Development and utilization of a visual loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for rapid detection of Echinostomatidae metacercaria in edible snail samples.

Trematodes belonging to the family Echinostomatidae are food-borne parasites which cause echinostomiasis in animals and humans. This is a global public health issue, particularly in East and Southeast Asia. A method to detect the infective stage of Echinostomatidae species is required to prevent transmission to humans. In this study, a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was developed for visual detection of the metacercarial stage in edible snails of the genus Filopaludina from local markets in Thailand. The LAMP-LFD method can be performed within 70 min at a consistent temperature of 66 °C, and the results can be interpreted with the naked eye. The detection limits of the assay using Echinostoma mekongi, E. macrorchis, E. miyagawai and Hypoderaeum conoideum genomic DNA were equal between the four species at 50 pg/µL. A specificity evaluation demonstrated that the LAMP-LFD assay had no cross-reaction with another parasite (Thapariella species) or with the snail host species (Filopaludina martensi martensi, F. sumatrensis speciosa, and F. s. polygramma). Clinical test assessments were compared to microscopic examination in 110 edible snail samples. The clinical sensitivity and specificity of the tests were 84.62 % and 100 %, respectively, with a strong level of agreement based on the kappa statistic and the results of both methods were not significantly different (p > 0.05) per McNemar's test. The test successfully developed in this study may be useful for the detection of the metacercarial stage in edible snails for epidemiological investigations, control, surveillance, and to prevent future echinostomiasis health issues.

Echinostoma miyagawai Ishii, 1932 (Echinostomatidae) from Ducks in Aceh Province, Indonesia with Special Reference to Its Synonymy with Echinostoma robustum Yamaguti, 1935.

Adult echinostomes having 37 collar spines collected from the intestine of Pitalah ducks in Aceh Province, Indonesia in 2018 were morphologically and molecularly determined to be Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae). Among 20 ducks examined, 7 (35.0%) were found to be infected with this echinostome, and the number of flukes collected was 48 in total with average 6.9 (1-17) worms per duck. The adult flukes were 7.2 (6.1-8.5) mm in length and 1.2 (1.0-1.4) mm in width (pre-ovarian or testicular level) and characterized by having a head collar armed with 37 collar spines (dorsal spines arranged in 2 alternating rows), including 5 end group spines, and variable morphology of the testes, irregularly or deeply lobed (3-5 lobes) at times with horizontal extension. The eggs within the worm uterus were 93 (79-105) µm long and 62 (56-70) µm wide. These morphological features were consistent with both E. miyagawai and Echinostoma robustum, for which synonymy to each other has been raised. Sequencing of 2 mitochondrial genes, cox1 and nad1, revealed high homology with E. miyagawai (98.6-100% for cox1 and 99.0-99.8% for nad1) and also with E. robustum (99.3-99.8% for nad1) deposited in GenBank. We accepted the synonymy between the 2 species and diagnosed our flukes as E. miyagawai (syn. E. robustum) with redescription of its morphology. Further studies are required to determine the biological characteristics of E. miyagawai in Aceh Province, Indonesia, including the intermediate host and larval stage information.

Presence of one ecto- and two endoparasite species of the black stork (Ciconia nigra) in Portugal.

BACKGROUND: The black stork (Ciconia nigra Linnaeus, 1758) is a recognized endangered species in Europe and most of the specimens from the Western Palearctic region breed in the Iberian Peninsula. Available works regarding parasites in black storks are scarce. This work reports the presence one ecto- and two endoparasite species from a black stork in Portugal. CASE PRESENTATION: A black stork was found in southern Portugal after colliding against electric cables. The specimen did not survive its sustained injuries and a post-mortem exam was performed. During the procedure, several ecto- and endoparasite specimens were found. The collected parasites were lice (Neophilopterus tricolor), nematodes (Desportesius sagittatus) and trematodes (Cathaemasia hians). CONCLUSIONS: Three different species of parasites are reported from a black stork in Portugal. Ecto- and endoparasites of C. nigra have not frequently been described in the literature, and this case report is a contribution to the field. Additional studies will be important to better understand the impact that parasites can have on C. nigra health and survival.

Multiplex PCR development for the differential detection of four medically important echinostomes (Trematoda: Echinostomatidae) in Thailand.

