Galactosomum nagasakiense n. sp. (Digenea: Heterophyidae) from the black-tailed gull, Larus crassirostris, with a description of metacercariae from some marine fishes developing trematode whirling disease.

Galactosomum nagasakiense n. sp. (Digenea: Heterophyidae), the causative parasite of trematode whirling disease of marine fish, is described, based on the specimens collected from the black-tailed gull, Larus crassirostris in Nagasaki Prefecture, Japan. The new species belongs to the cochleariformum group, and is different from the three species in the group: G. cochleare (Diesing, 1850) in the larger oral sucker and phaynx and smaller eggs, G. cochleariformum (Rudolphi, 1819) in the smaller oral sucker and pharynx and smaller eggs, and G. spinetum (Braun, 1901) in the uterus which extends anterior to the ventrogenital sac. Metacercariae collected from the brain of several marine fish developing trematode whirling disease are also described. Considering low host specificity of the second intermediate hosts, the endemic nature of the trematode whirling disease may be attributed to the infection of a particular population of the final host and/or a limited geographical distribution of the first intermediate host.

Innovative molecular and immunological approaches of heterophyiasis infecting some Egyptian marketed fishes.

Heterophyiasis is a highly endemic disease in the Nile Delta, Egypt, where people consume raw or undercooked Oreochromis niloticus and Mugil cephalus. Birds and rats play a crucial role in fish-borne zoonotic trematode transmission since they serve as natural and experimental hosts. This study aimed to update the epidemiological information, morphological description, molecular identification and gene expression of two distinct heterophyid metacercariae in Giza, Wadi Al-Rayan, and Lake Manzala, Egypt, whereas various heterophyid infections could be expected. The present Centrocestus formosanus, Heterophyes heterophyes, and Heterophyes nocens with accession numbers OR947651.1, OR947700.1, and OR947719.1, respectively, matched with those recorded in the GenBank. Findings of the current investigation indicated that various cytokines like IL-1ß, MHC-II, and TNF-α rapidly elevated in the infected pigeon's intestines. Additionally, the infection expanded due to the parasite's ejection from the host and the host's clinical affliction, which induced humoral immune responses. Interestingly, investigation of other trematode species is in extreme demand in terms of zoonoses. We suggest controlling snails, managing migratory birds, and examining and frying fishes to the point when the encysted metacercariae is destroyed.

Infection characteristics of Metagonimus species (Digenea: Heterophyidae) metacercariae in fish from major rivers of Korea.

This article analyzed the infection characteristics of metacercariae of Metagonimus spp. (MsMc) in fish from 9 major water systems in Korea. A total of 19,568 fish in 87 species were examined over a period of 10 years (2011-2020). MsMc were detected in fish from all 44 survey areas in 9 water systems. Most of the surveyed sites showed very low and low infection levels (66.7%), while 33.3% of the areas, such as Tamjin-gang and Seomjin-gang, revealed moderate and high infection levels. High endemicity depends on the abundance of susceptible fish species, especially sweet smelt (Plecoglosus altivelis). The susceptibility index (SI) with MsMc in index fish, Zacco spp., was very low and low levels in 62.0%, moderate in 28.0%, and high in 10.0% regions. The SI was highest in the following order: Yeongam-cheon (283.8), Hoeng-cheon (192.3), Togyo-jeosuji (131.2), Deokcheon-gang (119.1), and Joyang-gang (106.3). The recent infection status of MsMc in P. altivelis was analyzed by the survey localities. In addition, except for P. altivelis, 9 fish species were highly infected with MsMc in some survey areas, including Zacco platypus, Z. koreanus, Z. temminckii, Opsariichthys uncirostris, Rhynchocypris oxycephalus, Carassius auratus, Acheilognathus rhombeus, Onchorhynchus masou, and Tribolodon hakonensis. In Korea, 74 fish species (15 families) are collectively listed as second intermediate hosts of Metagonimus spp. This review provides several novel characteristics of MsMc infection and clarifies the fish species of second intermediate host of Metagonimus spp. in this country.

