Effects of a 100 metacercarial cyst inoculum on the host-parasite relationship of Echinostoma caproni and ICR mice

The host-parasite relationship of a 100 metacercarial cyst inoculum of Echinostoma caproni in the ICR mouse was examined. Three groups of mice, A, B and C, each with six mice per group were used and all mice were necropsied at 14 days postinfection (p.i.), at which time the worms were ovigerous. Group A consisted of uninfected controls, whereas group B received 25 cysts per mouse (low dose) and group C received 100 cysts per mouse (high dose). There was no significant difference in food consumption between any of the groups from 0 to 14 days p.i. Control mice increased their body weight by 12%, group B by 5%, and group C showed a less than 1% increase in body weight between 0 and 14 days p.i. Echinostome parasitism caused a significant increase in the diameter of the mouse gut, with the gut of group C being more significantly dilated than that of either group A or B. The average worm recovery from group B was 20 worms per host, compared to 72 worms per host from group C. The mean wet and dry weights per worm from group B were 2.4 and 0.4 mg, respectively as compared to 0.6 and 0.2 mg respectively for group C. The mean number of uterine eggs per worm from group B was 180 compared to 125 for worms from group C. Worms from group C were more widely distributed in the small intestine than those from group B. Crowding effects associated with the high dose infection were clearly demonstrated in E. caproni from ICR mice.     

Single and multiple worm infections of Echinostoma caproni (Trematoda) in the golden hamster

Six of 10 hamsters fed a single metacercarial cyst of Echinostoma caproni (single-worm infections) and 13 of 19 hamsters fed either 2 or 5 cysts (multiple-worm infections) were infected with adult echinostomes at necropsy 22 days post-infection. Considerable histopathological changes to the small intestine occurred in hamsters carrying single-worm infections. There were no differences in either mean length, width or wet weight of echinostomes in single- versus multiple-worm infections. The mean number of eggs/worm from single-worm infections (525) was significantly greater than that from multiple-worm infections (288). The average percentage of fully developed miracidia/worm from single worms (94%) was similar to that from worms in multiple infections (92-95%). Single worms of E. caproni were capable of self-fertilization and production of viable eggs. Miracidia derived from single worms were as capable of infecting laboratory-reared Biomphalaria glabrata and producing patent rediae as were those from multiple infections.     

Diurnal migration of Echinostoma caproni (Digenea: Echinostomatidae) in ICR mice

Twenty-four female ICR mice, 12 acclimated to a 12 ∶ 12 light-dark cycle and 12 to a 12 ∶ 12 dark-light cycle for 7 days, were each infected with 10 metacercariae of Echinostoma caproni. Infected mice were maintained on their respective lighting regimes for 28 days. Six mice (3 from each group) were necropsied at 4-hr intervals beginning at 0700 hr. The small intestine was removed, opened, and the position of individual worms and worm clusters was measured to the nearest 0.1 cm. Each intestine was subsequently divided into 20 equal segments and individual worms and worm clusters were assigned to the appropriate segment based on the original measurements. All worms were found in the posterior 55% of the intestine (ileum). All posterior segments (10-20), with the exception of segment 18, harbored at least 1 worm at some time. A Monte Carlo simulation of worm abundance in segments 10-17 over all time periods indicated a random distribution, while the same analysis of segments 10-20 indicated a non-random distribution due to large numbers of worms in segment 20 and to the absence of worms in segment 18. To analyze temporal changes in worm distribution, mice were grouped by time of necropsy as follows: night (1900 and 2300 hr), morning (0300 and 0700 hr), and day (1100 and 1500 hr). During the night and morning, E. caproni was heavily concentrated in segments 10-17 and, during the day, worms were located more posteriorly, with a heavy concentration in the last segment (20).     

