Use of an aceto-carmine procedure to examine the excysted metacercariae of Echinostoma caproni and E. trivolvis.

A simple aceto-carmine procedure was developed to relax, fix, stain, and clear excysted metacercariae of Echinostoma caproni and E. trivolvis. This procedure allowed for morphologic details on whole metacercariae comparable to those seen with more elaborate staining procedures. Differences in dimensions and staining intensities between the two species of metacercariae are described.     

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.     

Viability of the encysted metacercariae of Echinostoma caproni judged by light microscopy versus chemical excystation

The purpose of this study was to determine the viability of encysted metacercariae of Echinostoma caproni stored in Locke's solution 1:1 at 4 C for 1-24 wk. Viability was judged by light microscopy (LM) based on morphological characteristics of the encysted metacercariae versus chemical excystation of the cysts in a trypsin-bile salts excystation medium. The percent viability was very similar under both methods of assessment at 4, 8, and 16 wk poststorage. At 1 and 24 wk poststorage, viability was found to be about 2x greater based on excystation than using LM. We concluded that LM alone underestimated the viability of cysts and that determination of cyst viability was more accurate under assessment by chemical excystation.     

Effects of various physical and chemical factors on excystation of the encysted metacercariae of Echinostoma caproni

Various physical and chemical factors were studied to determine their effects on the viability of encysted metacercariae of Echinostoma caproni. Viability was equated with chemical excystation in an alkaline trypsin-bile salts (TB) medium. Control cysts showed excystation percentages of > 90% in TB. Excystation proved to be a more reliable criterion of cyst viability than observations by light microscopy. Isolated cysts and cysts left in the snail (in situ cysts) were studied. Generally, in situ cysts proved more resistant to various physical and chemical treatments than did isolated cysts. Cysts stored for 7 days at 28 C in a Locke's 1:1 solution showed 97% excystation, suggesting that cysts of this species would survive postal delays during shipment. Of numerous marinades tested, the one that was most harmful to isolated and in situ cysts was vinegar. Isolated and in situ cysts were killed by boiling (100 C) for 1 or 3 min, but freezing at -10 C did not kill all isolated or in situ cysts after 24 hr. Concentrations of potassium permanganate ranging from 300 to 1,200 mg/L killed most isolated cysts within 5 min, but in situ cysts survived these concentrations for 24 hr. Concentrated solutions of NaCl and sucrose had no effect on the viability of isolated and in situ cysts, suggesting that their use in food preparations for molluscs would not be effective in killing echinostomatid cysts in tainted snail tissues.     

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.     

Concurrent infections of Echinostoma caproni and Echinostoma trivolvis in ICR mice.

ICR female mice, 6- to 8-weeks old, were exposed concurrently to 25 metacercarial cysts of Echinostoma caproni and 25 metacercarial cysts of Echinostoma trivolvis and necropsied 10 and 14 days post-infection. Controls consisted of mice exposed singly to either 25 or 50 E. caproni or E. trivolvis cysts. All 23 mice exposed to E. caproni cysts were infected with a total of 331 worms (37.8%), whereas only 11 (37.9%) of 29 mice exposed to E. trivolvis cysts were infected with a total of 77 (6.4%) worms. In the concurrent infections, 13 (59.1%) of 22 mice were infected with both species and the percentage of worm recovery was 72.6% for E. caproni and 14.2% for E. trivolvis. There was no difference in worm distribution of either species in single vs concurrent infections. In concurrent infections at 14 days PI, there was a significant decrease in the body area of worms of both species, when compared to single worm species.     

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.     

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.     

The expulsion of Echinostoma trivolvis and retention of Echinostoma caproni in the ICR mouse: pathological effects

The infectivity and distribution of Echinostoma trivolvis were studied in female ICR mice each infected with 25 metacercarial cysts. At 7 and 10 days post-exposure worm recoveries were 58.8 and 58.4%, respectively. Worm recovery declined to 38.2% by day 14, to 6.4% by day 21, and 0% by day 28. The distribution of the parasites demonstrated an anteriad shift over time. Comparative histopathological studies were carried out on E. trivolvis and Echinostoma caproni in the mouse. Compared to control and E. trivolvis-infected intestine, mouse intestine infected with E. caproni showed marked dilation and villous atrophy. E. trivolvis-infected intestine showed a nearly two-fold increase in goblet cells compared to control intestine, whereas the intestine of E. caproni-infected mice showed almost complete goblet cell loss. Additionally, there was a marked increase in collagen in the intestinal musculature of the mice infected with E. trivolvis compared to control and E. caproni-infected mice.     

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.     

A comparison of Echinostoma caproni and Echinostoma trivolvis (Trematoda: Echinostomatidae) adults using isoelectrofocusing.

