Changes in resident microbiota associated with mice susceptibility or resistance to the intestinal trematode Echinostoma caproni.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode with no tissue phases in the definitive host that has been extensively used as an experimental model to study the factors that determine resistance against intestinal helminths. In E. caproni infections in mice, interleukin-25 (IL-25) plays a critical role and it is required for the resistance to infection. However, little is known on the factors that determine its production. Primary E. caproni infection in mice is characterized by the development of chronic infections and elevated worm recovery, in relation to a local Th1 response with elevated production of interferon-γ. However, partial resistance against secondary E. caproni infections in ICR (Institute of Cancer Research) mice is developed after the chemotherapeutic cure of a primary infection and the innately produced IL-25 after pharmacological treatment. In this paper, we analyse the potential role of intestinal microbiota in the production of IL-25, and the subsequent resistance to infection. For this purpose, we analysed the production of IL-25 under conditions of experimental dysbiosis and also the changes in the resident microbiota in primary infections, pharmacological curation and secondary infections. The results obtained showed that resident microbiota play a major role in the production of IL-25 and the appearance of members of the phylum Verrucomicrobia as a consequence of the curation of the primary infection could be related to the partial resistance to secondary infection.

Neglected food-borne trematodiases: echinostomiasis and gastrodiscoidiasis.

In the present paper, we review two of the most neglected intestinal food-borne trematodiases: echinostomiasis, caused by members of the family Echinostomatidae, and gastrodiscoidiasis produced by the amphistome Gastrodiscoides hominis. Both parasitic infections are important intestinal food-borne diseases. Humans become infected after ingestion of raw or insufficiently cooked molluscs, fish, crustaceans, amphibians or aquatic vegetables. Thus, eating habits are essential to determine the distribution of these parasitic diseases and, traditionally, they have been considered as minor diseases confined to low-income areas, mainly in Asia. However, this scenario is changing and the population at risk are currently expanding in relation to factors such as new eating habits in developed countries, growing international markets, improved transportation systems and demographic changes. These aspects determine the necessity of a better understanding of these parasitic diseases. Herein, we review the main features of human echinostomiasis and gastrodiscoidiasis in relation to their biology, epidemiology, immunology, clinical aspects, diagnosis and treatment.

Research Note: Genetic analysis, pathology, and vectors of echinostomiasis, a zoonotic helminth infection in chickens in Bangladesh.

Echinostomes (Trematoda: Echinostomatidae) are food-borne zoonotic flatworms that affect birds, animals and humans, and has been classified as neglected tropical diseases (NTDs) by the World Health Organization (WHO), which cause severe enteritis in poultry and hamper production. Here, we confirmed the species of echinostomes affecting chickens in Bangladesh along with their genetic analyses, pathology and vectors. We isolated and identified adult worms from chickens, cercariae from fresh water snails and metacerariae (MC) from some wild fishes. We recovered Echinostoma revolutum (10.3%) and Hypoderaeum conoideum (6.0%) from chickens. Zoonotic E. revolutum was confirmed by amplifying nad1 gene and subsequent sequencing. Several mutations were detected in nad1 gene and our isolates belonged to the Euro-Asian clade. We observed thickening of mucosal layer, hyperplasia of goblet cells, infiltration of eosinophils, lymphocytes and must cells in the infected intestine. About 5.3% snails were infected and the highest percentage of infection was found in Lymnaea luteola (12.1%). Echinostome infection in snails was the highest in November (9.6%) and lowest in February (3.1%) in Bangladesh. MC of echinostomes were identified from blue panchax (Aplocheilus panchax) and tank goby (Glossogobius giuris). In conclusion, echinostomiasis is a notable big problem in indigenous chickens in Bangladesh and people, especially, villagers are at risk.

Patterns of Sphaeridiotrema pseudoglobulus infection in sympatric and allopatric hosts (Bithynia tentaculata) originating from widely separated sites across the USA.

In circumstances where populations of invasive species occur across variable landscapes, interactions among invaders, their parasites, and the surrounding environment may establish local coevolutionary trajectories for the participants. This can generate variable infection patterns when parasites interact with sympatric versus allopatric hosts. Identifying the potential for such patterns within an invasive-species framework is important for better predicting local infection outcomes and their subsequent impacts on the surrounding native community. To begin addressing this question, we exposed an invasive snail (Bithynia tentaculata) from two widely separated sites across the USA (Wisconsin and Montana) to the digenean parasite, Sphaeridiotrema pseudoglobulus, collected from Wisconsin. Parasite exposures generated high infection prevalences in both sympatric and allopatric snails. Furthermore, host survival, host growth, the proportion of patent snails, and the timing of patency did not differ between sympatric and allopatric combinations. Moreover, passaging parasites through snails of different origins had no effect on transmission success to subsequent hosts in the life cycle. However, the number of parasites emerging from snails and the pattern of their release varied based on snail origin. These latter observations suggest the potential for local adaptation in this system, but subsequent research is required to further substantiate this as a key factor underlying infection patterns in the association between S. pseudoglobulus and B. tentaculata.

