Glycosomes are specialized peroxisomes found in all kinetoplastid organisms. The organelles are unique in harbouring most enzymes of the glycolytic pathway. Matrix proteins, synthesized in the cytosol, cofactors and metabolites have to be transported across the membrane. Recent research on Trypanosoma brucei has provided insight into how these translocations across the membrane occur, although many details remain to be elucidated. Proteins are imported by a cascade of reactions performed by specialized proteins, called peroxins, in which a cytosolic receptor with bound matrix protein inserts itself in the membrane to deliver its cargo into the organelle and is subsequently retrieved from the glycosome to perform further rounds of import. Bulky solutes, such as cofactors and acyl-CoAs, seem to be translocated by specific transporter molecules, whereas smaller solutes such as glycolytic intermediates probably cross the membrane through pore-forming channels. The presence of such channels is in apparent contradiction with previous results that suggested a low permeability of the glycosomal membrane. We propose 3 possible, not mutually exclusive, solutions for this paradox. Glycosomal glycolytic enzymes have been validated as drug targets against trypanosomatid-borne diseases. We discuss the possible implications of the new data for the design of drugs to be delivered into glycosomes.

Changes in population allele frequencies may be driven by several forces, including selection and drift, and are revealed only by sampling over many generations. Such studies, however, are rare for protist parasites. Microsatellite allele frequencies for 4 loci were followed in a population of Plasmodium mexicanum, a malaria parasite of lizards in California USA at 1 site from 1978 to 2010. Rapid turnover of the lizards indicates the parasite was studied for a minimum of 33 transmission cycles and possibly twice that number. Sample sizes ranged from 841 to 956 scored parasite clones per locus. DNA was extracted from frozen dried blood and blood removed from stained blood smears from the earliest years, and a verification study demonstrated DNA from the blood smears provided valid genetic data. Parasite prevalence and effective population size (Ne) dropped after 2000, remaining lower for the next decade. For 2 loci, allele frequencies appeared stable for the first 2 decades of the study, but changed more rapidly after the decline in prevalence. Allele frequencies changed more gradually for the other 2 loci. Genetic drift could account for changes in allele frequencies, especially after the drop in prevalence and Ne, but the force of selection could also have driven the observed patterns.

Current treatments for different clinical forms of leishmaniasis are unsatisfactory, highly toxic and associated with increasing failure rates resulting from the emergence of resistant parasites. Leishmania (Viannia) braziliensis is the main aetiological agent of different clinical forms of American tegumentary leishmaniasis, including the mucosal form for which treatment has high failure rates. The aim of this work was to investigate the activity of the Morita-Baylis-Hillman adduct, methyl 2-{2-[hydroxy(2-nitrophenyl)methyl])acryloyloxy} benzoate in vitro against isolates of L. (V.) braziliensis obtained from patients with different clinical manifestations of tegumentary leishmaniasis: localized cutaneous leishmaniasis, mucosal leishmaniasis and disseminated cutaneous leishmaniasis. The adduct effectively inhibited the growth of promastigotes of the different isolates of L. (V.) braziliensis (IC50 ⩽ 7·77 μg/ml), as well as reduced the infection rate of macrophages infected with these parasites (EC50 ⩽ 1·37 μg/ml). It is remarkable to state that the adduct was more effective against intracellular amastigotes (P ⩽ 0·0045). The anti-amastigote activity correlated with an immunomodulatory effect, since the adduct was able to decrease the production of IL-6 and IL-10 by the infected macrophages. However, its effect was independent of nitric oxide production. This work demonstrates the anti-leishmanial activity of methyl 2-{2-[hydroxy(2-nitrophenyl)methyl])acryloyloxy} benzoate and suggests its potential in the treatment of human infections caused by L. (V.) braziliensis.

Brugia malayi is one of the parasitic worms which causes lymphatic filariasis in humans. Its geographical distribution includes a large part of Asia. Despite its wide distribution, very little is known about the genetic variation and molecular epidemiology of this species. In this study, the internal transcribed spacer 1 (ITS1) nucleotide sequences of B. malayi from microfilaria-positive human blood samples in Northeast Borneo Island were determined, and compared with published ITS1 sequences of B. malayi isolated from cats and humans in Thailand. Multiple alignment analysis revealed that B. malayi ITS1 sequences from Northeast Borneo were more similar to each other than to those from Thailand. Phylogenetic trees inferred using Neighbour-Joining and Maximum Parsimony methods showed similar topology, with 2 distinct B. malayi clusters. The first cluster consisted of Northeast Borneo B. malayi isolates, whereas the second consisted of the Thailand isolates. The findings of this study suggest that B. malayi in Borneo Island has diverged significantly from those of mainland Asia, and this has implications for the diagnosis of B. malayi infection across the region using ITS1-based molecular techniques.

