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.     

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.     

Cultivation of Clinostomum marginatum (Digenea: Clinostomatidae) metacercariae in vitro, in chick embryo and in mouse coelom

Metacercariae of Clinostomum marginatum obtained from naturally infected Perca flavescens were cultured by 5 methods. In vitro cultivation at 37 C in twice daily changes of Tyrode's, Na-poor Tyrode's, and Eagle's media failed to produce ovigerous adults after 4-5 days. Metacercariae placed on the chorioallantois of week-old chick embryos at 37 C migrated to the albumen. Only 8 of 123 worms recovered were ovigerous after 4-7 days in ovo. Best success occurred with metacercariae injected in groups of 4 or 6 into the abdominal cavities of male A/J mice. Despite liver lesions and strong inflammatory responses in the host, all 174 worms recovered were ovigerous after 4-8 days. None of 41 mice died prematurely from the procedure or the parasites.     

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.     

Postmetacercarial changes in Echinostoma caproni maintained in a defined medium plus calf serum

The present study examined postmetacercarial changes in the excysted metacercariae of Echinostoma caproni maintained in the defined medium Mixture 199 plus 20% calf serum for 7 days at 41 degrees C. The gas phase was atmospheric air. Each culture was inoculated with 25 excysted metacerariae. Cultures were maintained upright in closed 15 ml plastic centrifuge tubes each containing 10 ml of medium plus 200 units of penicillin/ml and 200 micrograms of streptomycin/ml. By 4 days in culture, most metacercariae had voided their excretory concretions. Organisms were clumped or solitary at the bottom of the cultures. Many organisms showed flaring of the oral collar and extension of both the collar and tegumentary spines. By 4 days in culture, posterior protuberances or bumps were noted on many of the organisms and some organisms showed abnormal vesicular growths or blebs at their posterior ends. Some mortality was noted in culture by day 5, but most organisms were still alive when the cultures were terminated on day 7.     

Mallard ducklings (Anas platyrhynchos) experimentally infected with Echinostoma trivolvis (Digenea)

Of 8, day-old mallard ducklings, each fed 50 encysted metacercariae of Echinostoma trivolvis, 4 (50%) were infected 15-31 days postinfection (PI) with a total of 10 (2.5%) worms. The worms were attached loosely to the mucosa of the lower ileum and rectum-cloaca, and some were ovigerous by day 15 PI. Ducklings, 4-14 days old when fed encysted metacercariae, became infected with E. trivolvis adults, but ducks 150 days old were refractory to infection. Compared to our previous studies on experimental infections of echinostomes, mallard ducklings were less susceptible than golden hamsters to E. trivolvis.     

Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick

Fried B. and Fujino T. 1984. Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick. International Journal for Parasitology14: 75–81. Scanning electron microscopy (SEM) was used to study the development of chemically excysted metacercariae of Echinostoma revolutum on the chick chorioallantois. SEM studies were also made on preovigerous adults of E. revolutum grown in the domestic chick. During worm development on the chorioallantois the tegument changed from smooth to granular and sensory papillae on the suckers became well-defined. As worms developed on the chorioallantois the cephalic collar spines became thicker and more curved and the tegumentary spines showed marked changes in shape, size and distribution on both ventral and dorsal aspects of the body. Changes in the surface ultrastructure of worms grown on the chorioallantois were essentially similar to those observed in preovigerous worms from chicks.     

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.     

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.     

The effects of crowding on adults of Philophthalmus nocturnus grown in domestic chicks.

The effects of crowding on growth, reproduction, rate of recovery and site of infection of Philophthalmus nocturnus were investigated by infecting various groups of day-old chicks with 25, 50, 75, or 100 excysted metacercariae and examining them at 10 or 25 days post-infection. Considerable reduction in the body length and the size of the gonads was noticed in flukes grown under crowded conditions. Crowding also caused some change in the habitat of flukes, but had no effect on their rate of recovery. Very few flukes reached the ovigerous stage under overcrowded conditions and none reached the embryonated larval stage.     

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.     

Cultivation of excysted metacercariae of Echinostoma caproni (Trematoda) to ovigerous adults on the chick chorioallantois

Excysted metacercariae of Echinostoma caproni were cultivated on the chick chorioallantoic membrane (CAM) maintained at 38.5 +/- 1 C and a relative humidity of 60-65%. Of 59 6-day-old embryos, each inoculated with 25 metacercariae, 29 (49.2%) were infected 2-12 days postinoculation. The total number of worms recovered from the infected eggs was 163 or 22.5% of the 725 inoculated metacercariae. Eggs contained from 1 to 12 (average 5.6) worms per CAM. Worm length increased rapidly from an average of 0.5 mm at 2 days to about 3.0 mm at 6 days postinoculation. Ovigerous worms first were seen on day 8 PI, but fluke eggs did not develop embryos. Worm development in ovo lagged about 1 day behind that of in vivo worms. One worm maintained for 17 days on 2 successive CAMs reached 6 mm in length, contained about 100 eggs in its uterus, and laid an additional 100 eggs on the CAM surface.     

