Hookworms feed on blood, utilizing haemoglobin for nutrition, growth and reproduction. The haemoglobin digestion cascade has been partially elucidated, but the process immediately preceding this event, haemolysis, has received considerably less attention. We have cloned and expressed Ancylostoma caninum mRNAs encoding 2 proteins belonging to the saposin-like protein (SAPLIP) family, termed Ac-slp-1 and Ac-slp-2. The open reading frames of SLP-1 and SLP-2 were used to identify expressed sequence tags encoding SAPLIPs from the 4 major clades of animal parasitic nematodes. Both Ac-slp-1 and slp-2 mRNAs were shown to be expressed in all life stages assessed, with slp-1 predominantly being expressed in third-stage larvae (L3) before and after activation with dog serum. Recombinant SLP-1 and SLP-2 were expressed in insect cells and used to raise specific antisera in mice. These antisera were used as probes in fluorescence microscopy to localize the anatomic expression sites of both proteins to small, punctate organelles or vesicles within the intestinal cells of adult worms; weak staining was detected on the microvillar brush border of the intestine. Using transmission electron microscopy, both proteins were localized to similar vesicles in the intestinal cells of the L3. Recombinant proteins contained C-terminal purification tags that potentially precluded dimerization and possibly interfered with the subsequent detection of haemolytic activity. Their expression in the gut of the L3 and adult stages suggests a role for these hookworm SAPLIPs in the lysis of host cells during tissue migration and/or feeding.

Procyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in ΔScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of ΔScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in ΔScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

Expansion of human activities frequently results in habitat fragmentation, a phenomenon that has been widely recognized in the last decades as one of the major threats to world's biodiversity. The transformation of a continuous forest into a fragmented area results in a hyper-dynamic landscape with unpredictable consequences to overall ecosystem health. The effect of the fragmentation process on Trypanosoma cruzi infection among small wild mammals was studied in an Atlantic Rain Forest landscape. Comparing continous forest to fragmented habitat, marsupials were less abundant than rodents in the continuous landscape. An overall decrease in small wild mammal richness was observed in the smaller fragments. An anti-T. cruzi seroprevalence of 18% (82/440) was deteced by immunofluorescence assay. Moreover, this seroprevalence was higher in the fragmented habitat than in the continuous forest. According to the collected data, 3 main factors seem to modulate infection by T. cruzi in small wild mammals: (i) habitat fragmentation; (ii) biodiversity loss; (iii) increase of marsupial abundance in mammal communities. Furthermore, an extremely mild controlled infection by T. cruzi was detected since no patent parasitaemia could be detected in fresh blood samples, and no parasites were isolated by haemoculture.

The monogenean ectoparasite, Gyrodactylus salaris Malmberg, 1957, has had a devastating effect on wild Atlantic salmon (Salmo salar) since its introduction to Norway in the mid-1970s. In Lake Pålsbufjorden, southern Norway, upstream of the stretches of the River Numedalslågen with anadromous Atlantic salmon, a resident Arctic charr (Salvelinus alpinus) population has been reported to be infected with G. salaris which is viable in the absence of its normal host, the Atlantic salmon. Currently, there is no record of G. salaris infecting Atlantic salmon in the downstream sections of the River Numedalslågen. We studied experimentally the infectivity and reproductive capacity of G. salaris from Lake Pålsbufjorden on wild and hatchery-reared Atlantic salmon as well as on Arctic charr and rainbow trout (Oncorhynchus mykiss). Arctic charr and rainbow trout were moderately susceptible, whereas the Atlantic salmon stocks from River Numedalslågen and River Drammenselva were innately resistant to only slightly susceptible. Thus, the G. salaris from Arctic charr in Lake Pålsbufjorden is considered non-pathogenic to Atlantic salmon. This is the first observation of variation in host preference among Norwegian G. salaris populations. The observed differences in virulence between G. salaris populations could have important consequences for the international legislation and management of Atlantic salmon.