The effects of crowding on adults of Echinostoma revolutum (Digenea: Echinostomatidae) in experimentally infected golden hamsters, Mesocricetus auratus

All 30 female golden hamsters, Mesocricetus auratus, fed either 125 +/- 50 (group A), 300 +/- 50 (group B), or 500 +/- 50 (group C) metacercarial cysts of Echinostoma revolutum were infected 7-35 days postexposure. The mean number of worms in A, B, and C were 62, 96, and 212, respectively. Most of the worms in A were in the jejunum, but in C worms were about equally distributed in the duodenum, jejunum, and ileum, and some were in the cecum. The body area and wet and dry weights of worms from C were significantly less than that of A or B at 2, 4, and 5 wk postinfection. Echinostoma revolutum eggs were in the feces of 100% of the hamsters by days 12, 13, and 14 in A, B, and C, respectively.     

Survival and distribution of Echinostoma caproni in the small intestine of ICR mice up to 36 hours after the death of the host

This study examined survival and distribution of Echinostoma caproni in the small intestine of ICR mice at various times up to 36 hr following the death of the host. Adult worms were obtained at 2-wk postinfection of 21 ICR mice each infected with 50 metacercarial cysts. Mice were killed with light ether anesthetization and cervical dislocation and maintained at room temperature (22 +/- 1C) until examination at 0 (controls), 2, 4, 6, 12, 24, and 36 hr postmortem. Survival was based on worm activity and distribution was assessed on the basis of worm location in 1 of 5 equal intestinal segments numbered from the pylorus to the ileocecal valve. Worms were alive up to 36 hr post-mortem and were distributed mainly in segments 3 and 4 at all times postmortem. Histochemical Oil Red O studies on whole control and experimental worms showed neutral lipids localized in the protone-phridial tubules and the excretory bladder. Eggs from experimental worms at all times produced miracidia that infected Biomphlaria glabrata snails.     

Age of adult worms of Echinostoma caproni does not affect development of miracidia

The effect of the age of adult Echinostoma caproni on egg development was studied. The percentage of fully developed miracidia was determined in eggs derived from adult worms obtained from laboratory mice at 2, 4, 6, and 8 wk postinfection (PI). Regardless of the age of worms from which the eggs were obtained, the percentage of fully developed miracidia was always >90%, and 60-80% of the eggs hatched. Several previous studies have shown that eggs derived from 2- to 4-wk-old E. caproni yielded miracidia that infected Biomphalaria glabrata snails. Results of the present study on E. caproni were in marked contrast to previous results with Echinostoma friedi, for which viable eggs were not obtained at 2 and 3 wk PI and maximal infectivity of miracidia in snails was obtained from eggs derived from worms collected at 8 and 9 wk PI. Further studies are needed to determine if the egg viability of other species in the "revolutum" group follow that of E. caproni or E. friedi.     

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 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.     

Infectivity, growth, and distribution of Echinostoma caproni (Trematoda) in the ICR mouse

Host-parasite interactions of the intestinal trematode Echinostoma caproni were studied in ICR laboratory mice. All of 40 mice, each fed 25 metacercarial cysts of Echinostoma caproni, were infected 1-20 wk postinfection (PI) with a mean of 17.2 worms/host. At 24 and 29 wk PI only 2 of 6 mice (33%) were infected, with a mean of 4.2 worms/host. Mean body area of worms increased rapidly to about 5 mm2 by week 2, increased less rapidly to 8.8 mm2 by week 12, plateaued until week 24, and then declined. Mean dry weight of worms increased rapidly to about 0.5 mg by week 2, less rapidly to 1.4 mg by week 12, and then plateaued until week 24 PI. From 1 to 8 wk PI most worms localized in the jejunum and ileum; later most worms were in the jejunum and duodenum. Considerable differences were seen in the growth and distribution of E. caproni in the ICR mouse compared with previous studies on this echinostome species in the NMRI mouse.     

