The relevance of spatial variation in the environment and host communities for parasite community composition is poorly documented, creating a need for additional case studies from which general principles can be developed. Avian malaria in southern African waterfowl has not previously been studied. As a first step towards documenting and understanding its biogeography, we used PCR and molecular sequencing techniques to analyse 454 blood samples from Afrotropical ducks from 5 different locations (spread around the subregion) for avian malaria. Fifty-five blood samples were positive for one or more genera of haematozoa. The regional infection rate across all sites and sampling periods was 12·1%. Nine individuals carried dual infections containing multiple haematozoa. Fifteen different cytochrome b haplotypes among 52 positives (3 samples failed to sequence) and 61 total sequences were found. Eleven haplotypes closely matched Plasmodium, whereas 4 were more similar to Haemoproteus. Five distinct haematozoan clades were identified. Haemoproteus parasites appeared to be more host-specific than Plasmodium, which occurred at every sampling location and in every host species examined. There were no significant differences in overall parasite prevalence attributable to either site or species, although Plasmodium and Haemoproteus occurrences differed by site-species combination and the borderline significance of our test for between-site variation (P < 0·06) implied that with a larger sample size, differences in parasite prevalence among locations might be detectable.

We tested for the effect of age of a rodent host (Meriones crassus) on reproductive performance of fleas in terms of number and quality of offspring and predicted that fleas would perform better on juvenile and old than on subadult and adult hosts. The number of flea offspring was evaluated via egg and new imago production, while their quality was estimated via duration of development, resistance to starvation and body size. Although fleas produced more eggs when they exploited adults than when they exploited juvenile, subadult and old hosts, significantly more new imago emerged from fleas fed on juvenile and old hosts than on subadult and adult hosts. Fleas performed better when they fed on juvenile and/or old hosts than on subadult and adult hosts in 2 of 3 measures of offspring quality (duration of development and body size). Nevertheless, when offspring quality was estimated via resistance to starvation of a new imago, fleas demonstrated good performance in young (juvenile and subadult) hosts, while they performed poorly in old hosts. Thus, general reproductive performance of fleas was better when they exploited young and old hosts than when they exploited median age cohorts. However, the effect of host age on flea reproductive performance was manifested somewhat differently between (a) male and female hosts and (b) male and female flea offspring.

Temperature is an important factor influencing the biology of ectothermic organisms and is intrinsically linked to climate change. Trematodes are potentially susceptible to temperature changes and in order to develop predictive frameworks of their responses to climate change large-scale analyses are needed. The present study, using the Q10 value, analyses experimental data from the scientific literature on the effects of temperature on cercarial development and emergence across a wide range of temperature in low (⩽35°) and mid-latitude (36–60°) species. Temperature appears to have no significant effect on the rate of development of cercariae within molluscan hosts. Data on cercarial emergence, corrected to incorporate the minimum emergence temperature threshold (METT) and acclimation status, was found to be largely unaffected by temperature over optimum ranges of ≈20 °C (15–25 °C) for mid-latitude species and ≈25 °C (20–30 °C) for low-latitude species. In addition, a decline in emergence rates was shown at higher temperatures. These results are contrary to a previous study on the meta-analysis of cercarial emergence. Some evidence of strain-specific differences and thermostability over a wide temperature range for both cercarial development and emergence was apparent. The significance of these results in furthering our understanding of cercarial biology under natural conditions is discussed.

Leishmaniasis, like other neglected diseases is characterized by a small arsenal of drugs for its control. To safeguard the efficacy of current drugs and guide the development of new ones it is thus of utmost importance to acquire a deep understanding of the phenomenon of drug resistance and its link with treatment outcome. We discuss here how (post-)genomic approaches may contribute to this purpose. We highlight the need for a clear definition of the phenotypes under consideration: innate and acquired resistance versus treatment failure. We provide a recent update of our knowledge on the Leishmania genome structure and dynamics, and compare the contribution of targeted and untargeted methods for the understanding of drug resistance and show their limits. We also present the main assays allowing the experimental validation of the genes putatively involved in drug resistance. The importance of analysing information downstream of the genome is stressed and further illustrated by recent metabolomics findings. Finally, the attention is called onto the challenges for implementing the acquired knowledge to the benefit of the patients and the population at risk.

