Extensive connective tissue lysis is a common outcome of haplosporidian infection. Although such infections in marine invertebrates are well documented, they are relatively rarely observed in freshwater invertebrates. Herein, we report a field study using a comprehensive series of methodologies (histology, dissection, electron microscopy, gene sequence analysis, and molecular phylogenetics) to investigate the morphology, taxonomy, systematics, geographical distribution, pathogenicity, and seasonal and annual prevalence of a haplosporidian observed in zebra mussels, Dreissena polymorpha. Based on its genetic sequence, morphology, and host, we describe Haplosporidium raabei n. sp. from D. polymorpha – the first haplosporidian species from a freshwater bivalve. Haplosporidium raabei is rare as we observed it in histological sections in only 0·7% of the zebra mussels collected from 43 water bodies across 11 European countries and in none that were collected from 10 water bodies in the United States. In contrast to its low prevalences, disease intensities were quite high with 79·5% of infections advanced to sporogenesis.

LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in the protozoan of the genus Leishmania, which is associated with the immunopathogenesis and susceptibility of BALB/c mice to Leishmania major infection. We previously demonstrated that intranasal immunization with a plasmid DNA encoding the p36/LACK leishmanial antigen (pCI-neo-LACK) followed by challenge 7 days after a booster dose effectively protects BALB/c mice against both cutaneous and visceral leishmaniasis. In the present study, the correlation between systemic mRNA expression after nasal DNA uptake, and the duration of protective immunity was addressed. LACK mRNA transcripts were detected in the spleen, brain, cervical lymph nodes and popliteal lymph nodes as early as 7 days, lasting 3 months after vaccination with pCI-neo-LACK. The kinetics of transcript expression correlated with enhanced cutaneous hypersensitivity against parasite antigens. Leishmania chagasi infection at 7 days or 3 months, but not 6 months after vaccination resulted in significantly lower parasite loads as compared with non-vaccinated controls. Protection also correlated with enhanced spleen cell responsiveness to parasite antigens leading to increased IFN- γ and IL-4 and decreased IL-10 production. Together, these data demonstrate that the protection conferred by the intranasal DNA vaccine lasts at least 3 months and is associated with expression of vaccine mRNA in peripheral organs.

Psychodiella sergenti is a recently described specific pathogen of the sand fly Phlebotomus sergenti, the main vector of Leishmania tropica. The aim of this study was to examine the life cycle of Ps. sergenti in various developmental stages of the sand fly host. The microscopical methods used include scanning electron microscopy, transmission electron microscopy and light microscopy of native preparations and histological sections stained with periodic acid-Schiff reaction. Psychodiella sergenti oocysts were observed on the chorion of sand fly eggs. In 1st instar larvae, sporozoites were located in the ectoperitrophic space of the intestine. No intracellular stages were found. In 4th instar larvae, Ps. sergenti was mostly located in the ectoperitrophic space of the intestine of the larvae before defecation and in the intestinal lumen of the larvae after defecation. In adults, the parasite was recorded in the body cavity, where the sexual development was triggered by a bloodmeal intake. Psychodiella sergenti has several unique features. It develops sexually exclusively in sand fly females that took a bloodmeal, and its sporozoites bear a distinctive conoid (about 700 nm long), which is more than 4 times longer than conoids of the mosquito gregarines.

Trypanosomatids represent the causative agents of major diseases in humans, livestock and plants, with inevitable suffering and economic hardship as a result. They are also evolutionarily highly divergent organisms, and the many unique aspects of trypanosome biology provide opportunities in terms of identification of drug targets, the challenge of exploiting these putative targets and, at the same time, significant scope for exploration of novel and divergent cell biology. We can estimate from genome sequences that the degree of divergence of trypanosomes from animals and fungi is extreme, with perhaps one third to one half of predicted trypanosome proteins having no known function based on homology or recognizable protein domains/architecture. Two highly important aspects of trypanosome biology are the flagellar pocket and the nuclear envelope, where in silico analysis clearly suggests great potential divergence in the proteome. The flagellar pocket is the sole site of endo- and exocytosis in trypanosomes and plays important roles in immune evasion via variant surface glycoprotein (VSG) trafficking and providing a location for sequestration of various invariant receptors. The trypanosome nuclear envelope has been largely unexplored but, by analogy with higher eukaryotes, roles in the regulation of chromatin and most significantly, in controlling VSG gene expression are expected. Here we discuss recent successful proteomics-based approaches towards characterization of the nuclear envelope and the endocytic apparatus, the identification of conserved and novel trypanosomatid-specific features, and the implications of these findings.