Interleukin-25-mediated resistance against intestinal trematodes does not depend on the generation of Th2 responses.

BACKGROUND: The cytokine interleukin-25 (IL-25) is recognized as the most relevant initiator of protective T helper 2 (Th2) responses in intestinal helminth infections. This cytokine induces resistance against several species of intestinal helminths, including the trematode Echinostoma caproni. E. caproni has been extensively used as an experimental model to study the factors determining resistance to intestinal infections. In the study reported here, we assessed the role of IL-25 in the generation of resistance in mice infected with E. caproni. METHODS: The factors that determine the production of IL-25 in mice experimentally infected with E. caproni were determined, as were the consequences of IL-25 production in terms of polarization of the immune response and resistance to infection. RESULTS: Our results show that the role of IL-25 in the polarization of the immune response differs between the primary and secondary immune responses. IL-25 is required for the development of a Th2 phenotype in primary E. caproni infections, but it can also promote the differentiation to Th2 memory cell subsets that enhance type-2 immunity in memory responses. However, the development of Th2 responses does not induce resistance to infection. The Th2 phenotype does not elicit resistance, and IL-25 is responsible for the resistance regardless of its type-2 cytokine activity and activation of signal transducer and activator of transcription (STAT6). Alternative activation of macrophages induced by IL-25 can be implicated in the resistance to infection. CONCLUSIONS: In contrast to primary infection, secondary infection elicits a type-2 immune response even in the absence of IL-25 expression. Despite the development of a type-2 response, mice are susceptible to secondary infection associated with the lack of IL-25. Resistance to infection is due to the production of IL-25, which acts autonomously from Th2 response in terms of parasite clearance.

Ileal proteomic changes associated with IL-25-mediated resistance against intestinal trematode infections.

BACKGROUND: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, which has been extensively used to investigate the factors that determine the rejection of intestinal helminths. In this sense, several studies have shown that IL-25 is critical for the development of resistance against E. caproni in mice. In fact, treatment of mice with recombinant IL-25 generates resistance against primary E. caproni infection. However, the mechanisms by which IL-25 induces resistance remain unknown. METHODS: To study the mechanisms responsible for resistance elicited by IL-25, we analyzed the ileal proteomic changes induced by IL-25 in mice and their potential role in resistance. To this purpose, we compared the protein expression profiles in the ileum of four experimental groups of mice: naïve controls; E. caproni-infected mice; rIL-25-treated mice; and rIL-25-treated mice exposed to E. caproni metacercariae. RESULTS: Quantitative comparison by 2D-DIGE showed significant changes in a total of 41 spots. Of these, 40 validated protein spots were identified by mass spectrometry corresponding to 24 proteins. CONCLUSIONS: Our results indicate that resistance to infection is associated with the maintenance of the intestinal epithelial homeostasis and the regulation of proliferation and cell death. These results provide new insights into the proteins involved in the regulation of tissue homeostasis after intestinal infection and its transcendence in resistance.

Timing and order of exposure to two echinostome species affect patterns of infection in larval amphibians.

The study of priority effects with respect to coinfections is still in its infancy. Moreover, existing coinfection studies typically focus on infection outcomes associated with exposure to distinct sets of parasite species, despite that functionally and morphologically similar parasite species commonly coexist in nature. Therefore, it is important to understand how interactions between similar parasites influence infection outcomes. Surveys at seven ponds in northwest Pennsylvania found that multiple species of echinostomes commonly co-occur. Using a larval anuran host (Rana pipiens) and the two most commonly identified echinostome species from our field surveys (Echinostoma trivolvis and Echinoparyphium lineage 3), we examined how species composition and timing of exposure affect patterns of infection. When tadpoles were exposed to both parasites simultaneously, infection loads were higher than when exposed to Echinoparyphium alone but similar to being exposed to Echinostoma alone. When tadpoles were sequentially exposed to the parasite species, tadpoles first exposed to Echinoparyphium had 23% lower infection loads than tadpoles first exposed to Echinostoma. These findings demonstrate that exposure timing and order, even with similar parasites, can influence coinfection outcomes, and emphasize the importance of using molecular methods to identify parasites for ecological studies.

