Eurytrema coelomaticum: updated morphology of adult worms using advanced microscopy experiments.

Eurytrema coelomaticum is a digenean flatworm of ruminants that is the causative agent of eurytrematosis, a disease of veterinary health concern. Although modern techniques of morphological analysis have provided new insights about the morphology and anatomy of parasitic helminths, most studies on E. coelomaticum adults are based on conventional light microscopy. In the present study, a combined approach using brightfield, fluorescence, confocal and scanning electron microscopies (SEMs), together with the cryofracture technique, have updated morphological data on E. coelomaticum recovered from cattle in Rio de Janeiro State, Brazil. Light microscopy confirmed the presence of several structures present in the current description, such as suckers, pharynx, oesophagus, intestinal bifurcation and the cirrus-sac. Fluorescence stereomicroscopy revealed for the first time the cubic crystal protein inclusions in the forebody, which were further detailed by confocal and SEMs. Confocal microscopy provided detailed information of the muscular architecture associated with the attachment structures (suckers), digestive system (pharynx and oesophagus), egg-forming complex (ovary, Mehlis' gland and Laurer's canal) and male reproductive system, which are similar to those found in other digenean flukes. SEM images of cryofractured parasites showed mucus and developing eggs within uterine loops. It was demonstrated that the combination of advanced tools generated complementary information, confirming the importance of experimental morphology in parasitology. Therefore, the knowledge of the adult structural organization of E. coelomaticum was improved and this work has contributed to propose new morphological criteria to evaluate the effects of antiparasitic drugs on flukes of medical and veterinary importance.

Pygidiopsis cambodiensis n. sp. (Digenea: Heterophyidae) from experimental hamsters infected with metacercariae in mullets from Cambodia.

Pygidiopsis cambodiensis n. sp. is described based on adult flukes recovered from Syrian golden hamsters experimentally infected with metacercariae from mullets (Liza macrolepis) purchased at a local fish market in Phnom Penh, Cambodia. The specimens were examined by light and scanning electron microscopy. Among the 13 species so far assigned to Pygidiopsis, the new species belongs to the summa-type (including Pygidiopsis pelecani, Pygidiopsis phalacrocoracis, Pygidiopsis piclaumoreli, Pygidiopsis plana, and Pygidiopsis summa) which lack circumoral spines and have vitelline follicles extending posteriorly from the level of the ovary some distance into the post-testicular space and the uterus not exceeding the acetabulum anteriorly. The new species differs from the other five species of the summa-type particularly in the morphology of the ventrogenital complex, including the genital sac, gonotyl, and gonotyl spines (= rodlets). The genital sac is well developed, sucker-like, slightly larger than the ventral sucker, muscular, and equipped with two gonotyls on the ventral side of the sac. Gonotyls are protruding pad-like, and the number of rodlets on the left gonotyl is four to five and that on the right gonotyl is 10-11 in two rows. This is the fifth Pygidiopsis species reported in Asia, following P. summa (Japan, Korea, and Vietnam), P. phalacrocorasis (Japan), P. pelecani (China), and Pygidiopsis marivillai (Philippines).

Experimental centrocestiasis: Worm burden, morphology and fecundity of Centrocestus formosanus (Trematoda: Heterophyidae) in dexamethasone immunosuppressed mice.

Centrocestus formosanus is an intestinal foodborne trematode with medical and veterinary importance that remains with the pathological and immunological aspects of the infection in definitive host poorly studied. In the present study, we evaluated the effects of pharmacological immunosuppression by glucocorticoids in experimental centrocestiasis. Mice of the AKR/J strain were orally inoculated with 100 metacercariae of C. formosanus obtained in naturally infected fish (Australoheros facetus) collected in an urban reservoir from Brazil. Treatment with dexamethasone (25 mg/kg, via subcutaneous injection) was started 1h before infection of mice and then continued daily during 14 days post-infection. Untreated mice also infected with C. formosanus were used as control. At the end of the treatment course, all rodents were euthanized and adult parasites recovered from host intestines were subjected to morphological and morphometric analysis under optical microscopy. The worm burden in dexamethasone treated group [70±14 (41-85)] was significantly greater (p<0.0001) than that in the control group [15±4 (10-22)]. In addition, the parasites recovered from immunosuppressed mice were larger, with more developed reproductive structures and greater number of intrauterine eggs than in control mice. These parasite developmental changes induced by dexamethasone treatment are reported for the first time in experimental centrocestiasis. Moreover the higher parasite fecundity induced by glucocorticoid treatment had so far not been reported for any heterophyid species, which can have implications for the pathology and morbidity in infections caused by these parasites.