RNA interference (RNAi) has emerged as an invaluable gene-silencing tool for functional analysis in a wide variety of organisms, particularly the free-living model nematode Caenorhabditis elegans. An increasing number of studies have now described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when nematodes take up double stranded RNA (dsRNA) or short interfering RNAs (siRNAs) that elicit a systemic RNAi response. Despite many successful reports, there is still poor understanding of the range of factors that influence optimal gene silencing. Recent in vitro studies have highlighted significant variations in the RNAi phenotype that can occur with different dsRNA concentrations, construct size and duration of soaking. Discrepancies in methodology thwart efforts to reliably compare the efficacy of RNAi between different nematodes or target tissues. Nevertheless, RNAi has become an established experimental tool for plant parasitic nematodes and also offers the prospect of being developed into a novel control strategy when delivered from transgenic plants.

Two new myxosporean species in the gallbladders of frogs have recently spread across eastern Australia and cause disease. Cystodiscus axonis sp. n. and Cystodiscus australis sp. n. are species of Myxosporea (Myxozoa) identified from a range of Australian frogs and tadpoles including the introduced Cane toad (Rhinella marina). The new species are defined by their distinct genetic lineage, myxospore morphology and ultrastructure of the pre-sporogonic development. Spores of both species are produced in the gallbladder. Spores of C. axonis sp. n. possess distinct filiform polar appendages (FPA). The pre-sporogonic development of C. axonis sp. n. is within myelinated axons in the central nervous system of hosts, as well as bile ducts of tadpoles. Pre-sporogonic and sporogonic development of C. australis sp. n. is confined to tadpole bile ducts and myxospores of C. australis sp. n. are devoid of FPA. The genus Cystodiscus Lutz, 1889 introduced for Cystodiscus immersus Lutz, 1889 is emended to accompany myxosporean parasites affecting amphibians previously classified in the genus Myxidium sensu lato. A synopsis of described species within Cystodiscus is provided.

Microsporidia are intracellular parasites, frequently infecting the planktonic crustacean Daphnia. Questioning the ability to detect and identify microsporidia with conventional microscopic techniques, we applied molecular methods in order to investigate the distribution and co-infection patterns of this parasite among 8 communities of the Daphnia longispina hybrid complex. Eight microsporidian taxa were detected, including 3 that previously had not been characterized genetically. Microsporidian communities from nearby lakes were found to be more similar to each other, apparently due to short distance dispersal via secondary hosts. Moreover, we detected seasonal (but not interannual) changes in microsporidian community structure. With some microsporidia being host-specific, these changes might have resulted from seasonal changes in host taxon and clonal composition. The 2 dominant and closely related parasite species were found mainly in single infections, whereas another pair of related microsporidians was found predominantly in co-infections; suggesting species-level differences in the ability to colonize infected hosts. By applying molecular methods, we were not only able to unambiguously identify parasite taxa but also to reveal multiple infections that otherwise would have remained undetected. Given the increased level of accuracy and sensitivity, we highly recommend molecular approaches in future parasite surveys of Daphnia infections.

Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.

Trypanosoma evansi is the aetiological agent of trypanosomosis in domestic animals. In this pathology, an inflammatory response can be observed and, as a consequence, the increase of extracellular adenine nucleotides such as ATP. These nucleotide concentrations are regulated by ectoenzymes such as NTPDase (EC 3.6.1.5, CD39), which catalyses the hydrolysis of ATP and ADP into AMP. In this study, the activity of NTPDase in lymphocytes of rats experimentally infected with T. evansi was evaluated. The animals were inoculated with the parasite and monitored by blood smear on a daily basis. The animals were then were divided into 4 groups according to the degree of parasitaemia and period of infection. The blood collections for enzyme analysis and lymphocyte count were performed on the 3rd (beginning of infection), 5th (acute infection) and 15th (chronic infection) days post-infection (p.i.). The control group was composed of non-infected animals. In the infected group a decrease in ATP hydrolysis (36%) was observed on the 3rd day p.i. and a decrease in ADP hydrolysis (62%) was observed on the 5th day p.i. when compared to the control. On the 15th day p.i., an increase in ATP (94%) and ADP (50%) hydrolysis was observed in the infected group. Considering these data it is suggested that NTPDase activity is altered on the surface of lymphocytes of rats infected with T. evansi at different time-points of infection.

