Immunohistochemical analysis of skin and draining lymph nodes of sheep repeatedly infested with the ixodid tick Hyalomma anatolicum anatolicum were studied for different antigen-presenting cells and lymphocyte subpopulations. Infiltration of neutrophils, macrophages and lymphocytes adjacent to the tick bite site were observed. Skin biopsies showed significant increases in dermal infiltration of CD8+ and γδ+ T cells at 72 h and 8 days after both primary and secondary infestation. Infiltrations of MHC-II DR/DQ decreased at 72 h after tick infestation, whereas significant increases were recorded for 8-day skin biopsies. CD1+ cellular infiltrations were observed during secondary infestations at the dermis. Decreased ratios of CD4[ratio ]CD8 T cells and MHC-II[ratio ]CD1 antigen-presenting cells were observed in both infestations compared to healthy skin biopsies. Ratios of αβ[ratio ]γδ T cells increased gradually during infestation compared to uninfested skin. The regional lymph nodes from tick-infested sheep showed an increased CD8+, γδ+ T and CD1+ cellular infiltration compared to control lymph nodes. CD4+ T cells were decreased. There were no significant changes in CD45R+ cellular infiltration either at skin lesions or regional lymph nodes.

In this study the anti-leishmanial activity and anti-microtubule effects of paclitaxel, trifluralin and a combination of paclitaxel and trifluralin have been tested in a wild type and sodium arsenite-resistant strain of Leishmania donovani. Both paclitaxel and trifluralin have been shown to be effective in limiting parasite growth. Specific alterations in morphology, tubulin polymerization dynamics, post-translational modifications and cellular distribution of the tubulins have been confirmed to be a part of the intracellular anti-microtubule-events that occur in arsenite-resistant L. donovani in response to these agents, ultimately leading to death of the parasite. DNA analyses of the drug-treated wild type and arsenite-resistant strains revealed an apoptosis-like death in response to paclitaxel and the combination but not to trifluralin. Data provide valuable information for further development of chemotherapeutic strategies based on anti-microtubule agents against drug resistant Leishmania parasites.

To investigate the prevalence of a flea-borne protozoan (Trypanosoma (Herpetosoma) microti) in its field vole (Microtus agrestis) host, we monitored over a 2-year period a range of intrinsic and extrinsic parameters pertaining to host demographics, infection status and vector (flea) prevalence. Generalized Linear Mixed Modelling was used to analyse patterns of both flea and trypanosome occurrence. Overall, males of all sizes and ages were more likely to be infested with fleas than their female counterparts. Flea prevalence also showed direct density dependence during the winter, but patterns of density dependence varied amongst body mass (age) classes during the summer. Trypanosome prevalence did not vary between the sexes but was positively related to past flea prevalence with a lag of 3 months, with the highest levels occurring during the autumn season. A convex age-prevalence distribution was observed, suggesting that individuals develop a degree of immunity to trypanosome infection with age and exposure. An interaction between age and whether the individual was new or recaptured suggested that infected animals are less likely to become territory holders than their uninfected counterparts.

Aggregation is one of the distinctive features in parasite-host relationships, which has generally been studied at the level of host communities. Parasite aggregation at the infracommunity level may nevertheless be important for intraspecific interactions such as parasite mating success and opportunities for cross-fertilization. In the present paper, we studied the infracommunity aggregation of 3 highly abundant Dactylogyrus (Monogenea) species occurring on the gills of crucian carp (Carassius carassius). In line with the previous work on monogenean communities, we observed no competition between the species. At the species level, parasites were distributed unevenly on the gills showing aggregation in the majority of infracommunities. However, aggregation decreased with increasing parasite abundance, which supports the hypothesis that less aggregation may be needed to ensure successful mating when the distance to a potential mate decreases with increasing number of conspecifics. Lack of interspecific interactions, species specific site selection and the importance of study scale for aggregation patterns in dactylogyrids are discussed.

