Endocrine and immune responses of larval amphibians to trematode exposure.

In nature, multiple waves of exposure to the same parasite are likely, making it important to understand how initial exposure or infection affects subsequent host infections, including the underlying physiological pathways involved. We tested whether experimental exposure to trematodes (Echinostoma trivolvis or Ribeiroia ondatrae) affected the stress hormone corticosterone (known to influence immunocompetence) in larvae representing five anuran species. We also examined the leukocyte profiles of seven host species after single exposure to R. ondatrae (including four species at multiple time points) and determined if parasite success differed between individuals given one or two challenges. We found strong interspecific variation among anuran species in their corticosterone levels and leukocyte profiles, and fewer R. ondatrae established in tadpoles previously challenged, consistent with defense "priming." However, exposure to either trematode had only weak effects on our measured responses. Tadpoles exposed to E. trivolvis had decreased corticosterone levels relative to controls, whereas those exposed to R. ondatrae exhibited no change. Similarly, R. ondatrae exposure did not lead to appreciable changes in host leukocyte profiles, even after multiple challenges. Prior exposure thus influenced host susceptibility to trematodes, but was not obviously associated with shifts in leukocyte counts or corticosterone, in contrast to work with microparasites.

Evaluation of the mitochondrial system in the gonad-digestive gland complex of Biomphalaria glabrata (Mollusca, Gastropoda) after infection by Echinostoma paraensei (Trematoda, Echinostomatidae).

The effect of infection by Echinostoma paraensei on the mitochondrial physiology of Biomphalaria glabrata was investigated after exposure to 50 miracidia. The snails were dissected one, two, three and four weeks after infection for collection and mechanical permeabilization of the gonad-digestive gland (DGG) complex. The results obtained indicate that prepatent infection by this echinostomatid fluke significantly suppresses the phosphorylation state (respiratory state 3) and basal oxygen consumption of B. glabrata, demonstrating that the infection reduces the ability of the intermediate host to carry out aerobic oxidative reactions. Additionally, relevant variations related to the uncoupled mitochondrial (state 3u) of B. glabrata infected by E. paraensei were observed. Four weeks after exposure, a significant reduction in mitochondrial oxygen consumption after addition of ADP (3.68±0.26pmol O2/mg proteins) was observed in the infected snails in comparison with the respective control group (5.14±0.25). In the uncoupled state, the infected snails consumed about 62% less oxygen than the infected snails (7.87±0.84pmol O2/mg proteins) in the same period. These results demonstrate a reduction in oxidative decarboxylation rate of the tricarboxylic acid cycle and faster anaerobic degradation of carbohydrates in the infected snails. The possible mechanisms that explain this new metabolic condition in the infected organisms are discussed.

Echinostoma caproni (Trematoda): differential in vivo mucin expression and glycosylation in high- and low-compatible hosts.

Enhanced mucus production and release appears to be a common mechanism for the clearance of intestinal helminths, and this expulsion is normally mediated by Th2-type immune responses. To investigate the factors determining the expulsion of intestinal helminths, we have analysed in vivo expression of mucin genes at the site of infection in two host species displaying different compatibility with Echinostoma caproni (Trematoda). Surprisingly, a general down-regulation on mucin mRNA expression was detected in low-compatible hosts (rats) coinciding with the development of Th2/Th17 responses and the early rejection of the worms from the intestinal lumen. This suggests the existence of a mechanism by which the parasites can modulate the mucus barrier to favour their survival. In highly compatible hosts (mice), some mucin genes were found to be up-regulated throughout the infection, probably, to protect the intestinal epithelium against the infection-induced inflammation developed in this host species. Moreover, infection-induced changes on mucin glycans were also studied by lectin histochemistry. Similar alterations were detected in the ileum of infected mice and rats, except with SNA lectin, indicating that sylated mucins might play an important role in determining the evolution of the infection in each host species.