Small intestinal flukes of the genus Metagonimus (Digenea: Heterophyidae) in Europe and the Middle East: A review of parasites with zoonotic potential.

The heterophyid trematode Metagonimus romanicus (Ciurea, 1915) (Digenea) is redescribed on the basis of type material from domestic dogs (Canis familiaris) in Romania, vouchers from experimentally infected cats (Felis catus) and adults recovered from golden hamsters (Mesocricetus auratus) infected with metacercariae from scales of chub (Squalius cephalus) and common nase (Chondrostoma nasus) (Cypriniformes: Leuciscidae) in Hungary. This trematode, endemic to Europe and neighbouring regions (northwestern Türkiye), was previously misidentified as M. yokogawai (Katsurada, 1912), a zoonotic parasite of humans in East Asia. However, the two species differ considerably both genetically and morphologically, e.g., in the position of the ventral sucker, the presence of the prepharynx, the anterior extent of the vitelline follicles and the posterior extent of the uterus. Metagonimus ciureanus (Witenberg, 1929) (syn. Dexiogonimus ciureanus Witenberg, 1929), described from domestic cats and dogs in Israel, is a valid species distributed in the Middle East and Transcaucasia, which is also confirmed by molecular data. It differs from all Metagonimus species, including M. romanicus, in having symmetrical testes instead of the oblique testes of the other congeners. The zoonotic significance of M. romanicus and M. ciureanus is unclear, but appears to be low in Europe, mainly because raw or undercooked, whole fish with scales are generally not consumed. Accidental infection of fishermen by metacercariae in the scales when cleaning fish is more likely, but has never been reported. Remains of cyprinoids with scales infected with metacercariae of Metagonimus spp. can be an important natural source of infection for dogs, cats, and other carnivores, which can serve as a reservoir for these parasites.

Title: Petits trématodes intestinaux du genre Metagonimus (Digenea : Heterophyidae) en Europe et au Moyen-Orient : revue de parasites à potentiel zoonotique. Abstract: Le trématode Heterophyidae Metagonimus romanicus (Ciurea, 1915) (Digenea) est redécrit sur la base de matériel type provenant de chiens domestiques (Canis familiaris) en Roumanie, de vouchers issus de chats (Felis catus) infectés expérimentalement et d'adultes collectés chez des hamsters dorés (Mesocricetus auratus) infectés par des métacercaires provenant d'écailles de chevesne commun (Squalius cephalus) et de nase commun (Chondrostoma nasus) (Cypriniformes : Leuciscidae) de Hongrie. Ce trématode, endémique d'Europe et des régions voisines (nord-ouest de la Turquie), avait été précédemment identifié à tort comme étant M. yokogawai (Katsurada, 1912), un parasite zoonotique des humains en Asie de l'Est. Cependant, les deux espèces diffèrent considérablement sur le plan génétique et morphologique, par exemple par la position de la ventouse ventrale, la présence du prépharynx, l'étendue antérieure des follicules vitellins et l'étendue postérieure de l'utérus. Metagonimus ciureanus (Witenberg, 1929) (syn. Dexiogonimus ciureanus Witenberg, 1929), décrite chez des chats et des chiens domestiques en Israël, est une espèce valide répartie au Moyen-Orient et en Transcaucasie, ce qui est également confirmé par des données moléculaires. Cette espèce diffère de toutes les espèces de Metagonimus, y compris M. romanicus, par ses testicules symétriques au lieu des testicules obliques des autres congénères. L'importance zoonotique de M. romanicus et M. ciureanus n'est pas claire, mais semble faible en Europe, principalement parce que les poissons n'y sont généralement pas consommés crus ou insuffisamment cuits et entiers avec les écailles. L'infection accidentelle des pêcheurs par des métacercaires présents dans les écailles lors du nettoyage du poisson est plus probable mais n'a jamais été signalée. Les restes de poissons cyprinoïdes avec les écailles, infectés par des métacercaires de Metagonimus spp. peuvent être une source naturelle importante d'infection pour les chiens, les chats et autres carnivores, qui peuvent servir de réservoir à ces parasites.

