Trypanosomatid mitochondrial DNA is structured as a giant network of thousands of interlocked DNA molecules enclosed within the kinetoplast. The structure and replication mechanism of kinetoplast DNA (kDNA) is unique, thereby making it an excellent chemotherapeutic target. Alteration in the structural organization of kDNA can give rise to dyskinetoplastic (Dk) strains. In Dk cells, the kDNA is dispersed in clumps throughout the mitochondrial matrix and not organized into a network. In this work, Trypanosoma cruzi epimastigotes were treated with acriflavine, a DNA intercalating drug, which promoted a decrease in cell proliferation and induced the appearance of Dk protozoa. In treated cells, the kinetoplast lost its normal disc-shaped structure because the fibrillar arrangement was reduced to a compact, amorphous mass within the mitochondrion. Moreover, basic proteins associated with kDNA were redistributed throughout the Dk protozoal kinetoplast. We sought to understand how the disruption of the kDNA leads to the emergence of the Dk phenotype with atomic force microscopy (AFM) analysis of isolated networks. Our results demonstrate that the detachment of minicircles from the kDNA disk promotes the disassembly of the network, thereby generating Dk cells. Our data strongly suggest that acriflavine inhibits T. cruzi multiplication by interfering with kDNA replication.

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to indigenous European crayfish species. The North American invasive spiny-cheek crayfish, Orconectes limosus, is an important source of this pathogen in central and western Europe. We evaluated potential changes in A. astaci spore release rate from infected individuals of this species by experiments investigating the pathogen transmission to susceptible noble crayfish, Astacus astacus. We filtered defined volumes of water regularly to quantify spore concentration, and sampled crayfish tissues at the end of the experiment. The filters and tissues were then tested for the presence of A. astaci DNA by species-specific quantitative PCR. Additionally, we tested the efficiency of horizontal transmission to apparently uninfected O. limosus. The experiments confirmed that A. astaci can be transmitted to susceptible crayfish during intermoult periods, and that the pathogen was more frequently detected in noble crayfish recipients than in American ones. The pathogen spore concentrations substantially varied in time, and significantly increased during moulting of infected hosts. Our study strengthens the evidence that although the likelihood of crayfish plague transmission by water transfer from localities with infected American crayfish might increase when these are moulting or dying, no time-periods can be proclaimed safe.

The aim of this study was to evaluate the anti-trypanosomal effect of treatment with 3′-deoxyadenosine (cordycepin) combined with deoxycoformycin (pentostatin: inhibitor of the enzyme adenosine deaminase) in vitro by using mice experimentally infected with Trypanosoma evansi. In vitro, a dose-dependent trypanocidal effect of cordycepin was observed against the parasite. In the in vivo trials, the two drugs were used individually and in combination of different doses. The drugs when used individually had no curative effect on infected mice. However, the combination of cordycepin (2 mg kg−1) and pentostatin (2 mg kg−1) was 100% effective in the T. evansi-infected groups. There was an increase in levels of some biochemical parameters, especially on liver enzymes, which were accompanied by histological lesions in the liver and kidneys. Based on these results we conclude that treatment using the combination of 3′-deoxyadenosine with deoxycoformycin has a curative effect on mice infected with T. evansi. However, the therapeutic protocol tested led to liver and kidney damage, manifested by hepatotoxicity and nephrotoxicity.

The small intestines of 420 wild canids (111 corsac foxes, 191 red foxes and 118 wolves) from Mongolia, were examined for adult worms of the genus Echinococcus. The Mongolian genotype of Echinococcus multilocularis was found in fifteen red foxes and four wolves, whereas two genotypes (G6/7 and G10) of Echinococcus canadensis were found in two and three wolves, respectively. No adult Echinococcus worms were found in the corsac foxes examined. The genotypes of E. multilocularis and E. canadensis are discussed in terms of host specificity and distribution in Mongolia. The importance of wolves in the completion of the life cycle of Echinococcus spp. is also discussed.