Intestinal IFN-γ production is associated with protection from clinical signs, but not with elimination of worms, in Echinostoma caproni infected-mice.

In the present paper, we assess the relationship between the expression of IFN-γ and the development of clinical signs in Echinostoma caproni-infected mice. For this purpose, we studied the course of the infection in three mouse strains: ICR (CD-1®) (a host of high compatibility with E. caproni), BALB/c (a prototypical Th2 strain), and BALB/c deficient for IFN-γ mice (IFN-γ(-/-)). Infection in ICR mice is characterized by the elevated expression of IFN-γ and iNOS in the intestine concomitantly with the lack of clinical signs. In contrast, the infection was more virulent in BALB/c and IFN-γ-deficient mice that developed a severe form of the disease together with the absence of IFN-γ expression. The disease was more severe in IFNγ(-/-) mice in which the disease was lethal during the few first weeks of the infection. The analysis of different parameters of the infection in each host strain showed that most of the features were similar in the three mouse strains, suggesting the IFN-γ plays a central role in that protection against severe disease. Thus, IFN-γ seems to play a dichotomous role in the infection facilitating the parasite establishment, but it may also benefit mice since it protects the mice from morbidity and mortality induced by the parasite.

Differential expression and glycosylation of proteins in the rat ileal epithelium in response to Echinostoma caproni infection.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been used as experimental model to investigate the factors determining the expulsion of intestinal helminths. We analyze the changes in the protein expression and glycosylation induced by E. caproni in Wistar rat, a host of low compatibility in which the parasites are rapidly rejected. To determine the changes in protein expression, two-dimensional difference gel electrophoresis was employed using protein extracts from the intestine of naïve and infected rats. The patterns of glycosylation were analyzed by lectin blotting. Those spots showing differential expression or glycosylation were analyzed by mass spectrometry. A total of 33 protein spots differentially expressed were identified (26 were found to be over-expressed and 7 down-regulated). Moreover, E. caproni induced changes in the glycosylation status of 8 proteins that were successfully identified. Most of these proteins were related to the cytoskeleton and the maintenance of the functional integrity of the ileal epithelium. This suggests that the regeneration of the intestinal tissue is a major effector mechanism responsible for the early expulsion of this helminth. Furthermore, several proteins involved in the energy metabolism were also altered in the ileum of rats as a consequence of the E. caproni infection. BIOLOGICAL SIGNIFICANCE: Our analysis provides essential new insights in the factors determining the natural expulsion of intestinal parasitic helminths from their hosts. The results obtained contribute to a better understanding of the effective mechanisms involved in the defense against the intestinal helminths. The identification of proteins in the intestine that become modified in their expression or glycosylation in hosts in which the parasite is rapidly rejected may serve for the development of tools for the control of these infections.

The transcriptome of Echinostoma caproni adults: further characterization of the secretome and identification of new potential drug targets.

Echinostomes are cosmopolitan parasites that infect a large number of different warm-blooded hosts, both in nature and in the laboratory. They also constitute an important group of food-borne trematodes of public health importance mainly in Southeast Asia and the Far East. In addition, echinostomes are an ideal model to study several aspects of intestinal helminth biology, since they present a number of advantages. For example, echinostomes are large worms whose life cycle is relatively easy to maintain in the laboratory. Recently, several studies documented their great value in the study of intestinal helminth-vertebrate host relationship. Detailed knowledge of their genome, transcriptome and proteome is likely to have an important impact on the development of control strategies for intestinal helminths. We present the first transcriptome of the adult stage of Echinostoma caproni using 454 sequencing coupled to a semi-automated bioinformatic analyses. 557,236 raw sequence reads were assembled into 28,577 contiguous sequences using iAssembler. 23,296 putative proteins were characterized based on homology, gene ontology and/or biochemical pathways. Comparisons of the transcriptome of E. caproni with those of other trematodes revealed similarities in the transcription pattern of molecules inferred to have key roles in parasite-host interactions. Enzymatic proteins like kinases and peptidases were abundant. Of the 3415 predicted excretory/secretory proteins compiled (including non-classical secretory proteins), 180 different proteins were confirmed by proteomic analysis. Potential drug targets were also identified. BIOLOGICAL SIGNIFICANCE: In this study the first transcriptome of the adult stage of E. caproni is presented and compared to those of other trematodes revealing similarities in transcription for molecules inferred to have key roles in parasite-host interactions. 3415 predicted excretory/secretory proteins were compiled, being 180 different proteins confirmed by proteomic analysis. The current transcriptome data increased by nine times the number of previous protein identifications. In addition, potential drug targets for this parasite were identified. The present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of E. caproni, as well as a basis for applied outcomes, such as the development of novel methods of intervention against this model organism and related parasites.

Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells.

The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30-100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.

Panorganismal metabolic response modeling of an experimental Echinostoma caproni infection in the mouse.

Metabolic profiling of host tissues and biofluids during parasitic infections can reveal new biomarker information and aid the elucidation of mechanisms of disease. The multicompartmental metabolic effects of an experimental Echinostoma caproni infection have been characterized in 12 outbred female mice infected orally with 30 E. caproni metacercariae each, using a further 12 uninfected animals as a control group. Mice were killed 36 days postinfection and brain, intestine (colon, ileum, jejeunum), kidney, liver, and spleen were removed. Metabolic profiles of tissue samples were measured using high-resolution magic angle spinning (1)H NMR spectroscopy and biofluids measured by applying conventional (1)H NMR spectroscopy. Spectral data were analyzed via principal component analysis, partial least-squares-derived methods and hierarchical projection analyses. Infection-induced metabolic changes in the tissues were correlated with altered metabolite concentrations in the biofluids (urine, plasma, fecal water) using hierarchical modeling and correlation analyses. Metabolic descriptors of infection were identified in liver, renal cortex, intestinal tissues but not in spleen, brain or renal medulla. The main physiological change observed in the mouse was malabsorption in the small intestine, which was evidenced by decreased levels of various amino acids in the ileum, for example, alanine, taurine, glutamine, and branched chain amino acids. Furthermore, altered gut microbial activity or composition was reflected by increased levels of trimethylamine in the colon. Our modeling approach facilitated in-depth appraisal of the covariation of the metabolic profiles of different biological matrices and found that urine and plasma most closely reflected changes in ileal compartments. In conclusion, an E. caproni infection not only results in direct localized (ileum and jejenum) effects, but also causes remote metabolic changes (colon and several peripheral organs), and therefore describes the panorganismal metabolic response of the infection.

Moderate Echinostoma trivolvis infection has no effects on physiology and fitness-related traits of larval pickerel frogs (Rana palustris).

High intensity infections of Echinostoma trivolvis metacercariae decrease survival and growth of young amphibian larvae. However, in nature, parasites are highly aggregated, which results in a large proportion of the amphibian population being only moderately infected. Survival and growth responses at these more-common, low-infection levels remain poorly studied. Thus, we investigated the effects of moderate Echinostoma trivolvis metacercariae infection (following exposure to 0, 10, 30, or 90 cercariae) on the growth and development of pickerel frog (Rana palustris) tadpoles. We measured metabolism to determine whether increased energy expenditure is a potential physiological mechanism underlying previously documented reduced growth. Furthermore, we quantified tadpole intestine size, which can exhibit plasticity in response to changing metabolic demands, and we characterized metacercariae distribution in tadpole kidneys. Metacercariae encysted in the pronephros significantly more than in the mesonephros, but tended to occur equally in right and left kidneys. Two mo post-infection (PI), there were no changes in tadpole survival, development, intestine size, or growth related to metacercariae infection. Similarly, metacercariae did not significantly increase metabolic rates during encystment or at 1 mo PI. Our study demonstrated that modest E. trivolvis infections, representative of a large proportion of the host population, had no detectable effects on fitness-related traits in laboratory isolation from other ecological variables.

