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.     

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.     

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.     

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.     

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).     

Effects of a protein-free diet on worm recovery, growth, and distribution of Echinostoma caproni in ICR mice.

The effects of a protein-free diet on the host-parasite relationship of Echinostoma caproni in ICR mice were studied. The experimental diet was a customized protein-free diet (PFD) in pellet form containing 0% protein. The control diet consisted of a standard laboratory diet containing 23% casein as a source of protein. A total of 24 mice were each infected with 15 metacercarial cysts of E. caproni. Twelve mice were placed on the experimental diet (experimentals) and the remaining mice (controls) were placed on the control diet. Experimental and control mice were necropsied at 2, 3, and 4 weeks postinfection (p.i.). The weight of mice on the PFD was markedly lower than that of mice on the control diet. The length and circumference of the small intestine of infected mice on the PFD were significantly lower than those of the controls at 3 weeks p.i. (Student's t-test; P < 0.05). Worm recoveries from mice on the PFD were significantly lower than those of the controls at 3 weeks p.i. There was a significant decline in worm body area in worms from the mice on the PFD compared with those on the control diet at 2, 3, and 4 weeks p.i. Worm dry weights from mice on the PFD were significantly lower than those on the control diet at 2 weeks p.i. Worms from hosts on the PFD were located more posteriad in the gut than those recovered from mice on the control diet. The findings suggest that the PFD contributes to growth retardation of E. caproni in ICR mice.     

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.     

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 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.     

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.     

Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode

The surface antigens, which induce a serum antibody response during infection of mice with the intestinal trematode Echinostoma caproni, were examined. It was demonstrated that antigens are shed from the surface of juvenile and 4-week old adult E. caproni during in vitro culture. SDS-PAGE and Western blot analysis of in vitro shed and detergent solubilized surface antigens indicated that the four major antigens released from the surface of adult parasites had molecular masses of approximately 26,000, 66,000, 75,000 and 88,000. A modified ELISA technique showed the in vitro turn-over rate of the surface antigens to be very high, with a half-life of 8-15 min in both juvenile and adult E. caproni trematodes. Transmission electron microscopy of the surface of adult parasites revealed a highly active secreting tegument which was densely packed with membrane-bound vesicles, reflecting the high rate of shedding of the surface antigens. An attempt to immunize mice with detergent solubilized adult surface antigens failed to induce resistance to infection with metacercariae of E. caproni.     

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.     

Concurrent infections of the trematode Echinostoma caproni and the tapeworms Hymenolepis diminuta and Hymenolepis microstoma in mice

Superimposing the intestinal tapeworm Hymenolepis diminuta on an established infection with the trematode Echinostoma caproni or simultaneous infection of mice with H. diminuta and Hymenolepis microstoma caused destrobilation and expulsion of H. diminuta, whereas establishment and growth of H. microstoma under the same infection regimes were not affected. In contrast, simultaneous superimposition of H. diminuta and H. microstoma on an established E. caproni infection caused destrobilation and expulsion of both H. diminuta and H. microstoma.     

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: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode

The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritin-conjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretion along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior.     

Development and pathology of Echinostoma caproni in experimentally infected mice

In the present article, several parasitological features of mice, each experimentally infected with 75 metacercariae of Echinostoma caproni (Trematoda: Echinostomatidae), were studied during the first 12 wk postinfection. Moreover, the early pathological responses also were analyzed and compared with data previously published on other host species of E. caproni to gain further insight into the factors determining worm rejection or establishment of chronic infections. The results obtained show that the pattern of E. caproni infection in mice is consistent with a highly compatible host-parasite system. This combination is characterized by a high worm establishment, high egg output, and long survival of the worms. However, some differences with respect to other highly compatible hosts have been observed, particularly in relation to the survival of the adult worms. Histological studies suggest that the kinetics of goblet cells, mucosal neutrophils, and mononuclear inflammatory cells in the mesentery seem to be essential in determining the course of E. caproni infection in mice.     

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.     

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.     

Th17 responses in Echinostoma caproni infections in hosts of high and low compatibility

In order to investigate the factors determining the expulsion of intestinal helminths, we have analyzed the in vivo expression of IL-17, TGF-β and IL-23 in several tissues of two host species displaying different compatibility with Echinostoma caproni (Trematoda). We did not observe upregulation of these cytokines in any of the tissues of the high compatible host (mice). In contrast, the responses in the host of low compatibility (rats) with the parasite were markedly different. Significant increases in the expression of IL-17 and TGF-β were observed in the Peyer’s patches and the intestine from the 2 to 8 weeks post-infection. The expression of IL-23 was upregulated from 2 to 4 weeks post-infection in the spleen, Peyer’s patches and the intestine. Considering together our results with those published previously the development of chronic infections appears to be related with the development of local Th1 responses, whereas the early rejection of the worms is mediated by the development a biased Th17/Th2 phenotype. The Th17 response generated in rats may facilitate the worm expulsion via the suppression of the inflammatory Th1 responses and the increase in intestinal contractility.