[Characterization of Echinostoma miyagawai in domestic ducks in Wuhu City].

OBJECTIVE: To investigate the morphological characteristics of Echinostoma miyagawai in domestic ducks in Wuhu area, and to explore the feasibility of the cytochrome oxidase subunit-1 (Cox1) gene as a molecular marker for the identification of E. miyagawai. METHODS: E. miyagawai was isolated from free-ranged domestic ducks in Wuhu area, and the parasites were stained and identified. In addition, the mitochondrial Cox1 gene of E. miyagawai was amplified using a PCR assay, and the amplification product was sequenced and aligned with the GenBank database to yield the homology for the identification of parasite species in combination with morphological findings. Intra-species comparison was done based on the Cox1 gene sequence. RESULTS: The prevalence of E. miyagawai infection was 16.67% in domestic ducks in Wuhu area, and the adult E. miyagawai was 6.6 to 13.2 mm in length. The size of the E. miyagawai Cox1 gene was approximately 660 bp, which had a 99.68% homology to the E. miyagawai accessed in GenBank. The morphological findings were in agreement with molecular identification. CONCLUSIONS: E. miyagawai infection is common in domestic ducks in Wuhu area, and the mitochondrial Cox1 gene is a feasible marker of intra- and inter-species molecular identification of Echinostoma.

Ancient Echinostome Eggs Discovered in Archaeological Strata Specimens from a Baekje Capital Ruins of South Korea.

Echinostomiasis is prevalent in southeastern as well as northeastern Asian countries. This endemicity notwithstanding, no echinostome egg has as yet been reported by paleoparasitological studies conducted in Asian countries. Recently we analyzed geological-strata specimens retrieved from the ancient capital city of the Baekje Kingdom to uncover clues to the possible prevalence of echinostomiasis among contemporaneous populations of Korea. By means of archaeoparasitological technique, we found ancient Isthmiophora hortensis eggs in the specimens, thus revealing for the first time that ancient Korean people experienced isthmiophoriasis. Our report can be considered to have effectively broadened the spatial and temporal scope of research on echinostomiasis in human history.

Secreted cathepsin L-like peptidases are involved in the degradation of trapped antibodies on the surface of Echinostoma caproni.

Antibody trapping is a recently described strategy for immune evasion observed in the intestinal trematode Echinostoma caproni, which may aid to avoiding the host humoral response, thus facilitating parasite survival in the presence of high levels of local-specific antibodies. Parasite-derived peptidases carry out the degradation of trapped antibodies, being essential for this mechanism. Herein, we show that cathepsin-like cysteine endopeptidases are active in the excretory/secretory products (ESPs) of E. caproni and play an important role in the context of antibody trapping. Cysteine endopeptidase activity was detected in the ESPs of E. caproni adults. The affinity probe DCG-04 distinguished a cysteine peptidase band in ESPs, which was specifically recognized by an anti-cathepsin L heterologous antibody. The same antibody localized this protein in the gut and syncytial tegument of adult worms. Studies with cultured parasites showed that in vivo-bound antibodies are removed from the parasite surface in the absence of peptidase inhibitors, while addition of cathepsin L inhibitor prevented their degradation. These results indicate that cathepsin L-like peptidases are involved in the degradation of surface-trapped antibodies and suggest that cysteine peptidases are not only crucial for tissue-invading trematodes, but they can be equally relevant at the parasite-host interface in gut-dwelling flukes.

Sub-chronic exposure to a neonicotinoid does not affect susceptibility of larval leopard frogs to infection by trematode parasites, via either depressed cercarial performance or host immunity.

Little information is available on the effects of neonicotinoid insecticides on vertebrates. Previous work using amphibians found chronic exposure to some neonicotinoids had no detrimental effects on fitness-relevant traits. However, there is some evidence of more subtle effects of neonicotinoids on immune traits and evidence that other pesticides can suppress tadpole immunity resulting in elevated levels of parasitism in the exposed tadpoles. The objective of our study was to assess whether neonicotinoid exposure affected tadpole immunometrics and susceptibility to parasitic helminths. We assessed northern leopard frog tadpole (Lithobates pipiens) levels of parasitism and leukocyte profiles following exposure to environmentally relevant concentrations of clothianidin and free-living infective cercariae of a helminth parasite, an Echinostoma sp. trematode. When comparing tadpoles from controls to either 1 or 100 µg/L clothianidin treatments, we found similar measures of parasitism (i.e. prevalence, abundance and intensity of echinostome cysts) and similar leukocyte profiles. We also confirmed that clothianidin was not lethal for cercariae; however, slight reductions in swimming activity were detected at the lowest exposure concentration of 0.23 µg/L. Our results show that exposure to clothianidin during the larval amphibian stage does not affect leukocyte profiles or susceptibility to parasitism by larval trematodes in northern leopard frogs although other aspects such as length of host exposure require further study.

