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.     

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.     

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.     

Susceptibility of several strains of mice to Echinostoma hortense infection

Susceptibilities of 5 different mice strains, including C3H/HeN, BALB/c, C57BL6, FvB and ICR, to Echinostoma hortense infection, was evaluated. The worm expulsion rate, worm size and egg production were observed from 1 to 8 weeks after infection with 30 metacercariae. C3H/HeN and ICR mice showed the highest worm maturation rates. The worm recovery rate and the number of eggs per gram (EPG) of feces was also higher in C3H/HeN and ICR mice than in BALB/c, C57BL6, and FvB mice. It is suggested that E. hortense is highly infectious to ICR and C3H/HeN mice, but not to the other strains of mice. Based on the results obtained, we believe that the susceptibility of different mouse strains to E. hortense infection is dependent on the genetic and immunologic background of mice.     

Exposure of Dugesia tigrina (Turbellaria) to cercariae of Echinostoma trivolvis and Echinostoma caproni (Trematoda)

Laboratory-reared planarians, Dugesia tigrina, were exposed to cercariae of Echinostoma trivolvis or Echinostoma caproni. Of 100 D. tigrina exposed to 2,750 cercariae of E. trivolvis, 29 were infected with a total of 85 encysted metacercariae 24 hr postinfection (PI). None of 40 D. tigrina exposed to 1,100 cercariae of E. caproni was infected with metacercariae 24 hr PI. Cyst structure of E. trivolvis from the parenchyma of D. tigrina varied from normal to abnormal. Metacercariae removed from planarians could be excysted in an alkaline trypsin-bile salts medium. Cercariae of E. trivolvis were not attracted to dialysate material from D. tigrina. Cercariae of E. trivolvis that penetrated, but did not encyst in planarians, voided the contents of their paraesophageal glands. Cercariae of E. caproni lack paraesophageal glands. Secretions of those glands probably are involved in tissue penetration of D. tigrina by E. trivolvis cercariae.     

In vitro and in vivo encystment of the cercariae of Echinostoma caproni

In vitro and in vivo studies were conducted on the cercariae of Echinostoma caproni. Of the 15 media tried, 2 resulted in effective in vitro encystment in petri dish cultures maintained at 23 +/- 1 C. They were a Locke's--artificial springwater (ASW) (1:1) medium (67% encystment) and a Biomphalaria glabrata embryonic cell line medium (23% encystment). To obtain large numbers of in vitro--formed cysts, finger bowl cultures containing 40 ml of the Locke's-ASW (1:1) medium were used at 23 +/- 1 C. Of 3,000 cercariae tested, 1,890 (63%) were encysted in this medium by 48 hr. Most of these cysts looked similar to those formed in vivo, although some showed abnormalities in the outer cyst wall and other malformations. A total of 200 in vitro-formed cysts treated in an alkaline trypsin-bile salts (TB) medium for 2 hr at 41 C showed 94% excystation. In vitro-formed cysts fed to mice produced ovigerous adults within 2 wk postinfection (PI). Eggs from these worms gave rise to miracidia that produced patent intramolluscan infections in B. glabrata snails. In vivo encystment was studied in lab-raised juvenile Helisoma trivolvis (Colorado strain) snails, 1-3 mm in shell diameter. From 6 to 24 hr PI, 93-100% of the cercariae were recovered as metacercarial cysts in the snail tissue. Treatment of these cysts in the TB medium resulted in 96% excystation within 2 hr at 41 C.     

Failure of Echinostoma lindoense to reinfect snails already harboring that species.

In Biomphalaria glabrata snails already harboring Echinostoma lindoense, attempts at reinfection with the same trematode species failed when the interval between exposures was 6 days or more. With 2 days between exposures, the experimental snails were as susceptible to reinfection as the control snails. Between these limits, the reinfection failure rate increased with the exposure interval. Failure to reinfect the snails was probably due to cannibalism, young sporocysts from the second infection being swallowed by predatory rediae from the first infection. Development of sporocysts in successfully reinfected snails was normal.     