Metagonimoides oregonensis (Heterophyidae: Digenea) infection in Pleurocerid snails and Desmognathus quadramaculatus salamander larvae in Southern Appalachian streams.

Metagonimoides oregonensis (Heterophyidae) is a little-known digenetic trematode that uses raccoons and possibly mink as definitive hosts, and stream snails and amphibians as intermediate hosts. Some variation in the life cycle and adult morphology in western and eastern populations has been previously noted. In the southern Appalachians, Pleurocera snails and stream salamanders, e.g., Desmognathus spp., are used as intermediate hosts in the life cycle. We completed a series of studies in this system examining some aspects of larval trematode morphology and first and second intermediate host use. Molecular sequencing of the 28S rDNA of cercariae in our survey placed them clearly within the heterophyid family. However, light and scanning electron microscopy revealed both lateral and dorso-ventral finfolds on the cercariae in our region, whereas original descriptions of M. oregonensis cercariae from the west coast indicate only a dorso-ventral finfold, so further work on the systematics of this group may be warranted. A survey of first intermediate host, Pleurocera proxima, from 7 streams in the region identified only M. oregonensis, virgulate-type cercariae, and cotylomicrocercous-type cercariae in the streams, with M. oregonensis having the highest prevalence, and the only type present that use amphibians as second intermediate hosts. Based on clearing and staining of 6 Desmognathus quadramaculatus salamander larvae, we found that individual salamanders could have over 600 metacercariae, which form between muscle fibers throughout the body. Histological observations suggest that the metacercariae do not cause excessive tissue damage or inflammation, and likely persist through metamorphosis, thereby transmitting potentially large numbers of worms to definitive host raccoons foraging along streams.

Anthelmintic effect of artesunate in experimental heterophyid infection.

Artesunate is a water-soluble semi-synthetic drug derived from artemisinin which is extracted from leaves of the Chinese plant Artemisia annua. This successful basically-antimalarial drug has been proved to be also effective against trematodes like Schistosoma japonicum, S. mansoni, Fasciola hepatica and Clonorchis sinensis. The present work aimed to investigate the in-vivo efficacy of artesunate against heterophyids in mice, using praziquantel as a therapeutic control. Results showed that artesunate was effective in treatment of experimental heterophyidiasis as proved by a 100% reduction of intestinal adult worm count at a dose regimen of 200 mg/kg/day, given for 3 successive days. Besides, surface tegumental damage of the adult worm was observed by SEM in form of bleb formation, disruption, erosion and peeling. The proved therapeutic efficacy of artesunate together with its reported safety, favor its possible use as a new alternative therapy in human heterophyidiasis.

The life cycle of Ascocotyle (Phagicola) longa (Digenea: Heterophyidae), a causative agent of fish-borne trematodosis.

The complete life cycle of the trematode Ascocotyle (Phagicola) longa (Digenea: Heterophyidae) is elucidated by natural observation validated by experimental infections. The natural first intermediate host of A. (P.) longa, an agent of human heterophyiasis in Brazil, is the cochliopid snail Heleobia australis (new first intermediate host). Metacercariae were found encysted in the body musculature, heart, stomach, liver, kidney, spleen, gonads and mesentery of mullets Mugil liza. Hamsters Mesocricetus auratus were experimentally infected with metacercariae of A. (P.) longa obtained from the mullets, and the adults recovered were used to infect the snails H. australis. Rediae and cercariae of A. (P.) longa are described for the first time. The ultrastructure of the tegument of A. (P.) longa shows a change in spination pattern from the cercaria with single-pointed spines to the metacercaria and adult with multipointed, brush-shaped spines. The life cycle of A. (P.) longa is related to estuaries and coastal lagoons where the recruitment of mugilid juveniles occurs. The high prevalence (100%) of A. (P.) longa encysted in the mullets examined within the urban area of Rio de Janeiro indicates the potentially great public health impact of the consumption of raw mullets.

