. In contrast, increased TUNEL staining, abnormal nuclear chromatin condensation and kDNA disorganization was observed in venom-treated trypomastigotes, suggesting cell death by an apoptotic mechanism. On the other hand, intracellular amastigotes presented a heterogeneous cell death phenotype profile, where apoptosis-like death seemed to be predominant. Our findings confirm the great potential of A. mellifera venom as a source for the development of new drugs for the treatment of neglected diseases such as Chagas disease.

Leishmania are obligatory intracellular parasitic protozoa that cycle between sand fly mid-gut and phagolysosomes of mammalian macrophages. They have developed genetically programmed changes in gene and protein expression that enable rapid optimization of cell function according to vector and host environments. During the last two decades, host-free systems that mimic intra-lysosomal environments have been devised in which promastigotes differentiate into amastigotes axenically. These cultures have facilitated detailed investigation of the molecular mechanisms underlying Leishmania development inside its host. Axenic promastigotes and amastigotes have been subjected to transcriptome and proteomic analyses. Development had appeared somewhat variable but was revealed by proteomics to be strictly coordinated and regulated. Here we summarize the current understanding of Leishmania promastigote to amastigote differentiation, highlighting the data generated by proteomics.

The twin concepts of zooprophylaxis and the dilution effect originated with vector-borne diseases (malaria), were driven forward by studies on Lyme borreliosis and have now developed into the mantra “biodiversity protects against disease”. The basic idea is that by diluting the assemblage of transmission-competent hosts with non-competent hosts, the probability of vectors feeding on transmission-competent hosts is reduced and so the abundance of infected vectors is lowered. The same principle has recently been applied to other infectious disease systems – tick-borne, insect-borne, indirectly transmitted via intermediate hosts, directly transmitted. It is claimed that the presence of extra species of various sorts, acting through a variety of distinct mechanisms, causes the prevalence of infectious agents to decrease. Examination of the theoretical and empirical evidence for this hypothesis reveals that it applies only in certain circumstances even amongst tick-borne diseases, and even less often if considering the correct metric – abundance rather than prevalence of infected vectors. Whether dilution or amplification occurs depends more on specific community composition than on biodiversity per se. We warn against raising a straw man, an untenable argument easily dismantled and dismissed. The intrinsic value of protecting biodiversity and ecosystem function outweighs this questionable utilitarian justification.

Existing algorithms for predicting species' distributions sit on a continuum between purely statistical and purely biological approaches. Most of the existing algorithms are aspatial because they do not consider the spatial context, the occurrence of the species or conditions conducive to the species' existence, in neighbouring areas. The geostatistical techniques of kriging and cokriging are presented in an attempt to encourage biologists more frequently to consider them. Unlike deterministic spatial techniques they provide estimates of prediction errors. The assumptions and applications of common geostatistical techniques are presented with worked examples drawn from a dataset of the bluetongue outbreak in northwest Europe in 2006. Emphasis is placed on the importance and interpretation of weights in geostatistical calculations. Covarying environmental data may be used to improve predictions of species’ distributions, but only if their sampling frequency is greater than that of the species’ or disease data. Cokriging techniques are unable to determine the biological significance or importance of such environmental data, because they are not designed to do so.

Despite the utility of RNAi for defining gene function in Caenorhabditis elegans and early successes reported in parasitic nematodes, RNAi has proven to be stubbornly inconsistent or ineffective in the animal parasitic nematodes examined to date. Here, we summarise some of our experiences with RNAi in parasitic nematodes affecting animals and discuss the available data in the context of our own unpublished work, taking account of mode of delivery, larval activation, site of gene transcription and the presence/absence of essential RNAi pathway genes as defined by comparisons to C. elegans. We discuss future directions briefly including the evaluation of nanoparticles as a means to enhance delivery of interfering RNA to the target worm tissue.

Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1–T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (∼1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2 0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.