Four species of echinostomes, Echinostoma revolutum (Froelich, 1802), Echinostoma ilocanum (Garrison, 1908), Hypoderaeum conoideum (Bloch, 1872) Dietz, 1909, and Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 commonly infect humans in Thailand, but their eggs present similar morphologies resulting in difficult differentiation for diagnosis. Present molecular methods have a great potential to provide superior detection/diagnosis. DNA sequences, especially the mitochondrial NADH dehydrogenase subunit 1 (ND1) gene, have already been used to differentiate among echinostomes; thus, we aimed to develop species-specific primers for the differential detection of four medically important echinostomes by multiplex PCR. The species-specific reverse primers and a forward primer were based on variable regions and conserved regions of the ND1 gene, respectively. Four reverse primers and a forward primer were combined in a multiplex PCR reaction to amplify the ND1 fragment. Different ND1 fragment sizes were amplified: 108, 209, 384 and 419 bp of E. revolutum H. conoideum, E. ilocanum and A. malayanum, respectively. Specificity was tested with other medically important parasite DNA; no cross-reaction occurred. Sensitivity ranged between 0.1 and 0.05 ng. The species-specific primers developed in this study could be of further use in differential diagnosis for these medically important echinostomes infection in human and animal hosts.

The effect of trematode infection on the markers of oxidative stress in the offspring of the freshwater snail Lymnaea stagnalis.

Most invertebrate species exhibit immunological responses that can inactivate and eliminate penetrating parasites. Such immune responses in particular involve the formation of potentially toxic reactive oxygen species (ROS). We explored the immune capabilities of the first-generation (F1) offspring of naturally infected freshwater snails, Lymnaea stagnalis, in response to infection by trematode cercariae under laboratory conditions. The rates of ROS formation and peroxidase activity in the hemolymph of the F1 offspring of L. stagnalis parents infected by an asexual stage of trematodes were significantly higher than in F1 offspring of uninfected parents. Compared to offspring from uninfected parents, the growth rate of F1 snails from infected parents was higher, but survival was lower. After infection of F1 snails by trematode cercariae of Echinoparyphium aconiatum under laboratory conditions, the rate of ROS formation and peroxidase activity in the hemolymph of F1 offspring of uninfected parents increased compared to control snails. This pattern persisted throughout the entire 3-week observation period. In contrast, the rate of ROS formation in the hemolymph of F1 snails from infected parents after experimental infection by E. aconiatum cercariae did not differ from controls, and peroxidase activity even decreased. Thus, trematode parthenitae infection of parents could alter the immune response of their offspring.

Echinochasmus caninus n. comb. (Trematoda: Echinostomatidae) Infection in Eleven Riparian People in Khammouane Province, Lao PDR.

Adult specimens of Echinochasmus caninus n. comb. (Verma, 1935) (Trematoda: Echinostomatidae) (syn. Episthmium caninum Yamaguti, 1958) were recovered from 11 riparian people who resided along the Mekong River in Khammouane Province, Lao PDR. In fecal examinations done by the Kato-Katz technique, the cases revealed eggs of Opisthorchis viverrini/minute intestinal flukes, hookworms, and in 2 cases echinostome eggs. To recover the adult helminths, praziquantel 30-40 mg/kg and pyrantel pamoate 10-15 mg/kg in a single dose were given and purged with magnesium salts. Various species of trematodes (including O. viverrini and Haplorchis spp.), cestodes, and nematodes were recovered from their diarrheic stools. Among the trematodes, small echinostome flukes (n=42; av. 3.8 specimens per case) of 0.7-1.2 mm in length are subjected in this study. They are morphologically characterized by having 24 collar spines interrupted dorsally and anterior extension of vitellaria from the cirrus sac or genital pore level to the posterior end of the body. Particularly based on this extensive distribution of vitellaria, the specific diagnosis was made as Echinochasmus caninus. The cases were co-infected with various other helminth parasites; thus, clinical manifestations specific for this echinostome infection were difficult to determine. The present paper describes for the first time human E. caninus infections in Lao PDR. Our cases marked the 4-14th human infections with this echinostome around the world following the 3 previous cases reported from Thailand.

First molecular identification and phylogenetic tree of Petasiger exaeretus Dietz, 1909 (Digenea: Echinostomatidae) from an intermediate host Radix auricularia (L., 1758) in Greater Zab river, Iraq.

