Cultivation of excysted metacercariae of Echinostoma revolutum (Trematoda) in chick embryos

Three techniques were used to study post-metacercarial growth and development of chemically excysted metacercariae of Echinostoma revolutum on the chorioallantoic membrane of domestic chick embryos. The in ovo technique of Zwilling (1959) and the in vitro technique of Auerbach et al. (1974) provided for better worm recovery in chick embryos than the in ovo technique of Woodruff &; Goodpasture (1931). Regardless of the technique used, postmetacercarial development was obtained for this species, and 6- to 8-day-old chorioallantoic-wornis achieved sexual development to the coiled uterus stage comparable to worms grown in domestic chicks for 7 days. However, somatic growth of worms from the chorioallantois was stunted when compared to worms grown in chicks. Worms grown on the chorioallantois voided their excretory concretions and showed histochemical lipid staining identical to that seen in worms grown in chicks.     

Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda)

Fried B. and Huffman J. E. 1982. Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda). International Journal for Parasitology12: 427–431. Encysted metacercariae of Sphaeridiotrema globulus were obtained from naturally infected Goniobasis virginica snails in Morris County, New Jersey, U.S.A. These metacercariae were successfully excysted within l h in an alkaline bile salts trypsin medium maintained at 41°C in the absence of acid pepsin pretreatment. Encysted metacercariae treated in 3% sodium bicarbonate produced successful infections in day-old domestic chicks and worms became ovigerous in the lower ileum by day 4. Excysted metacercariae transferred to the chorioallantois of 6–10-day-old chick embryos maintained at 41°C developed into ovigerous adults by day 4.     

Growth, development, pathogenicity, and transplantation of Echinostoma caproni (Trematoda) on the chick chorioallantois.

Excysted metacercariae of Echinostoma caproni were cultivated on the chorioallantoic membrane (CAM) of 6-day-old domestic chick embryos for 2-13 days postinoculation. There was no significant difference in the body area of fixed and stained preovigerous worms from the CAM versus those grown in domestic chicks. However, ovigerous worms from the CAM were significantly smaller than those from chicks. Worm development, i.e., gonadal differentiation, uterine curling, vitellinogenesis, ovigerousness, and oviposition, took 1 day longer on the CAM than in the chick. Histopathologic studies of worms attached to the CAM were done on cryostat and paraffin sections stained with hematoxylin and eosin. Some worms attached to the CAM by their collar spines and acetabulum, whereas others penetrated the chorionic epithelium and encapsulated in the mesenchyme. Pathogenicity to the CAM included hyperplasia of the chorionic epithelium, hemorrhagia, reduced fibrocytes and blood vessels, but increased lymphocytes and eosinophils in the mesenchyme. Attempts to transplant 11-day-old CAM worms to new CAMs were unsuccessful.     

Cultivation of excysted metacercariae of Echinostoma caproni to ovigerous adults in the allantois of the chick embryo.

Chemically excysted metacercariae of Echinostoma caproni inoculated into the allantois of domestic chick embryos became ovigerous in that site within 9 days postinoculation. The egg preparation technique of Saville and Irwin was markedly better than that of a modified Zwilling procedure for obtaining large numbers of postinoculation embryos with worm infections. Adults of E. caproni from the allantois were larger and became ovigerous sooner than worms grown on the chorioallantois. Only worms from the allantois produced eggs with fully developed miracidia. Miracidia were released from these eggs, but an insufficient number was available to attempt infections in Biomphalaria glabrata snails.     

Growth and feeding of Echinostoma revolutum on the chick chorioallantois and in the domestic chick

Chemically excysted metacercariae of Echinostoma revolutum cultivated on the chick chorioallantois grew slowly until day 5, more rapidly between 5 and 7 days, and slowly between 7 and 10 days. Worms did not become ovigerous in this site by 12 days, at which time studies were terminated. In contrast, chemically excysted metacercariae reared in the domestic chick were ovigerous by day 9, at which time their mean body area was about 4 times greater than the largest chorioallantoic worms. Histochemical studies, solubility tests for hematin, and X-ray microanalysis of cecal contents showed that chorioallantoic-worms fed on blood from the vascular membrane, whereas chick-worms fed on host intestinal mucosa.     