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

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.

The aim of this study was to fractionate and partially characterize the antigenic extract of filariform larvae of Strongyloides venezuelensis in ion-exchange resin diethylaminoethyl sepharose (DEAE), to obtain antigenic fractions potentially applicable in immunoassays. Somatic antigen (SA) and its fractions DEAE S1 and DEAE S2 - which interacted with the resin - were evaluated by 1-dimensional electrophoresis to obtain protein profiles. SA and its fractions were tested in serum samples for IgG detection by ELISA. Serum samples (n = 155) were analysed: 50 from strongyloidiasis patients (G1), 55 from patients with other parasitic infections (G2) and 50 from healthy volunteers. Sensitivity (Se), specificity (Sp), area under curve (AUC) and likelihood ratios (LR) were calculated. The DEAE S2 fraction provided a high diagnostic value for IgG detection (Se 92·0%, Sp 91·4%, AUC 0·981, LR+ 10·75, LR − 0·09). In conclusion, the DEAE S2 fraction would probably be a source of immunodominant polypeptides for IgG detection in human strongyloidiasis serodiagnosis.

The name Paragonimus westermani (Kerbert, 1878) is commonly applied to members of a species complex that includes the well-known Asian lung fluke of medical and veterinary importance. Unambiguous molecular and morphological evidence showing the presence of a member of the complex in India has recently been published. In the present study we report the occurrence of 2 more members of the P. westermani complex in northeastern (NE) India. Surveys of the freshwater crabs Maydelliatelphusa lugubris in NE India revealed 2 morphologically distinct types of lung fluke metacercariae. Phylogenetic analyses, using DNA sequences from ITS2, 28S and cox1 gene regions indicate that these lung metacercariae belong to P. westermani complex. Type 1 metacercariae have a more basal position within the complex whereas type 2 metacercariae are closely related to the relatively derived forms of P. westermani from NE Asia (Japan, Korea, China) and Vietnam. A third type of metacercaria (type 3), detected in another crab host, Sartoriana spinigera in Assam, was phylogenetically close to P. siamensis, also a member of the P. westermani group. Molecular evidence has demonstrated the existence of 3 genotypes of lung flukes within the Paragonimus westermani complex in NE India. Two of these were previously unknown.

The immune response affects host's survival and reproductive success. Insurmountable immune function has not evolved because it is costly and there is a trade-off between other life-history traits. In previous studies several factors such as diet and temperature have been proposed to cause interpopulation differences in immune response. Moreover, the insect immune system may be functionally more protective upon secondary exposure, thus infection history may associate with the immune response. Here we measured how geographical location and parasite burden is related to variation in immune response between populations. We included 13 populations of the Northern Damselfly Coenagrion hastulatum (Odonata: Coenagrionidae) in Finland over a latitudinal range of 880 km to this study. We found that water mites associated strongly with the immune response at interpopulation level: the more the mites, the higher the immune response. Also, in an alternative model based on AIC, latitude and individual size associated with the immune response. In turn, endoparasitic gregarines did not affect the immune response. To conclude, a positive interpopulation association between the immune response and the rate of water mite infection may indicate (i) local adaptation to chronic parasite stress, (ii) effective ‘induced’ immune response against parasites, or (iii) a combined effect of both of these.

Resilin is a soft and elastic protein, which is found in many exoskeletal structures of arthropods. Proteins with similar chemical properties have been described for other invertebrates including monogenean fish parasites. However, for the latter taxon no clear microscopic evidence for a resilin-like protein has been shown so far. Here, we present the results of detailed microscopic analyses of the clamp sclerites (attachment devices) of the monogenean Diplozoon paradoxum. Toluidine blue, which is known to stain resilin, selectively stained the material in the clamp sclerites. In addition, when exposed to UV light, this material exhibited an intense blue autofluorescence. The emission spectrum of this autofluorescence has its maximum at 424 nm and is nearly identical to emission spectra of blue autofluorescences observed in 2 well-studied structures containing high proportions of resilin in the exoskeleton of the locust Schistocerca gregaria. The results strongly indicate that the sclerite material of D. paradoxum contains a resilin-like protein. The presence of such a protein likely enhances the attachment efficiency of the clamp sclerites and increases their lifetime.