Echinostoma caproni in mice: studies on the attachment site of an intestinal trematode

During infection in mice Echinostoma caproni is attached to the mucosa of the small intestine with the ventral sucker (acetabulum). The morphology, histology and dynamics of attachment sites from primary infections were examined. The sites were highly characteristic microscopically, and consisted of a plug of grasped mucosa occupying the cavity of the ventral suckers. The mucosa in the area of the small intestine where the parasite resided showed marked villous atrophy and crypt hyperplasia. The cellular composition of the attachment sites did not appear significantly different from what was seen in other parts of the mucosa in the residential area, and the study did not reveal any specific cellular host response at the attachment site. After mechanical removal of the parasites from unfixed tissue in saline, the attachment sites gradually reduced in size and disappeared. It is suggested that the attachment sites are only temporary structures, formed by the mechanical grasp of the ventral sucker as the parasites move about in the residential area of the intestine.     

Single- and five-worm infections of Echinostoma caproni (Trematoda) in the ICR mouse

Twenty-nine (64%) of 44 ICR mice fed a single metacercarial cyst of Echinostoma caproni and all of 23 mice each fed five cysts were infected with ovigerous worms at necropsy 2-4 weeks post-infection. Each host fed five cysts had two to five worms at necropsy, and all worms were either paired or clustered. Distribution of worms in the small intestine was similar in single- and five-worm infections and all worms were located 17-20 cm anterior to the ileo-cecal valve. Both single and multiple worms produced eggs with fully-developed miracidia. The number of eggs per uterus in 2-week-old multiple worms was almost twice that of single worms. The body area of 3- and 4-week-old multiple worms was significantly greater than that of single worms of the same age.     

Kinetics of Echinostoma caproni (Trematoda: Echinostomatidae) antigens in feces and serum of experimentally infected hamsters and rats

This study reports on the kinetics of antibody production to Echinostoma caproni and the dynamics of antigens in feces and sera in 2 experimental hosts (hamsters and rats) that display different degrees of susceptibility with this echinostome. Echinostoma caproni produced chronic infections in hamsters, whereas rats lost the infection at 49-56 days postinfection (DPI). Hamsters developed higher antibody responses than rats, probably in relation to different intestinal absorptions of worm antigens in each host species. The levels of coproantigens were indicative of the course of infection in each host. Positive coproantigen levels were detected at 1-2 DPI in both hosts, and the values remained positive until the end of the experiment in hamsters; in rats, the coproantigen levels reverted to negative values, coinciding with the loss of infection. High levels of circulating antigens were detected in hamsters from 21 DPI to the end of the study. In contrast, low levels of E. caproni seroantigens were detected in rats only. These observations may reflect the differences in local inflammatory responses induced by E. caproni in each host species.     

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.     

Effects of a diet deficient in the B complex vitamins on infectivity, growth and distribution of Echinostoma caproni in ICR mice

The effects of a diet deficient in the B vitamins on infectivity, growth, and distribution of Echinostoma caproni in ICR mice were studied. The vitamin-deficient diet (experimental) was isocaloric to the control diet but lacked the B vitamins. Thirty-six female, 6- to 8-week-old ICR mice were each infected with 25 metacercarial cysts. From the day of infection to the day of necropsy, 18 mice were fed the experimental diet and the remaining mice received the control diet. Equal numbers of experimental and control mice were necropsied at 2, 3 and 4 weeks postinfection (p.i.). Mice on the experimental diet showed a significant loss in body weight between 2 and 4 weeks p.i. There was no significant difference in worm recovery at 2 to 4 weeks p.i. from mice on either diet. Worms from hosts on the experimental diet were more dispersed and located more posteriad in the small intestine than those from mice on the control diet. Worm dry weight was significantly less in hosts on the experimental diet at all weeks p.i. compared with that of hosts on the control diet. The body area of worms on the experimental diet was significantly less at 2 and 3 weeks p.i. than that of worms on the control diet. An isocaloric diet deficient in the B vitamins had a detrimental effect on the growth of E. caproni in ICR mice.     

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.     