Over the last decade the need for new strategies and compounds to control parasitic helminths has become increasingly urgent. The neuromuscular systems of these worms have been espoused as potential sources of target molecules for new drugs which may address this need. One facet of helminth neuromuscular biology which has garnered considerable research interest is that of neuropeptidergic neurotransmission, particularly regarding parasites of humans and animals, as well as free-living nematode model species. This research interest has been piqued by the fact that neuropeptides have been demonstrated to be fundamentally important to nematode biology and thus may be of utility in this search for new drug targets. This review focuses on the neuropeptide biology of plant parasitic nematodes, a subject which has been comparatively neglected despite the fact that the search for alternative control measures also extends to these economically important parasites. We focus on the FMRFamide-like peptide (FLP) neuropeptides and the complexity and distribution of this peptide family in plant parasitic nematodes. Possible roles for FLPs in plant parasitic nematode behaviour, as elucidated by a combination of molecular imaging techniques and RNA interference (RNAi), are discussed. We propose that disruption of FLP neurosignalling in plant parasitic nematodes represents a novel form of pest control and speculate as to how this may be achieved.

We investigated variation in the abundance-prevalence relationships of fleas among 17 different flea-host associations as well as among different species of hosts and fleas in the Negev desert. We explored variation in the value of exponent of Taylor's power relationship with changes in flea community size and flea specialization (host specificity and seasonal pattern of activity). We tested if a simple epidemiological model can reproduce the pattern of the abundance-prevalence relationship. We confirmed aggregated distribution of fleas within a population of host species as well as across a whole host community and the existence of a positive relationship between local flea abundance and their prevalence. Prevalence, mean abundance and variance of abundance were significantly higher in host specific than host opportunistic fleas. When ecological specialization was considered, based on a seasonal pattern of activity, these parameters were higher in year-round-active than seasonal fleas. The degree of flea specialization and flea community richness affected the pattern of the relationship between mean abundance and its variance. Power law slopes decreased with increasing richness of flea community. A simple epidemiological model based on mean flea abundance and degree of aggregation, corrected for host sample size, can predict the observed pattern of prevalence. In some cases, observed flea prevalence was higher than that predicted from the epidemiological model. The discrepancy of the observed prevalence from that predicted by the model can be explained by either a relatively low negative effect of flea parasitism on a host (at least, in terms of pathology) or strong resistance of a host to flea parasitism or both.

This study established the mechanisms by which Microphallus primas cercariae penetrate the crab Carcinus maenas in which they form metacercarial cysts. Light and electron microscopy were used to investigate cercarial features and to follow the fate of cercariae released in close proximity to crabs. It was shown that cercariae were carried in respiratory currents into crabs' branchial chambers where each enveloped itself in a transparent penetration cyst on the gill lamellae. When cercariae were present the number of respiratory current reversals performed by crabs increased. Using an ‘artificial branchial chamber’ it was possible to observe how cercariae attached to crab gills during breaks in current flow that preceded each current reversal. Inside the penetration cysts the now tail-less larvae used their stylets to pierce holes through which they levered themselves into underlying haemolymph channels in the gills. Histochemical tests demonstrated that the penetration cysts were products of glands in the cercariae and that penetration of the crabs was achieved by mechanical means. The importance of crab respiratory current reversals to the success of cercarial penetration is discussed as it represents the exploitation by a parasite of a host behavioural response to an unrelated stimulus.

Trichomonas vaginalis is a parasitic protozoan that causes trichomonosis, a sexually-transmitted disease, with serious sequelae to women and men. As the host–parasite relationship is complex, it is important to investigate biochemical aspects of the parasite that contribute to our understanding of trichomonal biology and pathogenesis. Nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1), which hydrolyses extracellular ATP and ADP, and ecto-5′-nucleotidase, which hyrolyses AMP, have been characterized in laboratory isolates of T. vaginalis. Here we show that the extracellular ATP[ratio ]ADP hydrolysis ratio varies among fresh clinical isolates, which presented higher ATPase and ADPase activities than long-term-grown isolates. Growth of parasites in iron-replete and iron-depleted medium resulted in different, albeit minor, patterns in extracellular ATP and ADP hydrolysis among isolates. Importantly, some isolates had low or absent ecto-5′-nucleotidase activity, regardless of environmental conditions tested. For isolates with ecto-5′-nucleotidase activity, high- and low-iron trichomonads had increased and decreased levels of activity, respectively, compared to organisms grown in normal TYM-serum medium. This suggests a regulation in expression of either the enzyme amounts and/or activity under the control of iron. Finally, we found no correlation between the presence or absence of dsRNA virus infection among trichomonad isolates and NTPDase and ecto-5′-nucleotidase activities.

