The effects of crowding on adults of Philophthalmus gralli (Trematoda) grown in chickens

Chickens were infected with six, 10, 30, 50, or 100 metacercariae of Philophthalmus gralli per eye and the adults were harvested 9, 14, 21, 35, and 50 days later. The worms recovered were measured and assessed for maturity. Significantly more adults from infections of six and 10 metacercariae per eye were recovered than from infections of 100 per eye. When metacercariae were placed in one eye, over one-fourth of the resulting adults were found in the opposite eye. Worms recovered from groups of zero to 10 were significantly longer than those in groups of 41 to 50 and 51 to 60 at all ages sampled. The normal movement of worms from the conjunctival sac to the outside of the nictitating membrane was hindered by crowding; when more than 10 were present, some worms developed in the sac to mature adults. Worms in all groups produced eggs in which miracidia developed at the same rate.     

Optimization of culture conditions for in vitro fertilization and reproduction of Microphallus turgidus (Trematoda: Microphallidae)

Most attempts to culture adult digeneans in vitro are unsuccessful. Even progenetic digeneans typically fail to produce infective eggs in axenic culture. However, metacercariae of Microphallus turgidus grown in vitro mature into adults and release eggs infective to the hydrobiid snail Spurwinkia salsa. The objectives of the present study were to verify the reproducibility of the M. turgidus culture protocol, to define optimal culture conditions for M. turgidus further, and to investigate why the parasite can be grown successfully in the absence of the definitive host. In the original cultivation protocol, excysted M. turgidus metacercariae from grass shrimp (Palaemonetes pugio) were incubated overnight in a conical-bottom centrifuge tube containing Hank's balanced salt solution and then cultivated in flat-bottom culture plate wells containing RPMI-1640 plus 20% horse serum. The gas phase was air. Worms cultured under this protocol consistently deposited eggs infective to snails. Worms grown in anaerobic conditions deposited few eggs, and those cultured in a gas phase of 5% CO(2) survived longer and produced more eggs than those cultured in air. However, snails were less likely to become infected when fed eggs deposited by worms cultured in 5% CO(2). Additionally, worms incubated with conspecifics in conical-bottom tubes prior to cultivation were more likely to be inseminated than worms incubated in flat-bottom culture wells; the highest percentages of inseminated worms occurred when metacercariae were incubated 24 hr in conical-bottom tubes at a density of 50 worms/tube and at a temperature of 37 C. Worms incubated in the absence of conspecifics were not fertilized and failed to produce infective eggs.     

Endemic echinostome infections of candidate hosts

Wild Lymnaea tomentosa snails, recovered from Lake Wanaka, New Zealand, were established in the laboratory. Wild snails, naturally infected with echinostomes, provided metacercariae for infection of laboratory maintained snails. Metacercarial cysts from wild and laboratory snails were then used to attempt infection of definitive host candidates. Laboratory snails provided convenient packaging of known numbers of cysts. Metacercariae excysted in the small intestines of ducklings to mature in 6 days. Worms were expelled as they became gravid. Attempts to establish infections in experimental hosts other than ducklings were not successful. No worms were recovered from mice, white rats, guinea pigs, hamsters or immunosuppressed white rats.     

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.     

Susceptibility of experimental animals to reinfection with Clonorchis sinensis

The present study observed the resistance to reinfection with Clonorchis sinensis in various experimental animals including mice, guinea pigs, rabbits, and dogs, as well as rats and hamsters. The resistance rates to reinfection in rats, mice, hamsters, guinea pigs, rabbits, and dogs were 79.7%, 58.0%, -12.6%, 54.8%, 62.6%, and 6.0%, respectively. Worms recovered from reinfected rats and mice were immature, and significantly smaller than those from the primarily infected (P < 0.01), whereas those from other animals were fully matured to adults. These findings indicate that the protective response against reinfection with C. sinensis is prominent in rats and mice, and that they may be a good animal model to investigate the mechanism of resistance to reinfection with C. sinensis.     

Radioresistance of Anisakis simplex third-stage larvae and the possible role of superoxide dismutase

The radioresistance of Anisakis simplex third-stage larvae and the possible role of sublethal radiation on superoxide dismutase (SOD) were investigated. Larvae were isolated from the viscera of the sea eel Anago anago; irradiated with 10, 100, 200, 500, or 1,000 Gy; and then given orally to rats. Worms were recovered at 16 hr postinoculation. Most larvae were found to have invaded the gastric wall, omentum, and abdominal cavity, suggesting that their viability and infectivity were not controlled by irradiation with the doses used. To determine the relationship between SOD activities in parasites and their radiosensitivities, the larvae of A. simplex and the metacercariae of Neodiplostomum seoulense (a radiosensitive control) were irradiated with 0, 30, 100, or 500 Gy, and parasite SOD levels were measured. In nonirradiated A. simplex larvae, the average SOD level was 38.9 U/mg, and this increased to 51.3 U/mg at 500 Gy. However, at all radiation doses applied, SOD activities of N. seoulense metacercariae were significantly (P < 0.05) lower than those of A. simplex larvae. Our results demonstrate that A. simplex third-stage larvae are radioresistant, and suggest that SOD plays a role in this radioresistance.     