Life-cycle,delimitation and redescription of Echinostoma revolutum (Froelich, 1802) (Trematoda: Echinostomatidae)

The life-cycle of Echinostoma revolutum (Froelich, 1802) Dietz, 1909 has been completed experimentally beginning with infected snails collected at the type-locality, near Erlangen, Germany. Based on the specimens obtained, each stage of the life-cycle has been redescribed. Important taxonomic features are discussed and hitherto unknown characteristics are described. Synonyms for E. revolutum are: Fasciola revoluta Froelich, 1802; Echinostoma paraulum Dietz, 1909; E. audyi Lie & Umathevy, 1965; and E. ivaniosi Mohandas, 1973. Adults and larvae described as E. revolutum in other works are found to be identical with Echinostoma echinatum (Zeder, 1803), E. trivolvis (Cort, 1914), E. jurini (Skvortsov, 1924), E. caproni Richard, 1964, Moliniella anceps (Molin, 1859), Echinochasmus beleocephalus (Linstow, 1873) and other echinostome species. For nearly a century, incorrect morphological, biological, life-cycle and host information has been attributed to E. revolutum, and at times these data have contributed to the diagnoses of the species. Occasionally, authors actually working with E. revolutum have ascribed their results to other species. Based on extensive experimental life-cycle studies beginning with infected snails from type-localities, it is shown that (1) the first intermediate host is a lymnaeid snail; (2) the second intermediate hosts are various pulmonate and prosobranch snails, mussels, frogs and freshwater turtles; (3) the final hosts are birds; (4) E. revolutum cercariae and adults have 37 collar spines; (5) the species occurs only in Europe and Asia; (6) Cercaria echinata Siebold, 1937, Echinostoma echinatum (Zeder, 1803) and E. jurini (Skvortsov, 1924) are the closely related 37-spined allies in Europe; and (7) species specific characteristics are expressed only in the larvae and the host-parasite relationships. The adults of E. revolutum cannot be identified using morphological criteria and it is proposed that worms with 37 collar spines belonging to the genus Echinostoma and occurring in naturally infected birds in Europe and Asia be referred to an “E. revolutum group.”     

Cultivation of the cercaria of Himasthla quissetensis (Trematoda) on the chick chorioallantois

Cercariae of Himasthla quissetensis were cultivated on the chick chorioallantois maintained at 38 ± 1°C and a relative humidity of 70–75%. Of 68, 6-day-old eggs, each inoculated with 100 cercariae, 28 (41%) were examined 1–9 days post-inoculation. Of the 28 eggs, 23 (82%) were infected with a total of 224 live worms plus 10 encysted metacercariae. Worms contained blood or hematin-like material in their intestinal caeca and showed considerable development of reproductive structures. A total of 58, 6- or 7-day-old chorioallantoic-worms were serially transferred to new 6-day-old embryos, but only a single live worm was recovered after 13 days on two membranes. Based on fixed and stained specimens, 7-day-old chorioallantoic-worms increased their body area about 4× compared to cercariae and the 13-day-old worm increased its body area about 10×.     

Scanning electron microscopy of viable and calcified Onchocerca gibsoni

Holdsworth P. A. 1987. Scanning electron microscopy of viable and calcified Onchocerca gibsoni. International Journal for Parasitology17: 957–964. Scanning electron microscopy (SEM) of adult Onchocerca gibsoni revealed that the cephalic ends of male and female specimens possessed features of little value for differentiation from other species. The marked cuticular ridges of the mid-body region of female worms were highlighted using SEM, as were the cuticular annulations and the body constriction at the anal region. Male specimens revealed the uniform annulations of the worm body along with the caudal papillae and the spicules. Microfilariae (mff) possessed cephalic papillae, lateral lines and cuticular annulations similar to adult worms. The cephalic hook was the most prominent feature at the anterior end of the mff. Calcification of adult worms was found to be mainly internal, however, one specimen was recorded with external calcification. Energy dispersive X-ray analysis revealed the composition of the calcification to be of calcium phosphate.     