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.

Haplorchis popelkae N. SP. (Digenea: Heterophyidae) from short-necked turtles (Chelidae) in Northern Australia.

Haplorchis popelkae n. sp. is described from the intestine of the short-necked turtles Elseya dentata and Emydura victoriae in Northern Territory, Australia. This slipper-shaped heterophyid digenean differs from the 9 previously known species of Haplorchis in its larger overall size, a forebody that is wider than the hindbody, comparatively short ceca, and a ventral sucker that is less heavily spined than in many other species in the genus. This is the first report of Haplorchis in turtles.

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.

Scanning electron microscope study of ancient parasite eggs recovered from Korean mummies of the Joseon Dynasty.

We have previously shown that parasite eggs have been identified in the coprolites of Korean mummies. These eggs have shed light on parasitic infection patterns in Korean populations living several hundred years ago. We conducted a scanning electron microscopy (SEM) study on ancient Trichuris trichiura, Ascaris lumbricoides, Metagonimus yokogawai, Paragonimus westermani, and Gymnophalloides seoi eggs recovered from Korean mummies of the Joseon Dynasty. We anticipated that the taphonomic conditions of mummification would alter the eggs of certain species but not of others. Our SEM data show that each species of ancient egg exhibited different degrees of preservation. Thus, some of them, for example, M. yokogawai, exhibited a better preservation status than others, suggesting that they should be the first candidates considered when choosing subjects for future paleoparasitological studies.

Attachment and penetration of Centrocestus armatus (Digenea: Heterophyidae) cercariae to gills of secondary intermediate fish hosts.

The attachment and penetration of Centrocestus armatus cercariae into the fish host Zacco temmincki are described in this study. Light and scanning electron microscopy (SEM) were used to examine the topographical features and behavior of cercariae. Histochemistry and transmission electron microscopy (TEM) were employed to trace glandular products and secretions released by cercariae during penetration. Cercariae are first carried into the fish gill chambers via the respiratory currents. The frequency of respiratory-current reversals of fish increased when infected with cercariae. The behavior of cercariae, during breaks in current flow that preceded each current reversal, was observed using a specially devised apparatus. Cercariae produce a mucus-like secretion upon attachment, shed their tail, and employ a brief period of leech-like creeping behavior before penetration. In all cases, the site of penetration was via the surface of the primary gill lamellae. SEM revealed a well-developed anterior penetration apparatus, and a highly contractile body region, that created a driving force for penetration. TEM and histochemistry showed that the mucus observed on the surface tegument of cercariae during attachment were glandular secretions from the parasite. The significance of fish respiratory current reversals to the success of cercariae penetration nicely illustrates the exploitation by the parasite of a host response to a stimulus.

Effect of niclosamide on the tegumental surface of Haplorchis taichui using scanning electron microscopy.

The effect of niclosamide on the tegument of adult Haplorchis taichui (Trematoda: Heterophyidae) exposed in vitro was observed by scanning electron microscope. Adult worms were incubated in Tyrode's solution containing 0.01, 0.1, 1.0, and 10 microg ml(-1) of niclosamide for 30 min, 1, 6, 12 and 24 h. Control groups were incubated in Tyrode's solution without niclosamide and worms remained active until 24 h. In 0.01 microg ml(-1) of niclosamide, worms showed slightly active movements up to 1 h after incubation, while in 0.1 microg ml(-1) solution a few worms showed only slightly active movements after 30 min. Tegumental changes were determined by scanning electron microscopy. Swelling and blebbing of the tegument were observed on both ventral and dorsal sides. After longer periods, extensive swelling and blebbing of the tegument became more severe and there was a loss of the apical plasma membrane in some regions. Empty spine sockets occurred, and small perforations penetrated the basal lamina, followed by some lesions. Destruction of both surfaces was more pronounced on the posterior compared with the anterior regions.