Metabolic profiling of an Echinostoma caproni infection in the mouse for biomarker discovery.

BACKGROUND: Metabolic profiling holds promise with regard to deepening our understanding of infection biology and disease states. The objectives of our study were to assess the global metabolic responses to an Echinostoma caproni infection in the mouse, and to compare the biomarkers extracted from different biofluids (plasma, stool, and urine) in terms of characterizing acute and chronic stages of this intestinal fluke infection. METHODOLOGY/PRINCIPAL FINDINGS: Twelve female NMRI mice were infected with 30 E. caproni metacercariae each. Plasma, stool, and urine samples were collected at 7 time points up to day 33 post-infection. Samples were also obtained from non-infected control mice at the same time points and measured using (1)H nuclear magnetic resonance (NMR) spectroscopy. Spectral data were subjected to multivariate statistical analyses. In plasma and urine, an altered metabolic profile was already evident 1 day post-infection, characterized by reduced levels of plasma choline, acetate, formate, and lactate, coupled with increased levels of plasma glucose, and relatively lower concentrations of urinary creatine. The main changes in the urine metabolic profile started at day 8 post-infection, characterized by increased relative concentrations of trimethylamine and phenylacetylglycine and lower levels of 2-ketoisocaproate and showed differentiation over the course of the infection. CONCLUSION/SIGNIFICANCE: The current investigation is part of a broader NMR-based metabonomics profiling strategy and confirms the utility of this approach for biomarker discovery. In the case of E. caproni, a diagnosis based on all three biofluids would deliver the most comprehensive fingerprint of an infection. For practical purposes, however, future diagnosis might aim at a single biofluid, in which case urine would be chosen for further investigation, based on quantity of biomarkers, ease of sampling, and the degree of differentiation from the non-infected control group.

High-performance thin-layer chromatographic analysis of neutral lipids and phospholipids in Biomphalaria glabrata patently infected with Echinostoma caproni.

This study examined the effects of larval trematode infection on the neutral lipid and phospholipid content of Biomphalaria glabrata patently infected with the daughter rediae of Echinostoma caproni. Uninfected snails were used as matched controls. As determined by qualitative high-performance silica gel thin-layer chromatography (HPTLC), the major neutral lipids present in the whole bodies and digestive gland-gonad complexes in both snail populations were free sterols, free fatty acids, and triacylglycerols, and the major polar lipids were phosphatidylcholine and phosphatidylethanolamine. Quantitative analysis by HPTLC with visible and UV scanning reflectance densitometry showed no significant differences in the concentrations of these lipids in whole bodies of infected snails vs the controls, but the concentration of triacylglycerols in the infected digestive gland-gonad complex was significantly less than that of the uninfected. No qualitative differences in neutral lipids and phospholipids in shell or plasma samples were found between infected vs uninfected snails.

Identification of proteins in excretory/secretory extracts of Echinostoma friedi (Trematoda) from chronic and acute infections.

In the present study, we describe the investigation of Echinostoma friedi excretory/secretory products using a proteomic approach combined with the use of heterologous antibodies. We have identified 18 protein spots corresponding to ten proteins, including cytoskeletal proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase, and aldolase; detoxifying enzymes like GSTs; and stress proteins like heat shock protein (Hsp) 70. Among these proteins, both actin and, to a lesser extent, Hsp70, exhibited differential expression patterns between chronic and acute infections in the Echinostoma-rodent model, suggesting that these proteins may play a role in the survival within the host.

Hemozoin formation in Echinostoma trivolvis rediae.