The culture of Diplostomum spathaceum metacercariae on the chick chorioallantois

Metacercariae (2,828) obtained from the lens of naturally infected Aplodinotus grunniens were transplanted onto chorioallantoic membranes (CAM's) of 94 eggs (means 30/egg) 5 to 12 days old. Membranes were examined 2 to 8 days later and 381 flukes were recovered. Two hundred fifty-nine chorioallantoic-grown worms were transferred to 28 additional embryos 3 to 8 days after the initial inoculation. These eggs were examined 3 to 11 days later. Fifty-three serially transferred worms were recovered after 7 to 14 days on CAM's. Six stages of development were recognized: stage 1, immature; stage 2, genital rudiment; stage 3, testes; stage 4, follicular ovary; stage 5, vitellaria; stage 6, ovigerous. The intestinal ceca of many worms (except stage 1) contained ingested blood. Trematode eggs oviposited on CAM's were embryonated in tap water, and viable miracidia were observed in 15. No previous study has achieved gonadal development in Diplostomum spathaceum on the CAM. Furthermore, inasmuch as metacercaria matured and produced fertile eggs, this form is shown to be a useful model for the study of trematode differentiation. The development of metacercariae to ovigerous adults capable of producing viable miracidia suggests that the CAM shares characteristics with the intestine of the definitive host, a piscivorous bird.     

Choline acetyl transferase activity in chick embryo neuroretinas during development in ovo and in monolayer cultures

The specific activity of the enzyme choline acetyl transferase (CAT) in chick neuroretinas was investigated during in ovo development and in monolayer cultures. The enzyme activity was barely detectable on the 6th day of incubation but increased markedly between the 7th and 11th days. The activity increased sharply between the 15th and 17th days and then slowly until hatching. When cell suspensions from 6- to 7-day neuroretinas were cultured as monolayers, CAT specific activity increased rapidly. After 4–5 days in culture, the activity of the enzyme was identical to that found in the neuroretina on the 11th day of incubation. Cells from 9-day neuroretinas also differentiate in monolayer cultures, but with a more irregular pattern. These data show that cholinergic neurons from chick embryo neuroretina differentiate in monolayer cultures without a lag and at the same rate as in vivo.     

Antibody-dependent rat macrophage-mediated damage to the excysted metacercariae of Paragonimus westermani in vitro

An in vitro immune effector mechanism against the target excysted metacercariae of Paragonimus westermani was demonstrated in the rat system. Peritoneal exudate cells, mainly macrophages from normal rats, showed adherence to and killing of excysted metacercariae of P. westermani in the presence of complement-independent serum from rats infected with Paragonimus metacercariae. These reactions were specific for the excysted metacercariae, as tissue-migrating juvenile worms were not affected. Damage of excysted metacercariae of P. westermani due to antibody and macrophages was assessed by morphological observation, by cell adherence reaction and by the use of vital dyes. Trypan blue dye exclusion proved to be a reliable indicator of judging metacercarial viability. Electron microscopic studies demonstrated that macrophages reacted with fuzzy material on the tegumental surface and fine structures in the syncytium of the parasites. The tubular tunnels formed between the basement membrane and muscle layers of the damaged parasites were also noticeable. The relevance of these findings to cellular immunity in the early paragonimiasis was discussed.     

Effects of copper sulfate toxicity on cercariae and metacercariae of Echinostoma caproni and Echinostoma trivolvis and on the survival of Biomphalaria glabrata snails