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.

Effect of Mirazid (Commiphora molmol) on experimental heterophyidiasis.

Mirazid (MZ), an oleo-resin extract derived from Myrrh was reported in several experimental and clinical trails to be safe and effective against other trematodes like schistosomiasis and fascioliasis. This experimental work aimed at investigating the possible efficacy of MZ against heterophyids (Pygidiopsis genata), using praziquantel as a therapeutic control. Results showed that MZ in emulsion form is a promising drug for the treatment of heterophyidiasis, as proved by significant reduction of worm count, overt surface tegumental changes like deformity and erosion of tegumental spines observed by scanning electron microscope (SEM). The effective dose regimen was 500 mg/kg/d for 3 successive days, produced 100% reduction in worm load. The proved efficacy of the drug, together with reported low toxicity, relative to praziquantel, favours its use as a natural new alternative therapy for the treatment of human heterophyidiasis.

Food-borne intestinal trematodiases in humans.

Food-borne trematodiases still remain a public health problem world-wide, despite changes in eating habits, alterations in social and agricultural practices, health education, industrialization, environmental alteration, and broad-spectrum anthelmintics. Food-borne trematodiases usually occur focally, are still persistently endemic in some parts of the world, and are most prevalent in remote rural places among school-age children, low-wage earners, and women of child-bearing age. Intestinal fluke diseases are aggravated by socio-economic factors such as poverty, malnutrition, an explosively growing free-food market, a lack of sufficient food inspection and sanitation, other helminthiases, and declining economic conditions. Control programs implemented for food-borne zoonoses and sustained in endemic areas are not fully successful for intestinal food-borne trematodiases because of centuries-old traditions of eating raw or insufficiently cooked food, widespread zoonotic reservoirs, promiscuous defecation, and the use of "night soil" (human excrement collected from latrines) as fertilizer. This review examines food-borne intestinal trematodiases associated with species in families of the Digenea: Brachylaimidae, Diplostomidae, Echinostomatidae, Fasciolidae, Gastrodiscidae, Gymnophallidae, Heterophyidae, Lecithodendriidae, Microphallidae, Nanophyetidae, Paramphistomatidae, Plagiorchiidae, and Strigeidae. Because most of the implicated species are in the Echinostomatidae and Heterophyidae, emphasis in the review is placed on species in these families.

Surface ultrastructure of Pygidiopsis summa (Digenea: Heterophyidae) adult flukes.

A scanning electron microscopic study was performed on the surface ultrastructure of Pygidiopsis summa (Digenea: Heterophyidae) adults. Metacercariae were collected from gills and muscles of mullets (Mugil cephalus) caught in a known endemic area, and adult flukes were harvested from dogs after 8 weeks of experimental infection. The worm was calabash form with its posterior part broader than the anterior part. Tegumental spines were densely distributed over the body surface, except on the suckers and genital apparatus, and around the excretory pore. Well differentiated spines were observed on the anterior half of the body, with 14-16 tips ventrally, and 19-20 tips dorsally. On the oral sucker, three pairs of type I sensory papillae (uni-ciliated knob-like swellings) and one pair of type II sensory papillae (aciliated round-swellings) were observed on the anterior and posterior parts of the lip, respectively. On the lip of the ventral sucker, one pair of type II sensory papillae was distributed only on its posterior part. Sperms were seen emerging from or entering into the genital apparatus. The results showed that the surface ultrastructure of P. summa was unique among the heterophyid trematodes, especially in digitation of tegumental spines and in distribution of sensory papillae on oral and ventral suckers.