Rediae of the trematode Echinostoma trivolvis, from naturally infected Helisoma trivolvis snails, form a black pigment while inside the snail host. Here we examine the black pigment to show that the insolubility characteristics in detergent and weak base solution are identical to Plasmodium falciparum hemozoin. Laser desorption mass spectrometry of the purified pigment demonstrates the presence of heme. Examination of purified pigment under polarized light microscopy illuminates ordered birefringent crystals. Field emission in lens scanning electron microscopy reveals irregular ovoid crystals of 200-300 nm in diameter. The purified pigment crystals seeded extension of monomeric heme onto the crystal which by Fourier Transform Infrared analysis is beta-hematin. Rediae of a second echinostome parasite, Echinostoma caproni, from experimentally infected Biomphalaria glabrata, do not produce measurable or recoverable heme crystals. These observations are consistent with heme crystal formation by a hematophagous parasite within a non-vertebrate intermediate host.

Differential expression of FREP genes in two strains of Biomphalaria glabrata following exposure to the digenetic trematodes Schistosoma mansoni and Echinostoma paraensei.

Fibrinogen-related proteins (FREPs) are hypothesized to function in non-self-recognition in the snail Biomphalaria glabrata. To investigate this assumption, the expression of four members of the FREP gene family was studied using quantitative PCR at 0.5-16 days following exposure of M line and BS-90 strain B. glabrata to Echinostoma paraensei and Schistosoma mansoni. Both strains react to, but fail to eliminate E. paraensei. Only the BS-90 strain is immunologically resistant to S. mansoni. Both snail strains responded to E. paraensei with significantly elevated expression of FREP 2 and 4. Following exposure to S. mansoni, resistant BS-90 snails showed an increase in expression of FREP 2 and 4 (57-fold and 4.5-fold increase, respectively), susceptible M line snails did not display a FREP response. Expression of FREP 3 and 7 was not significantly elevated in any snail/trematode combination. These expression profiles support the hypothesis that some FREPs play a role in the anti-trematode responses in B. glabrata.

Effects of diet and larval trematode parasitism on lutein and beta-carotene concentrations in planorbid snails as determined by quantitative high performance reversed phase thin layer chromatography.

High performance thin layer chromatography (HPTLC) was used to quantify the concentrations of beta-carotene and lutein in Biomphalaria glabrata and Helisoma trivolvis (Colorado and Pennsylvania strains) snails under various conditions. These conditions were: snails fed a lettuce (L) vs. a yolk (Y) diet; B. glabrata infected with Echinostoma caproni vs. uninfected snails; and H. trivolvis (PA) infected with Echinostoma trivolvis vs. uninfected snails. The pigments were extracted from the snail whole bodies and digestive gland-gonad complexes, separated by reversed phase HPTLC, and quantified by densitometric scanning with standard calibration curves. Snails on the L-diet showed significant increases (Student's t-test, P<0.05) in the concentrations of beta-carotene and lutein compared to snails on the Y-diet. Snails infected with echinostomes showed no significant differences (Student's t-test, P>0.05) in the concentrations of lutein and beta-carotene compared to the uninfected cohorts. Our results were compared with previous studies that analyzed beta-carotene and lutein in snails infected with larval trematodes. Variations in the results of our study compared with others reflect intrinsic differences in the larval trematode-snail systems used.

Effects of Echinostoma caproni infections on metallic ions in the intestinal mucosa of ICR mice.

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to study metallic ions in the intestinal mucosa of ICR mice infected with Echinostoma caproni and the mucosa of uninfected control mice. Infected mucosa (n = 9 with about 100 mg wet weight per sample) were examined at 2 weeks p.i. in mice that were infected with about 25 worms per host. Uninfected mucosa (n = 9 with about 100 mg wet weight per sample) were examined in the same time frame as the infected mucosa. Five metals were measured in the mucosa by ICP-AES analysis, as follows: calcium, potassium, magnesium, sodium and zinc. There were no significant differences (Student's t-test, P > 0.05) in the concentrations of calcium, potassium or zinc in infected versus uninfected mucosa. The concentration of sodium was significantly greater (P < 0.05) in the mucosa of infected versus uninfected mucosa, but the situation was reversed in regard to magnesium.