Recently, a novel human rickettsiosis, namely Atlantic rainforest spotted fever, was described in Brazil. We herein report results of a survey led around the index case in an Atlantic rainforest reserve in Peruibe municipality, southeastern Brazil. A Rickettsia parkeri-like agent (Rickettsia sp. Atlantic rainforest genotype) and Ricketsia bellii were isolated from adult Amblyomma ovale ticks collected from dogs. Molecular evidence of infection with strain Atlantic rainforest was obtained for 30 (12·9%) of 232 A. ovale adult ticks collected from dogs. As many as 88·6% of the 35 examined dogs had anti-Rickettsia antibodies, with endpoint titres at their highest to R. parkeri. High correlation among antibody titres in dogs, A. ovale infestations, and access to rainforest was observed. Amblyomma ovale subadults were found predominantly on a rodent species (Euryoryzomys russatus). From 17 E. russatus tested, 6 (35·3%) displayed anti-Rickettsia antibodies, with endpoint titres highest to R. parkeri. It is concluded that Atlantic rainforest genotype circulates in this Atlantic rainforest area at relatively high levels. Dogs get infected when bitten by A. ovale ticks in the forest, and carry infected ticks to households. The role of E. russatus as an amplifier host of Rickettsia to A. ovale ticks deserves investigation.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was evaluated for the rapid identification of ceratopogonid larvae. Optimal sample preparation as evaluated with laboratory-reared biting midges Culicoides nubeculosus was the homogenization of gut-less larvae in 10% formic acid, and analysis of 0·2 mg/ml crude protein homogenate mixed with SA matrix at a ratio of 1:1·5. Using 5 larvae each of 4 ceratopogonid species (C. nubeculosus, C. obsoletus, C. decor, and Dasyhelea sp.) and of 2 culicid species (Aedes aegypti, Ae. japonicus), biomarker mass sets between 27 and 33 masses were determined. In a validation study, 67 larvae belonging to the target species were correctly identified by automated database-based identification (91%) or manual full comparison (9%). Four specimens of non-target species did not yield identification. As anticipated for holometabolous insects, the biomarker mass sets of adults cannot be used for the identification of larvae, and vice versa, because they share only very few similar masses as shown for C. nubeculosus, C. obsoletus, and Ae. japonicus. Thus, protein profiling by MALDI-TOF as a quick, inexpensive and accurate alternative tool is applicable to identify insect larvae of vector species collected in the field.

Cysticercosis caused by infection with the larval stage of Taenia solium is an important cause of neurological disease worldwide and immunodiagnosis is important for the control and elimination of cysticercosis. In the present study, we established a simple and reliable preparation of immunodiagnostic low-molecular-weight antigens (LMWAgs) from T. solium cyst fluids by a cation-exchange chromatography (CEC). Banding patterns of LMWAgs on SDS-PAGE were different between isolates from Ecuador and China. All cysticercosis patient sera and some echinococcosis patient sera recognized both LMWAgs by enzyme-linked immunosorbent assay (ELISA), but sera from healthy persons were not positive. There was no statistical difference in immunodiagnostic performance of LMWAgs prepared from different geographical isolates. These results indicated that these novel immunodiagnostic antigen preparations could contribute the control and prevention of cysticercosis in endemic areas, especially developing countries.

Circulating microRNAs (miRNAs) have received considerable attention as a novel class of biomarkers for the diagnosis of cancer and as signalling molecules in mediating intercellular communication. Schistosomes, the causative agents of schistosomiasis, live in the blood vessels of a mammalian host in the adult stage. In the present study, we characterized schistosome-specific small RNA populations in the plasma of rabbits infected with Schistosoma japonicum (S. japonicum) using a deep sequencing method and then identified five schistosome-specific miRNAs, including four known miRNAs (Bantam, miR-3479, miR-10 and miR-3096), and one novel miRNA (miR-0001, miRBase ID: sja-miR-8185). Four of the five schistosome-specific miRNAs were also detected by real-time RT–PCR in the plasma of S. japonicum-infected mice. In addition, our study indicated that schistosome Argonaute 2/3 may be an excretory-secretory (ES) protein. In summary, our findings are expected to provide useful information for further development of novel biomarkers for the diagnosis of schistosomiasis and also for deeper understanding of the mechanism of host–parasite interaction.

Hookworms represent a major infectious burden globally, especially in developing countries. The murine hookworm Nippostrongylus brasiliensis is normally cleared in a manner dependent on IL-13, IL4-R and STAT6 signalling. Here we have used STAT6-deficient animals to model a non-resistant population and describe 2 novel STAT6-independent processes for the clearance of N. brasiliensis. During primary infection STAT6−/− animals are able to clear gut-dwelling N. brasiliensis by a mechanism involving the trapping and degradation of worms in the gut mucosa. Here, a previously undescribed STAT6-independent up-regulation of Relm-β was observed which correlated with the mucosal trapping and degradation of worms. Previous studies have indicated that during secondary infection STAT6 deficient animals fail to expel adult worms and remain susceptible to re-infection and long-term colonization of the gut. We report here that an initial partially protective response occurs early upon re-infection in the absence of STAT6, and that a late-phase protective secondary response arises in the gut of STAT6-deficient mice leading to the clearance of the majority of N. brasiliensis, through their trapping and death in the mucosal layer of the lower region of the small intestine. These findings show that there are a number of redundant effector pathways which act to reduce worm burden in the gut which can be activated by mechanisms that do not work through the dominant STAT6 signalling pathway and may be useful as targets for future vaccination strategies against resistant hookworm strains.