A study was carried out to investigate whether the action of triclabendazole sulphoxide (TCBZ.SO) against the liver fluke, Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for this in vitro study and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. For experiments with the Oberon isolate, flukes were incubated for 24 h with either R(+)-VPL (1×10−4m) on its own, TCBZ.SO (15 μg mL−1) alone, a combination of R(+)-VPL (1×10−4m) plus TCBZ.SO (15 μg mL−1), TCBZ.SO (50 μg mL−1) on its own, or a combination of TCBZ.SO (50 μg mL−1) plus R(+)-VPL (1×10−4m). They were also incubated in TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive; and in TCBZ.SO (50 μg mL−1) alone for a time to match that of the combination inactivity time. Flukes from the Cullompton isolate were treated with either TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive, or with TCBZ.SO (50 μg mL−1) alone time-matched to the combination inactivity time. Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R(+)-VPL alone had a minimal effect on either isolate. TCBZ.SO treatment had a relatively greater impact on the TCBZ-susceptible Cullompton isolate. When R(+)-VPL was combined with TCBZ.SO in the incubation medium, however, the surface disruption to both isolates was more severe than that seen after TCBZ.SO treatment alone; also, the time taken to reach inactivity was shorter. More significantly, though, the potentiation of drug activity was greater in the Oberon isolate; also, it was more distinct at the higher concentration of TCBZ.SO. So, the Oberon isolate appears to be particularly sensitive to efflux pump inhibition. The results of this study suggest that enhanced drug efflux in the Oberon isolate may be involved in the mechanism of resistance to TCBZ.

Transmission dynamics of the actinospore stage of Ceratomyxa shasta to the salmonid host were investigated under field and laboratory conditions. The number of parasites transmitted and the transmission rate were compared between 2 different exposure durations and also among different water velocities, by means of field exposures. Under laboratory conditions, the number of parasites transmitted and the transmission rates were compared across a broader range of water velocities and also at different water temperatures. Transmission rate was not constant over time as the number of parasites transmitted increased non-linearly between the 2 exposure durations. Transmission was also inversely related to water velocity and there was a threshold to transmission between 0·2–0·3 m s−1. Lastly, transmission rate increased with water temperature up to 18 °C then decreased at 23 °C. These experiments provide a range of values of transmission that will be incorporated into an epidemiological model to simulate the effectiveness of different management strategies. Additionally, these experiments provided novel information on the effects of environmental conditions (i.e. water velocity and water temperature) on the transmission dynamics between the salmonid host and the actinospore stage.

Anguillicola crassus is the most invasive species of its genus and it is a successful colonizer of different eel species worldwide. It is so far the only species of the genus Anguillicola whose life cycle has been studied completely. To analyse whether differences in life cycle may explain differences in invasiveness, we infected European eels with Anguillicola novaezelandiae under laboratory conditions. Anguillicola novaezelandiae shows a synchronized development in the European eel. Eggs with second-stage larvae appeared 120 days after infection. No density-dependent effect in parasite development could be found for A. novaezelandiae. The present study shows that the life cycle of A. novaezelandiae differs on final host level compared with A. crassus in ways which result in a less successful invasion of new host species.

The aim of the present investigation was to determine whether temporal variation in environmental factors such as rainfall or temperature influence long-term fluctuations in the prevalence and mean abundance of the nematode Mexiconema cichlasomae in the cichlid fish Cichlasoma uropthalmus and its crustacean intermediate host, Argulus yucatanus. The study was undertaken in a tropical coastal lagoon in the Yucatan Peninsula (south-eastern Mexico) over an 8-year period. Variations in temperature, rainfall and monthly infection levels for both hosts were analysed using time series and cross-correlations to detect possible recurrent patterns. Infections of M. cichlasomae in A. yucatanus showed annual peaks, while in C. urophthalmus peaks were bi-annual. The latter appear to be related to the accumulation of several generations of this nematode in C. urophthalmus. Rainfall and temperature appear to be key environmental factors in influencing temporal variation in the infection of M. cichlasomae over periods longer than a year together with the accumulation of larval stages throughout time.

We have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

From continental to regional scales, the zoonosis alveolar echinococcosis (AE) (caused by Echinococcus multilocularis) forms discrete patches of endemicity within which transmission hotspots of much larger prevalence may occur. Since the late 80s, a number of hotspots have been identified in continental Asia, mostly in China, wherein the ecology of intermediate host communities has been described. This is the case in south Gansu, at the eastern border of the Tibetan plateau, in south Ningxia, in the western Tian Shan of Xinjiang, and in the Alay valley of south Kyrgyzstan. Here we present a comparative natural history and characteristics of transmission ecosystems or ecoscapes. On this basis, regional types of transmission and their ecological characteristics have been proposed in a general framework. Combining climatic, land cover and intermediate host species distribution data, we identified and mapped 4 spatially distinct types of transmission ecosystems typified by the presence of one of the following small mammal ‘flagship’ species: Ellobius tancrei, Ochotona curzoniae, Lasiopodomys brandtii or Eospalax fontanierii. Each transmission ecosystem had its own characteristics which can serve as a reference for further in-depth research in the transmission ecology of E. multilocularis. This approach may be used at fine spatial scales to characterize other poorly known transmission systems of the large Eurasian endemic zone, and help in consideration of surveillance systems and interventions.