Copper in the form of copper sulfate (CuSO4) decreases the survival of Biomphalaria glabrata snails, but the effects of this molluscicide on Echinostoma caproni and Echinostoma trivolvis, 2 species of digeneans that use B. glabrata as intermediate hosts, are not known. Studies were done on the effects of various concentrations of CuSO4 in artificial spring water (ASW) on the survival and infectivity of E. caproni and E. trivolvis cercariae. Solutions containing 1.0, 0.1, and 0.01% CuSO4 were 100% lethal within 2 hr of exposure for both species. Time to 50% mortality in 0.001% CuSO4 was 8 hr for E. caproni and 16 hr for E. trivolvis; at 24 hr, the controls showed 50 and 65% mortality, respectively. Treatment of cercariae of both species for 0.5 hr in 0.001% CuSO4 had no effect on the ability of cercariae to form normal cysts in juvenile B. glabrata snails. However, treatment with 0.01% CuSO4 for 0.5 hr caused a significant reduction in the ability of cercariae of both species to encyst in snails. Treatment of encysted metacercariae of both species in 0.001% CuSO4 for I hr had no effect on subsequent excystation of these echinostomes in a trypsin-bile salts medium, whereas concentrations of 1.0, 0.1, and 0.01% CuSO4 and 1.0 and 0.1% CuSO4 decreased chemical excystation of E. caproni and E. trivolvis cysts, respectively. Survival studies on the effects of CuSO4 in Locke's solution on chemically excysted metacercariae of both species were also done. Excysted metacercariae of both species were killed by 2 hr in either 0.1 or 0.01% CuSO4 in Locke's solution. However, time to 50% mortality for both species of excysted metacercariae in 0.001% CuSO4 was approximately 5 hr. Time to 50% mortality for the controls was about 12 hr. Survival of juvenile B. glabrata snails was also examined. All B. glabrata snails were dead by 6 hr in 1 and 0.1% CuSO4 in ASW. Biomphalaria glabrata snails showed 50% mortality by about 6 hr in 0.01% CuSO4 and about 80% were still alive at 24 hr in 0.001% CuSO4. All controls were alive at 24 hr, at which time the experiment was terminated. Concentrations greater than 0.001% CuSO4 increased snail mortality, as well as that of the cercariae and excysted metacercariae of E. caproni and E. trivolvis. Our findings suggest that concentrations of copper sufficient to eliminate juvenile B. glabratta snails are also sufficient to kill the cercariae and excysted metacercariae of these digeneans but not the encysted metacercariae, which may be protected by their cyst walls.     

Development, growth, and migration of Philophthalmus gralli (Trematoda) from Jordan in the eyes of chicks

The growth of Philophthalmus gralli from Jordan was studied experimentally by inoculating 10 excysted metacercariae around the right eye of 47 day-old chicks. The growth rate was slow in the first three days during which flukes did not exceed 0.30 mm long. Then growth became rapid reaching 2.70 mm long on day 24. This was followed by a low growth rate during which the flukes grew from 2.70 mm to 2.90 mm on day 40. The flukes started to appear in the left eye on day 12 post-inoculation of metacercariae in the right eye. The recovery of flukes in the left eye increased from 5% on day 12 to 30% on day 32. The development of P. gralli from Jordan was the same as of other species of Philophthalmus. However, a sixth stage, post-maturation stage, is suggested in addition to the 5 developmental stages of Philophthalmus. The ratio of the transverse diameter of the ovary to that of testes in the post-maturation stage was 1:1.1-1:1.2.     

The development of the genital system in Ichthyocotylurus variegatus (Trematoda: Strigeidae)

The development of Ichthyocotylurus variegatus from metacercariae to ovigerous adults in the natural definitive host (Larus ridibundus) was examined at 24 h intervals. The metacercariae develop to the adults through a somatic growth of hindbody; the gametogeny and vitellogenesis take 4-6 days. During the first day of development the pair of testes are separated from the genital anlage. The ovary differentiates one day later. Genital ducts are completely differentiated on 4th day after the beginning of spermatogenesis and cogenesis. The vitelline cells developed from 2nd to 5th day. They take an origin from the subtegumental cells of hindbody. The hind part of uterus in strigeoid adults is identical to the metraterm of other trematodes. The spermaduct along all its length works as a spermatic bladder.     

In vitro cultivation of Amblosoma suwaense (Trematoda: Brachylaimidae) from the metacercaria to the ovigerous adult

A variety of media were used to study the in vitro cultivation of Amblosoma suwaense (Brachylaimidae). Of these media most rapid development was achieved in NCTC 135 supplemented with 20% hen's egg yolk (NCTC 135-20Y). In this medium ovigerous adults were obtained in 4 days at 37·5°C and with a gas phase of air, and the eggs contained developing embryos. The mean body area of 7-day-old worms cultivated in NCTC 135-20Y was 20% less than that of the metacercariae, whereas the mean area of the gonads and vitellaria was 53 % greater than that of the metacercariae. The black pigment in the gut of metacercariae was egested during cultivation. The tegument of metacercariae was rugose, whereas that of cultured worms was smooth.     

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.