Following the recent description of microfilariae of a Cercopithifilaria sp. in a dog from Sicily, Italy, (herein after referred to as Cercopithifilaria sp. I), numerous skin samples were collected from dogs in the Mediterranean region. In addition to Cercopithifilaria sp. I (185·7 ± 7·2 μm long), microfilariae of 2 other species were identified, namely Cercopithifilaria grassii (651·7 ± 23·6 μm long) and a yet undescribed microfilaria, Cercopithifilaria sp. II (264·4 ± 20·2 μm long, with evident lateral alae). The morphological differentiation among the 3 species of dermal microfilariae was confirmed by differences in cytochrome c oxidase subunit 1 and ribosomal 12S sequences examined (mean level of interspecific pairwise distance of 11·4%, and 17·7%, respectively). Phylogenetic analyses were concordant in clustering these with other sequences of Cercopithifilaria spp. to the exclusion of Dirofilaria spp., Onchocerca spp. and Acanthocheilonema spp. Dermal microfilariae collected (n = 132) were morphologically identified as Cercopithifilaria sp. I (n = 108, 81·8%), Cercopithifilaria sp. II (n = 17, 12·9%), whereas only 7 (5·3%) were identified as C. grassii. Mixed infestations were detected in all sites examined. The great diversity of these neglected filarioids in dogs is of biological interest, considering the complex interactions occurring among hosts, ticks and Cercopithifilaria spp. in different environments.

Despite being the most prevalent nematode infections of man in Western Europe and North America, our knowledge of the genetic variability in Enterobius vermicularis is fragmented. We here report on a genetic study of pinworms in Denmark, performed using the cytochrome oxidase I (cox1) gene, with DNA extracted from individual eggs collected from clinical (human) samples. We collected cellophane-tape-test samples positive for pinworm eggs from 14 Departments of Clinical Microbiology in Denmark and surface-sterilized the eggs using a 1% hypochlorite solution before performing conventional PCR. Twenty-two haplotypes were identified from a total of 58 Danish patients. Cluster analysis showed that all Danish worms grouped together with human samples from Germany and Greece and with samples from Japanese chimpanzees designated as ‘type B’. Analysis of molecular variance showed no significant difference or trends in geographical distribution of the pinworms in Denmark, and several haplotypes were identical or closely related to samples collected in Germany, Greece and Japan. However, worms from the 4 countries were found to belong to different populations, with Fst values in the range of 0·16 to 0·47. This study shows pinworms in Denmark to be a homogenous population, when analysed using the cox1 mitochondrial gene.

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.

Trophically-transmitted parasites generally need to undergo a period of development in the intermediate host before reaching infectivity. During this vulnerable period, manipulation of the host to reduce susceptibility to predation would be advantageous for parasites, because it increases the probability of surviving until infectivity and thus the probability of transmission. We tested this ‘predation suppression’ hypothesis in 2 parasite species that use copepods as first hosts: the tapeworm Schistocephalus solidus and the nematode Camallanus lacustris. In a series of prey choice experiments, we found that copepods harbouring uninfective, still-developing worm larvae were less frequently consumed by stickleback predators than uninfected copepods. The levels of predation suppression were similar in the two parasite species, suggestive of convergent evolution. Additionally, copepods harbouring 2 worms of a given species were not more susceptible to predation than those with 1 worm, suggesting that excessive larval parasite growth does not increase host susceptibility to predation. Our results support the idea that parasites can suppress intermediate host susceptibility to predation while uninfective, but we also note that the available studies suggest that this effect is weaker than the frequently observed enhancement of host predation by infective helminth larvae.

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) is an essential regulatory enzyme of glycolysis in helminths in contrast to its role in gluconeogenesis in their host. Previously we have reported that phytochemicals from Flemingia vestita (Family: Fabaceae), genistein in particular, have vermifugal action and are known to affect carbohydrate metabolism in the cestode, Raillietina echinobothrida. In order to determine the functional differences of PEPCK from the parasite and its avian host (Gallus domesticus), we purified the parasite enzyme apparently to homogeneity, and characterized it. The native PEPCK is a monomer with a subunit molecular weight of 65 kDa. The purified enzyme displayed standard Michaelis-Menten kinetics with Km value of 42·52 μM for its substrate PEP. The Ki for the competitive inhibitors GTP, GMP, ITP and IMP for the carboxylation reaction were determined and discussed. In order to identify putative modulators from plant sources, phytochemicals from F. vestita and Stephania glabra were tested on the purified PEPCK, which resulted in alteration of its activity. From our results, we hypothesize that PEPCK may be a potential target site for anthelmintic action.