We previously observed that physalins have immunomodulatory properties, as well as antileishmanial and antiplasmodial activities. Here, we investigated the anti-Trypanosoma cruzi activity of physalins B, D, F and G. We found that physalins B and F were the most potent compounds against trypomastigote and epimastigote forms of T. cruzi. Electron microscopy of trypomastigotes incubated with physalin B showed disruption of kinetoplast, alterations in Golgi apparatus and endoplasmic reticulum, followed by the formation of myelin-like figures, which were stained with MDC to confirm their autophagic vacuole identity. Physalin B-mediated alteration in Golgi apparatus was likely due to T. cruzi protease perturbation; however physalins did not inhibit activity of the trypanosomal protease cruzain. Flow cytometry examination showed that cell death is mainly caused by necrosis. Treatment with physalins reduced the invasion process, as well as intracellular parasite development in macrophage cell culture, with a potency similar to benznidazole. We observed that a combination of physalins and benznidazole has a greater anti-T. cruzi activity than when compounds were used alone. These results indicate that physalins, specifically B and F, are potent and selective trypanocidal agents. They cause structural alterations and induce autophagy, which ultimately lead to parasite cell death by a necrotic process.

Hirstiella spp. are common ectoparasites of captive green iguanas (Iguana iguana). Suggested treatments are empirical and some of them are of low efficacy and potentially toxic. The objective of this open-label study was to investigate the short-term efficacy and safety of a single application of 0·25% fipronil solution for the treatment of hirstiellosis. The skin of 50 green iguanas was thoroughly examined with the aid of bright light and magnifying lenses. A total of 21 iguanas were found to be infested, harbouring 1–24 mites (median: 5). All 35 mites collected from 17 iguanas were identified as Hirstiella sp. Both infested and non-infested lizards, sharing the same enclosure, were carefully wiped with 0·25% fipronil solution. The safety and the efficacy of the treatment were evaluated after 2 days in 47/50 (94%) and 7 days in 29/50 (58%) iguanas. Compared with pre-treatment levels, the parasitic load did not changed significantly on the second day but was significantly lower on day 7 (P = 0·006). No adverse reactions were noticed. Based on these results a single whole-body application of 0·25% fipronil solution can be considered a safe and effective treatment for the reduction of parasitic burden in captive green iguanas infested by Hirstiella sp. mites.

Cestodes of the genus Taenia are parasites of mammals, with mainly carnivores as definitive and herbivores as intermediate hosts. Various medium-sized cats, Lynx spp., are involved in the life cycles of several species of Taenia. The aim of the present study was to identify Taenia tapeworms in the Eurasian lynx (Lynx lynx) from Finland. In total, 135 tapeworms from 72 lynx were subjected to molecular identification based on sequences of 2 mtDNA regions, the cytochrome c oxidase subunit 1 and the NADH dehydrogenase subunit 1 genes. Available morphological characters of the rostellar hooks and strobila were compared. Two species of Taenia were found: T. laticollis (127 samples) and an unknown Taenia sp. (5 samples). The latter could not be identified to species based on mtDNA, and the rostellar hooks were short relative to those described among other Taenia spp. recorded in felids from the Holarctic region. In the phylogenetic analyses of mtDNA sequences, T. laticollis was placed as a sister species of T. macrocystis, and the unknown Taenia sp. was closely related to T. hydatigena and T. regis. Our analyses suggest that these distinct taeniid tapeworms represent a putative new species of Taenia. The only currently recognized definitive host is L. lynx and the intermediate host is unknown.

Glucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0·25 and 0·014 mm respectively. The rBmG6PD exhibited an optimum pH of 8·5 and temperature, 40 °C. Adenosine 5′ [γ-thio] triphosphate (ATP-γ-S), adenosine 5′ [β,γ-imido] triphosphate (ATP-β,γ-NH), adenosine 5′ [β-thio] diphosphate (ADP-β-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0·02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% β-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