Chagas infection is a major endemic disease affecting Latin American countries. The persistence of Trypanosoma cruzi generates a chronic inflammatory reactivity that induces an immune response directed to the host's tissues. The effectiveness of the treatment in the chronic phase is still unsatisfactory due, amongst other reasons, to the collateral effects of the drugs used. We investigated the effect of clomipramine, a tricyclic antidepressant that, when used as a treatment of T. cruzi-chronically infected mice, inhibits trypanothione reductase, an exclusive and vital enzyme of T. cruzi. Clomipramine improved survival (P<0·05) by diminishing the parasite intensity as demonstrated by PCR studies in the heart and skeletal muscle, and significantly prevented the evolution to fibrosis of the inflammatory infiltrates. Clomipramine could be a good candidate for the treatment of chronic Chagas disease.

The aim of this study was to investigate neurochemical and enzymatic changes in rats infected with Trypanosoma evansi, and their interference in the cognitive parameters. Behavioural assessment (assessment of cognitive performance), evaluation of cerebral L-[3H]glutamate uptake, acetylcholinesterase (AChE) activity and Ca+2 and Na+, K+-ATPase activity were evaluated at 5 and 30 days post infection (dpi). This study demonstrates a cognitive impairment in rats infected with T. evansi. At 5 dpi memory deficit was demonstrated by an inhibitory avoidance test. With the chronicity of the disease (30 dpi) animals showed anxiety symptoms. It is possible the inhibition of cerebral Na+, K+-ATPase activity, AChE and synaptosomal glutamate uptake are involved in cognitive impairment in infected rats by T. evansi. The understanding of cerebral host–parasite relationship may shed some light on the cryptic symptoms of animals and possibly human infection where patients often present with other central nervous system (CNS) disorders.

Many protease genes have previously been shown to be involved in parasitism and in the development of Steinernema carpocapsae, including a gene predicted to encode an aspartic protease, Sc-ASP110, which was cloned and was analysed in this study. A cDNA encoding Sc-ASP110 was cloned based on an expressed sequence tag (EST) fragment from our EST library. The full-length cDNA of Sc-ASP110 consists of 1112 nucleotides with a catalytic aspartic domain (aa18–337). The putative 341 amino acid residues have a calculated molecular mass of 37·1 kDa and a theoretical pI of 4·7. BLASTp analysis of the Sc-ASP110 amino acid sequence showed 45–77% amino acid sequence identity to parasitic and non-parasitic nematode aspartic proteases. An expression analysis showed that the sc-asp110 gene was upregulated during the late parasitic stage, L4, and 24 h after induction of in vitro nematodes. A sequence comparison revealed that Sc-ASP110 was a member of an aspartic protease family; additionally, a phylogenetic analysis indicated that Sc-ASP110 was clustered with the closely related nematode Steinernema feltiae. In situ hybridization showed that sc-asp110 was expressed in the body walls of dorsal cells. The upregulated Sc-ASP110 expression revealed that this protease could play a role in the late parasitic process. In this study, we have cloned and analysed the gene transcript of Sc-ASP110 in S. carpocapsae.

Physical habitat structure can influence the distribution and abundance of organisms. In rivers, stream drift, a common process originating from the unidirectional water flow, favours the displacement and downstream dispersion of invertebrates. This process could also generate a gradient in infection levels, leading to decreasing numbers of parasites per host as one moves upstream from the river mouth. We tested this hypothesis using 4 trematode species infecting the fish Gobiomorphus breviceps in the Manuherikia River (New Zealand). We analysed the abundance of each trematode infrapopulation as a function of distance from the river junction and fish size by generalized linear models. Our results supported the existence of a longitudinal gradient in trematode abundance along the river with a decreasing downstream-to-upstream continuum. This applied to 3 out of the 4 trematode species studied, suggesting that this might be a common pattern in river populations. Thus, the unidirectional river flow and a major process like drift in lotic systems, that influences the dynamics and distribution of invertebrate hosts, can also affect trematodes. Host properties like habitat preference, and parasite traits, particularly those related to transmission mode can influence the strength of the observed gradient, as may other environmental and biotic factors.