A potential role of protein extractions from Metagonimus yokogawai in amelionating inflammation in patients with ankylosing spondylitis.

Helminth infections and their components has been recognized to have a positive impact on the immune system. This study aimed to investigate the potential of Metagonimus yokogawai-derived proteins (MYp) to provide protection against ankylosing spondylitis (AS) through modulation of immune responses. The cytotoxicity of MYp at various doses was first assessed using MTS and flow cytometry. Peripheral blood mononuclear cells (PBMCs) were collected from AS patients, and the production of inflammatory cytokines was analyzed through flow cytometry. In the experiments with SKG mice, MYp or vehicle was administered and inflammation was evaluated through immunohistochemistry and enzyme-linked immunosorbent assay. The results showed that MYp did not decrease cell viability of PBMCs even after 48 h. Additionally, the frequencies of IFN-γ and IL-17A producing cells were significantly reduced after MYp treatment in the PBMC cultures. Furthermore, MYp treatment significantly suppressed arthritis and enthesitis in the SKG mouse model. The results suggest the first evidence that MYp can effectively alleviate clinical symptoms and restore cytokine balance in patients with AS.

Ultra-structure, genetic characterization and Immunological approach of fish borne zoonotic trematodes (Family: Heterophyidae) of a redbelly tilapia.

Fish-borne zoonotic trematodes (FBZT) are highly significant zoonotic trematodes that can infect humans by eating raw or undercooked fish harboring active metacercaria. In this investigation, FBZT was found in samples of widely cultivated redbelly tilapia (Tilapia zillii) obtained from the Fayum governorate. Encysted metacercaria (EMC) infection was identified in fish belonging to the heterophyid family morphologically. The prevalence of heterophyid EMC was 30.5%. EMC was identified and implemented in a subsequent study on domestic pigeons (Columba livia domestica) carried out to allow adult flukes of Pygidiopsis (P.) genata; P. summa; and Ascocotyle (A.) pindoramensis species in their small intestine. This study presents the first report that combines ultra-structure, molecular approach of three species of heterophyid flukes, ultra-structure using transmission electron microscope in P. genata, and the study of host immunological responses and associated cytokines during Pygidiopsis species infection of pigeons in Egypt. Using Quantitative Real-time PCR (qRT- PCR), the gene expression levels of six cytokines (IL-1, IL-2, IL-6, IL-10, IFN-γ and TGF-ß3) were assessed. The molecular confirmation of P. genata, P. summa, and A. pindoramensis have a registration in the GenBank under accession number MT672308.1, OR083433.1, and OR083431.1, respectively. Throughout the infection, the gut produced cytokines in considerably variable amounts. As a result of the Pygidiopsis species infection in pigeons, our data showed distinctive cytokine alterations, which could aid in figuring out the immunological pathogenesis and host defense mechanism against this infection. This study focused on different types of fish-borne trematodes, particularly the zoonotically important ones.

Scaphanocephalus spp. (Trematoda: Opisthorchiidae) in intermediate and definitive hosts of the Yucatán Peninsula, Mexico, with a re-description of Scaphanocephalus expansus.

Scaphanocephalus is a small trematode genus belonging to the family Opistorchiidae. The genus currently contains only three species associated with marine fish as intermediate hosts and fish-eating birds as definitive hosts. Here, specimens of Scaphanocephalus were collected from the Osprey, Pandion haliaetus, and the White mullet, Mugil curema in the Yucatán Peninsula, Mexico. We report for the first-time DNA sequences of adult specimens of Scaphanocephalus, particularly S. expansus, as well as a sequence of a different species sampled as metacercaria. Morphological comparisons of Scaphanocephalus expansus confirmed the identity of the adult specimens, with minor morphological variations; Scanning electron photomicrographs were included, and the species was re-described. Phylogenetic analysis based on 28S rDNA sequences showed that Scaphanocephalus is monophyletic within Opisthorchiidae and consists of three independent lineages. Sequences of adults are identical to those of S. expansus. Instead, the sequence of the metacercaria sampled from the mesentery of Mugil curema nested with specimens reported as Scaphanocephalus sp. from a labrid fish in the Mediterranean Sea, herein named it as Scaphanocephalus sp. 2.