The aim of the study was the construction of risk maps for exposure to Phlebotomus sergenti, the main vector of Leishmania tropica, with a view to identifying hot spots for the potential establishment of this parasite in the southwest of Europe. Data were collected on the presence/absence of this vector and the ecological and climatic characteristics of 662 sampling sites located in the southeast, centre and northeast of the Iberian Peninsula (south-western Europe). The environmental factors associated with the distribution of P. sergenti were determined. The best predictors for the presence of this dipteran were ‘altitude’, ‘land use’, ‘land surface temperature’, ‘aspect’, ‘adjacent land cover’, ‘absence of vegetation in wall’ and the ‘absence of PVC pipes in the drainage holes of retaining walls’. Risk maps for exposure to the vector were drawn up based on these variables. The validation of the predictive risk model confirmed its usefulness in the detection of areas with a high risk of P. sergenti being present. These locations represent potential hot spots for an autochthonous focus of L. tropica becoming established. The risk maps produced for P. sergenti presence revealed several areas in the centre and south of the Iberian Peninsula to be the most prone to this process, which would make it possible for the disease to enter south-western Europe.

The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.

Parasite-mediated apparent competition occurs when one species affects another through the action of a shared parasite. One way of controlling the parasite in the more susceptible host is to manage the reservoir host. Culling can cause issues in terms of ethics and biodiversity impacts, therefore we ask: can treating, as compared to culling, a wildlife host protect a target species from the shared parasite? We used Susceptible Infected Recovered (SIR) models parameterized for the tick-borne louping ill virus (LIV) system. Deer are the key hosts of the vector (Ixodes ricinus) that transmits LIV to red grouse Lagopus lagopus scoticus, causing high mortality. The model was run under scenarios of varying acaricide efficacy and deer densities. The model predicted that treating deer can increase grouse density through controlling ticks and LIV, if acaricide efficacies are high and deer densities low. Comparing deer treated with 70% acaricide efficacy with a 70% cull rate suggested that treatment may be more effective than culling if initial deer densities are high. Our results will help inform tick control policies, optimize the targeting of control methods and identify conditions where host management is most likely to succeed. Our approach is applicable to other host-vector-pathogen systems.

Recent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of ‘cysts’ produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

In Russia, both alveolar and cystic echinococcoses are endemic. This study aimed to identify the aetiological agents of the diseases and to investigate the distribution of each Echinococcus species in Russia. A total of 75 Echinococcus specimens were collected from 14 host species from 2010 to 2012. Based on the mitochondrial DNA sequences, they were identified as Echinococcus granulosus sensu stricto (s.s.), E. canadensis and E. multilocularis. E. granulosus s.s. was confirmed in the European Russia and the Altai region. Three genotypes, G6, G8 and G10 of E. canadensis were detected in Yakutia. G6 was also found in the Altai region. Four genotypes of E. multilocularis were confirmed; the Asian genotype in the western Siberia and the European Russia, the Mongolian genotype in an island of Baikal Lake and the Altai Republic, the European genotype from a captive monkey in Moscow Zoo and the North American genotype in Yakutia. The present distributional record will become a basis of public health to control echinococcoses in Russia. The rich genetic diversity demonstrates the importance of Russia in investigating the evolutionary history of the genus Echinococcus.

When investigating parasite–host dynamics in wild populations, a fundamental parameter to investigate is prevalence. This quantifies the percentage of individuals infected in the population. Investigating how prevalence changes over time and space can reveal interesting aspects in the parasite–host relationship in natural populations. We investigated the dynamic between a common avian parasite (Syngamus trachea) in a host metapopulation of house sparrows (Passer domesticus) on the coast of Helgeland in northern Norway. We found that parasite prevalence varied in both time and space. In addition, the parasite prevalence was found to be different between demographic groups in the local populations. Our results reveal just how complex the dynamic between a host and its parasite may become in a fragmented landscape. Although temperature may be an important factor, the specific mechanisms causing this complexity are not fully understood, but need to be further examined to understand how parasite–host interactions may affect the ecological and evolutionary dynamics and viability of host populations.

Habitat fragmentation can adversely impact biodiversity, although where remnant fragments of natural vegetation provide favourable conditions the negative effects of fragmentation may be mitigated. Host-parasite systems in fragmented areas have only recently been examined, with parasites generally showing higher prevalence and richness in fragments, mediated by changes in host density. However, the effect of fragmentation on parasite body size and fecundity remains poorly investigated. Thus, here we compared the body size and condition of a generalist rodent host, Rhabdomys pumilio and the body size of 2 common flea species between pristine natural areas and remnant fragments within agriculture areas. Host body length, weight and body condition values were significantly larger in fragments than in pristine natural vegetation. Listropsylla agrippinae fleas showed the same pattern, being significantly larger in fragments, while Chiastopsylla rossi fleas did not differ in size between fragments and natural areas. The differential response of the 2 flea species may reflect the strength of association between the host and parasite, with the former spending a greater proportion of its lifespan on the host. Therefore, in this study agriculture fragments provide better conditions for both an opportunistic rodent and a closely associated flea species.