Suburbanization Increases Echinostome Infection in Green Frogs and Snails.

An important contribution to infectious disease emergence in wildlife is environmental degradation driven by pollution, habitat fragmentation, and eutrophication. Amphibians are a wildlife group that is particularly sensitive to land use change, infectious diseases, and their interactions. Residential suburban land use is now a dominant, and increasing, form of land cover in the USA and globally, contributing to increased pollutant and nutrient loading in freshwater systems. We examined how suburbanization affects the infection of green frog (Rana clamitans) tadpoles and metamorphs by parasitic flatworms (Echinostoma spp.) through the alteration of landscapes surrounding ponds and concomitant changes in water quality. Using sixteen small ponds along a forest-suburban land use gradient, we assessed how the extent of suburban land use surrounding ponds influenced echinostome infection in both primary snail and secondary frog hosts. Our results show that the degree of suburbanization and concurrent chemical loading are positively associated with the presence and burden of echinostome infection in both host populations. This work contributes to a broader understanding of how land use mediates wildlife parasitism and shows how human activities at the household scale can have similar consequences for wildlife health as seemingly more intensive land uses like agriculture or urbanization.

Characterization of Echinostoma revolutum and Echinostoma robustum from ducks in Bangladesh based on morphology, nuclear ribosomal ITS2 and mitochondrial nad1 sequences.

Precise discrimination of Echinostoma species within the 'revolutum' group is quite difficult because of their morphological similarities. The objective of this study was to precisely characterize the echinostomes of ducks from Bangladesh based on both morphological and molecular characteristics. Two Echinostoma species were identified: E. revolutum and E. robustum. In the phylogenetic trees (ITS2 and nad1), E. revolutum and E. robustum belonged to their respective Eurasian clade, which is distinct from the American clade. These results suggest that both species have two distinct and geographically separated lineages, Eurasian and American. Our molecular and morphological data combined with previously published data supports the synonymy of E. robustum, E. miyagawai, and E. friedi previously based on either molecular or morphological evidence. This study thus improves our understanding of species diversity of the 'revolutum' group, particularly in Asia.

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.

Trematode Parasite Infection Affects Temperature Selection in Aquatic Host Snails.

Animals infected by parasites or pathogens can exhibit altered behaviors that may reduce the costs of infection to the host or represent manipulations that benefit the parasite. Given that temperature affects many critical physiological processes, changes in thermoregulatory behaviors are an important consideration for infected hosts, especially ectotherms. Here we examined the temperature choices of freshwater snails (Helisoma trivolvis) that were or were not infected by a trematode (flatworm) parasite (Echinostoma trivolvis). Active snails that explored the experimental temperature gradient differed in their thermal preference based on their infection status, as parasitized snails chose to position themselves at a significantly higher temperature (mean: 25.4°C) compared to those that were uninfected (mean: 23.3°C). Given that snails rarely eliminate established trematode infections, we suggest that this altered thermal preference shown by infected hosts likely benefits the parasite by increasing the odds of successful transmission, either through enhanced production and emergence of infectious stages or by increasing spatial overlap with the next hosts of the complex life cycle. Further studies that employ experimental infections to examine temperature selection at different time points will be needed to understand the extent of altered host thermal preferences, as well as the possible benefits to both host and parasite.

Echinostoma revolutum: Development of a high performance DNA-specific primer to demonstrate the epidemiological situations of their intermediate hosts.

Echinostomiasis caused by the Echinostoma group, in particular E. revolutum are a significant problem for both humans and other animals. This group has a large number of morphological similarities that are difficult and time-consuming to identify. The present study aimed to develop high-performance tools for the detection of the prevalence of E. revolutum and to reveal the prevalence of E. revolutum infections in intermediate snail hosts in Lopburi province, Thailand. The snail specimens were collected by stratified sampling method and examined to collect trematodes in the larval stage. The specific primer was manually designed and based on 18 s rDNA and verified the specificity and sensitivity for use as an identification tool to compare with classical method, constructed by epidemic mapping. The overall prevalence value of E. revolutum was found to be 16.26%. Tha Luang district had the highest prevalence (70.14%), followed by Chai Badan, Phatthana Nikhom, Tha Wung, Ban Mi, Khok Samrong, Nong Muang and Sa Bot at 42%, 25.14%, 2.52%, 1.73%, 2%, 1.33% and 0.40%, respectively. With regard to the specific primer, it can amplify both cercarial and metacercarial DNA (90 pg/µl.) and discriminated E. revolutum from its hosts, other trematodes and other echinostome larvae with no cross-reactions. Therefore, the developed specific primer can be used as a species-specific identification tool with a high degree of sensitivity and specificity. Consequently, this data is important for monitoring the outbreak of E. revolutum. It can be applied for initiating surveillance programs of snail-borne diseases in both medical and veterinary studies.