Echinostoma revolutum: Freshwater Snails as the Second Intermediate Hosts in Chiang Mai,Thailand

The occurrence of 37-collar spined echinostome metacercariae in freshwater snails was investigated in 6 districts of Chiang Mai Province, Thailand, from October 2011 to April 2012. A total of 2,914 snails that belong to 12 species were examined, and 7 snail species (Clea helena, Eyriesia eyriesi, Bithynia funiculata, Bithynia siamensis siamensis, Filopaludina doliaris, Filopaludina sumatrensis polygramma, and Filopaludina martensi martensi) were found infected with echinostome metacercariae. The prevalence of metacercariae was the highest in Filopaludina spp. (38.5-58.7%) followed by B. funiculata (44.0%), E. eyriesi (12.5%), B. siamensis siamensis (8.2%), and C. helena (5.1%). Metacercariae were experimentally fed to hamsters and domestic chicks, and adult flukes were recovered from both hosts at days 15 and 20 post-infection. The adult flukes were identified based on morphological features, morphometrics, host-parasite relationships, and geographical distribution. They were compatible to Echinostoma revolutum or Echinostoma jurini, with only minor differences. As the adults were recovered from both hamsters and chicks, our specimens were more compatible to E. revolutum rather than E. jurini (reported only from mammals). This is the first report for metacercariae of E. revolutum in the snail host, C. helena, and also confirmed that Filopaludina spp., E. eryresi, and Bithynia spp. act as the second intermediate hosts of E. revolutum under natural conditions, which are indigenously distributed in Chiang Mai province.     

Site-finding and pairing of Echinostoma revolutum (Trematoda) on the chick chorioallantois

Site-finding of 14-day-old Echinostoma revolutum from the domestic chick was studied by inoculating single worms into various sites on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. Regardless of the site of inoculation, single worms were attracted significantly to the area of the CAM above the embryo. More worms were found in this site at 24 than at 1 hr postinoculation. Worm-pairing was studied in chick embryos by inoculating 2 worms in separate windows, 2 cm apart. Worm-pairing, i.e., worms in contact or within 5 mm of each other, was very evident at 24 hr. The percentage of paired worms on the CAM above the embryo was considerably less than single worms.     

Thin layer chromatographic analyses of amino acids in Echinostoma revolutum (Trematoda) adults

Bailey R. S. Jr. and Fried B. 1977. Thin layer Chromatographic analyses of amino acids in Echinostoma revolutum (Trematoda) adults. International Journal for Parasitology7: 497–499. Thin layer Chromatographic analyses were made on free pool amino acids and those obtained from the incubate fluid of Echinostoma revolutum adults maintained in a non-nutrient salt solution. The major free pool amino acids detected were alanine, proline, serine and methionine. Alpha-amino-n-butyric acid was also tentatively identified. Amino acids detected in the incubate fluid were leucine-isoleucine, valine, methionine, tyrosine, proline, alanine, glutamic acid, serine, arginine, and aplha-amino-n-butyric acid was also tentatively identified. Amino acids presumably isolated from either worm egesta or the protonephridial system were qualitively similar to those obtained from worm incubate fluid.     

Echinostoma caproni: Differential tegumental responses to growth in compatible and less compatible hosts

The topography of the tegument of Echinostoma caproni adults collected from high (mice) and low (rats) compatible hosts was compared by SEM. In the oral (OS) and the ventral sucker (VS) areas, a worm age-host species interaction was found with regard to the density of spines. There was a decrease in the density of spines in the adults collected from mice, whereas an increase occurred in the OS area in worms from rats over time. The tegumentary spines in adults from mice became larger and blunter. Some spines from the VS area in adults from mice at 4 wpi were multipointed. The spines of adults from rats were sharper, not covered by the tegument and no multipointed spines were observed. We detected a greater level of actin gene expression in the adults collected from rats. These facts suggest that the low compatible host induces an increased turnover of tegumentary spines.     

Comparative growth and development of the metacercariae of Fibricola seoulensis (Trematoda: Diplostomidae) in vitro, in vivo and on the chick chorioallantois

The growth and development of the metacercariae of F. seoulensis cultivated in vitro or on the chick chorioallantois were assessed by comparison with the optimum process of maturation in albino rats and new born chickens. The process of maturation was divided for convenience into six stages: Stage 1; cell multiplication, Stage 2; body shaping, Stage 3; separation of genital anlagen, Stage 4; organogeny, Stage 5; gametogony, and Stage 6; oviposition. In Hank's and Tyrode's solutions, the metacercariae were alive up to 200 days or more at 4 degrees C without any development. The in vivo maturation process in rats or chicks was as follows: stage 1 from 6 hours; stage 2 from 24 hours; stage 3 from 48 to 72 hours; stage 4 from 3 to 4 days; stage 5 from 4 to 5 days; and stage 6 from 5 to 8 days. Despite unsuccessful infection of the metacercariae to 12 day old chicks, fully mature worms of stage 5 or 6 were recovered from new born chicks (1 to 2 days old). The metacercariae of F. seoulensis grown in vitro were up to stage 3 and no further maturation was observed. Of various media employed, the medium NCTC 109 (Gibco) or NCTC 135 (Gibco) supplemented with 20% egg yolk or 20% whole egg macerate or 0.5% yeast was basically required for the earlier development of the fluke. It took 16.1 days (in average) to reach the stage 3 after cultivation. The metacercariae cultivated on the chorioallantoic membranes of 6-13 day old chick embryo at 37-38 degrees C showed their full development up to stage 5 or 6. However, the worms were in general remarkably retarded, compared with those grown in rats or chickens. In the experiments of worm transplant, although the transfer was failed from in vitro culture to in vivo of rats (per os), the transplants from in vitro culture to the chorioallantois and from the chorioallantois to in vivo of rat host were successful with or without development of the transferred worms. In the present study, it was observed that the metacercariae of F. seoulensis can be maintained in vitro media with poor development as well as fully matured in 1 to 2 day-old chicks or on the chorioallantois at a very low rate.     