Since its early introduction as a marker for the immunodiagnosis of hydatid disease, antigen 5 (Ag5) has been regarded as one of the more relevant antigens of Echinococcus granulosus, and it is still widely used in different confirmation techniques. In this work we prepared 2 recombinant forms of the antigen, namely, rAg5 (corresponding to the unprocessed polypeptide chain of the antigen) and rAg5-38s (corresponding to its 38 kDa subunit). Their antigenicities were compared to that of the native antigen using a human serum collection. There was a major drop in the reactivity of the sera, particularly against rAg5-38s, which was confirmed by analysis of the cross-reactivity of 2 panels of monoclonal antibodies specific for rAg5-38s and the native antigen. Using the chemically deglycosylated native antigen, we demonstrated that the reduced antigenicity of the recombinants is due to the loss of the sugar determinants, and not to their misfolding. Inhibition experiments using phosphorylcholine confirmed that this moiety also contributes to the reactivity of the antigen, but to a much lesser extent. The presence of immunodominant highly cross-reactive glycan moieties in the Ag5 molecule may involve a parasite evasion mechanism.

The goal of this work is to report a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors. A great reduction of proteolytic activity in the insoluble chloroform/methanol and heated amoebic fraction (IC[ratio ]MC) was obtained by this method, even in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. The substrates azo-casein and the hide powder azure were used to determine the reduction of proteolytic activity of IC[ratio ]MC compared with the activity of crude extract and crude extract with iodoacetamide. The IC[ratio ]MC SDS-PAGE pattern shows a higher quantity of bands than extract with the inhibitor iodoacetamide or than crude extract. In addition, anti-Entamoeba histolytica antibodies from amoebic liver abscess patients recognized a richer antigenic Western blot pattern in the IC[ratio ]MC fraction than in crude extract alone or with inhibitor. The described method has proved to be suitable to preserve amoebic antigens for its use in diagnostic tests and it can be used for immunological response studies against E. histolytica antigens. Furthermore we propose that this method to obtain the IC[ratio ]MC fraction can be applied for the study of other microorganisms or cells with high enzymatic content.

We examined the relative merits of mitochondrial DNA loci and ribosomal DNA internal transcribed spacers for their use in prospecting for cryptic species of platyhelminth parasites. Sequence divergence at ITS1 and ITS2 was compared with divergence at 2 mtDNA loci (NADH dehydrogenase-1 and cytochrome c oxidase I) between closely related species of trematodes and cestodes. Both spacers accumulated substitutions substantially more slowly than mtDNA, which clearly shows a higher level of divergence among species relative to intra-specific variation. Besides a slow rate of substitution, other caveats that may be encountered when using ITS sequences as a prospecting marker are discussed. In particular, we note recent studies that suggest the existence of substantial levels of intra-individual variation in ITS sequences of flatworms. Because it is likely that closely related species share this phenomenon, it may confound the detection of cryptic species, especially if small sample sizes are studied. Although potential limitations of mtDNA are also recognized, the higher rate of evolution and smaller effective population size of this marker increases the probability of detecting diagnostic characters between cryptic species.

Accurate identification of aspermic Fasciola forms in Japan remains difficult because of their morphological variations. In order to characterize the forms genetically, nucleotide sequences of ribosomal internal transcribed spacer (ITS1 and ITS2) and mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NDI) genes in 34 liver flukes from 16 prefectures in Japan were analysed. Two major forms represented by Fsp 1 and Fsp 2 had sequences identical to or closely resembling those of F. hepatica and F. gigantica, respectively, in all the 4 DNA markers and were mainly distributed in northern and eastern-western parts of Japan, respectively. Fsp 1 and Fsp 2 would have been introduced into Japan with infected cattle of 2 distinct lineages via the Korean Peninsula and spread through limited parts of Japan (northern and eastern-western parts) together with the movement of each cattle lineage. The Japanese form (Fsp 1/2), which showed heterozygosity in ribosomal DNA and Fsp 2 haplotype in mitochondrial DNA, may have originated in interspecific cross hybridization between paternal F. hepatica and maternal F. gigantica.