Paleoparasitology research on ancient helminth eggs and larvae in the Republic of Korea.

Paleoparasitology is a discipline that applies existing conventional and molecular techniques to study parasites found in ancient ruins. This review focuses on the history of the discovery of parasites (mostly helminth eggs and larvae) in archaeological soil samples and mummies in Korea from the Three Kingdoms Period to the Joseon Dynasty (100 BCE-1910 CE). We also briefly review important milestones in global paleoparasitology. The helminth species reported so far in Korea included Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis (larva), Trichostrongylus sp. (larva), Paracapillaria philippinensis (syn. Capillaria philippinensis), Enterobius vermicularis, Fasciola hepatica, dicrocoeliids, Paragonimus westermani, Clonorchis sinensis, Metagonimus yokogawai, Pygidiopsis summa, Gymnophalloides seoi, Isthmiophora hortensis, Dibothriocephalus nihonkaiensis (syn. Diphyllobothrium nihonkaiense), and Taenia spp. tapeworms. The findings obtained by Korean paleoparasitologists/archaeologists have brought about deep insight into the status of helminthic infections in Korea's past populations. Continued paleoparasitological research is essential for further understanding of ancient parasites and parasitic diseases in Korea.

Molecular evidence of the absence of Metagonimus yokogawai (Katsurada, 1912) in Europe: report of Metagonimus sp. in cyprinoid fish from the River Danube in Hungary.

Trematodes of the genus Metagonimus Katsurada, 1912 (Digenea: Heterophyidae) are zoonotic parasites that cause infections in humans, with most cases reported in Southeast Asia. Larvae from the second intermediate host, called metacercariae, of one of human-infecting species, M. yokogawai (Katsurada, 1912), have been reported from cyprinoid fish in Europe. In the present study, we provided DNA-based evidence that metacercariae of Metagonimus, which are commonly found in the scales of various cyprinoids in Central Europe (Danube River in Hungary) do not belong to M. yokogawai. Sequence analysis of the ITS region, 28S rDNA, and cox1 genes showed that this species is clearly distinct from all Asian species, including M. yokogawai, which probably does not occur in Europe. Metacercariae from cyprinoids might belong to Metagonimus romanicus (Ciurea, 1915), an insufficiently known species described from Romania.

Trematode metacercariae and adults in cyprinoid fish from Khun Thale Swamp in Surat Thani province, Thailand.

The present study aimed to determine the infection status of trematode metacercariae and adults in cyprinoid fish from the Khun Thale Swamp in Surat Thani, Southern Thailand, with epidemiologic and faunistic viewpoints. In 2020, 577 fish in 15 species were collected in the summer (February-April) and rainy (September-November) seasons. Fish were individually examined for trematode metacercariae in the whole body and adults in the gastrointestinal tract using a stereomicroscope. Three species of digenetic trematode metacercariae, i.e., Haplorchis taichui, Haplorchoides mehrai, and Centrocestus formosanus, were detected in the muscle, fin, and/or scale of fish. Two species of adult flukes, including Rohdella siamensis and Helostomatis cyprinorum, were collected in the intestines. The prevalence of overall trematode infections was 32.4% (187/577 fish), which was higher in the rainy season (41.4%; 118/285) than in the summer season (23.6%; 69/292). The metacercariae of H. taichui and H. mehrai were detected in 7 fish species each, and those of C. formosanus were found only in Rasbora toneri. The aspidogastrean trematode R. siamensis (adult) was detected in Babonymus gonionotus. A digenean species, H. cyprinorum (adult), was found in Labiobarbus siamensis and Osteochilus vittatus. The present study has first confirmed that the metacercariae of heterophyid flukes, including H. taichui, H. mehrai, and C. formosanus, and adults of R. siamensis (Aspidogastrea) and H. cyprinorum (Digenea) are infected in some species of the cyprinoid fish from the Khun Thale Swamp in Surat Thani, Thailand.

