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.     

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.     

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.     

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.     

In vitro cultivation of Paragonimus miyazakii and P. ohirai

Excysted metacercariae of Paragonimus miyazakii and P. ohirai were cultured in various media at 37.5 C in a 5% CO2 atmosphere. Paragonimus miyazakii grew rapidly and showed a well-developed ovary, uterus, and testes at 172 days in NCTC 109 supplemented with 30% rabbit serum, 50% egg yolk-109, and rabbit red blood cells (RBC's). However, none of the worms formed yolk or eggs in these cultures. On the other hand, P. ohirai grew to the adult stage, in which vitellaria and imperfect ova were formed, in NCTC 109 supplemented with 30% dog serum, 10% yeast extract Earle's solution (YLE), and dog RBC's at 252 days. The maximum body length of these worms measured 7.0 mm (mean 5.5 mm) at 252 days. The dog RBC's were an essential ingredient of the culture medium for the development of P. ohirai. Additions of liver concentrate, chick embryo extract (CEE), and egg yolk-109 in the medium did not provide any additional benefits for the development of worms. Using this supplemented medium, adult worms of P. ohirai removed from rats were maintained in vitro to examine their ability to lay eggs. Egg laying occurred during the first 10-13 days for worms that survived more than 60 days. The number of eggs deposited in this medium was about 2 times that found when Hanks' BSS and NCTC 109 were used.     

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.     

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.     

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.     

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×.     

Fasciola hepatica in rats: Effects of age and infective dose

Groups of 6 male rats, aged either 5, 10, 15 or 25 weeks old, were infected with either 1, 5, 10, 15 or 20 metacercariae of Fasciola hepatica. The prepaient period among all groups ranged from 57 to 60 days, and the size of worms recovered was inconsistent. Within each age group of rats the proportion of metacercariae which developed to maturity was the same irrespective of the dose of metacercariae. These results indicate that neither a crowding effect nor competitive inhibition occurred. Five-week-old rats were generally more susceptible to infection than other age groups. Some variation in the expression of age resistance in 10-week-old animals suggests that it is at about this age that factors involved in age resistance develop.     

Growth and development of Metorchis orientalis in chicks and its adult morphology]

In order to observe the infectivity, growth and development and adult morphology of Metorchis orientalis, a total of 40 chicks were experimentally infected with 100 metacercariae respectively, collected from Pseudorasbora parva. The worms of various developmental stages were recovered from chicks at 1.5, 3, 5, 7, 9, 11, 14 and 21 days after infection, and they were prepared for morphological observations and measurements. All of the worms were found in the gallbladders of chicks, and their recovery rate was 32% in average. The growth of the body was rapid from 9 to 11 days after infection. The genital primordia appeared in 1.5 and 3-day old worms, and ovary and testes were first observed in 5-day old worms. Thereafter, genital organs gradually matured and completed up to 11 days after infection. The adult worm was leaf-like, and possessed a convoluted tubular seminal vesicle, an ovoid ovary, a sac-like seminal receptacle, 2 lobed-testes and follicular vitellaria. Eggs were 31.9 x 15.3 microns in average size, ellipsoid to elliptical in shape and possessed abopercular thickenings. From the above results, it is concluded that M. orientalis grows in sigmoid pattern in chicks, and their genital organs fully mature between days 9 and 11. It is also confirmed that a chick is a new definitive host of M. orientalis.     

Recovery,growth, and development of Acanthoparyphium tyosenense (Digenea: Echinostomatidae) in experimental chicks

Chicks were experimentally infected with Acanthoparyphium tyosenense (Digenea: Echinostomatidae) metacercariae per os, and the growth and development of worms in this host were observed from days I to 38 postinfection (PI). The worms grew rapidly and matured sexually in the small intestine (chiefly in the jejunum) of chicks by day 5 PI. and survived at least up to day 38 Pi, although worm recovery decreased after day 5 PI. Both parenchymal and reproductive organs increased greatly in size from day 2 to day 10 PI and then continued to increase gradually in size up to day 38 PI. The number of uterine eggs reached a peak on days 10 and 15 PI and then decreased gradually. The results suggest that chicks are a fairly suitable definitive host for experimental infection with A. tyosenense.     

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.     