Surface ultrastructure of Pygidiopsis summa (Digenea: Heterophyidae) adult flukes

A scanning electron microscopic study was performed on the surface ultrastructure of Pygidiopsis summa (Digenea: Heterophyidae) adults. Metacercariae were collected from gills and muscles of mullets (Mugil cephalus) caught in a known endemic area, and adult flukes were harvested from dogs after 8 weeks of experimental infection. The worm was calabash form with its posterior part broader than the anterior part. Tegumental spines were densely distributed over the body surface, except on the suckers and genital apparatus, and around the excretory pore. Well differentiated spines were observed on the anterior half of the body, with 14-16 tips ventrally, and 19-20 tips dorsally. On the oral sucker, three pairs of type I sensory papillae (uni-ciliated knob-like swellings) and one pair of type II sensory papillae (aciliated round-swellings) were observed on the anterior and posterior parts of the lip, respectively. On the lip of the ventral sucker, one pair of type II sensory papillae was distributed only on its posterior part. Sperms were seen emerging from or entering into the genital apparatus. The results showed that the surface ultrastructure of P. summa was unique among the heterophyid trematodes, especially in digitation of tegumental spines and in distribution of sensory papillae on oral and ventral suckers.

Surface ultrastructure of Metagonimus takahashii metacercariae and adults.

A scanning electron microscopic study was performed on the surface ultrastructure of metacercariae and adults of Metagonimus takahashii. Metacercariae were collected from the scale of crucian carp (Carassius auratus), and adult flukes were harvested 1-4 weeks after infection to rats. In excysted metacercariae, the oral sucker had type I (numerous) and type II (seven in total) sensory papillae. Tegumental spines were dense and digitated into 5-7 points on the surface anterior to the ventral sucker, but became sparse and less digitated posteriorly toward the end of the body. In adults, seven type II sensory papillae were characteristically arranged around the lip of the oral sucker, and on the inner side of the lip four small and two large type I sensory papillae were symmetrically seen on each side (12 in total). Tegumental spines on anterior two-thirds of the body, were digitated with 9-12 tips ventrally and 8-13 tips dorsally. Sperms entering into the Laurer's canal were observed. The results show that the surface ultrastructure of M. takahashii is generally similar to those of M. yokogawai and M. miyatai except for the digitation of tegumental spines.

Surface ultrastructure of Stictodora tridactyla (Trematoda: Heterophyidae) from Kuwait Bay.

The surface morphology of Stictodora tridactyla recovered from a kitten that was fed the killifish, Aphanius dispar, naturally infected with the metacercariae was studied using scanning electron microscopy. The body comprised a rounded head, elongate neck, and widely pyriform hind-body. The head bore a circular oral sucker on the ventral side, and concentric rows of peg-like cephalic spines on the dorsal side. The oral sucker was armed with pre-oral spines similar in shape and size to the cephalic spines. The neck and hind-body were densely covered with scale-like multipointed spines, the size and density of which decreased from anterior to posterior parts of the body. Ciliated dome-shaped papillae were found solitarily or as conjugated groups on the head apex and lips of the oral sucker. Non-ciliated dome-shaped papillae were restricted to the lower lip of the oral sucker. The body was devoid of a ventral sucker. The genital opening appeared as a round depression of the tegument at about the anterior third of the body. This is the first record of the occurrence of S. tridactyla in the Arabian Gulf region and A. dispar is a new second intermediate host.

Ultrastructure of metacercarial cysts of six heterophyid trematodes from fish.

The ultrastructure of metacercarial cysts of Ascocotyle sexidigita. A. (Phagicola) diminuta, and Pygidiopsoides spindalis (herein described for the first time) collected from natural hosts in California was compared with that of cysts from A. tenuicollis (also described for the first time), A. pachycystis, and A. leighi collected from natural fish hosts in Mississippi. The results show that none of the cysts studied approximated the thick cyst produced by A. pachycystis, but all cysts comprised several layers of putative glycoprotein except for A. (P.) diminuta, which was associated with a single layer of spongy cyst wall.