Molecular Detection of Haplorchis pumilio Eggs in Schoolchildren, Kome Island, Lake Victoria, Tanzania.

A survey of intestinal helminths targeting 1,440 schoolchildren in 12 primary schools on Kome Island (Lake Victoria), Tanzania, revealed small trematode eggs in 19 children (1.3%), seemingly of a species of Haplorchis or Heterophyes. The eggs were molecularly confirmed to be Haplorchis pumilio on the basis of 18S and 28S rDNA sequences.

Resolution of cryptic species complexes within the genus Metagonimus (Trematoda: Heterophyidae) in Japan, with descriptions of four new species.

A nationwide fish survey was conducted in Japan to detect metacercariae of the genus Metagonimus (Trematoda: Heterophyidae). The metacercariae were subjected to DNA barcoding for molecular species identification. A phylogeny inferred from the sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) prompted us to recognize three cryptic species complexes (i.e., the M. miyatai complex, the M. takahashii complex, and the M. katsuradai complex). Each complex included one or two undescribed species. For morphological description, adult flukes of each species were raised through the experimental infections of immunosuppressed mice. We propose M. saitoi n. sp., M. kogai n. sp., M. shimazui n. sp., and M. kinoi n. sp., based on their phylogeny, morphology, biogeography, and ecology (host-parasite relationships). The originally described species, M. miyatai, was split into M. miyatai sensu stricto and M. saitoi n. sp. The former is distributed mainly in eastern Japan and uses the sweetfish (Plecoglossus altivelis) and daces (Pseudaspius hakonensis and Ps. sachalinensis) as principal second intermediate hosts, while the latter is in western Japan and its principal fish hosts are the dark chub (Nipponocypris temminckii) and the pale chub (Opsariichthys platypus). The present survey resolves a long-standing controversy on the microtaxonomy of Metagonimus in Japan since the first discovery of Metagonimus yokogawai in 1912, and shows that 10 species of Metagonimus are still distributed in Japan, although human metagonimiasis is almost eradicated.

General overview of the current status of human foodborne trematodiasis.

Foodborne trematodes (FBT) of public health significance include liver flukes (Clonorchis sinensis, Opisthorchis viverrini, O. felineus, Fasciola hepatica and F. gigantica), lung flukes (Paragonimus westermani and several other Paragonimus spp.) and intestinal flukes, which include heterophyids (Metagonimus yokogawai, Heterophyes nocens and Haplorchis taichui), echinostomes (Echinostoma revolutum, Isthmiophora hortensis, Echinochasmus japonicus and Artyfechinostomum malayanum) and miscellaneous species, including Fasciolopsis buski and Gymnophalloides seoi. These trematode infections are distributed worldwide but occur most commonly in Asia. The global burden of FBT diseases has been estimated at about 80 million, however, this seems to be a considerable underestimate. Their life cycle involves a molluscan first intermediate host, and a second intermediate host, including freshwater fish, crustaceans, aquatic vegetables and freshwater or brackish water gastropods and bivalves. The mode of human infection is the consumption of the second intermediate host under raw or improperly cooked conditions. The major pathogenesis of C. sinensis and Opisthorchis spp. infection includes inflammation of the bile duct which leads to cholangitis and cholecystitis, and in a substantial number of patients, serious complications, such as liver cirrhosis and cholangiocarcinoma, may develop. In lung fluke infections, cough, bloody sputum and bronchiectasis are the most common clinical manifestations. However, lung flukes often migrate to extrapulmonary sites, including the brain, spinal cord, skin, subcutaneous tissues and abdominal organs. Intestinal flukes can induce inflammation in the intestinal mucosa, and they may at times undergo extraintestinal migration, in particular, in immunocompromised patients. In order to control FBT infections, eating foods after proper cooking is strongly recommended.

Assessing the population structure of trematode Metagonimus suifunensis using three mitochondrial markers.

