Morphological and Molecular Identification of Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae) in Thailand.

We tried a series of morphological and molecular approaches to identify a new species of Stellantchasmus (Digenea: Heterophyidae) originating from the wrestling half-beaked fish, Dermogenys pusillus of Thailand. Adult worm samples of the new species were recovered from hamsters experimentally infected with the metacercariae from D. pusillus in Thailand. Two isolates (Thai and Korean) of Stellantchasmus falcatus were used as comparative control groups. Worm samples of 3 Stellantchasmus groups were morphologically observed and molecularly analyzed with the mitochondrial cytochrome c oxidase 1 gene. The morphological characteristics of S. dermogenysi n. sp. are similar to S. falcatus originating from brackish water fish, but minor difference was noted including the absence of the prepharynx, position of the ovary near the ceca end, smaller body size, and shorter esophageal length. A phylogenetic tree derived from neighborjoining and maximum-likelihood methods suggests that S. dermogenysi n. sp. is separated from S. falcatus supported by high bootstrap values. The relative divergences persist between these host-specific trematodes, which we suggest should be recognized as 2 distinct species. Comparisons of S. dermogenysi n. sp. with S. falcatus isolated from mullets in Thailand and Korea indicate a genetic divergence of mitochondrial DNA of 19.4% and 21.7%, respectively. By the present study, a new species, Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae), is proposed in Thailand based on molecular evidences, in addition to minor morphological differences between S. falcatus and the new species.

The life cycle of Neocladocystis intestinalis (Vaz, 1932) (Digenea: Cryptogonimidae), in Aylacostoma chloroticum (Prosobranchia: Thiaridae), and Salminus brasiliensis (Characiformes: Characidae), in Argentina.

The life cycle of Neocladocystis intestinalis (Vaz, 1932) was resolved experimentally. The prosobranchiate snail Aylacostoma chloroticum Hylton Scott (Thiaridae) collected in the Yacyretá Dam, Province of Misiones, Argentina, was found naturally infected with cercariae that possessed pigmented eye spots, 7 pairs of penetration glands, 12 pairs of flame cells, and a V-shaped, or Y-shaped excretory vesicle with very short stem. The cercariae developed in oval cysts, which were found on fin rays, and under scales of naturally and experimentally exposed tetragonopterid fish species and of experimentally exposed poecilid and prochilodont fish species. Adults were obtained experimentally from juvenile Salminus brasiliensis (Characidae), bred in captivity, and infected with metacercariae from albino Gymnocorymbus ternetzi (Tetragonopteridae), which had been exposed to emerging cercariae.

Epidemiological and molecular data on heterophyid trematode metacercariae found in the muscle of grey mullets (Osteichthyes: Mugilidae) from Sardinia (western Mediterranean Sea).

This study is a contribution to the molecular taxonomy and epidemiology of heterophyid (Trematoda: Heterophyidae) metacercariae found in the muscle of Mugilidae (Osteichthyes) from Sardinia (western Mediterranean Sea). Sixty specimens of mugilids (13 Chelon labrosus, 18 Liza aurata, 6 Liza ramada, 8 Liza saliens, 15 Mugil cephalus) were examined and 17,899 metacercariae isolated in 95 % of the hosts. Four types of metacercariae were identified: Heterophyes sp. (n = 14,113), Heterophyes sp. -small (1225), Stictodora sp. (1606), and Ascocotyle (Phagicola) sp. (955). The experimental infection of a hamster with Heterophyes sp. metacercariae produced six adults identified as Heterophyes heterophyes and two as Heterophyes cf. nocens. The morphology of Heterophyes sp. -small metacercariae matched with that of Heterophyes dispar. The sequence analysis of the ITS2 and 28S portions of rDNA confirmed the morphological identification of metacercariae, showing four clusters. All adults grouped together with the Heterophyes sp. metacercariae, whereas adults of Heterophyes nocens from Korea clustered separately, showing that this species is distinguished from H. heterophyes, and suggesting caution in the exclusive use of the number of rodlets of the genital sucker to separate the two species. The presence of metacercariae was high in all hosts; the highest prevalence is of Heterophyes sp. (prevalence ≥78 %; mean intensity ≥135 metacercariae/100 g muscle), and the most heavily infected host is M. cephalus (prevalence = 100 %; mean intensity = 841 metacercariae/100 g muscle).

Stellantchasmus falcatus (Digenea: Heterophyidae) in Cambodia: Discovery of Metacercariae in Mullets and Recovery of Adult Flukes in an Experimental Hamster.