INTRODUCTION: Radix auricularia (Linnaeus, 1758) is a freshwater gastropod belongs to the Lymnaeidae (pond snails) family which act as intermediate hosts or vectors of various parasitic flukes. No study has yet been undertaken on the prevalence of Petasiger spp. infection in R. auricularia. Species of Petasiger (Dietz, 1909) are a cosmopolitan parasite that utilize snails as the first intermediate host, with vertebrates like amphibians larvae and fish as the second intermediate host, followed by fish-eating birds. The current paper is considered to be the first report of Petasiger exaeretus parasitized R. auricularia in Iraq, which is supported with molecular and phylogenetic analysis. MATERIAL AND METHODS: Freshwater snails R. auricularia were collected during October 2016 - September 2017 from different locations of Sufaia village on the Greater Zab river, Erbil province, Iraq. RESULTS: A total of 307 freshwater snails R. auricularia were collected, only five of them were infected with a prevalence of Petasiger exaeretus (1.62%). CONCLUSIONS: The current study agrees with the opinion of Selbach, Soldánová (26), which suggested the possibility of a much higher morphological diversity within Petasiger species, based on the number of described cercariae, compared with adult forms.It is clear that P. phalacrocoracis specimens have often been erroneously designated as P. exaeretus by many authors (Nasincová et al., 1994). Certain morphological similarities and dissimilarities between P. exaeretus and P. phalacrocoracis can be detected: the pear-shaped body resembles P. exaeretus, whereas, P. phalacrocoracis have an elongated body.

Infections of Two Isthmiophora Species (Digenea: Echinostomatidae) in Wild Mammals from Republic of Korea with Their Morphological Descriptions.

Isthmiophora hortensis (Digenea: Echinostomatidae) is a dominant echinostome in animal reservoir hosts and humans in the Republic of Korea (Korea). We intended to investigate the infection status with this echinostome species in the several species of wild animals and describe the morphological characteristics in the faunistic view point. A total of 175 animal carcasses belonging to 3 families, i.e., Canidae, Felidae and Mustelidae, were collected from the southern regions of Korea from March 2010 to July 2017. Isthmiophora spp. worms were recovered from the small intestines of each animal under a stereomicroscope after washing of intestinal contents. Isthmiophora hortensis was recovered from 4 species of wild carnivores, i.e., Nyctereutes procyonoides (3/107: 2.8%), Mustela sibirica (11/31: 35.5%), Meles lucurus (2/3: 33.3%) and Martes flavigula (1/2: 50%). The other 3 carnivores comprising stray dogs, cat and leopard cat were negative for I. hortensis infection (0/2, 0/10 and 0/12, respectively). Specimens obtained from the Lutra lutra (6/8: 75%) were identified as a distinct species, I. inermis, by morphological comparison. Isthmiophora inermis has thinner body, elongate testes and different anterior limits of vitelline fields. Detailed morphological descriptions and comparisons with the morphological characteristics are provided. Conclusively, it was confirmed for the first time that 3 species of mustelid mammals, i.e., M. sibirica, M. lucurus, and M. flavigula, are to be the new definitive hosts of I. hortensis in Korea. Additionally, I. inermis is to be newly added in the Korean echinostome fauna.

Infection Status of Isthmiophora hortensis Metacercariae in Dark Sleepers, Odontobutis Species, from Some Water Systems of the Republic of Korea.

Present study was performed to survey on infection status of Isthmiophora hortensis (formerly Echinostoma hortense) metacercariae (IhMc) in dark sleepers, Odontobutis spp., from some water systems of the Republic of Korea. A total of 237 Odontobutis spp. was collected in the water systems of 5 rivers, i.e., Mangyeong-gang (gang means river), Geum-gang, Tamjin-gang, Seomjin-gang, and Nakdong-gang. They were all examined with artificial digestion method for 5 years (2013-2017). A total of 137 (57.8%) Odontobutis spp. were infected with 14.8 IhMc in average. The prevalence was the highest in Nakdong-gang areas (62.9%) and followed by in Mangyeong-gang (57.1%), Geum-gang (56.3%), Tamjin-gang (54.8%), and Seomjin-gang (53.9%) areas. Metacercarial densities were 28.1 (Geum-gang), 13.9 (Mangyeong-gang), 13.3 (Nakdong-gang), 13.1 (Tamjin-gang), and 2.3 (Seomjin-gang) per infected fish. Especially, in case of Yugucheon (cheon means stream), a branch of Geum-gang, IhMc were detected in all fish (100%) examined and their density was about 48 per fish. By the present study, it was confirmed that the infection status of IhMc is more or less different by the surveyed areas and the dark sleepers, Odontobutis spp., are suitable fish hosts of I. hortensis.

Multilocus phylogenetic analysis and morphological data reveal a new species composition of the genus Drepanocephalus Dietz, 1909 (Digenea: Echinostomatidae), parasites of fish-eating birds in the Americas.