Echinostoma macrorchis Metacercariae in Cipangopaludina chinensis malleata from Xiengkhuang Province, Lao PDR and Morphologies of Adults from Experimental Animals.

We identified the echinostome metacercariae in Chinese mystery snails, Cipangopaludina chinensis malleata, from Xiengkhuang Province, Lao PDR with morphologies of adult worms recovered. Total 20 snails were examined with artificial digestion method and then the collected metacercariae were orally infected to a mouse and a rat. Adult worms recovered from experimental animals were observed with a light microscope and a SEM. The metacercariae were round, 125×123 µm in average size, with a moderately thick cyst wall, collar spines distributed in the head collar and excretory granules in 2 canals of excretory tube. Adult flukes (3-week-old in a rat) were elongated, ventrally curved and 5.310×1.023 mm in average size. Head collar distinct, bearing 43 collar spines with 5 end group ones on each side. Oral sucker subterminal, prepharynx very short, pharynx well developed, and esophagus relatively short. Cirrus sac well developed, with a saccular seminal vesicle, and ventral sucker very large. Ovary round and on the median line of the body. Testes tandom and elongated. Eggs operculated, elliptical and 90×57 µm in average size. In the SEM observation, the head crown prominent, with 43 collar spines resembled with horns of younger stag. Scale-like tegumental spines were densely distributed on the surface between the head collar and ventral sucker, and their densities were decreased posteriorly. Conclusively, the metacercariae detected in C. chinensis malleata from Lao PDR were identified as those of Echinostoma macrorchis based on the morphological characteristics of adult worms.

Echinostoma ilocanum Infection in Two Residents of Savannakhet Province, Lao PDR.

Adult specimens of Echinostoma ilocanum (Garrison, 1908) Odhner, 1911 (Trematoda: Echinostomatidae) were recovered from 2 riparian people who resided along the Mekong River in Savannakhet Province, Lao PDR. In fecal examinations done by the Kato-Katz technique, they revealed echinostome eggs together with eggs of Opisthorchis viverrini (and minute intestinal fluke eggs) and hookworms. To recover the adult flukes, they were treated with praziquantel 30-40 mg/kg in a single dose and purged with magnesium salts. A total of 658 adult fluke specimens were recovered from the 2 people; 456 from case 1 and 202 from case 2. Specimens from case 1 consisted of 335 echinostomes (301 E. ilocanum and 34 species undetermined), 120 O. viverrini, and 1 Haplorchis taichui, and those from case 2 consisted of 36 E. ilocanum, 134 O. viverrini, and 32 H. taichui. Thus, the number of E. ilocanum specimens was 337 in total (average per person, 168.5). From this study, it is suggested that foodborne intestinal flukes and liver flukes are highly prevalent along the Mekong River in Savannakhet Province. The present report describes for the first time human infections with E. ilocanum in Lao PDR.

Adaptation of the secretome of Echinostoma caproni may contribute to parasite survival in a Th1 milieu.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, broadly employed to study the host-dependent mechanisms that govern the evolution of intestinal helminth infections. Resistance against E. caproni homologous secondary infections has been reported in mice and appears to be related to the generation of a local Th2 response, whereas Th1 responses promote the development of chronic primary infections. Herein, the ability of E. caproni to modulate its secretome according to the host environment is investigated. A two-dimensional differential in gel electrophoresis (2D-DIGE) analysis was performed to elucidate changes in the excretory/secretory products of E. caproni adults after primary and secondary infections in mice. A total of 16 protein spots showed significant differences between groups, and 7 of them were successfully identified by mass spectrometry. Adult worms exposed to a primary infection appear to upregulate proteins involved in detoxification (aldo-keto reductase), stress response (GroEL), and enhancement of parasite survival (acetyl-CoA A-acetyltransferase and UTP-glucose-1-phosphate urydyltransferase). In contrast, any protein was found to be significantly upregulated after secondary infection. Upregulation of such proteins may serve to withstand the hostile Th1 environment generated in primary infections in mice. These results provide new insights into the resistance mechanisms developed by the parasites to ensure their long-term survival.