Stellantchasmus falcatus (Digenea: Heterophyidae) is first reported from Cambodia through recovery of the metacercariae from mullet fish and adult flukes from an experimentally infected hamster. We purchased 7 mullets, Chelon macrolepis, in a local market of Phnom Penh, Cambodia, and each of them was examined by the artificial digestion method on May 2010. The metacercariae of S. falcatus were detected in all mullets (100%) examined, and their average density was 177 per fish. They were elliptical, 220×168 µm in average size. They were orally infected to an hamster to obtain adult flukes. Adults recovered at day 10 post infection were observed with a light microscope and a scanning electron microscope (SEM). They were small, 450×237 µm in average size, had a small oral sucker (41×50 µm), subglobular pharynx (29×21 µm), slender esophagus (57 µm), long and thick-walled expulsor (119×32 µm), spherical ovary (58×69 µm), and 2 ovoid testes (right: 117×74 µm; left: 114×63 µm). Eggs were small, yellow, and 23×12 µm in average size. In SEM observations, tegumental spines were densely distributed on the whole tegument, and single small type I sensory papillae were distributed around the lip of oral sucker. The small ventral sucker was dextrally located and had 8 type I sensory papillae on the left margin. It has been first confirmed in the present study that the mullet, C. macrolepis, is playing the role of a second intermediate host of S. falcatus in Cambodia.

Molecular characterization of Stictodora tridactyla (Trematoda: Heterophyidae) from Kuwait Bay using rDNA ITS and mtCO1.

Stictodora tridactyla is an intestinal fluke in the family Heterophyidae that parasitizes shorebirds and mammals, including humans. Its metacercarial cyst stage was reported in the Arabian killifish, Aphanius dispar, at Kuwait Bay. In the present study, Cerithidea cingulata was found to serve as the first intermediate host of S. tridactyla. In order to establish the snail-fish link in the life cycle of S. tridactyla, complete sequences of ribosomal DNA internal transcribed spacer region 1 and 2 (rDNA ITS1 and ITS2) and partial sequence of cytochrome oxidase subunit 1 were obtained for metacercarial cysts isolated from the fish A. dispar and rediae isolated from the snail C. cingulata. Sequence alignment demonstrated that these larval stages belong to the same heterophyid species, S. tridactyla. Phylogenetic analysis based on rDNA ITS1, ITS2, and mtCO1 confirmed the position of S. tridactyla within the Heterophyidae and found it to cluster with Haplorchis spp. The present study represents the first molecular study correlating the larval stages of S. tridactyla using rDNA ITS1, ITS2, and mtCO1 and examining the phylogenetic relationships of S. tridactyla with different heterophyid species.

Susceptibility, metacercarial burden, and mortality of juvenile silver barb, common carp, mrigal, and tilapia following exposure to Haplorchis taichui.

Fish-borne zoonotic trematodes (FZT) including heterophyids and opisthorchiids are prevalent in native and aquaculture fish in Southeast Asia. FZT are reported highly prevalent in juvenile aquaculture fish that belonged to the family Heterophyidae, particularly Haplorchis taichui. Aquaculture fish are reported to have varying levels of natural infection with H. taichui, but data for their susceptibility as well as resistance to infection are not available. The aim of this study was to determine the susceptibility of five aquaculture fish to H. taichui. Experimental infections were performed by exposing fish individually to 0 (control), 50, 100, or 200 H. taichui cercariae for 12 h. Metacercarial burden was measured at 45 days postinfection. Three out of five fish species, silver barb (Barbonymus gonionotus), common carp (Cyprinus carpio), and mrigal (Cirrhinus mrigala), were highly susceptible to H. taichui infection with percentage ranging from 93.33 to 100%. The Nile and red tilapia were not susceptible to infection. Among the susceptible fish species, silver barb had higher intensity of H. taichui metacercariae than common carp and mrigal (P < 0.001). Metacercarial burden significantly correlated with the dose of cercarial infection (P < 0.001). Our findings that common aquaculture fish species have varying degrees of susceptibility to H. taichui infection provide important information to reduce parasite transmission in aquaculture fish.

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand.

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

[A woman with long-lasting diarrhoea and fatigue]. / En ung kvinne med langvarig diaré og slapphet.