Effects of Echinostoma caproni infection on the phospholipid and sphingolipid content of the intestinal mucosa of ICR mice.

High performance thin layer chromatography (HPTLC) was used to determine phospholipids and sphingolipids in the intestinal mucosa of ICR mice infected with Echinostoma caproni for two weeks. The major phospholipids detected in both infected and non-infected mucosa were phosphatidylcholine (PC) and phosphatidylethanolamine (PE). HPTLC-densitometric analysis showed that there was a significant decrease in the weight of both PC and PE in the intestinal mucosa of infected mice compared to that of the uninfected controls. Cerebrosides and sulphatides, but not sphingomyelin, were identified in the intestinal mucosa of both infected and uninfected hosts. There was an apparent increase in the cerebroside content of the mucosa of infected versus control mice. The pathobiochemical changes seen in the polar lipid content of infected hosts probably reflect the feeding and behavioural activities of E. caproni in the mouse intestine.

A family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection.

After infection with the digenetic trematode Echinostoma paraensei, hemolymph of the snail Biomphalaria glabrata contains lectins comprised of 65-kDa subunits that precipitate polypeptides secreted by E. paraensei intramolluscan larvae. Comparable activity is lacking in hemolymph of uninfected snails. Three different cDNAs with sequence similarities to peptides derived from the 65-kDa lectins were obtained and unexpectedly found to encode fibrinogen-related proteins (FREPs). These FREPs also contained regions with sequence similarity to Ig superfamily members. B. glabrata has at least five FREP genes, three of which are expressed at increased levels after infection. Elucidation of components of the defense system of B. glabrata is relevant because this snail is an intermediate host for Schistosoma mansoni, the most widely distributed causative agent of human schistosomiasis. These results are novel in suggesting a role for invertebrate FREPs in recognition of parasite-derived molecules and also provide a model for investigating the diversity of molecules functioning in nonself-recognition in an invertebrate.

Effects of diet on the lipid composition of Echinostoma caproni (Trematoda) in ICR mice.

High-performance thin-layer chromatography (HPTLC) was used to determine neutral lipids and phospholipids in the intestinal trematode Echinostoma caproni from experimentally infected ICR mice fed a high-fat diet (hen's egg yolk) as compared with worms from mice fed a standard laboratory diet. Worms were removed from the hosts at 2, 3, and 4 weeks postinfection (p.i.). Analysis by TLC-densitometry showed significantly greater amounts of triacylglycerols and free sterols at 2, 3, and 4 weeks p.i. in worms from mice on the high-fat diet as compared with worms from mice on the standard laboratory diet. Significantly greater amounts of phosphatidylcholine and phosphatidylethanolamine were found in worms from mice on the high-fat diet as compared with worms from those on the standard diet at 2 weeks p.i. but not at 3 and 4 weeks p.i. The results of this study suggest that the host diet influences the lipid content of E. caproni adults.

Effects of Echinostoma trivolvis (trematoda) infection on metallic ions in the host snail Helisoma trivolvis (gastropoda).

Flame and graphite-furnace atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry were used to study the metallic ions in the digestive gland-gonad complex (DGG) of Helisoma trivolvis snails infected with the daughter rediae of Echinostoma trivolvis and in uninfected DGG. Seven metals were found to be present in infected and uninfected DGG at concentrations above the detection limits of the analytical methods. Of these, sodium was present in significantly higher amounts (Student's t-test, confidence level of 95%) in the infected versus uninfected DGG; magnesium and manganese occurred in significantly lower amounts in the infected DGG. Our results were compared with those from a Bulgarian study in which neutron activation analysis was used to determine elements in Lymnaea stagnalis snails infected with the intramolluscan stages of the 37-collar-spined echinostome E. revolutum. The Bulgarian study also reported a significant elevation of sodium but reduction of zinc in the hepatopancreas (i.e., digestive gland) of infected snails. Other differences between the two studies are discussed.