Flatworm, nematode and arthropod parasites have proven their ability to develop resistance to currently available chemotherapeutics. The heavy reliance on chemotherapy and the ability of target species to develop resistance has prompted the search for novel drug targets. In view of its importance to parasite/pest survival, the neuromusculature of parasitic helminths and pest arthropod species remains an attractive target for the discovery of novel endectocide targets. Exploitation of the neuropeptidergic system in helminths and arthropods has been hampered by a limited understanding of the functional roles of individual peptides and the structure of endogenous targets, such as receptors. Basic research into these systems has the potential to facilitate target characterization and its offshoots (screen development and drug identification). Of particular interest to parasitologists is the fact that selected neuropeptide families are common to metazoan pest species (nematodes, platyhelminths and arthropods) and fulfil specific roles in the modulation of muscle function in each of the three phyla. This article reviews the inter-phyla activity of two peptide families, the FMRFamide-like peptides and allatostatins, on motor function in helminths and arthropods and discusses the potential of neuropeptide signalling as a target system that could uncover novel endectocidal agents.

Anthelmintic bioactivity against gastrointestinal nematodes has been associated with leguminous forages supporting the hypothesis of a role of condensed tannins. However, the possibility that other compounds might also been involved has received less consideration. Using bio-guided fractionation, the current study aimed at characterizing the biochemical nature of the active compounds present in sainfoin (Onobrychis viciifolia), previously identified as an anthelmintic leguminous forage. The effects of sainfoin extracts were evaluated on 3rd-stage larvae (L3) of Haemonchus contortus by using a larval migration inhibition (LMI) assay. Comparison of extracts obtained with several solvent systems showed that the bioactivity was associated with the 70[ratio ]30 acetone/water extract. Further fractionation of the later allowed the separation of phenolic compounds. By use of a dialysis method, compounds were separated with a molecular weight cut-off of 2000 Da. The in vitro anthelmintic effect of the fraction with condensed tannins was confirmed. In the fraction containing molecules of MW <2000 Da, 3 flavonol glycosides were identified as rutin, nicotiflorin and narcissin. At 1200 μg/ml, each inhibited significantly the migration of larvae. Addition of polyvinyl pyrrolidone (PVPP) to both fractions before incubation restored larval migration. These results confirmed the role of both tannins and flavonol glycosides in the anthelmintic properties of sainfoin.

In order to reveal the mechanisms underlying nurse cell formation during Trichinella spiralis infection, the expression of the factors of tumor necrosis factor-alpha (TNF-α)/TNF receptor 1 (TNFR-1) signalling pathway mediating apoptosis was investigated. The analysed factors included TNF-α, TNFR-1, TNF receptor-associated death-domain (TRADD), caspase 3, caspase 8, TNF receptor associated factor-2 (TRAF2) and receptor interactive protein (RIP), all of which are involved in the TNF-α/TNFR-1 signalling pathway-mediated apoptosis. The quantitative RT-PCR indicated that the infected muscle tissues up-regulate the expression of pro-apoptosis genes (TNF-α, TNFR-1 and TRADD, caspase 3 and caspase 8), and anti-apoptosis genes (TRAF2 and RIP) at the beginning of cyst formation. The expression returned to the normal level after cyst formation. The quantitative RT-PCR analysis of mRNA from tissue samples isolated by laser capture micro-dissection confirmed that the up-regulation of these genes was restricted in infected muscle cells, was not in the inflammation cells around infected muscle cells nor in normal muscle cells. The in situ localization study of pro-apoptosis (TRADD, caspase 3) and anti-apoptosis gene products (TRAF2) indicated that these were expressed in the basophilic cytoplasm (infected muscle cell origin) of the nurse cells. Thus the present study suggests that the TNF-α/TNFR-1 signalling pathway is involved in nurse cell formation.

Paleohaemoproteus burmacis gen. n., sp. n. (Haemospororida: Plasmodiidae) is described from the abdominal cavity of a female biting midge (Diptera: Ceratopogonidae) preserved in 100 million year old amber from Myanmar (Burma). The description is based on the developmental stages of oocysts and sporozoites. The fossil species differs from extant species of Haemoproteus by its wide range of oocyst sizes, small sporozoites and occurrence in an extinct species of biting midge. Numerous sporozoites in the abdominal cavity suggest that the biting midge was an effective vector of this malarial parasite. Characters of the biting midge suggest that the host was a large, cold-blooded vertebrate. This is the earliest record of a malaria parasite and first indication that Early Cretaceous reptiles were infected with haemosporidial parasites.