BACKGROUND: Intestinal trematodes are common parasites in man and many mammals. Infection is often asymptomatic and unrecognised. CASE PRESENTATION: A woman in her twenties presented with loose stools of variable intensity over six months. Additionally, she had experienced considerable fatigue during this period. There was no weight loss and initial blood tests were normal. Further testing at the second visit included stool microscopy, and small trematode eggs consistent with H. heterophyes infection were found. A more thorough anamnesis revealed the onset of symptoms on the day she returned from a week's holiday, and the probable exposure occurred from eating sushi twice during this holiday. After one day of treatment with praziquantel 40 mg/kg administered in three doses, the patient recovered completely within two to four weeks. Her asymptomatic partner had consumed the same food and had the same eggs in his stool sample. He was successfully treated with the same treatment dose. INTERPRETATION: A detailed travel history may provide important information relating to the diagnosis of diarrhoea and fatigue. Symptoms of H. heterophyes infection are variable. A single day's dose of 40 mg/kg of praziquantel was sufficient to eradicate infection in the two cases presented.

Survival of heterophyid metacercaria in common carp (Cyprinus carpio).

Heterophyidae are small intestinal trematodes that infect vertebrates worldwide. Common carp (Cyprinus carpio) is one of the most preferred freshwater fish species by consumers in Asia, the region where fish-borne trematodes like Heterophyidae are most prevalent. How long Heterophyidae survive in common carp is unknown. The objective of this study was to quantify survival of Heterophyidae in common carp after experimental exposure. Fish of 0.18 g were either used as controls or exposed to 250 heterophyid cercaria for 24 h. Control fish did not become infected. Percentage infection of exposed fish at 0-2 (n = 53), >2-10 (n = 15), >10-20 (n = 11), and >20-27 (n = 33) weeks post exposure was 98, 80, 100, and 100 % respectively. The number of metacercaria per fish did not significantly decrease (P = 0.19) during 27 weeks after exposure: exp [3.6200-0.0193 × weeks post exposure]. All developed metacercaria were identified as Haplorchis spp. It was concluded that Heterophyidae may persist in carp for a long time, implying that harvestable carp are a risk to human health.

Metagonimus yokogawai: metacercariae survey in fishes and its development to adult worms in various rodents.

A parasitological survey for Metagonimus yokogawai metacercariae was carried out by examining a total of 321 freshwater fish comprising of 7 species. Of the 321 fish samples examined, 182 (56.7%) were found to be infected with M. yokogawai metacercariae. The prevalence of M. yokogawai metacercariae in Opsariichthys pachycephalus was 93.4% (86/92), Zacco platypus 75.0% (30/40), Distoechodon turmirostris 61.3% (38/62), Varicorhinus barbatulus 56.5% (13/23), Hemibarbus labeo 33.3% (1/3), Acrossocheilus formosanus 15.9% (14/88), and 0% in Sinibrama macrops (0/13), respectively. This is the first record of M. yokogawai infection in Z. platypus, D. turmirostris, V. barbatulus, and H. labeo in Taiwan. The major site of predilection of the metacercariae in the fishes was in the scale, but some metacercariae were also observed in the flesh and fins. The M. yokogawai metacercariae were orally inoculated into mice, rat, gerbil, and golden hamster to study their infectivity and also to obtain the adult worms for taxonomic study. Worm recovery in hamsters was 75.3%, in mice was 70.0%, in rats was 23.3%, and in gerbils was 6.0%, respectively. Moreover, larger worms were recovered from the golden hamster. Golden hamster was thus found to be the most susceptible experimental rodent host for the infectivity study of Metagonimus. Besides M. yokogawai, metacercariae of Centrocestus formosanus was also observed in the fishes examined.

The Life Cycle of Heterophyes yacyretana n. sp. (Digenea, Heterophyidae), Parasitic in the Endemic Snail Aylacostoma chloroticum (Caenogastropoda, Thiaridae) in Argentina.

Here we describe a new heterophyid species, Heterophyes yacyretana n. sp., and resolve its life cycle experimentally. We found the prosobranch snail Aylacostoma chloroticum in Candelaria, Province of Misiones, Argentina (a sector of the High Paraná River affected by the Yacyretá Dam), naturally infected with opisthorchioid cercariae. These cercariae lacked pigmented eyespots as well as body pigment and possessed 7 pairs of penetration glands arranged in 2 lateral bands, together with 18 pairs of flame cells and a V-shaped excretory vesicle. We exposed specimens of 21 fish species to emerging cercariae and obtained metacercariae from the muscles of the caudal peduncle of 3 species of siluriform fish, and adults from chicks infected with experimentally obtained metacercariae from the albino variety of the bronce corydoras, Corydoras aeneus. The new species differs from other species in the genus by the number of sclerites on the genital sac, the distribution of the vitelline follicles, and the combination of the size relationship of the suckers and the genital sac with respect to the posterior extent of intestinal ceca. Heterophyes yacyretana is the first species of the genus reported from the Americas.

The life history of Pygidiopsis macrostomum Travassos, 1928 (Digenea: Heterophyidae).