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.     

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.     

Experimental infection of Paragonimus iloktsuenensis to albino rats, dogs and cats

This study was performed to observe the susceptibility of dogs and cats as definitive hosts of Paragonimus iloktsuenensis. The metacercariae of this fluke were obtained from Sesarma dehaani collected at a focus near the mouth of Sumjin river in November, 1986 and February, 1987. The larvae isolated from the crabs were introduced per os into 7 albino rats, 2 dogs and 3 cats. The adults were recovered from the experimental animals, and they were morphologically observed and measured. The results were as follows: 1. The recovery rate of adult worms at 42 days after infection was 53.3% from three albino rats, 21.0% from a dog and 12.7% from two cats. Most of the worms were recovered from the worm capsules in the lungs. 2. The size of worms recovered from albino rats, a dog, and cats 42 days after infection averaged 6.3 x 3.2 mm, 6.3 x 3.0 mm, or 6.2 x 3.5 mm, respectively. There were little differences in the morphology of worms by different experimental animals. 3. The size of eggs from a dog was 88.9 x 49.3 microns, and that from cats was 84.3 x 53.7 microns on average. Dogs and cats were good definitive hosts of P. iloktsuenensis. This fact suggests that human infection by this fluke may be possible if the metacercariae were ingested.     

The effects of gamma irradiation on the survival and development of Clonorchis sinensis metacercariae

The effects of gamma irradiation on the survival and development of C. sinensis metacercariae were studied to evaluate the feasibility of irradiation as a control measure for clonorchiasis. Pseudorasbora parva were collected at an endemic river of clonorchiasis and were used for irradiation of the fluke in three schemes. The first (Scheme 1) was irradiation of the isolated metacercariae from the fish followed by infection to experimental rats. The second (Scheme 2) was irradiation of the fish, and then the metacercariae were isolated and infected to rats. The third (Scheme 3) was irradiation on the rat livers after infection with normal metacercariae. Irradiation doses varied from 5 to 100 Gy for Schemes 1 and 2, and 10 to 25 Gy for Scheme 3. The rats were sacrificed 2 to 6 weeks after infection. In Scheme 1, the metacercariae irradiated at 50 Gy failed to survive in the rats after 2 or 6 weeks. However, 1 to 44% of the metacercariae irradiated at 5-30 Gy survived. The estimated LD50 of Scheme 1 was 16.5 Gy. The flukes irradiated in Scheme 2 survived better than those in Scheme 1. The average worm recovery rate in 50 Gy was 28%(7-39% individually). Increasing the dose up to 100 Gy brought a remarkably low survival rate of an average 1%(0-3% individually). The LD50 of Scheme 2 was 47.5 Gy. Worm recovery rates in the 10 Gy group of Scheme 3 were 21-39%, and those in the 25 Gy group were 2% and 34%. Although the metacercariae were irradiated, all of the recovered worms were morphologically normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early stage biliary and intrahepatic migration of Opisthorchis viverrini in the golden hamster

In Thailand, infection with the liver fluke Opisthorchis viverrini is a serious health problem, with over 8.6 million human infections each year. Early stage biliary intrahepatic migration and infection of O. viverrini in the Syrian golden hamster were used to study the growth and development of this fluke. Fifty metacercariae of O. viverrini were introduced into each hamster by gastric intubation. Worms were found to migrate rapidly from the stomach to the gall bladder and hepatic duct, where they remained in relatively constant numbers until the end of week 8. Sexual development of worms was rapid, with full development of the uterus and testes by one and one half weeks and the appearance of eggs in the uterus by the beginning of the third week of infection. Worm growth as indicated by body length had ceased by week 8. Hamsters demonstrated development of a full reproductive cycle with in three to four weeks, and this generation continued until 8 weeks.     

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.     

Growth and development of Gymnophalloides seoi in immunocompetent and immunosuppressed C3H/HeN mice

The growth and development of Gymnophalloides seoi were studied in C3H/HeN mice and effects of immunosuppression of the host on the worm development were observed. Two hundred metacercariae of G. seoi were orally administered to each mouse, and worms were recovered on days 1, 3, 5, 7, 14 and 21 post-infection (PI). The worm recovery rate was significantly higher in immunosuppressed (ImSP) mice than in immunocompetent (ImCT) mice except on days 1 and 3 PI. The worms attained sexual maturity by day 3 PI with eggs in the uterus, and worm dimensions and the number of uterine eggs continuously increased until day 14 PI in ImSP mice. Worms recovered from ImSP mice were significantly larger in size than those from ImCT mice on days 1 and 3 PI, and the number of uterine eggs was significantly larger in ImSP mice on days 5 and 7 PI. Genital organs such as the ovary, testes, and vitellaria, that were already developed in the metacercarial stage, grew a little in size until day 14 PI. The results show that the C3H/HeN mouse is, though not excellent, a suitable laboratory host for G. seoi.     