An isoenzymatic analysis using thin-layer agarose gel isoelectrofocusing on laboratory strains of Echinostoma trivolvis and Echinostoma caproni adults showed characteristic monomorphic phenotypes for phosphoglucomutase and glucose phosphate isomerase. The fixed allelic variation observed between these 2 taxa is consistent with their current classification as distinct species.     

Electrophoretic analysis of proteases in Echinostoma Caproni and Echinostoma trivolvis

Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.     

Excystation in vitro of Echinostoma liei and E. revolutum (Trematoda) metacercariae

Metacercariae of Echinostoma liei and E. revolutum were excysted in an alkaline bile-trypsin medium at 41 C in the absence of acid-pepsin pretreatment. After 60 min at a pH of 7.8 or 8.0, excystation of E. liei reached 98%; optimal excystation of E. revolutum occurred at pH 8.2 and was 70% after 60 min. The rate of excystation was very rapid in E. liei, reaching 91% at 30 min, and less rapid in E. revolutum reaching 40% at 30 min. Almost 100% of the E. liei cysts stored for 5.5 mo at 4 C in Locke's 1:1 excysted in the medium, compared to 40% for E. revolutum treated identically.     

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.     

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.     

Effects of host age on infection of ICR mice with Echinostoma caproni or E. trivolvis

There was no significant difference in the number of E. caproni recovered 21 days post-infection from 1-, 2-, 4-, 5-, 6-, 7-, 12-, or 21-month-old ICR mice infected with 25 metacercarial cysts. A range of 10.8-17.3 worms was recovered from mice in the age groups. In a similar experiment with E. trivolvis, there was no significant difference in worms recovered in young vs old mice. A range of 0-1.7 worms was recovered from mice in each age group. Contrary to a previous study on E. caproni in NMRI mice, our results indicate that host age does not affect establishment of E. caproni or E. trivolvis in the ICR mouse.     

The influence of Echinostoma caproni metacercariae (Trematoda) on the survival of biomphalaria molluscs (Pulmonata)

The infrapopulation of the Echinostoma caproni partenites has a development of prolong character (Ataev et al, 2005). However, in laboratory conditions, Biomphalaria molluscs infested with this parasite die within 1--3 weeks after the beginning of cercariae emission. It has been suggested that autoinvasion of the mollusc host with the cercariae, which use it as second intermediate host, is the cause of this phenomenon. Studying the dynamics of metacercariae accumulation in the host (both infected and non-infected with the Echinostoma caproni rediae) and experiments where quantity of cercariae around molluscs reduced by different ways, confirmed this hypothesis. Evidently, pathogenicity of metacercariae for molluscs is lesser in nature, because the concentration of cercariae reduces to the values, which do not result in lethal effect: some part of cercariae dies, but another part uses other animals as a host (Haas, 2000).     

The crowding effect and morphometric variability in Echinostoma caproni (Digenea: Echinostomatidae) from ICR mice

Population density, or crowding, was examined to determine its effect on the morphometric variability of Echinostoma caproni (Digenea) in ICR mice. Six mice were infected with 25 and 100 metacercariae, and a single mouse was infected with 300 metacercariae. All mice were infected at necropsy 22 days postinfection with recoveries of 77%, 69%, and 7.3%, respectively. Whole mounts were prepared, and 31 characters were evaluated (25 direct measurements and 6 ratios). Univariate and multivariate statistical analysis revealed significant differences between adult worms from all 3 groups. Twenty-seven of 31 characters showed significant within-group differences, with the primary differences between worms from 25/100 versus 300 metacercariae infections. Discriminant function analysis yielded a 100% correct classification based on infection size, which is consistent with studies on distinct species of Echinostoma. The low recovery from the mouse infected with 300 metacercariae suggests inflammatory expulsion of juvenile worms and the possibility of immunity as a factor in the crowding effect. These results suggest that external factors may affect morphometric variability of digenetic trematodes to a larger degree than previously recognized.     

Longevity of Echinostoma caproni in Balb/c mice

This study used Balb/c mice to examine the longevity of Echinostoma caproni. Five mice each exposed to 75 encysted metacercariae (cysts) were necropsied at 23 weeks postinfection (PI) (160 days PI). Two of the 5 were infected with a total of 33 worms; 23 in one mouse and 10 in the other. Body and organ area measurements showed that these worms were robust and normal in appearance. No signs of atrophy of any of the genital structures were observed. The mean +/- SE of eggs/uterus per worm (n = 10) was 243 +/- 6. This strain of mouse will be suitable to study the effect of long-term survival on the host-parasite relationship of E. caproni in Balb/c mice.     

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.