Cercarial tail loss in Echinostoma caproni: the influence of in vivo encystment and copper sulphate

Echinostoma caproni tail loss was studied in vitro in the presence of the toxicant copper sulphate (CuSO4) in concentrations ranging from 1 to 10 000 mg l(-1) in standardized artificial spring water (pH 7.4, osmolarity 34 mOsm kg(-1) H2O, Ca(2+) 20 mg l(-1)) at 23 degrees C. Tail loss was also studied in the absence of toxicants during in vivo encystment of the cercariae in juvenile Biomphalaria glabrata. As the concentration of CuSO4 increased, the percentage of cercarial tail loss increased. By 2 h in 10 000 mg l(-1), 1000 mg l(-1) and 100 mg l(-1) CuSO4, 50%, 23% and 13%, respectively, of the cercariae had lost their tails. In the in vivo studies, by 1 h PI, 59+/-5% of cercariae had lost their tails and only 4+/-1% of the cercariae were actively swimming in the multi-well dishes. At 3 h PI, 72+/-3% of the cercariae began to form cysts within the snails.     

Observations on the host-parasite relations between Echinostoma revolutum and lymnaeid snails.

Echinostoma revolutum from Taiwan was studied in lymneid snails at 29 +/- 0.5 degrees C. Given 3-5 miracidia, 95% of Lymnaea ollula and 40% of Lymnaea swinhoei became positive; the prepatent periods were 18 and 25 days, respectively. The following are based on the observations in Lymnaea ollula: The time required for miracidial penetration was about one hour. The sporocysts developed only in the ventricle of the snail but mother rediae developed in the heart and other organs. Mature daughter rediae were not found in the heart cavity. The sporocysts reached the ventricle within 3 days. Mother rediae were released after 6 days and daughter rediae after 8 days. Given 5 miracidia, 1-3 sporocysts reached the heart and 2-20 mother rediae were found per snail. The number of mature daughter rediae was usually 100-200 although more than a thousand may develop in a snail. The sporocysts and mother rediae attained maximal size 9 days postinfection and started degeneration 13 days postinfection. Daughter rediae were largest at the beginning of cercarial emergence and decreased in size thereafter. Simultaneous production of daughter rediae and cercariae by the mother redia was seen only once in this snail mature cercariae were obtained in 10 days postinfection. The cercariae emerged from a small area of mantle collar near the posterior corner of shell aperture. They were negatively phototactic and positively geotactic. An estimation showed that each snail shed about 350 cercariae a day. The cercariae reached the pericardial cavity of snail in one hour via the renal orifice and metacercariae were seen 4-5 hours after exposure. The infectivity of cercariae at various times after shedding, as expressed by cyst recovery rates, were: 51.6%, O-hr old; 76.1%, 2-hr; 68% 4-hr; 32%, 6-hr; 3%, 8 and 10-hr. Cyst recovery rates were not different within the dosage of 50-500 cercariae per snail. Most metacercariae recovered 1-2 days after cercarial exposure were viable; only 5 among 6,533 cysts were dead.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Echinostoma caproni

High performance thin-layer chromatography (HPTLC) was used to analyse the neutral lipids in the rediae, cercariae, and encysted metacercariae of Echinostoma caproni from Biomphalaria glabrata snails. Visual observations of the chromatograms showed that the most abundant lipid fraction in all stages was free sterol. Quantification of the free sterol revealed mean weights of 2.7 +/- 0.64 ng per redia, 0.53 +/- 0.023 ng per cercaria, and 0.081 +/- 0.0098 ng per encysted metacercaria. Oil Red O staining of the larval stages confirmed the presence of lipids within the rediae and cercariae but did not show lipids in the encysted metacercariae. The dimunition in neutral lipids from the cercarial to the encysted metacercarial stage does not support a previous observation that fat increases in successive phases of the digenean life cycle.     

Host specificity of Pisidium coreanum (Bivalvia: Sphaeriidae) to larval infection with a human intestinal fluke Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

The fingernail clam, Pisidium coreanum, has been traditionally consumed raw as a so-called drug therapy by patients with bone fractures in Korea. The present study was designed to determine the possible occurrence and, if present, the prevalence of Echinostoma cinetorchis in P. coreanum collected at a local site, and to determine the susceptibility of the clams in the laboratory to infection with miracidia and cercariae of E. cinetorchis. No cercariae or metacercariae of E. cinetorchis were observed in field-collected P. coreanum clams. In susceptibility experiments with laboratory-reared clams, individuals exposed to miracidia of E. cinetorchis did not release cercariae by 20 days after exposure; necropsy of exposed clams failed to show development of any sporocysts or rediae. To confirm the possibility of these clams serving as an experimental second intermediate host of E. cinetorchis, 20 of them were exposed to E. cinetorchis cercariae from experimentally infected Segmentina hemisphaerula that had been previously exposed to miracidia of E. cinetorchis; all exposed clams became infected. Metacercariae from clams at 14 days postinfection were fed to rats, and adult worms were recovered from the ileocecal regions. This is the first report of P. coreanum serving as second intermediate host of E. cinetorchis.     

