Form and Function in the Digenea.

This review aims at covering the general aspects of the form and function of the major body systems in digenetic trematodes, with emphasis on the advances that have been made on this topic since the publication of the first edition of this book; this is since 2012. In particular, the advancement in the knowledge of form and function of tegumentary, sensory, neuromuscular, alimentary, respiratory, excretory, and reproductive systems is covered. Furthermore, a brief mention of the current trends in the targeting of trematode body systems for developing novel opportunities of treatment and control is provided.

Trematoda (flukes).

The class Trematoda is the largest group of Platyhelminths and includes two subclasses: Aspidogastrea and Digenea. Trematodes, and particularly Digeneans, is a large group of organisms with significant medical and veterinary interest. Over 100 species of digenetic trematodes have been reported infecting humans. Although the significant mortality and morbidity that some of these infections cause, they are among the most neglected tropical diseases. Apart from their impact in public and animal health, the Digenea constitutes an intriguing group of organisms that has a vast interest in experimental biology. Systematics and taxonomy of this group constitute a challenge for biologists in relation to the difficulty entailed in the establishment of phylogenetic relationships between trematodes and the determination of valid diagnostic features. Moreover, their complex life cycles, using at least two hosts and alternating free-living and parasitic stages or sexual and asexual multiplication, constitute a paradigm of how organisms can evolve to become adapted to different biotic and abiotic environments to enhance survival. In this review, we briefly summarize the major features of trematodes in relation to both biological and medical areas.

Reduced [14C]-methionine uptake and fecundity in Schistosoma mansoni females treated with recombinant tumor necrosis factor α in vitro.

Recombinant human TNFα (rhTNFα) has previously been shown to reduce fecundity in Schistosoma mansoni adult females maintained in vitro without males, and adversely affect the uptake of [14C]-tyrosine, an amino acid required for schistosome vitellogenesis. Here we report on the effect of rhTNFα on [14C]-methionine uptake in both separated and paired females, and the effect of three different preparations of rTNFα on schistosome oviposition in vitro. In the absence of rhTNFa, separated females incorporated only 30% of the [14C]-methionine incorporated by paired females in a dose and time-dependent manner, suggesting low metabolic activity of females in the absence of males. Separated females and worm-pairs were treated with increasing doses of rhTNFα for 2 or 4 hr and then incubated in RPMI 1640 containing 10% fetal calf serum (FCS) and 5 µCi ml-1[14C]-methionine for 1 hr. Separated females treated with rhTNFa for 4 hr incorporated less methionine than those treated for 2 hr. In contrast, paired females treated with rhTNFa incorporated significantly smaller amounts of [14C]-methionine in a TNFa dose-dependent but time-independent manner [2 hr (P = 0.001) or 4 hr (P = 0.027) One-Way ANOVA]. Worm-pairs maintained in RPMI 1640 containing 10% FCS and 100 ng ml-1 of any of the three rTNFa preparations laid significantly fewer eggs than the worms cultured without rTNFα(P = 0.001; Kruskal-Wallis Test). We also observed that among rTNFα-treated worm-pairs, females were sluggish and tended to separate from their male partners. These observations suggest that TNFa inhibits [14C]-methionine uptake and reduces fecundity in females paired with males. Since paired females incorporate substantially greater amounts of [14C]-methionine, the role of males in stimulating metabolic activity in females is affirmed. Reduced amino acid uptake, and possibly other nutrients, may contribute to the diminished fecundity observed in TNFa-treated females.

Coinfection of Schistosoma Species with Hepatitis B or Hepatitis C Viruses.