Understanding the population dynamics and co-evolution of host–parasite systems requires detailed knowledge of their phenology which, in turn, requires a deep knowledge of the effect of abiotic factors on the life cycles of organisms. Temperature is known to be a key environmental influence that participates in the regulation of diapause. Yet, not much is known about the effect of temperature on the free-living stages of true parasites and how it may influence host–parasite interactions. Here we experimentally study the effect of ambient temperature on overwintering pupae of Carnus hemapterus (Diptera, Carnidae), an ectoparasitic fly of various bird species. We also test whether chilling is a prerequisite for completion of diapause in this species. In the course of three winter seasons we experimentally exposed carnid pupae from nests of various host species to spring temperatures with and without chilling and recorded the emergence patterns in experimental and control groups. Experimental groups showed an advanced emergence date, a lower emergence rate and, consequently, a protracted emergence period. Chilling had no obvious effect on the start of emergence but it did advance the mean emergence date, shortened the length of the emergence period when compared with the control treatment and increased the emergence rate when compared with the spring treatment. This study identifies an environmental cue, namely temperature during the free-living stage, affecting the emergence of a widespread parasite and demonstrates the plasticity of diapause in this parasite. Our findings are of potential significance in understanding host–parasite interactions.

Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed.

This paper reports the effects of BnSP-7 toxin, a catalytically inactive phospholipase A2 from Bothrops pauloensis snake venom, on Leishmania (Leishmania) amazonensis. BnSP-7 presented activity against promastigote parasite forms both in the MTT assay, with IC50 of 58·7 μg mL−1 of toxin, and a growth curve, inhibiting parasite proliferation 60–70% at concentrations of 50–200 μg mL−1 of toxin 96 h after treatment. Also, the toxin presented effects on amastigotes, reducing parasite viability by 50% at 28·1 μg mL−1 and delaying the amastigote–promastigote differentiation process. Ultrastructural studies showed that BnSP-7 caused severe morphological changes in promastigotes such as mitochondrial swelling, nuclear alteration, vacuolization, acidocalcisomes, multiflagellar aspects and a blebbing effect in the plasma membrane. Finally, BnSP-7 interfered with the infective capacity of promastigotes in murine peritoneal macrophages, causing statistically significant infectivity-index reductions (P < 0·05) of 20–35%. These data suggest that the BnSP-7 toxin is an important tool for the discovery of new parasite targets that can be exploited to develop new drugs for treating leishmaniasis.

Anthelmintic resistance is a major problem for the control livestock parasites and a potential threat to the sustainability of community-wide treatment programmes being used to control human parasites in the developing world. Anthelmintic resistance is essentially a complex quantitative trait in which multiple mutations contribute to the resistance phenotype in an additive manner. Consequently, a combination of forward genetic and genomic approaches are needed to identify the causal mutations and quantify their contribution to the resistance phenotype. Therefore, there is a need to develop genetic and genomic approaches for key parasite species identified as relevant models. Haemonchus contortus, a gastro-intestinal parasite of sheep, has shown a remarkable propensity to develop resistance to all the drugs used in its control. Partly because of this, and partly because of its experimental amenability, research on this parasite has contributed more than any other to our understanding of anthelmintic resistance. H. contortus offers a variety of advantages as an experimental system including the ability to undertake genetic crosses; a prerequisite for genetic mapping. This review will discuss the current progress on developing H. contortus as a model system in which to study anthelmintic resistance.

We evaluated the effect of chemotherapy with a sequential combined treatment of a low dose of benznidazole and allopurinol, in different schedules of administration, in experimental models of acute and chronic Trypanosoma cruzi infection. Mice were infected with Nicaragua T. cruzi isolate, a virulent parasite from an endemic area of Nicaragua, genotyped as TcI (Grosso et al. 2010). We assessed survival rate, IgG levels, histopathological studies and quantified parasitaemia. A 15% survival rate was recorded in untreated mice during the acute phase of T. cruzi infection. Allopurinol administered immediately after benznidazole treatment was able to reduce parasitaemia and attenuate tissue damage by reducing inflammation. Trypanosoma cruzi-specific antibodies also decreased in 40–50% of the treated mice. The addition of allopurinol during the chronic phase showed the highest beneficial effect, not only by reducing parasitaemia but also by lowering the degree of inflammation and fibrosis.