Refugia-based treatment strategies aim to prolong anthelmintic efficacy by maintaining a parasite population unexposed to anthelmintics. Targeted selective treatment (TST) achieves this by treating only animals that will benefit most from treatment, using a determinant criterion (DC). We developed a mathematical model to compare various traits proposed as DC, and investigate impacts of TST and drenching frequency on sheep performance and anthelmintic resistance. Short term, decreasing the proportion of animals drenched reduced benefits of anthelmintic treatment, assessed by empty body weight (EBW), but decreased the rate of anthelmintic resistance development; each consecutive drenching had a reduced impact on average EBW and an increased impact on the rate of anthelmintic resistance emergences. The optimal DC was fecal egg count, maintaining the highest average EBW when reducing the proportion of animals drenched. Long-term, reducing the proportion of animals drenched had little impact on total weight gain benefits, across animals and years, whilst reducing drenching frequency increased it. Decreasing the frequency and proportion of animals drenched were both predicted to increase the duration of anthelmintic efficacy but reduce the total number of drenches administered before resistance was observed. TST and frequency of drenching may lead to different benefits in the short versus long term.

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.

A total of 20 749 bulk tank milk (BTM) samples was collected in November 2008 from all over Germany, corresponding to 20·9% of all German dairy herds. The BTM samples were analysed for antibodies against Fasciola hepatica using the excretory–secretory (ES) ELISA. A geospatial map was drawn to show herd prevalences per postal code area. Various spatial risk factors were tested for potential statistical associations with the ELISA results in logistic regression supported by a geographical information system (GIS). The mean seroprevalence was 23·6% and prevalences in different German federal states varied between 2·6% and 38·4%. GIS analysis revealed statistically significant positive associations between the proportion of grassed area and water bodies per postal code area and positive BTM ELISA results. This can be explained by the biology of the intermediate host, the amphibious snail Galba (Lymnea) truncatula and the pasture-borne nature of fasciolosis. The full logistic regression model had a Pseudo-R2 of 22%, while the final model obtained by controlled stepwise model building revealed a Pseudo-R2 of 14%, indicating that additional, unrecorded factors and random effects contributed substantially to the occurrence of positive ELISA results. Considering the high seroprevalences in some areas and the economic impact of fasciolosis, farmers and veterinarians should be strongly advised to implement effective liver fluke control programmes.

In the present study we detected Schellackia haemoparasites infecting the blood cells of Lacerta schreiberi and Podarcis hispanica, two species of lacertid lizards from central Spain. The parasite morphometry, the presence of a refractile body, the type of infected blood cells, the kind of host species, and the lack of oocysts in the fecal samples clearly indicated these blood parasites belong to the genus Schellackia. Until now, the species of this genus have never been genetically characterized and its taxonomic position under the Lankesterellidae family is based on the lack of the exogenous oocyst stage. However, the phylogenetic analysis performed on the basis of the 18S rRNA gene sequence revealed that species of the genus Schellackia are clustered with Eimeria species isolated from a snake and an amphibian species but not with Lankesterella species. The phylogenetic analysis rejects that both genera share a recent common ancestor. Based on these results we suggest a revision of the taxonomic status of the family Lankesterellidae.

The parasitic roundworms Strongyloides stercoralis (in man) and Strongyloides ratti (in rats) employ environmentally controlled XX/XO sex determination with a pair of X chromosomes and two pairs of autosomes. Strongyloides papillosus (in sheep) has only two pairs of chromosomes, one of which combines the genetic material homologous to the S. ratti chromosomes X and I. This species creates males through the elimination of one copy of the portion related to the X chromosome (chromatin diminution). It is not clear which one of these two sex-determining mechanisms is ancestral. We demonstrate that Strongyloides vituli (in cattle) has two pairs of chromosomes like its very close relative S. papillosus whereas Parastrongyloides trichosuri, a closely related out-group to Strongyloides spp. in Australian brushtail possums, has three chromosome pairs and employs XX/XO sex determination. The X chromosome of P. trichosuri is homologous to the X chromosome of S. ratti. Our data strongly suggest that the last common ancestor of Strongyloides spp. and Parastrongyloides spp. had two pairs of autosomes along with two or one X chromosome in females and males, respectively. The situation with two pairs of chromosomes is likely derived and occurred through the fusion of the X chromosome with an autosome.