Observation of mucosal pathology after praziquantel treatment in experimental Fibricola seoulensis infection in rats

It is well known that the duodenum of mice or rats infected with Fibricola seoulensis shows atrophy of villi (shortening, blunting, widening, fusion) and hyperplasia of crypts. This study was performed to observe healing process of these pathological changes after deworming with anthelmintic treatment. Albino rats infected each with 1,000 metacercariae of F. seoulensis were treated with single dose of 10 mg/kg praziquantel on day 15 post-infection. On day 1, 3, 5, 7, 15, 21 and 28 after the treatment, they were sacrificed and their duodenums were histopathologically studied. Control (uninfected) rats showed their normal finger-like projections of duodenal villi and well arranged crypts. In comparison, untreated (infected) controls revealed severe mucosal changes characteristic of villous atrophy and crypt hyperplasia in their duodenum. The damaged duodenal mucosa was found to restore its normal morphology after praziquantel treatment; until day 3 post-treatment the mucosa was severely atrophied; on day 5 long and slender villi sometimes appeared among the fused and stout ones; after day 15 the villi were in their normalizing process. From this experiment, it was shown that the mucosal changes in the duodenum of rats caused by F. seoulensis infection were completely reversible in 21-28 days after anthelmintic treatment.     

Recovery rate, growth and development of Heterophyopsis continua in experimental chicks

The growth and developmental pattern of H. continua was observed after experimental infection of their metacercariae to chicks. The recovery rate of worms from the chicks at 1 to 28 days post-infection (PI) was 12.8% in average. The rate remained fairly high for early 4 days of infection but decreased thereafter rapidly till 28 days PI. Most of the flukes, 91.9%, were recovered from the ileum of the chicks. In metacercariae, genital organs such as the ovary, testes, seminal vesicle, seminal receptacle and genital sucker were recognizable. At one day PI Mehlis' gland appeared, and at 2 days follicular vitellaria were observed. At 3 days PI, eggs were formed in the uterine tubule and increased in number as the worm grew old. The worms reached 2,990 microns in length and 525 microns in width at 28 days PI. Genital organs developed rapidly in early stages of infection but slowly thereafter to 28 days PI, whereas non-genital organs developed steadily through the infection period. It was proved by this experiment that chicks should be a moderately suitable final host of H. continua.     

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.     

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.     

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.     

Optimization of culture conditions for the maintenance of Onchocerca gutturosa adult worms in vitro

A series of experiments examined the effects of various media, serum supplements, gas phases and the incorporation of mammalian cell feeder layers on the survival of Onchocerca gutturosa adult worms in vitro. The survival of male worms was poor in all media tested that were not supplemented with inactivated foetal calf serum (IFCS), with improved but variable survival in media supplemented with 10-30% IFCS. Using a cell-free system in an atmosphere of 5% CO2 in air, good results were obtained in medium NCTC 135 + 10% IFCS (median survival time 39 days, range 25-41). Marginally better survival was obtained with the same medium in an atmosphere of 95% N2/5% CO2 (median 45 days, range 25-56) and with a 1:1 mixture of media NCTC 135 and IMDM + 10% IFCS (median 38 days, range 38-51). Survival was enhanced in culture systems which incorporated bovine kidney (MDBK) cells, bovine trachea (EBTR) cells and monkey kidney (LLCMK2) cells. Exceptionally long survival was obtained using medium MEM + 10% IFCS + LLCMK2 cells under a gas phase of 5% CO2 in air, in which male worms survived from approximately 6 to over 7 months. Under similar conditions, female worms were also maintained for periods of up to 6 months and 5 out of 18 specimens released microfilariae into the culture system. The long-term culture described in this study will be useful for basic biochemical, chemotherapeutic and immunological studies in vitro.     

Effect of praziquantel treatment on pulmonary lesions of rats infected with Paragonimus iloktsuenensis

An experimental pathological study was performed to observe the effect of praziquantel treatment on the pulmonary lesions of the rat lung fluke, Paragonimus iloktsuenensis. The metacercariae were obtained from the freshwater crab, Sesarma dehaani, and 40 rats (wistar) were fed each with 10 metacercariae. On 20 rats praziquantel treatment (100mg/kg/day x 5 days) was done at 5 weeks after the infection while remaining 20 rats were kept untreated for use as controls. The drug-treated rats and the untreated ones were sacrificed 3, 7, 14, 21 or 28 days later for the observation of lung pathology. The rats infected with P. iloktsuenensis showed remarkable pulmonary changes; gross features of hemorrhagic and nodular worm capsules protruded on to the surface of the lung, and histologically local atelectasis, inflammatory cell infiltration, and egg granuloma around the worm capsules each containing one or two worms. Praziquantel treatment of the rats was shown to be highly effective in killing the worms and to lead them to degenerate, as early as in 3 days post-treatment. Almost all worms in the lung were dead and absorbed by the host cells in 21 days post-treatment, except a few living ones seen in a rat of 14-day post-treatment group. In most of the rats treated the pulmonary lesions showed the signs of resolution; regression of worm capsules with mummification of worms, decrease of inflammatory cell infiltration, improvement in the degree of atelectasis, and decreases in the frequency and size of the egg granuloma. From the results it is concluded that praziquantel is highly effective for the treatment of rat P. iloktsuenensis infection in the lung, not only by its direct killing effect of the worms but also due to the excellent resolution capacity of the pulmonary tissues.