Scanning electron microscopy of the eggs of three human schistosomes

Ford J. W. and Blankespoor H. D. 1979. Scanning electron microscopy of the eggs of three human schistosomes. International Journal for Parasitology9: 141–145. The surface of the eggs of Schistosoma haematobium, S. japonicum and S. mansoni, examined by scanning electron microscopy, are covered with microspines. The spines of S. mansoni and S. haematobium are essentially similar; however, in S. japonicum they are smaller and more densely distributed. A fibrous matrix, present on the surface of the eggs, is not host derived. This matrix may account for the stickiness of the eggs and the micospines may function to hold the matrix in place.     

The life-cycle of Echinostoma friedi n. sp. (Trematoda: Echinostomatidae) in Spain and a discussion on the relationships within the `revolutum' group based on cercarial chaetotaxy

The morphology of the different stages and life-cycle of Echinostoma friedi n. sp. are described and figured. The freshwater snail Lymnaea peregra (Gastropoda: Lymnaeidae) serves as the natural and experimental first intermediate host and L. corvus and Gyraulus chinensis (Gastropoda: Planorbidae) as experimental first intermediate hosts. These, and Physella acuta (Gastropoda: Physidae), also serve as second intermediate hosts. Adult worms, possessing 37 collar spines, were obtained from naturally infected Rattus norvegicus and experimentally from albino rats, golden hamsters and chickens. Mice were not suitable experimental definitive hosts. E. friedi differs from the most closely related species in the `revolutum' group mainly in terms of several morphological and biological features of the life-cycle stages and in its cercarial chaetotaxy. The chaetotaxy patterns of the species of the `revolutum' group are analyzed and the results show that a taxonomic comparison of these species may be carried out on the basis of the number of sensilla in the clusters CIII V₁, CIII V₂ (or CIII V₁ + CIII V₂), CIV DL and UV_b. These clusters appear adequate to establish taxonomic relationships between different species within the `revolutum' group.     

Tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis]

The tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis (Trematoda: Echinostomatidae) was observed by scanning electron microscopy. Three-day (juvenile) and 16-day (adult) worms were harvested from rats (Sprague-Dawley) experimentally fed the metacercariae from the laboratory-infected fresh water snail, Hippeutis cantori. The worms were fixed with 2.5% glutaraldehyde, processed routinely, and observed by an ISI Korea DS-130 scanning electron microscope. The 3-day old juvenile worms were elongated and ventrally curved, with their ventral sucker near the anterior two-fifths of the body. The head crown was bearing 37-38 collar spines arranged in a zigzag pattern. The lips of the oral and ventral suckers had 8 and 5 type II sensory papillae respectively, and between the spines, a few type III papillae were observed. Tongue or spade-shape spines were distributed anteriorly to the ventral sucker, whereas peg-like spines were distributed posteriorly and became sparse toward the posterior body. The spines of the dorsal surface were similar to those of the ventral surface. The 16-day old adults were leaf-like, and their oral and ventral suckers were located very closely. Aspinous head crown, oral and ventral suckers had type II and type III sensory papillae, and numerous type I papillae were distributed on the tegument anterior to the ventral sucker. Scale-like spines, with broad base and round tip, were distributed densely on the tegument anterior to the ventral sucker but they became sparse posteriorly. At the dorsal surface, spines were observed at times only at the anterior body. The results showed that the tegument of E. cinetorchis is similar to that of other echinostomes, but differs in the number and arrangement of collar spines, shape and distribution of tegumenal spines, and type and distribution of sensory papillae.     