Members of the genus Drepanocephalus are endoparasites of fish-eating birds of the families Phalacrocoracidae and Sulidae distributed across the Americas. Currently, Drepanocephalus contains three species, i.e. D. spathans (type species), D. olivaceus and D. auritus. Two additional species, D. parvicephalus and D. mexicanus were transferred to the genus Petasiger. In the current study, available DNA sequences of D. spathans, D. auritus and Drepanocephalus sp., were aligned with newly generated sequences of D. spathans and Petasiger mexicanus. Phylogenetic analyses inferred with three nuclear (LSU, SSU and ITS1, 5.8S, ITS2) and two mitochondrial (cox1, nad1) molecular markers showed that the sequences of D. spathans and D. auritus are nested together in a single clade with very low genetic divergence, with Petasiger mexicanus as its sister species. Additionally, P. mexicanus was not a close relative of other members of the genus Petasiger, showing that P. mexicanus actually belongs to the genus Drepanocephalus, suggesting the need to re-allocate Petasiger mexicanus back into the genus Drepanocephalus, as D. mexicanus. Morphological observations of the newly sampled individuals of D. spathans showed that the position of the testes is variable and testes might be contiguous or widely separated, which is one of the main diagnostic traits for D. auritus. Our results suggest that D. auritus might be considered a synonym of D. spathans and, as a result, the latter represents a species with a wide geographic range across the Americas, parasitizing both the Neotropical and the double-crested cormorant in Argentina, Brazil, Paraguay, Venezuela, Colombia, Mexico, USA and Canada.

New Definitive Hosts and Differential Body Indices of Isthmiophora hortensis (Digenea: Echinostomatidae).

The present study was performed to record new definitive hosts of Isthmiophora hortensis, and to describe morphological characteristics derived from a variety of worm samples for clarification of its taxonomic validity. Morphological characteristics with dimensions were observed in worm samples (n=21) from naturally infected wild animals, including a raccoon dog Nyctereutes procyonoides from Gimhae-si (City), Gyeongsangnam-do, stray cats and a striped field mouse from several localities, and a wild boar Sus scrofa, from Gurye-gun (County), Jeollanam-do. In addition, adult flukes (n=45) recovered in albino rats experimentally infected with the metacercariae from a freshwater fish species were also subjected to morphological studies. The mean ratios of the body length (BL) to body width (BW) were 5.86 and 5.76 in worms from wild animals and experimental rats, respectively. Those of the ventral sucker to oral sucker were 2.92 and 3.01 in worms from 2 groups. The mean percentages of the hindbody length (HBL) to BL were 42.1 and 41.2 in 2 groups. Those of uterine fields to BL were 9.8 and 12.2 in the 2 worm groups. By the present study, the 2 species of wild animals, the raccoon dog and wild boar, have been added as new definitive hosts for I. hortensis. The morphological characteristics of adult flukes derived from a variety of host source were redescribed to support the taxonomic validity of this echinostome species.

Occurrence of Paratanaisia confusa Freitas, 1951 in free-living guira cuckoo (Guira guira, Cuculiformes: Crotophagidae).

This report presents a description of natural infection of an adult free-living guira cuckoo with Paratanaisia confusa. Histological and morphological evaluations of renal and parasite samples were performed. The morphological analysis of the parasites revealed spiny tegument, characteristic of P. confusa helminthes. Although macroscopic alterations were absent in the kidneys, the histopathology revealed parasites inside the collecting ducts causing dilatation and destruction of the lining epithelial cells in addition to small focal inflammatory infiltrates in the renal parenchyma. However, as the bird was free-living and naturally infected it was not possible to confirm if all these alterations were directly caused by the parasite presence or if they were related to causes other than the parasitism. Nonetheless, such findings indicate that these trematodes may have pathogenic potential in this host.

Occurrence of Paratanaisia confusa Freitas, 1951 in free-living guira cuckoo (Guira guira, Cuculiformes: Crotophagidae) / Ocorrência de Paratanaisia confusa Freitas, 1951 em anu-branco de vida livre (Guira guira, Cuculiformes: Crotophagidae)

Abstract This report presents a description of natural infection of an adult free-living guira cuckoo with Paratanaisia confusa. Histological and morphological evaluations of renal and parasite samples were performed. The morphological analysis of the parasites revealed spiny tegument, characteristic of P. confusa helminthes. Although macroscopic alterations were absent in the kidneys, the histopathology revealed parasites inside the collecting ducts causing dilatation and destruction of the lining epithelial cells in addition to small focal inflammatory infiltrates in the renal parenchyma. However, as the bird was free-living and naturally infected it was not possible to confirm if all these alterations were directly caused by the parasite presence or if they were related to causes other than the parasitism. Nonetheless, such findings indicate that these trematodes may have pathogenic potential in this host.