A deterministic, dynamic simulation model is developed to account for the interactions between gastrointestinal parasitism and host nutrition, and predict their consequences on performance and level of parasitism of sheep. Larval intake and established adult worms are assumed to result in nutrient loss for the host. In order to reduce this loss the host will mount an immune response, which will affect the establishment rate of incoming larvae, mortality rate of adult worms, and fecundity of female worms, as well as nutrient loss caused by larval intake per se. Host anorexia is modelled as a function of worm mass. Parasitism is also assumed to affect the allocation of ingested nutrients to the host's bodily functions, with maintenance getting absolute priority, and protein allocated to immunity and production proportionally to their requirements. Inputs to the model include the expected growth attributes of the animal, feed quality, various parasitological parameters and daily larval intake. Outputs include feed intake, growth rate and body composition, as well as worm burden and faecal egg counts. The model allows exploration of the consequences of gastrointestinal parasitism on sheep of different growth characteristics, kept under environments that vary in the provision of nutrients and exposure to parasites.

Gyrodactylus salaris is a serious pest of wild pre-smolt Atlantic salmon (Salmo salar) in Norway. The closely related G. thymalli, originally described from grayling (Thymallus thymallus), is assumed harmless to both grayling and salmon. The 2 species are difficult to distinguish using traditional, morphometric methods or molecular approaches. The aim of this study was to explore whether there is a consistent pattern of morphometrical variation between G. salaris and G. thymalli and to analyse the morphometric variation in the context of ‘diagnostic realism’ (in natural populations). Specimens from the type-material for the 2 species are also included. In total, 27 point-to-point measurements from the opisthaptoral hard parts were used and analysed by digital image processing and uni- and multivariate morphometry. All populations most closely resembled its respective type material, as expected from host species, with the exception of G. thymalli from the Norwegian river Trysilelva. We, therefore, did not find clear support in the morphometrical variation among G. salaris and G. thymalli for an a priori species delineation based on host. The present study also indicates an urgent need for more detailed knowledge on the influence of environmental factors on the phenotype of gyrodactylid populations.

Trichinella spiralis is the nematode causative agent of trichinellosis, an intestinal and tissular parasitosis. Even though an early diagnosis during the intestinal phase is essential to limit the infection in humans, to date, there are no available tests to achieve this goal. Based on the immune response generated by the host's intestinal mucosa, the aim of this work was to develop ELISAs to assess the presence of coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes in stool samples of 18 individuals belonging to different outbreaks that have arisen in Argentina. By the methodologies developed in this work it was found that anti-muscle larva excretory-secretory products (ML-ESP) CAbs were detected in 89% of the samples analysed regardless of the time p.i. Anti-ML-ESP IgA, IgG, IgE and IgM were detected in 56%, 56%, 28% and 22% of the individuals respectively. Those samples negative for anti-ML-ESP total immunoglobulins proved positive for anti-adult worm-ESP CAbs. No CAgs were detected in any of the samples. The results obtained in this work indicate that the intestinal immune response in human trichinellosis is featured by all the isotypes of specific immunoglobulins. Furthermore, the detection of antibodies in stool samples, in either the free or complexed form, could be applied to confirm early human trichinellosis.

Newborn larvae (NBL) and adult (Ad) stage-specifically expressed genes or members of gene families of Trichinella spiralis were identified by suppression subtractive hybridization (SSH)†. Six cDNA clones were identified as NBL stage-specific, including 1 member of the T. spiralis gene family encoding glutamic acid-rich proteins, 2 clones encoding novel serine proteases, 2 closely related clones encoding proteins that are members of a deoxyribonuclease II (DNase II)-like family and 1 clone with no similarity to known genes. Four stage-specific clones encoding homologues of retinoid X receptor, caveolin, C2H2 type zinc finger protein and a putative protein with no homology to known sequences were obtained from 3-day-old adult worms. One gene specifically up-regulated in the 5-day-old adult worms encoding a putative cuticle collagen was also identified.

Specimens of Contracaecum spp. from Phalacrocorax auritus and Pelecanus occidentalis from Florida were characterized by sequencing of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) and by PCR-based RFLP analysis of the same gene and of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Analyses of the rrnS sequence data using the MP and UPGMA approaches yielded trees with similar topologies, delineating 3 main clusters. Specimens from Ph. auritus, morphologically assigned to C. rudolphii (s.l.), were part of the cluster comprising also the other 2 species of the C. rudolphii complex (A and B), but representing a genetically distinct group, potentially corresponding to a distinct lineage within the complex, provisionally named as C. rudolphii C. The second cluster comprised 5 individuals from P. occidentalis, which formed a genetically relatively homogeneous group. The rrnS data indicate that these specimens (indicated as Contracaecum sp. 1) are clearly genetically different from the morphologically most closely related species, i.e. C. rudolphii (s.l.) and C. microcephalum, and could represent a new species. The third cluster comprised 7 specimens from P. occidentalis morphologically assigned to C. multipapillatum (s.l.). These were shown to be genetically homogeneous and related to but quite distinct from C. multipapillatum from Greece, although additional studies are needed to assess their status. PCR-RFLP based markers for the quick identification of these taxa are provided.