The life history of the trematode Pygidiopsis macrostomum Travassos, 1928 is described for the first time. Rediae and cercariae were obtained from naturally infected snails Heleobia australis (d Orbigny), a new first intermediate host. Metacercariae were found encysted in the mesenteries of three naturally infected guppies, Phalloptychus januarius (Hensel), Jenynsia multidentata (Jenyns) (new host records) and Poecilia vivipara Bloch and Schneider. Experimental infections were successfully completed in the intermediate hosts H. australis and Poe. vivipara reared in the laboratory and hamsters Mesocricetus auratus Waterhouse were utilised as a definitive host.

Survey for fishborne zoonotic metacercariae in farmed grouper in Vietnam.

Because of the common and growing practice of consuming raw grouper in Vietnam, the potential for transmission of zoonotic parasites is unclear. An investigation of the prevalence of zoonotic parasites in cage-reared grouper (Epinephelus coioides and Epinephelus bleekeri) from marine waters of Cat Ba Island, Hai Phong city, Northern Vietnam, revealed the presence of two zoonotic trematode metacercariae species, Procerovum varium and Heterophysopsis continua. The identity of the metacercariae was confirmed from adult flukes recovered from mice experimentally inoculated with metacercariae. The prevalence of zoonotic parasites in 40 E. coioides was 10.0% and 1.2% in 172 E. bleekeri. Since little is known of their ecology and epidemiology, further investigation of these zoonotic parasites is needed to develop prevention guidelines.

Haplorchis taichui: worm recovery rate and immune responses in infected rats (Rattus norvegicus).

Worm recovery rate, mucosal mast cells (MMCs), eosinophils and serum IgE concentration in rats were investigated after orally feeding 300 Haplorchis taichui metacercariae to male rats. The duodenal, jejunal and ileal tissue sections were stained with 1% alcian blue and 0.5% safranin-O for MMC count. Eosinophil count and the serum IgE concentration assay were measured from cardiac puncture blood. The average worm recovery rates were 20.00%, 13.00%, 0.67%, 1.67% and 0.00% on day 3, 7, 14, 21 and 28 post-infection (PI), respectively. The number of MMCs in the infected rats were significantly higher than in the controls (P<0.01), reaching a peak on day 21 PI. They decreased thereafter, with the decline in worm recovery. Eosinophil count and Serum IgE concentration were also increased but not significantly higher than the controls. However, they showed a positive relationship to worm recovery. It could be concluded from the results that MMCs, eosinophils and IgE may play an important role in the expulsion of H. taichui from rat intestine. However, the mechanism by which the MMC result in the helminth expulsion still need to be understood, and it is recommended that other cells such as goblet cells be studied further.

Redescription of Ascocotyle (Ascocotyle) felippei Travassos, 1928 (Digenea: Heterophiydae) with new synonymies.

The heterophyid trematode Ascocotyle (Ascocotyle) felippei Travassos, 1928, is redescribed and new data on its life cycle are provided, based on types and metacercariae found in the heart bulb and gills of naturally infected guppies, Poecilia vivipara (new fish intermediate host), from a coastal lagoon in Rio de Janeiro, Brazil. Examination of the type and all voucher specimens of A. (A.) felippei collected by Travassos in the type host and locality in Brazil has shown that they possess only 32 (16 + 16) circumoral spines, rather than 36 (18 + 18) spines as previously reported. Based on the identical number and arrangement of circumoral spines, shape of the body, the presence of a long preoral lobe and posterior muscular prolongation of the oral sucker, short and wide ceca, a simple gonotyl lacking refractile bodies, and the site of infection of metacercariae (predominantly heart bulb), A. (A.) puertoricensis Price, 1932 and A. (A.) tenuicollis Price, 1935, are proposed as new synonyms of A. (A.) felippei.

Centrocestus formosanus (Trematoda: Heterophyidae) in Melanoides tuberculata (Gastropoda: Thiaridae) from Vila do Abraão, Ilha Grande, Rio de Janeiro, Brazil.

Pleurolophocercous cercariae found in the invasive gastropod Melanoides tuberculata (Müller, 1774) collected in a stream of the Vila do Abraão, Ilha Grande, Angra dos Reis, Rio de Janeiro, Brazil were used for experimental infection that enabled the identification of the heterophyid trematode Centrocestus formosanus (Nishigori, 1924). The parasite has been found in the locality since 2007, after two years of the introduction of M. tuberculata. Recently, from a sample of 483 specimens collected in June 2013, 101 (21%) were found infected with parasite. The potential environmental impacts caused by the parasite occurrence could be underestimated in the country, and actions to monitor and control both the parasite and the mollusk are necessary.