Resumo A infecção de um anu-branco, de vida livre, por Paratanaisia confusa é descrita. Avaliações histológicas e morfológicas de amostras renais e dos parasitas foram realizadas. A análise morfológica dos parasitas revelou a presença de tegumento espinhoso, que é característico de P. confusa. Embora alterações renais macroscópicas estivessem ausentes, a histopatologia revelou a presença de parasitas no interior de ductos coletores, provocando dilatação destes e destruição das células epiteliais, além de discretos infiltrados inflamatórios focais no parênquima renal. Entretanto, como a ave era proveniente de vida livre e naturalmente infectada, não foi possível confirmar se todas as alterações observadas foram diretamente provocadas pela presença dos parasitas, ou se estavam relacionadas a outras causas. Todavia, esses achados indicam que os trematódeos dessa espécie possam ter potencial patogênico no hospedeiro estudado.

A Case of Chaunocephalosis by Chaunocephalus ferox (Digenea: Echinostomatidae) in an Oriental White Stork, Ciconia boyciana, in Korea.

We intended to describe a case of chaunocephalosis and morphological characteristics of its causative agent, Chaunocephalus ferox, recovered from an oriental white stork, Ciconia boyciana, in the Republic of Korea. An oriental white stork was referred to the Wildlife Center of Chungbuk in Korea in February 2014 for severe depression with cachexia and it died the next day. At necropsy, the stomach was severely expanded and 7 thick-walled nodules were observed in the upper part of the intestine. Although the stomach was filled with full of foreign materials, the intestine was almost empty. The nodules were globular and total 9 flukes were recovered. They were 8,030-8,091 µm in length and 3,318-3,333 µm in maximum width. Because the flukes had bulbous forebody with short narrow subcylindrical hindbody, 27 collar spines, and vitelline follicles not reaching to the posterior end, the specimens were identified as being C. ferox. The cyst formation induced thickening of the intestinal wall with narrowing of the lumen that could have contributed to the gastric impaction to the death of the host. This is the first described case of chaunocephalosis and its causative agent C. ferox found from an oriental white stork in Korea.

Genomic diversity of cercarial clones of Himasthla elongata (Trematoda, Echinostomatidae) determined with AFLP technique.

The aim of this study was to reveal genomic diversity formed during parthenogenetic reproduction of rediae of the trematode Himasthla elongata in its molluskan host Littorina littorea. We applied amplification fragment length polymorphism (AFLP) to determine the genomic diversity of individual cercariae within the clone, that is, the infrapopulation of parthenogenetic progeny in a single molluskan host. The level of genomic diversity of particular cercariae isolates from a single clone, detected with EcoR1/Mse1 AFLP reaction, was significantly lower than the variability of cercariae from different clones. The presence of intraclonal genomic diversity indicates a nonsexual shuffle of alleles during parthenogenesis in the rediae of H. elongata. The obtained polymorphic AFLP fragments were long enough to detect the sequences that may be responsible for clonal genomic variability. Based on this, AFLP can be recommended as a tool for the study of genetic mechanisms of this variability.

Two Echinostome Species, Pegosomum bubulcum and Nephrostomum ramosum (Digenea: Echinostomatidae), from an Eastern Cattle Egret, Bubulcus ibis coromandus, in Republic of Korea.

We describe 2 echinostome species recovered from an Eastern cattle egret, Bubulcus ibis coromandus, from Cheongju-si (city), Chungcheongbuk-do (province), Korea. Total 72 Pegosomum bubulcum specimens were recovered from the bile duct. They were 7,566×2,938 µm in average size and had 27 collar spines with vitelline extension from anterior 1/3 level of the esophagus to mid-level of the posterior testis as characteristic features. Total 9 specimens of Nephrostomum ramosum were recovered in the small intestines of the bird. They were ribbon-shaped, 11,378×2,124 µm in average size, and morphologically variable in some organs, i.e., the number of collar spines (47-50), the shape of ovary and testes, and the extension of vitelline follicles. These morphological variations observed in a single host indicated that these features are not critical for the classification of Nephrostomum species and thus were reconsidered taxonomically as synonym of N. ramosum. This study is the first report documenting and describing both flukes and their associated genera in Korea.