Although a considerable number of studies have been undertaken to date, it is still controversial as to whether or not coinfection with schistosomiasis increases the susceptibility to or progression from Hepatitis B virus (HBV) or Hepatitis C virus (HCV) infection. This review is a closer examination of the key studies conducted on human populations on clinical factors that were published in English between 1975 and January 2015. Our review is mainly based on tables containing the salient information, which are arranged first by study population, country of study and publication date. We provide further explanation, clarification and discussion in the text. As such, it includes both studies that have been conducted on general populations who are largely asymptomatic for clinical disease (Table 3), as well as those focussing on special populations, which are usually comprised of clinical patients. These special populations have been presented as follows: subjects with chronic liver disease or related conditions such as cirrhosis, Table 4; subjects with primary liver cancer, Table 5; subjects with schistosomiasis, Table 6; subjects with acute or chronic hepatitis resulting from HBV, Table 7 and, subjects with HCV, Table 8. We have presented studies that compared two mono-infected groups with one that is coinfected separately in Table 9, as these offer us the best basis from which to evaluate if any synergistic effects accompany coinfection. A number of factors contributed to the results reported in our tables. These included, but are not limited to: subject selection (i.e. asymptomatic cases typically drawn from the general population vs subjects presenting to a hospital or clinic with clinical disease); study design, which directly impacts our ability to infer causality (i.e. case series, cross-sectional, case-control, cohort study); use and choice of control population (i.e. apparently healthy subjects vs other hospital patients vs none); sample size, which directly impacts statistical power and can result in a Type II error; geographic area, which may reflect differences in population genetics, public health history, environmental differences or any number of other important factors (i.e. Egypt, Brazil, China); method of testing for schistosomal infections (i.e. stool vs antibody test); method of testing to determine if advanced schistosomal disease was present (i.e. ultrasound, liver biopsy vs none); method of serological testing for HBV (i.e. use of HBsAg alone or with other markers or DNA testing); method of serological testing for HCV (i.e. use of anti-HCV alone or with RNA testing) and, year of the study, which reflects among other things, technological improvements between tests as well as possible changes in the frequency of exposure in the populations under study (i.e. use of parenteral antischistosomal therapy vs the oral antischistosomal medication). Despite all these differences, throughout this review we have observed general patterns that seem largely consistent with one another. Studies conducted on general, largely asymptomatic populations tend to support the view that having one of the diseases in question (i.e. schistosomiasis) does not necessarily predispose one to becoming coinfected with another (i.e. HBV or HCV). Rather, the probability of becoming coinfected seems most closely associated with modes of transmission for either HBV or HCV in schistosome-endemic areas, such as the past use of parenteral antischistosomal therapy or frequent blood transfusion. Once coinfected, however, the clinical course of illness for those with Schistosoma-HBV or Schistosoma-HCV infections are typically much more severe than for mono-infected subjects. The strongest evidence for this was found in the half-dozen or so prospective cohort studies that systematically monitored disease progression in their subjects. With respect to HBV infection, coinfection with Schistosoma prolonged the carriage state and more often resulted in chronic hepatitis with greater cirrhosis as well as higher mortality. Much of the same was also observed with respect to HCV, where coinfection with Schistosoma was associated with a reduced ability to spontaneously resolve the viral infection and more often resulted in rapid fibrosis as well as higher mortality. Furthermore, two of these studies which were fully comparative in nature, support the supposition that there is a synergistic association between Schistosoma-HCV for both liver fibrosis and mortality. Immunological studies, all conducted on HCV, also generally seem to support this. The results of our research argue for greater primary prevention for both HBV and HCV in Schistosoma-endemic populations. Although no vaccine currently exists for HCV as it does for HBV, additional steps can still be taken to reduce transmission in high-risk populations. Greater use of the HBV vaccine is particularly advisable. Finally, additional observational, longitudinal studies conducted on human populations that are fully comparative in nature could help answer some of the remaining questions on both Schistosoma-HBV as well as Schistosoma-HCV coinfections. Some of these include the role of active versus past schistosomal infections, the role of genetic variants, as well as the effect of coinfection on treatment. Future studies should make a particular effort to use a sufficient sample size to ensure adequate statistical power, which was not often properly considered in many of the studies we reviewed for this paper.

Effects of Echinostoma caproni miracidia dose on the amino acid contents of Biomphalaria glabrata as determined by high-performance thin-layer chromatography.

The effects of 5, 20, and 40 miracidia dose exposures of Echinostoma caproni on the amino acid contents of Biomphalaria glabrata were studied using high performance thin-layer chromatography-densitometry. Amino acids were identified and quantified in whole bodies of exposed snails and in the uninfected matched controls at 2 and 4 weeks post-exposure. Using cellulose layers with the mobile phase 2-butanol-pyridine-glacial acetic acid-deionized water (39:34:10:26) and ninhydrin detection reagent [2% ninhydrin in acetone-n-butanol (1:1)], five amino acids were identified, i.e., leucine/isoleucine, valine, alanine, glycine, and ornithine, by hRF value comparison and color differentiation. Quantitatively, there was a marked elevation in the amounts of four of these five amino acids (isoleucine/leucine, valine, alanine, and ornithine) across dose levels at 4 weeks post-infection (P<0.05). Elevation of the amino acid content in the high dose snail group suggested that some changes occurred in the amino acid metabolism of the snails in that group as a function of miracidia dose.

Altered Protein Expression in the Ileum of Mice Associated with the Development of Chronic Infections with Echinostoma caproni (Trematoda).