The objective of this experiment was to investigate the potential influence of inulin on the establishment of new and patent infections of Trichuris suis in growing pigs. Two experimental diets were formulated based on barley flour with either added insoluble fibre from oat husk (Diet 1) or a pure inulin (16%) supplementation (Diet 2). Twenty-eight 10-week-old pigs were divided randomly into 4 groups (Groups 1–4) each of 7 pigs. After 3 weeks adaptation to the experimental diets all pigs were infected with a single dose of 2000 infective T. suis eggs. Group 1 was fed Diet 1 until 7 weeks post-infection (p.i.) and Group 3 until 9 weeks p.i., Group 2 was fed Diet 2 until 7 weeks p.i., Group 4 was fed Diet 1 until week 7 p.i. and was switched-over from Diet 1 to Diet 2 until week 9 p.i. Seven weeks p.i. pigs in Groups 1 and 2 were slaughtered, and pigs in Groups 3 and 4 were slaughtered at 9 weeks p.i. Trichuris suis worm burdens were determined for all pigs. Inulin-fed pigs (Group 2) exhibited an 87% reduction in EPG, compared to the pigs on standard diet (Group 1) (P<0·0001). The number of worms recovered at week 7 p.i. from pigs on the inulin diet (Group 2) was significantly reduced by 71%, compared to the pigs on standard diet (Group 1) (P<0·01). At week 9, worm recovery in pigs on the inulin diet switch protocol (Group 4) was reduced by 47% compared to the control pigs in Group 3 (P<0·01). Further, the inulin-fed pigs exhibited a significant reduction in female worm fecundity and worm large intestine location was more distal compared to those from pigs on standard diet. These results demonstrate that inclusion of the highly degradable fructose polymer inulin in the diet leads to significant reductions in T. suis establishment, egg excretion, and female worm fecundity and can be used as a treatment for patent infections.

Potential hosts for infective juveniles of entomopathogenic nematodes can vary considerably in quality based on the characteristics of the host species/stage, physiological status (e.g. stress, feeding on toxins), and infection status (heterospecific or conspecific infection). In this study, we investigated responses of the entomopathogenic nematode Steinernema riobrave to hosts (Galleria mellonella or Tenebrio molitor) that were previously parasitized with conspecifics or injected with the nematode-symbiotic bacterium, Xenorhabdus sp., to determine if there is a preference for previously parasitized/injected hosts and when this preference might occur. In no-choice bioassays, the number of juveniles infecting both host species decreased with increasing time post-infection. However, infective juveniles continued to infect previously parasitized hosts up to 72 h. Significant preference was exhibited by S. riobrave for 24 h post-infection G. mellonella larvae over uninfected, and by 24 h post-injection G. mellonella larvae over 48 h post-injection larvae. No significant preference was exhibited by S. riobrave for T. molitor hosts previously parasitized with conspecifics or those injected with bacteria in any treatment combination. Such preference for, or continued infection of parasitized insects, has the potential to impact nematode efficacy.

The migratory stage of Trichinella spiralis, the newborn larva, travels along the pulmonary microvascular system on its way to the striated muscle cells. In the present study, an important inflammatory reaction was observed on days 5 and 14 post-infection (p.i.) in the lungs of infected rats. This inflammation was characterized by a Th2 cell phenotype of hyperplastic bronchus-associated lymphoid tissue and by goblet cell hyperplasia. Among the inflammatory cells were eosinophils and mast cells scattered over the pulmonary parenchyma. On day 5 p.i. the number of IgE+, CD4+ and CD5+ cells in the bronchus-associated lymphoid tissue were increased and IgE-secreting lung cells were also detected. At the end of the migratory phase of the infection (day 14 p.i.), only IgE+ cells were detected in high numbers and in the bronchoalveolar lavage fluid, an increment in the total IgE levels as well as the presence of IgE and IgA anti-larvae surface were also detected. In cytotoxicity assays, cells from the bronchoalveolar lavage had considerable biological activity since they were able to kill the larvae even in the absence of specific antibodies. These results show that the lung is an organ involved in the immune response developed early during a T. spiralis infection and suggest its importance in the protection of the host.