Many of the fundamental concepts in studying infectious diseases are rooted in population ecology. We describe the importance of population ecology in exploring central issues in infectious disease research including identifying the drivers and dynamics of host-pathogen interactions and pathogen persistence, and evaluating the success of public health policies. The use of ecological concepts in infectious disease research is demonstrated with simple theoretical examples in addition to an analysis of case notification data of pertussis, a childhood respiratory disease, in Thailand as a case study. We stress that further integration of these fields will have significant impacts in infectious diseases research.

Ixodes ricinus horizontal movement within a humidity gradient and the influence of infection by Borrelia burgdorferi sensu lato (s.l.) on tick walking were investigated. Nymphs were placed within an arena containing a humidity gradient ranging from 45 to 95% relative humidity (RH). After 1 h of acclimation at 70% RH ticks were released so that they could either stay, or walk towards either the wet or the dry end. Their position was recorded 2 h post-release. Fat content was quantified and Borrelia infection was detected using real-time PCR and PCR followed by Reverse Line Blotting. Among the 1500 ticks tested, 29·85% were infected. More low-fat nymphs walked inside the arena than high-fat individuals. When nymphs walked, more low-fat ticks walked towards wetter than drier air, whereas more high-fat individuals walked towards drier than wetter air. Among high-fat nymphs, a lower proportion of Borrelia-infected ticks walked inside the arena compared to uninfected individuals, as though spirochetes manipulated their arthropod vector to stay. However, Borrelia infection had no effect on walking direction towards the dry or the wet end. Hence, it appears that I. ricinus nymphs walk horizontally over short distances within a humidity gradient depending on both energy resources and Borrelia infection.

Cysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with Ki's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.

Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.

We asked if and how feeding performance of fleas on an auxiliary host is affected by the phylogenetic distance between this host and the principal host of a flea. We investigated the feeding of 2 flea species, Parapulex chephrenis and Xenopsylla ramesis, on a principal (Acomys cahirinus and Meriones crassus, respectively) and 8 auxiliary host species. We predicted that fleas would perform better (higher proportion of fleas would feed and take larger bloodmeals) on (a) a principal rather than an auxiliary host and (b) auxiliary hosts phylogenetically closer to a principal host. Although feeding performance of fleas differed among different hosts, we found that: (1) fleas did not always perform better on a principal host than on an auxiliary host; and (2) flea performance on an auxiliary host was not negatively correlated with phylogenetic distance of this host from the principal host. In some cases, fleas fed better on hosts that were phylogenetically distant from their principal host. We concluded that variation in flea feeding performance among host species results from interplay between (a) inherent species-specific host defence abilities, (b) inherent species-specific flea abilities to withstand host defences and (c) evolutionary tightness of association between a particular host species and a particular flea species.

The microsporidian parasite Thelohania contejeani causes porcelain disease and has been implicated in mass mortalities in populations of the endangered European crayfish Austropotamobius pallipes. However, the route of parasite transmission is not known. This paper investigates the horizontal transmission of T. contejeani between A. pallipes hosts as well as its transmissibility to the invasive signal crayfish (Pacifastacus leniusculus). Field collected juvenile A. pallipes and P. leniusculus were assigned to 1 of 3 experimental treatments; fed heavily infected A. pallipes tissue, exposed to water from tanks housing heavily parasitized A. pallipes, and a control group to provide an estimate of the baseline infection levels in the field. After 26 weeks, abdominal muscle samples were screened by PCR for T. contejeani. Infection was significantly higher in the treatment groups (83% in the cannibalism treatment, 42% in the water exposure treatment) than in the control group (4%), providing evidence for horizontal transmission of the parasite between A. pallipes hosts. Cannibalism and scavenging are common amongst crayfish, providing transmission opportunities in the field. The study also provides the first direct evidence for transmission of the parasite from an indigenous European crayfish species to the invasive signal crayfish, with 50% of P. leniusculus in each treatment, and 8% of control animals infected. We discuss the possibility that high density populations of the invasive signal crayfish may serve either as reservoirs or sinks for the parasite.