BACKGROUND: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been extensively used as experimental model to investigate the factors determining the expulsion of intestinal helminths or, in contrast, the development of chronic infections. Herein, we analyze the changes in protein expression induced by E. caproni infection in ICR mice, a host of high compatibility in which the parasites develop chronic infections. METHODOLOGY/PRINCIPAL FINDINGS: To determine the changes in protein expression, a two-dimensional DIGE approach using protein extracts from the intestine of naïve and infected mice was employed; and spots showing significant differential expression were analyzed by mass spectrometry. A total of 37 spots were identified differentially expressed in infected mice (10 were found to be over-expressed and 27 down-regulated). These proteins were related to the restoration of the intestinal epithelium and the control of homeostatic dysregulation, concomitantly with mitochondrial and cytoskeletal proteins among others. CONCLUSION/SIGNIFICANCE: Our results suggests that changes in these processes in the ileal epithelium of ICR mice may facilitate the establishment of the parasite and the development of chronic infections. These results may serve to explain the factors determining the development of chronicity in intestinal helminth infection.

History of echinostomes (Trematoda).

Echinostomatidae (Trematoda) is the largest family within the class Trematoda. Members of this family have been studied for many years in relation to their utility as basic research models in biodiversity and systematics and also as experimental models in parasitology since they offer many advantages. Echinostomes have contributed significantly to numerous developments in many areas studied by parasitologists and experimental biologists. In this review, we examine the history of the echinostomebased studies from the beginnings to the present. For this purpose, we have divided the history of echinostomes into four periods (i.e. 18(th) and 19(th) centuries, first half of the 20(th) century, second half of the 20(th) century and the late 20(th) and 21(th) century) according to the types of studies performed in each of them. Moreover, we also briefly review the history of echinostome infections in humans.

The effect of glycosylation of antigens on the antibody responses against Echinostoma caproni (Trematoda: Echinostomatidae).

In the present study, we analyse the effect of glycosylation in Echinostoma caproni (Trematoda: Echinostomatidae) antigens in antibody responses against the parasite in experimentally infected mice. It has been previously demonstrated that the mouse is a host of high compatibility with E. caproni and develops elevated responses of IgG, IgG1, IgG3 and IgM as a consequence of the infection, though the role of glycans in these responses remains unknown. To this purpose, the responses generated in mice against non-treated excretory/secretory antigens of E. caproni were compared with those observed after N-deglycosylation, O-deglycosylation and double deglycosylation of the antigens by indirect ELISA and western blot. Our results suggest that E. caproni-expressed glycans play a major role in the modulation of the immune responses. The results obtained indicate that IgG subclass responses generated in mice against E. caproni are essentially due to glycoproteins and may affect the Th1/Th2 biasing. The reactivity significantly decreased after any of the deglycosylation treatments and the N-glycans appears to be of greater importance than O-glycans. Interestingly, the IgM response increased after N-deglycosylation suggesting that carbohydrates may mask peptide antigens.

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.

Effects of Echinostoma caproni miracidia dose on the neutral and polar lipids of Biomphalaria glabrata as determined by high-performance thin-layer chromatography.

The effects of a 5 versus 25 miracidia exposure of Echinostoma caproni on the lipid composition of Biomphalaria glabrata was studied using high performance thin layer chromatography (HPTLC)-densitometry. A 50 miracidia dose was not used because such a high level of exposure caused severe snail mortality by 3 weeks post-exposure (PE). Lipids were determined in the digestive-gland gonad complex (DGG) of the exposed snails and in the uninfected matched controls at 2 and 4 weeks PE. Extraction of lipids from DGGs was carried out by the Folch method with chloroform-methanol (2:1), and extracts were analyzed on Analtech HPTLC-HLF pre-adsorbent silica gel plates with measurement of separated bands using a CAMAG Scanner 3. For neutral lipids the mobile phase was petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and the detection reagent was 5% ethanolic phosphoric acid, and for polar lipids chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used. No significant differences in the concentrations of free sterols, free fatty acids, triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were seen at 2 weeks PE in any of the groups. At 4 weeks PE, the free fatty acid concentration increased significantly in the snails exposed to 25 miracidia compared to that of the 5 miracidia/snail group or the controls. Elevation of the free fatty acid fraction in the high dose snail group suggested that some changes occurred in the lipid metabolism of the snails in that group as a function of miracidia dose.

Global status of fish-borne zoonotic trematodiasis in humans.

Fishborne zoonotic trematodes (FZT), infecting humans and mammals worldwide, are reviewed and options for control discussed. Fifty nine species belonging to 4 families, i.e. Opisthorchiidae (12 species), Echinostomatidae (10 species), Heterophyidae (36 species) and Nanophyetidae (1 species) are listed. Some trematodes, which are highly pathogenic for humans such as Clonorchis sinensis, Opisthorchis viverrini, O. felineus are discussed in detail, i.e. infection status in humans in endemic areas, clinical aspects, symptoms and pathology of disease caused by these flukes. Other liver fluke species of the Opisthorchiidae are briefly mentioned with information about their infection rate and geographical distribution. Intestinal flukes are reviewed at the family level. We also present information on the first and second intermediate hosts as well as on reservoir hosts and on habits of human eating raw or undercooked fish.