We studied patterns of variation in parasite communities of 2 closely related species of Mastomys rodents. These 2 species live in sympatry in South-eastern Senegal, but differ drastically in their habitat choice. We asked (a) whether the host species have the same parasites; (b) whether there is any observable pattern relative to the host species/habitat type in the structure of parasite communities; (c) whether the variability in parasite community for each host species is related to habitat characteristics. We analysed 220 and 264 individuals of each host species, sampled respectively in 10 and 11 trap sites. Twenty parasite taxa were recorded, and the majority were nematodes. Between-host species comparisons showed that helminth communities were slightly more diversified in M. natalensis. Many parasite species were found in both Mastomys. However, various helminth taxa varied in frequency and abundance between host species. Within each host species, helminth diversity, prevalence and/or abundance of some parasites were correlated with habitat or host population factors that may influence parasite life-cycles, such as village structure, or the presence/absence of a pool. Our results suggest that habitat characteristics have a strong impact on helminth community structure.

In this study the lead and cadmium concentrations in the liver, intestine, muscle and gonad of the shark Carcharhinus dussumieri and its parasites, Anthobothrium sp. and Paraorigmatobothrium sp. (Cestoda), were measured and compared. Tissue samples were collected from Iranian coastal waters of the Persian Gulf. After tissue preparation, the cadmium and lead concentrations were analysed using the Inductively Coupled Plasma technique. The results revealed that lead and cadmium concentrations in both parasite species were many times higher than in the host tissues. The results strongly support the view that helminth parasites are extremely sensitive early warning bioindicators, particularly in sensitive environments under threat but where pollution levels are presently low. They may also have a beneficial effect on the health of their hosts by acting as heavy metal filters.

Transmission of parasites may be mediated by their habitat, consisting of abiotic and biotic components. I investigated the effect of 2 important habitat components in intertidal ecosystems, tidal level (abiotic) and density of upstream hosts (biotic), on the transmission of trematode cercariae to cockle (Cerastoderma edule) hosts. A field survey showed no general trend in metacercarial loads of cockles regarding tidal level but species-dependent reactions. Parasites originating from Littorina littorea (Himasthla elongata, Renicola roscovita) showed highest infection levels in the low intertidal while parasites originating from Hydrobia ulvae (H. continua, H. interrupta) showed highest infection levels in the mid-intertidal. This reflected the density of upstream hosts at both tidal levels and positive relationships between the density of upstream hosts and metacercarial load in cockles suggested the biotic habitat component to be the dominant factor in transmission. This was confirmed by a field experiment, manipulating tidal level and the density of infected upstream snail hosts. While tidal level had no significant effect on the number of metacercariae of H. elongata acquired by cockles, the effect of upstream host density was strong. In conclusion, although tidal level usually is a very important abiotic habitat component in intertidal ecosystems leading to conspicuous zonation patterns in free-living organisms, it seems of minor importance for trematode transmission. In contrast, the biotic component upstream host density is suggested to be the dominant predictor for trematode transmission to second intermediate hosts. Assessing the relative importance of abiotic and biotic habitat components in transmission is vital for the understanding of transmission processes in the field.

Taenia solium cysticercosis is a parasitic disease frequently affecting human health and the pig industry in many developing countries. A synthetic peptide vaccine (designated S3Pvac) against porcine cysticercosis has been developed previously as an aid to interrupt transmission and has been shown to be effective. The results of the present study support the effectiveness of the vaccine under endemic field conditions. However, given the time-frame of the vaccination trial, no changes in the local levels of transmission were detectable before and after vaccination using sentinel pigs. Thus, this investigation shows the limited usefulness of single vaccination as the sole means of interrupting Taenia solium transmission in an endemic region.