In this work, for the first time, the genetic variability of the Metagonimus suifunensis population in the Russian southern Far East was estimated based on the full-length sequences of the nad1 gene of mitochondrial DNA. In addition, for a sample of the same size, the sequences of cox1 and cytb genes, previously used for population studies for M. suifunensis, were reanalysed. Three markers were combined to a common sequence, and the obtained data were studied. Despite the higher level of variability, nad1 and cox1 mtDNA genes did not reveal subdivisions within the population. The combined dataset made it possible to determine that the sample from the Odyr River was the centre of the species' range formation and clarified the continental migration route of the parasite from south to north. According to the data obtained, it was presumed that piscivorous birds participate in the life cycle of the parasite. The subdivision within population revealed that using all three mitochondrial markers is consistent with the features of differentiation within populations of related species, but the reasons for its formation remain unclear due to the insufficient amount of data and the use of different markers in studies of different species.

Ancient DNA of Metagonimus yokogawai Recovered from Joseon Period Human Remains Newly Discovered at Goryeong County in South Korea.

PURPOSE: Metagonimiasis, commonly seen in East Asian countries, is a parasitic disorder caused by definitive hosts' ingestion of undercooked freshwater fishes. Recently, genetic analysis has proved 28S rRNA and cytochrome c oxidase subunit I (COI) mtDNA gene to be a successful marker differentiating species of the genus Metagonimus. In the present study, using specimens from the newly discovered Joseon Dynasty human remains of Goryeong, we obtained updated genetic data on genus Metagonimus, which was also prevalent during the Joseon period. METHODS: The ancient DNA (aDNA) was retrieved from the coprolite sample of the seventeenth century, half-mummified individual discovered at Goryeong Country, South Korea. Cloning and sequencing were performed on PCR-amplified amplicons for M. yokogawai 28S rRNA and COI mtDNA gene. The consensus sequences were used for species identification and phylogenetic analysis using NCBI/BLAST and MEGA X software. RESULTS: Based on the COI mtDNA gene region, the Goryeong sequence was confirmed as belonging to M. yokogawai, as it was shown to form a separate cluster with other M. yokogawai taxa that are distinct also from M. takahashii and M. miyatai. CONCLUSION: In a series of our genetic analyses on genus Metagonimus using samples retrieved from Joseon-period cases, aDNA sequences of genus Metagonimus revealed in South Korea thus far are those of M. yokogawai, but not of M. miyatai or M. takahashii yet.

Distribution of cysts holding Ascocotyle (Phagicola) longa metacercariae in tissues and organs of mugilid.

Ascocotyle (Phagicola) longa is an etiological agent of human phagicolosis. Mugilids are the second intermediate host, the first being Heleobia australis, and mugilids predatory birds and mammals are its definitive hosts. The occurrence of cysts holding A. longa metacercariae is described in mugilids with a prevalence of up to 100%. The wide geographical distribution of A. longa and its intermediate hosts coupled with the rise in the consumption of raw or poorly cooked fish may elevate the risk of human infection. Therefore, in this study, we aimed to verify the distribution pattern of cysts holding A. longa in mugilids. The tissue and organ samples of these fish were processed in a domestic blender and examined under a stereoscopic microscope to identify the cysts holding the digenetic metacercariae. Of the 24 (100%) fish samples that were analyzed, 12 of Mugil curema and 12 of Mugil liza possessed cysts holding A. longa metacercariae. Digenetic cysts were identified to be present in the gills, heart, stomach, liver, intestines, mesentery, and muscular tissues collected from M. curema and M. liza. Conclusively, in M. curema, the cysts holding A. longa metacercariae were found to be distributed randomly throughout the fish body in almost every tissue and organ that was examined.

The first record of Centrocestus formosanus (Trematoda: Heterophyidae) in southern Brazil supported by molecular data.