Arid zones of northern Venezuela are represented by isolated areas, important from an ornithological and ecological perspective due to the occurrence of restricted-range species of birds. We analysed the prevalence and molecular diversity of haemosporidian parasites of wild birds in this region by screening 527 individuals (11 families and 20 species) for parasite mitochondrial DNA. The overall prevalence of parasites was 41%, representing 17 mitochondrial lineages: 7 of Plasmodium and 10 of Haemoproteus. Two parasite lineages occurred in both the eastern and western regions infecting a single host species, Mimus gilvus. These lineages are also present throughout northern and central Venezuela in a variety of arid and mesic habitats. Some lineages found in this study in northern Venezuela have also been observed in different localities in the Americas, including the West Indies. In spite of the widespread distributions of some of the parasite lineages found in northern Venezuela, several, including some that are relatively common (e.g. Ven05 and Ven06), have not been reported from elsewhere. Additional studies are needed to characterize the host and geographical distribution of avian malaria parasite lineages, which will provide a better understanding of the influence of landscape, vector abundance and diversity, and host identity on haemosporidian parasite diversity and prevalence.

The genetic diversity of haematozoan parasites in island avifauna has only recently begun to be explored, despite the potential insight that these data can provide into the history of association between hosts and parasites and the possible threat posed to island endemics. We used mitochondrial DNA sequencing to characterize the diversity of 2 genera of vector-mediated parasites (Plasmodium and Haemoproteus) in avian blood samples from the western Indian Ocean region and explored their relationship with parasites from continental Africa. We detected infections in 68 out of 150 (45 3%) individuals and cytochrome b sequences identified 9 genetically distinct lineages of Plasmodium spp. and 7 lineages of Haemoproteus spp. We found considerable heterogeneity in parasite lineage composition across islands, although limited sampling may, in part, be responsible for perceived differences. Two lineages of Plasmodium spp. and 2 lineages of Haemoproteus spp. were shared by hosts in the Indian Ocean and also on mainland Africa, suggesting that these lineages may have arrived relatively recently. Polyphyly of island parasites indicated that these parasites were unlikely to constitute an endemic radiation and instead probably represent multiple colonization events. This study represents the first molecular survey of vector-mediated parasites in the western Indian Ocean, and has uncovered a diversity of parasites. Full understanding of parasite community composition and possible threats to endemic avian hosts will require comprehensive surveys across the avifauna of this region.

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the ‘essential’ genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

The tick-borne bacterium Rickettsia rickettsii is the aetiological agent of Brazilian spotted fever (BSF). The present study evaluated tick infestations on wild and domestic animals, and the rickettsial infection in these animals and their ticks in 7 forest areas adjacent to human communities in the São Paulo Metropolitan Area (SPMA). The results were compared to ecological traits of each sampled area. Two main tick species, Amblyomma aureolatum and Rhipicephalus sanguineus, were collected from dogs. The major ticks found on small mammals and birds were Ixodes loricatus and Amblyomma longirostre, respectively. Both anti-R. rickettsii antibodies and R. rickettsii-infected ticks were detected on dogs from only 2 areas in the southern part of the SPMA, which were considered to be endemic for BSF; the remaining 5 areas were considered to be non-endemic. Ecologically, the BSF-endemic areas clearly differed from the non-endemic areas by the presence of significantly more degraded forest patches in the former. The present results corroborate historical observations that have indicated that all human cases of BSF in the SPMA were contracted in the southern part of this metropolitan area. However, not all forest patches in the southern part of the SPMA were shown to be associated with BSF endemism.

This study aimed to investigate myocardial injuries in mice infected with Schistosoma mansoni and fed a high-fat chow. Sections of myocardial tissue from S. mansoni-infected mice, and controls that had been killed 9 and 17 weeks post-infection, were stained with H&E and Picrosirius red. Histopathological examination, stereological design-based method (optical disector) and morphometry (vessels, cardiomyocytes and an amount of collagen) were used. Data were analysed using two-way ANOVA. Regardless of time of infection, myocardial tissue from the infected mice fed high-fat chow showed myocarditis characterized by a higher number of inflammatory foci, several areas displaying coagulation of cardiac fibres, a greater loss of cardiomyocytes and fibroblast proliferation than in the standard chow control. Comparing infected mice from acute and chronic infections, a higher cardiomyocyte hyperplasia (P < 0·0001) and higher amounts of collagen (P < 0·05) were observed than in standard chow control. In addition, all animals fed high-fat chow showed lower numerical density and total number of cardiomyocytes (P < 0·05), thicker vessel walls and narrowed luminal intramyocardial vessels (P > 0·05) than in the standard chow control. Altogether the data supported the view that a double burden has a synergistic deleterious effect on the myocardial tissue.