We assessed the distribution of Trypanosoma cruzi Discrete Typing Units (DTUs) in domestic and peridomestic Triatoma infestans and Triatoma sordida specimens collected in a well-defined rural area in Pampa del Indio, northeastern Argentina. Microscopically-positive bugs were randomly selected with a multi-level sampling design, and DTUs were identified using direct PCR strategies. TcVI predominated in 61% of 69 T. infestans and in 56% of 9 T. sordida. TcV was the secondary DTU in T. infestans (16%) and was found in 1 T. sordida specimen (11%). Three T. sordida (33%) were found infected with TcI, a DTU also identified in local Didelphis albiventris opossums. Mixed DTU infections occurred rarely (5%) and were detected both directly from the bugs' rectal ampoule and parasite cultures. The identified DTUs and bug collection sites of T. infestans were significantly associated. Bugs infected with TcV were almost exclusively captured in domiciles whereas those with TcVI were found similarly in domiciles and peridomiciles. All mixed infections occurred in domiciles. TcV-infected bugs fed more often on humans than on dogs, whereas TcVI-infected bugs showed the reverse pattern. T. sordida is a probable sylvatic vector of TcI linked to D. albiventris, and could represent a secondary vector of TcVI and TcV in the domestic/peridomestic cycle.

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

We studied the distribution of ectoparasite species (an ixodid tick, a chigger mite, 7 mesostigmate mites, 5 fleas and 3 lice) on bodies of 5 species of rodent hosts from the marshlands in Argentina to establish whether arthropod ectoparasites are segregated across body parts of the same host individual. We asked (a) whether an individual ectoparasite species prefers certain parts of the body of its host and, if yes, whether these preferences overlap among ectoparasite species; (b) whether ectoparasite species composition differs among different parts of a host's body; and (c) whether co-occurrences of ectoparasite species within pre-defined body parts of a host are non-random and, if yes, whether ectoparasites co-occur in the same body part of a host either less or more often than expected by chance. It was found that, in general, ectoparasite species were not segregated across body parts of a host. Although some ectoparasites preferred certain body parts, these preferences were similar among ectoparasites belonging to different species and/or higher taxa resulting in similarity among host body parts in ectoparasite species composition. In addition, ectoparasite species demonstrated a tendency to co-occur on the same body parts of a host and not to be segregated among them. It was concluded that the distribution of ectoparasites on the body of a small mammalian host is driven mainly by their interaction with the host rather than by distinct preferences or interactions among ectoparasite species.

The epidemiology of Bartonella species infecting Apodemus flavicollis and Myodes glareolus in a forest in Eastern Poland was followed for 2 years using mark-recapture. Infections could be acquired in any month, but prevalence, and probability of infection, peaked in the summer. There were significant differences in the pattern of infections between the two species. Both hosts were primarily infected as juveniles, but the probability of infection was highest for A. flavicollis, which, evidence suggests, experienced longer-lasting infections with a wider range of Bartonella genotypes. There was no evidence of increased host mortality associated with Bartonella, although the infection did affect the probability of recapture. Animals could become re-infected, generally by different Bartonella genotypes. Several longer lasting, poorly resolved infections of A. flavicollis involved more than 1 genotype, and may have resulted from sequential infections. Of 22 Bartonella gltA genotypes collected, only 2 (both B. grahamii) were shared between mice and voles; all others were specific either to A. flavicollis or to M. glareolus, and had their nearest relatives infecting Microtus species in neighbouring fields. This heterogeneity in the patterns of Bartonella infections in wild rodents emphasizes the need to consider variation between both, host species and Bartonella genotypes in ecological and epidemiological studies.

The 3 post-marsupial juvenile stages of the gnathiid isopod, Paragnathia formica, are haematophagous ectoparasites of fishes that may, in heavy infestations, cause host mortality. Protein digestion in fed stage 3 juveniles is accomplished by cysteine proteinases, but what bioactive compounds attenuate host haemostatic, inflammatory and immunological responses during feeding is unknown. Trypsin inhibitory activity and anticoagulant activity were detected in crude extracts of unfed P. formica stage 1 juveniles; fractionation of stage 1 crude extracts by ion exchange chromatography resulted in 3 preparations each displaying these bioactivities. Further characterization revealed anti-thrombin activity in 2 of these preparations, whilst the third displayed the strongest anticoagulant activity that targeted a factor of the intrinsic coagulation pathway. Three trypsin inhibitors (18 kDa, 21 kDa, and 22 kDa) were also detected using reverse zymography. In parallel, homogenates of fed stage 2 and 3 juveniles were used to identify their fish hosts by amplifying the 16S mitochondrial rDNA and 18S genomic rDNA vertebrate gene regions. Blood from at least 4 fish families had been ingested by separate individuals during feeding. This study demonstrates that trypsin inhibitors and anticoagulants are present in P. formica juveniles which could suppress host haemostatic, inflammatory and immunological responses during feeding, and that juveniles are not host specific.