Centrocestus formosanus is a digenean parasite first described from Asia, which parasitizes Melanoides tuberculata (Gastropoda: Thiaridae) at the first intermediate stage, and different fish species as second intermediate host. C. formosanus was previously recorded in Brazilian states, but never before in the southern region of this country. Recording and identification of digenean species through morphological identification is a taxonomic challenge. In light of this, we use an integrative taxonomic approach to report the occurrence of cercariae and metacercariae of C. formosanus in molluscs and fish, respectively, in an urban park located in southern Brazil. Specimens of M. tuberculata and the fishes Poecilia reticulata and Xiphophorus sp. (Poeciliidae) were collected for screening for parasites and molecular analyses using partial fragments of the 28S rDNA gene. The identification of C. formosanus obtained from molluscs and fish specimens permitted us to partially solve the life cycle of this parasite for the first time in the studied environment, demonstrating the necessity of monitoring and controlling molluscs populations. Nevertheless, our results will support future studies aiming to elucidate the life-cycle of C. formosanus in this region, since all sampled hosts' species are invasive in this environment.

Euryhelmis squamula (Digenea: Heterophyidae) Recovered from Korean Raccoon Dog, Nyctereutes procyonoides koreensis, in Korea.

In this study, we intended to describe an unrecorded species of heterophyid trematode recovered from the small intestine of a Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. A total of 13 small flukes were collected from a deceased Korean raccoon dog which was found in Chuncheon-si, Gangwon-do, Korea in May 2017. The trematode body were covered with many small spines, rectangular, broader than long, 807-1,103 µm long and 1,270-1,550 µm wide. Oral sucker in the anterior end slightly smaller than acetabulum. Pharynx muscular and well developed. Esophagus relatively long and sigmoid. Acetabulum small and located at median in anterior 2/5 portion. Ceca bifurcated at the anterior of genital pore and acetabulum and terminated at testis level. Testes larger, deeply lobed and located at the near of posterior end of body. Ovary small, triangular and located at the slight left of median and the anterior of left testis. Vitelline follicles dendritic and extend from the middle level of esophagus to the posterior portion of body. Eggs embryonated, operculated, small and 33-35×15-16 µm in size. Based on the morphological characteristics, the small heterophyid flukes recovered from the small intestines of Korean raccoon dog, N. procyonoides koreensis, were identified as Euryhelmis squamula (Digenea: Heterophyidae). Accordingly, this species of heterophyid flukes is to be a new trematode fauna in Korea by this study.

Optimization of tools for the detection and identification of Cryptocotyle metacercariae in fish: Digestion method and viability studies.

Some trematode metacercariae, including marine digeneans belonging to the genus Cryptocotyle, induce black spots in target tissues due to the attraction of fish host melanophores. To promote precise quantification of infection, the counting of black spots has to be confirmed by reliable quantification of metacercariae after tissue digestion. This process ensures the isolation of undamaged parasites for morphological and molecular identification. The aim of this work was to optimize the pepsin digestion protocol and to assess the duration of viability of Cryptocotyle metacercariae in fish post-mortem (pm). Four digestion protocols were compared by measuring the viability rate of metacercariae. The present study shows that the orbital digestion method was the least destructive for metacercariae and allowed better quantification of Cryptocotyle infection. Moreover, morphological identification seemed reliable up to 8 days pm for Cryptocotyle infection.

A review of molecular identification tools for the opisthorchioidea.

The superfamily Opisthorchioidea encompasses the families Cryptogonimidae, Opisthorchiidae and Heterophyidae. These parasites depend on the aquatic environment and include marine and freshwater species. Some species, such as Clonorchis sinensis and Opisthorchis viverrini, have a high impact on public health with millions of infected people worldwide and have thus been the object of many studies and tool developments. However, for many species, tools for identification and detection are scarce. Although morphological descriptions have been used and are still important, they are often not efficient on the immature stages of these parasites. Thus, during the past few decades, molecular approaches for parasite identification have become commonplace. These approaches are efficient, quick and reliable. Nonetheless, for some parasites of the superfamily Opisthorchioidea, reference genomic data are limited. This study reviews available genetic data and molecular tools for the identification and/or the detection of this superfamily. Molecular data on this superfamily are mostly based on mitochondrial and ribosomal gene sequence analyses, especially on the cytochrome c oxidase subunit 1 gene and internal transcribed spacer regions respectively.