Effects of snail size and diet on encystment of Echinostoma caproni cercariae in juvenile Helisoma trivolvis (Colorado strain) and observations on survival of infected snails

The effects of snail size and diet on encystment of Echinostoma caproni cercariae in juvenile Helisoma trivolvis (Colorado strain) snails were studied. Encystment in neonatal (<1-mm shell diameter) and juvenile (2- to 3-mm shell diameter) snails was compared 24 hr postinfection (PI) after individual exposure of snails of each size to 1, 5, 10, 25, or 50 cercariae. Significantly more cysts were recovered from juvenile snails exposed to 10, 25, or 50 cercariae than from neonatals with comparable exposure. The maximum number of cysts recovered from juveniles exposed to 50 cercariae was 42, compared with a maximum of 15 cysts in neonatals comparably exposed. Size of H. trivolvis was a major factor in determining cyst burden in this planorbid. A diet of either Romaine lettuce leaf or hen's egg yolk did not have a significant effect on the number of cysts recovered at 24 hr PI from juvenile snails exposed to 25 or 75 cercariae. Survival of infected versus uninfected neonatals was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 6 and 7 days PI when compared with uninfected controls.     

Histopathology of experimental Schistosoma incognitum infection in mice following exposure to normal and irradiated cercariae.

The tissue reactions in mice, experimentally infected with normal and irradiated cercariae of S. incognitum were studied. The lesions observed in the skin, liver, lungs and the intestine of mice infected with normal cercariae are briefly described, and compared with those observed with cercariae irradiated at 3000 r of gamma rays. In general, the reactions in mice exposed to normal cercariae were more intense than in those infected with irradiated cercariae. The severity of the reactions appeared largely due to the deposition of eggs in the tissues of the mice infected with normal cercariae. The experimental evidence suggested that most of the flukes from the irradiated cercariae are destroyed in the liver by tissue reaction.     

A stock of infected snails for the mass breeding of cercariae and schistosomules

A routine to maintain infected snails Biomphalaria glabrata able to produce 1-2 million cercariae per week and a mean shedding of about 3,500 cercariae per snail was quantitatively described. To a monthly production of about 5.3 and 6.5 million cercariae during the years of 1982 and 1983 it was necessary to expose an average of 1,557 and 1,957 snails, respectively. The efficiency described was maintained by infecting 2,000 snails per month (infectivity index of about 60%). A maximal production of 6,000 cercariae/snail was obtained in the period between 56-70 days after the snail infection by miracidia. By this period, however, 90% of the infected snails were dead. In the summer of 1982-1983 it was observed an impairment on the daily cercarial harvests which was related to ambient temperature increase, and the presence of rotifers in the aquaria water. When the cercariae were transformed into schistosomula in vitro, a yield of 55% schistosomula was obtained, with 7% of tail contamination. Lyophilization of cercariae produced 50.9 +/- 63 g dry weight per 1,000 cercariae.     

Effects of copper sulphate on in vitro encystment of the cercariae of Echinostoma caproni

The effects of various concentrations of copper sulphate were studied on in vitro encystment of Echinostoma caproni in a Locke's-artificial spring water (ASW) (1:1) medium. Cercariae were killed in 10,000 mg l(-1) CuSO4 in Locke's-ASW (1:1) within 24 h and extruded cystogenous material to produce an abnormal cyst wall. The 'emergency response' of encystment to high concentrations of copper reported for Parorchis acanthus cercariae did not occur in E. caproni. Concentrations of 1000 mg l(-1) and 100 mg l(-1) CuSO4 in Locke's-ASW (1:1) also killed the cercariae without encystment by 48 h. A concentration of 10 mg l(-1) CuSO4 in Locke's-ASW (1:1) allowed for normal in vitro encystment within 48 h and these cysts were capable of excystation in a trypsin-bile salts medium.