Histochemical and thin layer chromatographic analyses of neutral lipids in Echinostoma revolutum metacercariae cultured in vitro

Excysted metacercariae of Echinostoma revolutum cultured in vitro in the defined medium, NCTC 135 supplemented with 20% hens' egg yolk, doubled their mean relative body area and showed significant sucker growth within 14 days. Histochemical Oil Red O staining showed neutral fat mainly in the excretory system of excysted metacercariae and in adults grown in the domestic chick. In vitro cultured worms showed neutral fat in the intestine, parenchyma, and excretory system. As detected by TLC the major neutral lipid fractions were free fatty acids for excysted metacercariae; free sterols for adults grown in chicks; and triglycerides, free fatty acids, and free sterols for cultured worms. Excysted metacercariae excreted free fatty acids into a nonnutrient incubation medium, whereas cultured worms excreted diglycerides, triglycerides, and free fatty acids into the medium.     

Echinostoma revolutum: Labeling of miracidia with radioselenium in vivo and assay for host finding

Radioactivity was incorporated into Echinostoma revolutum worms and eggs when ⁷⁵Semethionine was administered intraperitoneally to mice infected with the fluke parasites. The levels of incorporation of radioactivity increased in proportion to the amounts of radioselenium used. During the period 3–10 days p.i. the maximum egg-bound radioactivity was, in general, achieved 3 days after the administration of the radioisotope, but substantial radiolabeling was obtained at all isotope levels until Day 10. The radioactivity of the miracidium constituted 29–34% of that of the egg. The radiolabeling procedure did not interfere with the biological characteristics (behavioral activity, infectivity) of the radiolabeled miracidia. Thus, the use of such labeled miracidia for host-finding studies seems acceptable. A radioisotope tracer system for assaying E. revolutum miracidial host finding was described. This system employs exposure of the first intermediate host snail, Biomphalaria alexandrina, to radiolabeled miracidia. A linear proportionality was found to exist between the number of penetrating miracidia and the amount of snail-bound radioactivity. Thus, snail-bound radioactivity retained after exposure to radiolabeled miracidia can be used to measure miracidial host-finding capacity under various experimental conditions.     

A Case of Echinostoma cinetorchis (Trematoda: Echinostomatidae) Infection Diagnosed by Colonoscopy

Human cases of echinostomiasis have been sporadically diagnosed by extracting worms in the endoscopy in Korea and Japan. Most of these were caused by Echinostoma hortense infection. However, in the present study, we detected 2 live worms of Echinostoma cinetorchis in the ascending colon of a Korean man (68-year old) admitted to the Gyeongsang National University Hospital with complaint of intermittent right lower quadrant abdominal pain for 5 days. Under colonoscopy, 1 worm was found attached on the edematous and hyperemic mucosal surface of the proximal ascending colon and the other was detected on the mid-ascending colon. Both worms were removed from the mucosal surface with a grasping forceps, and morphologically identified as E. cinetorchis by the characteristic head crown with total 37 collar spines including 5 end-group ones on both sides, disappearance of testes, and eggs of 108×60 µm with abopercular wrinkles. The infection source of this case seems to be the raw frogs eaten 2 months ago. This is the first case of endoscopy-diagnosed E. cinetorchis infection in Korea.     

Zoonotic Echinostome Infections in Free-Grazing Ducks in Thailand

Free-grazing ducks play a major role in the rural economy of Eastern Asia in the form of egg and meat production. In Thailand, the geographical location, tropical climate conditions and wetland areas of the country are suitable for their husbandry. These environmental factors also favor growth, multiplication, development, survival, and spread of duck parasites. In this study, a total of 90 free-grazing ducks from northern, central, and northeastern regions of Thailand were examined for intestinal helminth parasites, with special emphasis on zoonotic echinostomes. Of these, 51 (56.7%) were infected by one or more species of zoonotic echinostomes, Echinostoma revolutum, Echinoparyphium recurvatum, and Hypoderaeum conoideum. Echinostomes found were identified using morphological criteria when possible. ITS2 sequences were used to identify juvenile and incomplete worms. The prevalence of infection was relatively high in each region, namely, north, central, and northeast region was 63.2%, 54.5%, and 55.3%, respectively. The intensity of infection ranged up to 49 worms/infected duck. Free-grazing ducks clearly play an important role in the life cycle maintenance, spread, and transmission of these medically important echinostomes in Thailand.