Bat flies are obligate ectoparasites of bats and it has been hypothesized that they may be involved in the transmission of Bartonella species between bats. A survey was conducted to identify whether Cyclopodia greefi greefi (Diptera: Nycteribiidae) collected from Ghana and 2 islands in the Gulf of Guinea harbour Bartonella. In total, 137 adult flies removed from Eidolon helvum, the straw-coloured fruit bat, were screened for the presence of Bartonella by culture and PCR analysis. Bartonella DNA was detected in 91 (66·4%) of the specimens examined and 1 strain of a Bartonella sp., initially identified in E. helvum blood from Kenya, was obtained from a bat fly collected in Ghana. This is the first study, to our knowledge, to report the identification and isolation of Bartonella in bat flies from western Africa.

Dientamoeba fragilis is an intestinal protozoan in humans that is commonly associated with diarrhoea and other gastrointestinal complaints. Studies conducted to investigate the biology of this parasite are limited by methods for in vitro cultivation. The objective of this study was to improve a biphasic culture medium, based on the Loeffler's slope, by further supplementation in order to increase the yield of trophozoites in culture. The current in vitro culture of D. fragilis is a xenic culture with a mix of bacteria. Three different liquid overlays were evaluated including Earle's balanced salt solution (EBSS), PBS and Dulbecco's modified PBS (DPBS), for their ability to support the in vitro growth of D. fragilis trophozoites. Out of these 3 overlays EBSS gave the highest increase in the trophozoite numbers. The effect of supplementation was analysed by supplementing EBSS with ascorbic acid, ferric ammonium citrate, L-cysteine, cholesterol and alpha-lipoic acid and quantification of in vitro growth by cell counts. A new liquid overlay is here described based upon EBSS supplemented with cholesterol and ferric ammonium citrate that, in conjunction with the Loeffler's slope, supports the growth of D. fragilis trophozoites in vitro. This modified overlay supported a 2-fold increase in the numbers of trophozoite in culture from all 4 D. fragilis isolates tested, when compared to a PBS overlay. These advances enable the harvest of a larger number of trophozoites needed for further studies on this parasite.

The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.

The distributions of parasitic diseases are determined by complex factors, including many that are distributed in space. A variety of statistical methods are now readily accessible to researchers providing opportunities for describing and ultimately understanding and predicting spatial distributions. This review provides an overview of the spatial statistical methods available to parasitologists, ecologists and epidemiologists and discusses how such methods have yielded new insights into the ecology and epidemiology of infection and disease. The review is structured according to the three major branches of spatial statistics: continuous spatial variation; discrete spatial variation; and spatial point processes.

Animals frequently host organisms on their surface which can be beneficial, have no effect or a negative effect on their host. Ectoparasites, by definition, are those which incur costs to their host, but these costs may vary. Examples of avian ectoparasites are chewing lice which feed exclusively on dead feather or skin material; therefore, costs to their bird hosts are generally considered small. Theoretically, many possible proximate effects exist, like loss of tissue or food, infected bites, transmission of microparasitic diseases or reduced body insulation due to loss of feathers, which may ultimately also have fitness consequences. Here, we experimentally examined a possible negative impact of 2 feather-eating louse species (Meropoecus meropis and Brueelia apiastri) on male and female European bee-eaters (Merops apiaster) by removing or increasing louse loads and comparing their impact to a control group (lice removed and immediately returned) after 1 month. A negative effect of chewing lice was found on body mass and sedimentation rate and to a lesser extent on haematocrit levels. Males and females lost more weight when bearing heavy louse loads, and were more susceptible to infestations as indicated by the higher sedimentation rate. Our results further suggest differences in sex-specific susceptibility.