Aggregated distributions among individual hosts are a defining feature of metazoan parasite populations. Heterogeneity among host individuals in exposure to parasites or in susceptibility to infection is thought to be the main factor generating aggregation, with properties of parasites themselves explaining some of the variability in aggregation levels observed among species. Here, using data from 410 samples of helminth parasites on fish hosts, I tested the contribution of (i) within-sample variation in host body size, taken as a proxy for variability in host susceptibility, and (ii) parasite taxon and developmental stage, to the aggregated distribution of parasites. Log-transformed variance in numbers of parasites per host was regressed against log mean number across all samples; the strong relationship (r2 = 0·88) indicated that aggregation levels are tightly constrained by mean infection levels, and that only a small proportion of the observed variability in parasite aggregation levels remains to be accounted for by other factors. Using the residuals of this regression as measures of ‘unexplained’ aggregation, a mixed effects model revealed no significant effect of within-sample variation in host body size or of parasite taxon or stage (i.e. juvenile versus adult) on parasite aggregation level within a sample. However, much of the remaining variability in parasite aggregation levels among samples was accounted for by the number of individual hosts examined per sample, and species-specific and study-specific effects reflecting idiosyncrasies of particular systems. This suggests that with most differences in aggregation among samples already explained, there may be little point in seeking universal causes for the remaining variation.

Behavioural alterations induced by parasites in their intermediate hosts can spatially structure host populations, possibly resulting in enhanced trophic transmission to definitive hosts. However, such alterations may also increase intermediate host vulnerability to non-host predators. Parasite-induced behavioural alterations may thus vary between parasite species and depend on each parasite definitive host species. We studied the influence of infection with 2 acanthocephalan parasites (Echinorhynchus truttae and Polymorphus minutus) on the distribution of the amphipod Gammarus pulex in the field. Predator presence or absence and predator species, whether suitable definitive host or dead-end predator, had no effect on the micro-distribution of infected or uninfected G. pulex amphipods. Although neither parasite species seem to influence intermediate host distribution, E. truttae infected G. pulex were still significantly more vulnerable to predation by fish (Cottus gobio), the parasite's definitive hosts. In contrast, G. pulex infected with P. minutus, a bird acanthocephalan, did not suffer from increased predation by C. gobio, a predator unsuitable as host for P. minutus. These results suggest that effects of behavioural changes associated with parasite infections might not be detectable until intermediate hosts actually come in contact with predators. However, parasite-induced changes in host spatial distribution may still be adaptive if they drive hosts into areas of high transmission probabilities.

Separation of pig slurry into solid and liquid fractions is gaining importance as a way to manage increasing volumes of slurry. In contrast to solid manure and slurry, little is known about pathogen survival in separated liquid slurry. The viability of Ascaris suum eggs, a conservative indicator of fecal pollution, and its association with ammonia was investigated in separated liquid slurry in comparison with raw slurry. For this purpose nylon bags with 6000 eggs each were placed in 1 litre bottles containing one of the two fractions for 308 days at 5 °C or 25 °C. Initial analysis of helminth eggs in the separated liquid slurry revealed 47 Ascaris eggs per gramme. At 25 °C, egg viability declined to zero with a similar trend in both raw slurry and the separated liquid slurry by day 308, a time when at 5 °C 88% and 42% of the eggs were still viable in separated liquid slurry and raw slurry, respectively. The poorer survival at 25 °C was correlated with high ammonia contents in the range of 7·9–22·4 mm in raw slurry and 7·3–23·2 mm in liquid slurry compared to 3·2–9·5 mm in raw slurry and 2·6–9·5 mm in liquid slurry stored at 5 °C. The study demonstrates that at 5 °C, A. suum eggs have a higher viability in separated liquid slurry as compared to raw slurry. The hygiene aspect of this needs to be further investigated when separated liquid slurry is used to fertilize pastures or crops.

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.