Effects of Schistosoma mansoni infection on lutein and ß-carotene concentrations in Biomphalaria glabrata snails as determined by quantitative high performance reversed phase thin-layer chromatography.

High performance thin-layer chromatography was used to determine the concentration of ß-carotene and lutein in the whole body and digestive gland-gonad complex (DGG) of uninfected Biomphalaria glabrata snails and those infected with Schistosoma mansoni for 6 and 8 weeks. Pigments were extracted from the snails using acetone and separated on EMD Millipore reversed phase C-18 plates with concentration zone using petroleum ether-acetonitrile-methanol (1:1:2) mobile phase. After development, two yellow pigment zones, lutein and ß-carotene, were identified with respective Rf values of 0.55 and 0.13 and then quantified by densitometry. Statistical analysis of the weight percentages of each pigment showed a significant decrease (P < 0.05) in the concentration of ß-carotene in the DGGs of infected B. glabrata at 6 and 8 weeks post-infection compared to the uninfected snails. No significant differences were seen in the concentrations of ß-carotene in the whole body of the uninfected versus infected snail samples. Changes in the lutein concentration of the infected DGG and whole snail bodies were insignificant compared to the uninfected controls. In conclusion, larval S. mansoni infection caused a significant decrease in the ß-carotene concentration of the DGG at 6 and 8 weeks post infection.

Metabolic profiling of Echinostoma caproni and Schistosoma mansoni in their definitive and intermediate hosts.

This review examines metabolic profiling of Schistosoma mansoni and Echinostoma caproni in their definitive and intermediate hosts. The earlier coverage of the literature on metabolic profiling was reviewed by Wang et al. 2010, Advances in Parasitology, 73, 373-404 and covered mainly studies using proton nuclear magnetic resonance spectroscopy. The methods focused upon in our review are mainly chromatographic. In the studies reviewed, various metabolites were analyzed in hosts infected with either E. caproni or S. mansoni and compared to the uninfected controls.

Protective immunity against Echinostoma caproni in rats is induced by Syphacia muris infection.

Syphacia muris (Nematoda: Oxyuridae) is a ubiquitous nematode that commonly infects rats in the laboratory and can interfere with the development of unrelated biological assays. In this context, we analysed the effect of a patent S. muris infection in Wistar rats on a superimposed infection with the intestinal trematode, Echinostoma caproni (Trematoda: Echinostomatidae). The results indicate that in the rats, infection with S. muris induces an immunity against a subsequent infection with E. caproni, although each parasite occupies different niches in the host. Echinostoma caproni worm recovery was significantly decreased in the rats primarily infected with S. muris and, at 3 and 4 weeks post-primary infection, the rats primarily infected with S. muris were refractory to the challenge infection with E. caproni. We observed that the main alterations induced by S. muris in the niche of E. caproni (ileum) that may be the cause of the resistance are: (i) a local antibody response with elevated levels of mucosal IgA, IgE, IgG, IgG1 and IgG2a that cross-react with E. caproni antigens; (ii) development of a biased Th17/Th2 phenotype; and (iii) changes in the glycosylation of ileal mucins. This indicates that S. muris induces distant alterations to the ileum of rats affecting the development of other helminth species. Apart from the interest of these results in the study of the interactions between helminths in a single host, it has been demonstrated that pinworm infections may interfere in non-related experiments.

Infection of neonatal Biomphalaria glabrata with the miracidia of Echinostoma caproni.

We used a Biomphalaria glabrata snail model for our studies and investigated the suitability of B. glabrata neonates, reared on a Nostoc sp. diet, for infection with Echinostoma caproni miracidia. We found that neonatal snails could become infected with E. caproni miracidia with 31 ± 11% standard error (SE) of our exposed snails containing rediae infections at 4 wk post-exposure (PE). However, the survival of exposed neonates was significantly (P < 0.05, Student's t-test) less than that of the unexposed controls at 1, 2, 3, and 4 wk PE.

The biology of Echinoparyphium (Trematoda, Echinostomatidae).

Echinoparyphium species are common, widely distributed intestinal parasites causing disease in animals worldwide. Intermediate hosts include snails, bivalves, and fish, whereas the definitive hosts are mainly birds and mammals. This review examines the significant literature on Echinoparyphium. Descriptive studies, life cycle, experimental and manipulative studies, and biochemical and molecular studies are presented. The influence of environmental factors, and toxic pollutants, are reviewed as well as studies on the pathology of Echinoparyphium.