Invasion of Flukes of the Echinostomatidae Family in Racing Pigeon ( Columba livia var. domestica) Lofts.

Over 4 years, only two known cases of fluke invasions were diagnosed in racing pigeons ( Columba livia ) originating from different regions of Poland. In both cases, the invasion was characterized by a very high mortality (approximately 70%), and the source of the infestation was snails of the Lymnaeidae family eaten by pigeons. Fluke invasions in pigeons are extremely rare and to date have not been described in Poland. Therefore, the occurrence of the symptoms of hemorrhagic diarrhea and sudden deaths of either adult pigeons or nestlings were suspected to be associated with poisoning. Autopsy revealed an invasion of flukes causing hemorrhagic enteritis. Renal failure and spleen atrophy were also found in the birds. Using molecular biology techniques, infestation with the fluke Echinostoma revolutum was determined in the second case.

Echinostomes in Felid Coprolites from Brazil.

The first record of Echinostoma (Trematoda: Echinostomatidae) in coprolites was from a mummified human body in Minas Gerais State, Brazil. The finding raised questions on this parasite's incidence in prehistoric populations and the natural hosts of each species in remote times. Echinostomes occur worldwide and, despite the wide range of hosts, there is no record of Echinostomatidae in felines in Brazil. This study reports the finding of Echinostomatidae eggs in felid coprolites in the Furna do Estrago Archaeological Site, located in Pernambuco State in the Brazilian semiarid. Despite the possibility of false parasitism, the finding expands the distribution of this Digenea in remote times and raises the hypothesis of other cases of echinostomiasis in pre-Colombian populations.

Molecular phylogeny and systematics of the Echinostomatoidea Looss, 1899 (Platyhelminthes: Digenea).

The Echinostomatoidea is a large, cosmopolitan group of digeneans currently including nine families and 105 genera, the vast majority parasitic, as adults, in birds with relatively few taxa parasitising mammals, reptiles and, exceptionally, fish. Despite the complex structure, diverse content and substantial species richness of the group, almost no attempt has been made to elucidate its phylogenetic relationships at the suprageneric level based on molecules due to the lack of data. Herein, we evaluate the consistency of the present morphology-based classification system of the Echinostomatoidea with the phylogenetic relationships of its members based on partial sequences of the nuclear lsrRNA gene for a broad diversity of taxa (80 species, representing eight families and 40 genera), including representatives of five subfamilies of the Echinostomatidae, which currently exhibits the most complex taxonomic structure within the superfamily. This first comprehensive phylogeny for the Echinostomatoidea challenged the current systematic framework based on comparative morphology. A morphology-based evaluation of this new molecular framework resulted in a number of systematic and nomenclatural changes consistent with the phylogenetic estimates of the generic and suprageneric boundaries and a new phylogeny-based classification of the Echinostomatoidea. In the current systematic treatment: (i) the rank of two family level lineages, the former Himasthlinae and Echinochasminae, is elevated to full family status; (ii) Caballerotrema is distinguished at the family level; (iii) the content and diagnosis of the Echinostomatidae (sensu stricto) (s. str.) are revised to reflect its phylogeny, resulting in the abolition of the Nephrostominae and Chaunocephalinae as synonyms of the Echinostomatidae (s. str.); (iv) Artyfechinostomum, Cathaemasia, Rhopalias and Ribeiroia are re-allocated within the Echinostomatidae (s. str.), resulting in the abolition of the Cathaemasiidae, Rhopaliidae and Ribeiroiinae, which become synonyms of the Echinostomatidae (s. str.); and (v) refinements of the generic boundaries within the Echinostomatidae (s. str.), Psilostomidae and Fasciolidae are made.

Surface topography of two trematodes parasites infecting grey heron Ardea cinerea Jouyi (Aves, Ciconiiformes) in Qena, Egypt.

Apharyngostrigea ardeolina and Echinoparyphium recurvatum are two important digenean parasites that were recovered from small intestine of grey heron with an infection rate (16.2%) and (8.8%) respectively. The surface topography of two species was redescribed by both light and scanning electron microscopy. Using SEM studies showed that the body surface of two trematodes were covered by contact receptors, several types of sensory tegumental papillae which may have useful function in orientation and feeding through increasing the surface area of absorption, could also play a role in sensation or in selection of the materials for ingestion by the fluke. The head collar of E. recurvatum is reniform in shape, bearing uninterrupted double row of 41 collar finger-like spines, a total including 4 end group ones on both ventral corners., tegumental spines were tongue-shaped without a terminal tip.