A review of the Giardia duodenalis sequences currently available on the GenBank database was completed to compare the different genotyping loci (small subunit ribosomal DNA, glutamate dehydrogenase, triose-phosphate isomerase and beta giardin) for their ability to discern assemblage and subassemblage groups and infer phylogenetic relationships. In total, 405 Giardia duodenalis sequences were sorted and aligned to examine the substitutions within and between the assemblages – A and B (zoonotic), C and D (dogs), E (livestock), F (cats) and G (rodents). It was found that all of the genes could reproducibly group isolates into their assemblages and that the AI/AII subassemblage groups were robust and identifiable at all loci. However, the assemblage B subgroups were not reproducible at half of the loci (small subunit ribosomal DNA and beta giardin), not due to their conserved nature, but because there was insufficient sequence data of reference isolates available for comparison. It is anticipated that further investigation of these loci may reveal the core subgroups of this medically important and zoonotic assemblage and also those of others. The closer, more recent, phylogenetic relationships amongst the assemblages appear to be resolved; however, more sequence data from the current loci, and possibly new loci, will be required to establish the remaining relationships.

This paper describes sensitivity analyses and expectations obtained from a mathematical model developed to account for the effects of host nutrition on the consequences of gastrointestinal parasitism in sheep. The scenarios explored included different levels of parasitic challenge at different planes of nutrition, for hosts differing only in their characteristics for growth. The model was able to predict the consequences of host nutrition on the outcome of parasitism, in terms of worm burden, number of eggs excreted per gram faeces and animal performance. The model outputs predict that conclusions on the ability of hosts of different characteristics for growth to cope with parasitism (i.e. resistance) depend on the plane of nutrition. Furthermore, differences in the growth rate of sheep, on their own, are not sufficient to account for differences in the observed resistance of animals. The model forms the basis for evaluating the consequences of differing management strategies and environments, such as breeding for certain traits associated with resistance and nutritional strategies, on the consequences of gastrointestinal parasitism on sheep.

This study determined whether the timing of re-feeding of protein-deficient mice restored functional protection against the gastrointestinal nematode, Heligmosomoides bakeri. Balb/c mice were fed a 3% protein-deficient (PD) diet and then transferred to 24% protein-sufficient (PS) diet either on the day of primary infection, 10 days after the primary infection, on the day of challenge infection, or 7 days after the challenge infection. Control mice were fed either the PD or PS diet. Onset of challenge, but not primary, infection caused short-term body weight loss, anorexia and reduced feed efficiency. Weight gain was delayed in mice when re-feeding commenced on the day of challenge infection; alkaline phosphatase (ALP) was also elevated in these mice on day 28 post-challenge. In contrast, other re-feeding groups attained similar body weights to PS mice within 4 days and had similar ALP at day 28. Serum leptin was higher in PD than PS mice and positively associated with food intake. As expected, worm survival was prolonged in mice fed the PD diet. However, egg production and worm burdens were similar in all re-feeding groups to the PS mice, indicating that protein re-feeding during either the primary or challenge infection rapidly restored normal parasite clearance.

This study analyses the anti-proliferative effect of lemongrass essential oil and its main constituent (citral) on all 3 evolutive forms of Trypanosoma cruzi. Steam distillation was used to obtain lemongrass essential oil, with chemical composition determined by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). The IC50/24 h (concentration that reduced the parasite population by 50%) of the oil and of citral upon T. cruzi was determined by cell counting in a Neubauer chamber, while morphological alterations were visualized by scanning and transmission electron microscopy. Treatment with the essential oil resulted in epimastigote growth inhibition with IC50=126·5 μg/ml, while the IC50 for trypomastigote lysis was 15·5 μg/ml. The IC50/48 h for the Association Index (% macrophage infection×number of amastigotes per cell) was 5·1 μg/ml, with a strong inhibition of intracellular amastigote proliferation. Ultrastructural analysis demonstrated cytoplasmic and nuclear extraction, while the plasma membrane remained morphologically preserved. Our data show that lemongrass essential oil is effective against T. cruzi trypomastigotes and amastigotes, and that its main component, citral, is responsible for the trypanocidal activity. These results indicate that essential oils can be promising anti-parasitic agents, opening perspectives to the discovery of more effective drugs of vegetal origin for treatment of parasitic diseases. However, additional cytotoxicity experiments on different cell lines and tests in a T. cruzi-mouse model are needed to support these data.