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).

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.

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.

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.

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.

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.

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.

Surface ultrastructure of the developmental stages of Heterophyopsis continua (Trematoda: Heterophyidae).

Ultrastructural observations were made on the tegument of juvenile and adult stages of Heterophyopsis continua using scanning electron microscopy. On the surface posterior to the ventral sucker, the tegumental processes were bandlike in the metacercariae, cobblestonelike in the flukes 2 days postinfection (PI), and velvety at 3 days PI. The anterior surface between the oral and ventral suckers of the metacercariae was packed densely with tegumental spines having a 10- to 14-pointed tip. In flukes 6 days PI, the number of points increased to 15-17. The tegumental spines immediately behind the ventral sucker on the metacercariae surface possessed 5-7 points; posteriorly the points were reduced in size and in number. Ciliate sensory papillae (type I), as single or clumped forms of 2 or 3, were abundant around the oral and ventral suckers of metacercaria and adult worms. The clumped papillae appeared bilaterally symmetrical on the dorsal and ventral surfaces. The ciliate papillae may function in tango-, rheo-, and/or chemoreception. On the lip of the ventral sucker, 6-7 aciliate domed papillae (type II) were arranged in an equidistant manner. At 2 days PI each type II papilla became a clumped form having 2 or 3 papillae. Type II papillae may function as tango- and/or pressure-receptors. The structure and distribution of papillae suggest that the ventral sucker likely functions as a holdfast organ and the oral sucker as a probing organ involved in feeding.

Phagicola longa (Heterophyidae) in dogs from Chile: morphological findings and taxonomical problems.

Trematodes found in the intestines of dogs from Chillan, VIII region of Chile, were studied. The species identified was Phagicola longa, a fluke of the Heterophyidae family, parasites of fish-eating birds or mammals in several countries. P. longa is very similar to P. arnaldoi, a species described in dogs from South America. The number of vitelline follicles and the distribution of cuticular spines are the only distinctive features for the two species. However, many authors report the same number of vitelline follicles for P. arnaldoi and P. longa. Furthermore, the distribution of cuticular spines is very similar. P. longa has a small area devoid of spines when compared to P. arnaldoi but a wide variability of this generic character in the Heterophyidae was observed. As previously reported, P. arnaldoi could be a recent synonym of P. longa, which is the only species of Phagicola that can be found in South American dogs. This is in agreement with the study of metacercariae found in Mugilidae, the intermediate hosts of Phagicola, from American waters in which P. longa is the only species found. The authors emphasize the finding of this trematode in dogs, since it could possibly cause also human infection.

Comparative morphology of eggs of the Haplorchiinae (Trematoda: Heterophyidae) and some other medically important heterophyid and opisthorchiid flukes.

The egg morphology of the following medically important small flukes from Southeast Asia and Far East were studied: Opisthorchis viverrini, Clonorchis sinensis (Opisthorchiidae), Haplorchis taichui, H. pumilio, H. yokogawai, Stellantchasmus falcatus and Metagonimus sp. (Heterophydiae). This study revealed a great intraspecific variability and interspecific similarity in size and shape of eggs. The eggs shape does not seem to be suitable for species identification. On the other hand, biometrical analysis of egg size enabled us to divide eggs from the species studied into four distinct groups according to the Faust-Meleney index (FMI) characterizing egg size rather than the length and width of eggs. The surface structures of eggs, delineated by using a scanning electron microscope (SEM), appeared to be a suitable morphological feature for distinguishing some groups of small flukes. Eggs from the Haplorchiinae were typified by the characteristic filamentous mesh structure. The problems of identification of eggs in human stool samples and suitability of using morphological criteria such as shape and size of eggs are discussed herein.

Light and scanning electron microscopy of Stellantchasmus sp (Trematoda : Heterophyidae).

Light and scanning electron microscopy of heterophyid fluke, Stellantchasmus sp were studied. The adults were obtained from Rattus norvegicus, 7 days after experimental infections with the metacercariae from the body cavity of Dermogenus pusillus. Living specimens and stained flukes were examined by light microscope. The present worm differed from S. batillans having a single testis while testes of S. gallinte were tandam. It is closed to S. falcatus and S. aspinosus but can not be identified to species because of acetabular spines. The tegumental surface by SEM was investigated on encysted metacercariae and adults. It was nearly smooth with one end shallow wide groove of the metacercarial surface. The entire surface of adult worms were almost covered with scale-like spines, pectinate, 7-9 teeth. The size of scale was largest in the middle of body and decreased both anterior and posterior. No spines were around the excretory pore but has a wrinkled surface appearance. The dorsal surface appeared to have scale-like spines and pectination on the ventral surface. The papillae consisted of a single club-like cilium, solitary or grouped on the anterior part of the body especially anterior of oral sucker. Acetabulum located slightly right of the middle and invagination.

Study of the body surface of Haplorchis yokogawai (Katsuta, 1932) and H. taichui (Nishigori, 1924) (Trematoda:Heterophyidae).

The morphology of the surface of two heterophyid flukes, Haplorchis yokogawai and H. taichui, that can infect humans eating raw fish flesh, has been studied using scanning and transmission electron microscopy. The body surface of both species is covered with scale-like, serrated spines with the exception of areas around the oral sucker, ventrogenital opening and excretory pore. The species slightly differ by the dentation pattern of tegumental spines: H. yokogawai has finer and more numerous spine teeth (maximum tooth number 14-16) while H. taichui has more serrated spines, mostly with a lower number (up to 10-12) of somewhat more robust teeth. However, because of the variability, the shape and dentation of spines do not seem to be reliable diagnostic features enabling mutual differentiation of both species.

Potassium permanganate staining for differentiation the surface morphology of Opisthorchis viverrini, Haplorchis taichui and Phaneropsolus bonnei eggs.

Potassium permanganate staining method was developed for differentiation Opisthorchis viverrini, Haplorchis taichui and Phaneropsolus bonnei eggs. The surfaces of O. viverrini, H. taichui and P. bonnei eggs stained permanently and temporarily were similar in appearance even the staining procedures were varied both in concentration and time. Determined under light microscope set at 400x, all of these eggs were oval-shaped, operculated at one pole and indistinct small knob at posterior end. O. viverrini eggs showed the distinct musk-melon-like prominent ridges on the surface. Haplorchis taichui eggs had a light striae pattern while P. bonnei eggs had a smooth egg shell. Length of these trematode eggs were significant different (chi2 test, p < 0.05). Mean +/- SD of O. viverrini, H. taichui and P. bonnei eggs were 26.34 +/- 1.65 microm, 29.03 +/- 1.48 microm and 23.00 +/- 1.49 microm, respectively. Regarding of their width, the mean +/- SD of O. viverrini, H. taichui and P. bonnei eggs were 15.54 +/- 0.69 microm. 14.94 +/- 0.91 microm and 12.25 +/- 1.02 microm, respectively. The means of width of O. viverrini and H. taichui eggs were not significantly different (chi2 test, p > 0.05), however, they were significantly different from those of P. bonnei (chi2 test, p < 0.05). Temporary staining using 1% w/v concentration and only 1 minute of time is useful in the mass fecal examination survey for the prevalence and intensity of truly Opisthorchis infection.

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.

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.

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.