Studies on host specificity in Paragonimus westermani: II. Histochemical and cytochemical characterization of metacercariae and worms from rats and dogs

Histochemical tests were done on newly excysted metacercariae and worms recovered from an abnormal host (rat) and the definitive host (dog) for some oxidoreductases, phosphatases and glycosidases. The results demonstrate that rat worms have enzymatic distribution and intensities more similar to those of metacercariae than to adult worms from dogs. Ultracytochemical examination of acid and alkaline phosphatase and Mg-ATPase activity was also carried out. Acid phosphatase activity occurred exceptionally in the excretory bladder and caeca of dog worms. No activity was observed in rat worms except for lysosomal granules in the tegument. Alkaline phosphatase activity was exhibited in the excretory bladder in both dog and rat worms. Mg-ATPase activity occurred in the tegument and parenchymal cells in dog worms and in the excretory bladder in rat worms. In metacercariae, little or no reaction for these enzymes was present except for Mg-ATPase activity on the excretory ducts. These observations, together with the histochemical results, indicate that metabolic activity in rat worms is higher than in metacercariae although it is strongly reduced compared with dog worms.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

Encystment and metacercariae development of Echinostoma cinetorchis cercariae in an in vitro culture system

For some species of 37-collar-spined Echinostoma, their cercariae successfully encyst and develop to metacercariae in vitro. In our study, we cultured Echinostoma cinetorchis cercariae in 12 different media to study the formation of metacercariae. Locke's solution, medium 199, and RPMI 1640 were used as media for culture. RPMI 1640 produced the highest encystment and normal metacercariae development. The osmolality of the media was related to their ability to encyst and develop. The 0.5 X media induced higher encystment and normal metacercaria formation than the 1x media. The addition of fetal bovine serum to RPMI 1640 increased the level of encystment and normal metacercariae development. In the mixture of 0.5 x RPMI 1640 and 10% fetal bovine serum, encystment was highest, at 96.0%, and the development ratio of normal metacercariae was also the highest, at 91.5%, after 48 hr of cultivation. In a viability test, 7 day- and 14 day-cultured metacercariae were successfully matured to adult worms in experimentally infected rats. These results showed that E. cinetorchis cercariae could be cultured to viable metacercariae in an in vitro culture system and that 0.5 x RPMI 1640 plus 10% fetal bovine serum was the most useful medium for cultivation. This culture system can be adapted for additional studies on the E. cinetorchis life cycle, especially to supply large numbers of metacercariae for other studies on this echinostome.     

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×.     

Clonorchis sinensis metacercarial infection in the pond smelt Hypomesus olidus and the minnow Zacco platypus collected from the Soyang and Daechung Lakes

The pond smelt Hypomesus olidus and minnow Zacco platypus were collected from the Soyang and Daechung Lakes in January 2003, and their metacercarial infections was examined by the muscle compression and artificial digestion techniques. In the Soyang Lake, 161 metacercariae of Clonorchis sinensis (0.35 per fish) were harvested from 459 pond smelts examined. Also, 13 metacercariae of C. sinensis (0.43 per fish), 1 of Metagonimus sp., 4 of Echinostoma sp., 148 of Centrocestus armatus and 44 unidentified species were collected from 30 minnows. In the Daechung Lake, 369 metacercariae of C. sinensis (3.69 per fish) and 51 unidentified species were recovered from 100 pond smelts. The metacercariae of C. sinensis were fed to experimental rats, in which the adult flukes were identified. The pond smelts and minnows collected from the Soyang and Daechung Lakes were verified to be the second intermediate hosts and the sources of human C. sinensis infection.     

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.     

Paragonimus paishuihoensis Metacercariae in Freshwater Crabs,Potamon lipkei,in Vientiane Province,Lao PDR

Among Paragonimus species, P. paishuihoensis is one of the most mysterious and poorly understood species. Metacercariae are characterized by having a unique dendritically branched excretory bladder. However, the morphology of the adult worm remains unknown. To date, metacercariae of this species have been reported only in China and Thailand. In this study, we first found P. paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Hinheub District, Vientiane, Lao PDR, with a prevalence of 77.7% and the average intensity of 10.3 (range 1-28) metacercariae per crab. The molecular data based on ITS2 and CO1 markers indicated that P. paishuihoensis from Laos and Thailand were almost completely identical and were close to members of the Paragonimus bangkokensis/Paragonimus harinasutai complex. Attempts to infect experimental animals (cats, dogs, and rats) with P. paishuihoensis were unsuccessful, suggesting that these animals might be unsuitable definitive hosts for the species. Further studies are necessary to elucidate the taxonomic status and life cycle of P. paishuihoensis.