Metagonimus suifunensis sp. n. (Trematoda: Heterophyidae) from the Russian Southern Far East: Morphology, life cycle, and molecular data.

The trematode from the Russian Southern Far East, earlier defined as M. yokogawai Katsuradai, 1912, was determined as a representative of the new species Metagonimus suifunensis sp. n. The life cycle of this trematode was experimentally studied and morphometric data for the cercariae, metacercariae and adult worm stages were obtained. The molecular data for samples from six Russian localities were received using the ITS1-5.8S-ITS2 region and 28S gene nuclear ribosomal DNA. The results revealed that M. suifunensis sp. n. does not have significant morphometric differences with M. yokogawai, M. takahashii and M. miyatai. However, the new species validity was confirmed by the molecular data. The phylogenetic relationship analysis of Metagonimus representatives showed that M. suifunensis sp. n. is well-differentiated specie with low variability in the whole area.

Susceptibility of inbred mouse strains to infection with three species of Metagonimus prevalent in the Republic of Korea.

Susceptibility of inbred mouse strains to Metagonimus yokogawai, Metagonimus miyatai, and Metagonimus takahashii infections was studied using BALB/c, ddY, C57BL/6J, C3H/HeN, and A/J mice, with H-2 haplotypes d, s, b, k, and a, respectively. Two hundred metacercariae were orally fed to each mouse, and the worm recovery rates (WRR), worm dimensions, and intrauterine egg numbers were measured at days 3, 7, 14, 21, and 28 postinfection (PI). On day 14 PI, the WRR of M. yokogawai was highest in ddY mice (average, 62.2%); those of M. miyatai and M. takahashii were highest in ddY (19.5%) and BALB/c mice (10.4%), respectively; worm maturation was best in C3H/HeN (M. yokogawai), C57BL/6J (M. miyatai), and ddY mice (M. takahashii). All mouse strains showed higher susceptibility to infection with M. yokogawai than with M. miyatai or M. takahashii. The results show that susceptibility of mice to Metagonimus infection varies according to mouse strain and parasite species but is suggested to be independent of the mouse H-2 haplotype.

Host preference of an introduced 'generalist' parasite for a non-native host.

Parasites can invade new ecosystems if they are introduced with their native hosts or if they successfully infect and colonise new hosts upon arrival. Here, we ask to what extent an introduced parasite demonstrates specialisation among novel host species. Infection surveys across three field sites in Gatun Lake, Panama, revealed that the invasive peacock bass, Cichla monoculus, was more commonly infected by the introduced trematode parasite Centrocestus formosanus than were three other common cichlid fishes. Laboratory infection experiments were conducted to determine whether parasitism might be driven by differential encounter/exposure to parasites or by differential infection susceptibility/preference across different host species. These experiments were performed by controlling for parasite exposure in single host (compatibility) experiments and in mixed host (preference) experiments. In all cases, the peacock bass exhibited higher infection rates with viable metacercariae relative to the other potential fish hosts. Our experiments thus support that an introduced generalist parasite shows apparent specialisation on a specific novel host. Further studies are needed to determine whether these patterns of specialisation are the result of local adaptation following invasion by the parasite.

First molecular identification of Ascocotyle (Phagicola) longa in its first intermediate host the mud snail Heleobia australis.

This is the first study that used species-specific DNA primers to confirm the presence of the heterophyid Ascocotyle (Phagicola) longa Ransom, 1920 in its first intermediate host. The larval stages (rediae and cercariae) of this parasite were morphologically and genetically identified in the gonad of the intertidal mud snail Heleobia australis (d'Orbigny, 1835) (Cochliopidae) in the Bahía Blanca estuary, Argentina. In addition, we asked whether the prevalence in H. australis varied between seasons. Mullets - the second intermediate host of this heterophyid - migrate in estuaries during the warmer seasons and it is expected that piscivorous birds and mammals - the definitive hosts - prey more intensively on this species at those times. Thus, the number of parasite eggs released into the tidal flat within their feces should be higher, thereby increasing the ingestion of the parasite by H. australis.We therefore expected a higher prevalence of A. (P.) longa in H. australis in the Bahía Blanca estuary during spring and summer than autumn and winter. We found that 16 out of 2,744 specimens of H. australis had been infected with A. (P.) longa (total prevalence of 0.58%). Nonetheless, the prevalence showed no significant variation between seasons. Hence, we discuss an alternative scenario where the lack of seasonal changes might be mostly related to the permanent residence of definitive hosts in the estuary and not to the seasonal recruitment of mullets. Finally, we highlight the need for more experimental and comparative approaches in order to understand the diagnosis and geographical distribution of this worldwide heterophyid.