Excystation of the encysted metacercariae of Echinostoma trivolvis and Echinostoma caproni in a trypsin-bile salts-cysteine medium and morphometric analysis of the excysted larvae

A trypsin-bile salts-cysteine (TBC) medium was used to excyst the encysted metacercariae of Echinostoma caproni and Echinostoma trivolvis, 2 allopatric species of Echinostoma. This medium was used to replace a previously used trypsin-bile salts (TB) medium that was no longer effective because of the unavailability of the original stocks of trypsin. The TBC medium maintained at 41 C allowed for 68.6% excystation of E. caproni at 1 hr and 57.5% excystation of E. trivolvis at 2 hr. The cysteine reductant in the TBC medium was necessary; if it was omitted, excystation was nil. Morphometric analysis was done on the excysted metacercariae following fixation of the larvae in hot, 5% neutral buffered formalin and mounting them on slides in glycerin jelly. Body and organ measurements were made on these larvae. The diameter of the acetabulum of E. caproni was significantly greater than that of E. trivolvis. Likewise, the number and diameter of the excretory concretions found in E. caproni were significantly larger than those of E. trivolvis. Morphometric analysis can be used to distinguish structural differences in closely related allopatric species of Echinostoma.     

Antibodies in the serum of golden hamsters experimentally infected with the intestinal trematode Echinostoma caproni.

The serum antibody response in golden hamsters (Mesocricetus auratus) infected with the intestinal trematode Echinostoma caproni was examined with ELISA, SDS-PAGE and Western blot, and IFAT techniques. All methods showed that the hamsters responded slowly but developed a clear positive humoral response to the infection. In most hamsters, an antibody response to infection could not be detected earlier than 11-13 weeks after infection with 6 or 25 metacercariae, and responses were weak when compared to previous results from mice infected with the same parasite. IFAT with positive hamster sera on live juvenile E. caproni showed only fluorescence at the posterior tip, which is a different pattern from that seen using from infected mice, indicating a different response to antigens on the juvenile parasites by these two hosts. The results are discussed in relation to the limited selfcure and development of resistance which is observed in golden hamsters infected with E. caproni.     

Infectivity, growth, and development of Echinostoma revolutum (Digenea: Echinostomatidae) in the golden hamster, Mesocricetus auratus

All 30 female golden hamsters, Mesocricetus auratus, each fed 100 +/- 25 metacercarial cysts of Echinostoma revolutum were found to be infected 2 to 105 days post-infection with 3 to 103 (avg. 38) flukes in the small intestine. Worm wet weights averaged 0.6 mg at 9 days, 3.5 mg at 14 days, and 19 mg at 42 days; average dry weights for the identical days were 0.2, 1.2 and 5.8 mg, respectively. The average body length of worms fixed in hot (80 C) alcohol-formalin-acetic acid was 0.33 mm on day 2, 5.11 mm on day 10, 9.30 mm on day 42 and 8.56 mm on day 105. Body and gonadal area increased rapidly from about day 5 to 15 and then less rapidly. Eggs of E. revolutum were seen in the feces of 10% of the hamsters by day 9, 89% by day 10 and 100% by day 11. Eggs teased from worms, embryonated in tap water, and produced miracidia which infected lab-reared Helisoma trivolvis.     

Echinostoma caproni: mating behavior and the timing of development and movement of reproductive cells

The development and movement of reproductive cells were determined in Echinsostoma caproni on autoradiograms by labeling nuclei of stem cells during exposure to [3H]thymidine and then transplanting the worms to mice for various times. The development and movement of sperm, primary oocytes, and vitelline cells were much more rapid in E. caproni than other digenetic trematodes investigated previously. Mating behavior was determined by labeling the sperm of 1 adult by in vitro exposure to [3H]tyrosine and transplanting alone or with unlabeled worms to mice for 4 and 6 days. Echinostoma caproni adults self-inseminated when isolated and self- and cross-inseminated when in groups. This behavior is similar to that found for the frog rectal fluke Megalodiscus temperatus but unlike that determined for eyeflukes in the genus Philophthalmus. Worm size was not a barrier to insemination in E. caproni. Cross-insemination could not be detected in 6-day transplants probably because of dilution or elimination of radioactive sperm due to rapid turnover or frequent sperm transfer. An increased number of structural anomalies was noted in the transplanted worms. The most common anomaly was an accumulation of vitelline cells in the vitelline reservoir and ducts.     

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.