The relationships between populations of the Deer Mouse (Peromyscus maniculatus) and the White-footed Mouse (P. leucopus) and their respective Cuterebra parasites were examined. Population genetic structure of hosts and parasites was inferred using cytochrome oxidase mitochondrial sequences of specimens from 7 populations. Genetic analyses revealed that isolation-by-distance applies for P. maniculatus and its associated parasite (C. grisea). A significant correlation was also observed between the genetic distances of these host and parasite species. Furthermore, populations of P. maniculatus and C. grisea from the North and South shores of the St Lawrence River were found to be significantly different. This structure may be explained by the St Lawrence River being a dispersal barrier for both species. A robust analysis of the other species pair (P. leucopus and C. fontinella) could not be performed because of limited sample sizes.

Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.

Strongyloides ratti is a parasitic nematode of rats. The host immune response against S. ratti affects the development of its free-living generation, favouring the development of free-living adult males and females at the expense of directly developing, infective 3rd-stage larvae. However, how the host immune response brings about these developmental effects is not clear. To begin to investigate this, we have determined the effect of non-immune stresses on the development of S. ratti. These non-immune stresses were subcurative doses of the anthelmintic drugs Ivermectin, Dithiazanine iodide and Thiabendazole, and infection of a non-natural host, the mouse. These treatments produced the opposite developmental outcome to that of the host immune response. Thus, in infections treated with subcurative doses of Ivermectin, Dithiazanine iodide and in infections of a non-natural host, the sex ratio of developing larvae became more female-biased and the proportion of female larvae that developed into free-living adult females decreased. This suggests that the mechanism by which the host immune response and these non-immune stresses affect S. ratti development differs.

Thelazia callipaeda, commonly known as the ‘oriental eyeworm’, has been recently reported in Italy and other European countries. The insect/s that act as intermediate hosts and details of larval development inside the vector remain unclear. In order to (1) demonstrate the species of fly that may act as vector/s for T. callipaeda in southern Italy (Site A) and China (Site B) and (2) describe the larval development of the nematode in the body of flies, 847 Phortica (Drosophilidae) flies were collected from the above two sites, each with a history of human and/or canine thelaziosis. Flies were identified as Phortica variegata (245 – site A) and Phortica okadai (602 – site B), experimentally infected by 1st-stage larvae (L1), kept at different temperatures and dissected daily until day 180 post-infection (p.i.). Dead flies from site A were subjected to specific polymerase chain reaction (PCR) assay to detect T. callipaeda. To demonstrate the role of Phortica as vectors of T. callipaeda, 3rd-stage larvae (L3) recovered from the proboscis of flies were deposited onto the cornea of the eyes of dogs and rabbits. Following dissection, 3 (2·9%) of P. variegata in site A were found to be infected by L3 in the proboscis on days +14, +21 and +53 p.i., compared with 26 (18·4%) of Phortica flies recorded as being positive by PCR. Sequences from positive PCR products were 99% identical to sequences of the corresponding species available in GenBank (AY207464). At site B, 106 (17·6%) of 602 dissected P. okadai were found to be infected by T. callipaeda larvae (different stages) and in total 62 L3 were recovered from the proboscis of 34 (5·6%) flies. The shortest time in which L3 were found was at day +14, +17, +19, and +50 p.i. respectively, depending on the environmental temperatures. Of 30 flies overwintered for 6 months, 6 L3 were detected at day +180 p.i. in 3 flies (10%). The biology of larval development was reconstructed on the basis of the dissection of 602 P. okadai-infected flies and the morphology of larval stages in the insect body described. The present work provides evidence that P. variegata and P. okadai act as vectors for T. callipaeda in southern Europe and in China, respectively. The phenomenon of overwintering is described here for the first time for T. callipaeda and discussed. Finally, the relationship between T. callipaeda and its fly vector is considered in light of disease prophylaxis and to model its dissemination into habitats and environments favourable to Phortica flies.