Cyathostomins are the most prevalent equine intestinal parasites and resistance has been reported in these nematodes against all 3 licensed anthelmintic classes. Strategies need to be developed that are less dependent upon drugs and more reliant on management-based control. To develop these we need to understand natural transmission patterns better. Here, we analysed longitudinal fecal egg count (FEC) data from 5 pony populations used for conservation purposes. We tested how egg excretion varied amongst populations and individuals, and how this was affected by age and climate. There was evidence for consistency in FECs over time at the individual level; this was generally weak and accounted for <10% of the total variance. Animals <5 years old had higher FECs and there was profound seasonal variation in FECs, with highest levels recorded in spring/summer. Effects of monthly temperature and rainfall explained most, but not all, of the observed seasonal variation and associations between climate measures and FECs were stronger in younger versus adult animals. One population was occasionally treated with anthelmintics and analysis of this population suggested that treatment substantially altered the seasonal dynamics. This paper highlights the variability in strongyle egg excretion amongst individuals and the factors involved in this variation.

Angiostrongylus cantonensis is a parasitic nematode of rodents and a leading aetiological agent of eosinophilic meningitis in humans. Definitive diagnosis is difficult, often relying on immunodiagnostic methods which utilize crude antigens. New immunodiagnostic methods based on recombinant proteins are being developed, and ideally these methods would be made available worldwide. Identification of diagnostic targets, as well as studies on the biology of the parasite, are limited by a lack of molecular information on Angiostrongylus spp. available in databases. In this study we present data collected from DNA random high-throughput sequencing together with proteomic analyses and a cDNA walking methodology to identify and obtain the nucleotide or amino acid sequences of unknown immunoreactive proteins. 28 080 putative ORFs were obtained, of which 3371 had homology to other deposited protein sequences. Using the A. cantonensis genomic sequences, 156 putative ORFs, matching peptide sequences obtained from previous proteomic studies, were considered novel, with no homology to existing sequences. Full-length coding sequences of eight antigenic target proteins were obtained. In this study we generated not only the complete nucleotide sequences of the antigenic protein targets but also a large amount of genomic data which may help facilitate future genomic, proteomic, transcriptomic or metabolomic studies on Angiostrongylus.

Yellow-eyed penguins (YEPs) have suffered major population declines over the past 30 years, with no single cause established. Leucocytozoon was first identified in yellow-eyed penguins in 2005. During the 2008/09 breeding season, a high mortality was seen in both mainland yellow-eyed penguins as well as those on Enderby Island of the Auckland Islands archipelago. A high overall prevalence of Leucocytozoon spp. in association with a high incidence of chick mortality was observed during this period on Enderby Island. One chick had histological evidence of leucocytozoonosis with megaloschizonts in multiple organs throughout its body. In addition, a high prevalence (73·7%) of Leucocytozoon was observed by PCR in the blood of adult Enderby yellow-eyed penguins taken during the 2006/07 season. These findings were different from the low prevalence detected by PCR on the coast of the South Island (11%) during the 2008/2009 breeding session and earlier on Campbell Island (21%) during the 2006/2007 breeding session. The Leucocytozoon spp. sequences detected lead us to conclude that the Leucocytozoon parasite is common in yellow-eyed penguins and has a higher prevalence in penguins from Enderby Island than those from Campbell Island and the mainland of New Zealand. The Enderby Island yellow-eyed penguins are infected with a Leucocytozoon spp. that is genetically distinct from that found in other yellow-eyed penguin populations. The role of Leucocytozoon in the high levels of chick mortality in the yellow-eyed penguins remains unclear.

Two new microsporidia, Anostracospora rigaudi n. g., n. sp., and Enterocytospora artemiae n. g., n. sp. infecting the intestinal epithelium of Artemia parthenogenetica Bowen and Sterling, 1978 and Artemia franciscana Kellogg, 1906 in southern France are described. Molecular analyses revealed the two species belong to a clade of microsporidian parasites that preferentially infect the intestinal epithelium of insect and crustacean hosts. These parasites are morphologically distinguishable from other gut microsporidia infecting Artemia. All life cycle stages have isolated nuclei. Fixed spores measure 1·3×0·7 μm with 5–6 polar tube coils for A. rigaudi and 1·2×0·9 μm with 4 polar tube coils for E. artemiae. Transmission of both species is horizontal, most likely through the ingestion of spores released with the faeces of infected hosts. The minute size of these species, together with their intestinal localization, makes their detection and identification difficult. We developed two species-specific molecular markers allowing each type of infection to be detected within 3–6 days post-inoculation. Using these markers, we show that the prevalence of these microsporidia ranges from 20% to 75% in natural populations. Hence, this study illustrates the usefulness of molecular approaches to study prevalent, but cryptic, infections involving microsporidian parasites of gut tissues.