To date no experimental infection studies have been conducted in laboratory mice using Mus spp. bartonella strains. Therefore we designed a study to evaluate the in vivo infection characteristics of 2 Bartonella tribocorum strains from wild Mus spp. in laboratory mice with the aim of developing a mouse model that reproduces characteristics of naturally acquired bartonella infections in rodents. Groups of outbred CD1 female mice were subcutaneously inoculated with low doses of 2 mouse bartonella strains (10, 100, and 1000 bacteria/mouse). Blood was collected weekly for 27 weeks to evaluate bacteraemia kinetics in infected mice. Mouse urine collected during weeks 3–6 post-inoculation was also tested for viable bacteria to determine whether urine might serve as a source of bacterial transmission. Mice were susceptible to infection with both strains. Bacteraemias in mice lasted up to 25 weeks, sometimes with abacteraemic intervals, and achieved levels up to 107 cfu/ml of blood. Temporal lags in bacteraemia onset of up to 19 weeks in length were noted at different inoculum doses. No viable bacteria were detected in mouse urine. Bacteraemic mice displayed characteristics of infection similar to those observed in natural rodent hosts during longitudinal field studies. This mouse model of persistent bacteraemia should be suitable for a variety of experimental uses.

Protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. A large number of protein kinases within the protein kinome of Entamoeba histolytica strongly suggest that protein phosphorylation is a key component of pathogenesis regulation by this parasite. PI3 K and Src are kinases previously described in this parasite, but their role is poorly understood. Here, the effect of Src-1-inhibitor and PI3 K inhibitor (Wortmannin) on the virulence factors of E. histolytica was evaluated. Results show that both inhibitors affect the actin cytoskeleton and the amoebic movement. Also, the proteolytic activity is diminished by Wortmannin, but not by Src-inhibitor-1; however, the phagocytic capacity is diminished by Wortmannin and Src-1-inhibitor. Finally, we found that the virulence in vivo of E. histolytica is affected by Wortmannin but not by Src-1-inhibitor. This study opens the way for the design of anti-amoebic drugs based on kinase inhibition.

The respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH : ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH : ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH : ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD+ regeneration in the mitochondrion of procyclic T. brucei.

Trichomonas gallinae is an emerging pathogen in wild birds, linked to recent declines in finch (Fringillidae) populations across Europe. Globally, the main hosts for this parasite are species of Columbidae (doves and pigeons); here we carry out the first investigation into the presence and incidence of Trichomonas in four species of Columbidae in the UK, through live sampling of wild-caught birds and subsequent PCR. We report the first known UK cases of Trichomonas infection in 86% of European Turtle Doves Streptopelia turtur sampled, along with 86% of Eurasian Collared Doves Streptopelia decaocto, 47% of Woodpigeons Columba palumbus and 40% of Stock Doves Columba oenas. Birds were more likely to be infected if the farm provided supplementary food for gamebirds. We found three strains of T. gallinae and one strain clustering within the Trichomonas tenax clade, not previously associated with avian hosts in the UK. One T. gallinae strain was identical at the ITS/5.8S/ITS2 ribosomal region to that responsible for the finch trichomonosis epizootic. We highlight the importance of increasing our knowledge of the diversity and ecological implications of Trichomonas parasites in order further to understand the sub-clinical impacts of parasite infection.

Partial sequences of the DNA polymerase delta (pold) gene from Taenia saginata-like adult worms were sequenced. Phylogenetic analysis revealed that pold gene sequences were clearly divided into two clades, differing from each other in five to seven nucleotides. There is little doubt that T. saginata and Taenia asiatica were once separated into two distinct taxa as has been concluded in previous studies. On the other hand, most of the adult worms, which were identified as T. asiatica using mitochondrial DNA, were homozygous for an allele that originated from the allele of T. saginata via single nucleotide substitution. These results indicate that most of the adult worms, which had been called T. asiatica, are not actually ‘pure T. asiatica’ but instead originated from the hybridization of ‘pure T. saginata’ and ‘pure T. asiatica’.

Leishmania parasites can escape the immune response by invading cell types lacking leishmanicidal mechanisms. Silent persistence of Leishmania parasites in the host organism is responsible for asymptomatic carriage and relapses after cured leishmaniasis. Here, we studied the interaction between Hepatic Stellate Cells (HSC) and Leishmania. An original model of human HSC in primary culture infected with L. donovani was developed. The presence of intracellular parasites was studied and quantified using optical and confocal microscopy. HSC characteristics were studied using microscopy, methylene blue assay, long-term cultures and qPCR. We showed for the first time that human HSC are permissive to L. donovani infection, with no modification of HSC survival, growth rate and proinflammatory and fibrogenic characteristics. Intracellular parasites did not replicate but HSC had no effect on their survival. Indeed, after a 40-day culture, infected HSC cultures transferred on NNN medium yielded new promastigotes that were able to proliferate and efficiently infect new cells. HSC are permissive to L. donovani, with neither parasite killing nor apparent cell damage. Thus, HSC could act as potent sanctuary cells for Leishmania in the liver, which could partially explain parasite reactivation after an asymptomatic carriage or a cured visceral leishmaniasis.