Intestinal Trematode Echinostoma Research Articles

Secreted cathepsin L-like peptidases are involved in the degradation of trapped antibodies on the surface of Echinostoma caproni.

Antibody trapping is a recently described strategy for immune evasion observed in the intestinal trematode Echinostoma caproni, which may aid to avoiding the host humoral response, thus facilitating parasite survival in the presence of high levels of local-specific antibodies. Parasite-derived peptidases carry out the degradation of trapped antibodies, being essential for this mechanism. Herein, we show that cathepsin-like cysteine endopeptidases are active in the excretory/secretory products (ESPs) of E. caproni and play an important role in the context of antibody trapping. Cysteine endopeptidase activity was detected in the ESPs of E. caproni adults. The affinity probe DCG-04 distinguished a cysteine peptidase band in ESPs, which was specifically recognized by an anti-cathepsin L heterologous antibody. The same antibody localized this protein in the gut and syncytial tegument of adult worms. Studies with cultured parasites showed that in vivo-bound antibodies are removed from the parasite surface in the absence of peptidase inhibitors, while addition of cathepsin L inhibitor prevented their degradation. These results indicate that cathepsin L-like peptidases are involved in the degradation of surface-trapped antibodies and suggest that cysteine peptidases are not only crucial for tissue-invading trematodes, but they can be equally relevant at the parasite-host interface in gut-dwelling flukes.

Resistance against Echinostoma caproni (Trematoda) secondary infections in mice is not dependent on the ileal protein production

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, which has been widely employed to investigate the factors determining the rejection of intestinal helminths. Protein production patterns of intestinal epithelial cells are related to the infection-induced changes that determine the course of E. caproni infections. Herein, we compare the protein production profiles in the ileum of four experimental groups of mice: control; infected; dewormed and reinfected. Worm burdens were significantly lower in secondary infections, confirming the generation of partial resistance to homologous secondary infections in mice. However, quantitative comparison by 2D-DIGE showed that the protein production profile is similar in control and dewormed mice, and after primary and secondary E. caproni infections. These results showed that, unexpectedly, protein production changes in E. caproni infections are not responsible of resistance development. Fifty-one protein spots were differentially produced between control/treated and infected/reinfected mice and 37 of them were identified by mass spectrometry. The analysis of differentially abundant proteins indicate that cell metabolism and the regulation of proliferation and cell death are the most affected processes after primary and secondary E. caproni infections. These results provide new insights into the proteins involved in the regulation of tissue homeostasis after intestinal infection. SignificanceIntestinal helminthiases are highly prevalent parasitic infections with about 1 billion people infected worldwide. In this scenario, better understanding of host-parasite relationships is needed to elucidate the factors that determine intestinal helminth rejection. The intestinal trematode Echinostoma caproni has been broadly employed in this field, with resistance against secondary homologous infections reported in mice. In this paper, new insights are provided in the regulation of tissue homeostasis after intestinal infection. The unexpected lack of an altered pattern of ileal protein production associated to resistance development suggests that this resistance depends on rapid changes, affecting the early establishment of worms, rather than the activation of later effector mechanisms. These results may contribute to the development of new control tools for the management of these parasitic infections.

Differential alterations in the small intestine epithelial cell turnover during acute and chronic infection with Echinostoma caproni (Trematoda).

BackgroundThe intestinal epithelium plays a multifactorial role in mucosal defense. In this sense, augmented epithelial cell turnover appears as a potential effector mechanism for the rejection of intestinal-dwelling helminths.MethodsA BrdU pulse-chase experiment was conducted to investigate the infection-induced alterations on epithelial cell kinetics in hosts of high (mouse) and low (rat) compatibility with the intestinal trematode Echinostoma caproni.ResultsHigh levels of crypt-cell proliferation and tissue hyperplasia were observed in the ileum of infected mice, coinciding with the establishment of chronic infections. In contrast, the cell migration rate was about two times higher in the ileum of infected rats compared with controls, with no changes in tissue structure, indicating that an accelerated cell turnover is associated with worm expulsion.ConclusionOur results indicate that E. caproni infection induces a rapid renewal of the intestinal epithelium in the low compatible host that may impair the establishment of proper, stable host-parasite interactions, facilitating worm clearance.

Biochemical and histological responses of Rattus novergicus (Wistar) infected by Echinostoma paraensei (Trematoda: Echinostomatidae)

Tests were performed to evaluate the biochemical alterations in Rattus norvegicus after infection by the intestinal trematode Echinostoma paraensei. The rodents received 150 metacercariae each, serum samples were collected and the parasite load was quantified weekly until the fifth week of infection. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), gamma-glutamyl transferase (GGT), bilirubin, glucose, total proteins and fractions and hepatic glycogen were determined. All the animals exposed to the metacercariae were infected in the first week and worms were recovered up to the third week after infection. The levels of AST, ALT, GGT, bilirubin and globulin rose in the first and/or second week and declined thereafter to levels near those of the control group. In contrast, the level of total proteins in the plasma fell significantly in the first week while the ALKP activity went down only in the fourth and fifth weeks in relation to the control group. There was no significant difference in the levels of albumin, glycogen and glucose. Infection by E. paraensei in R. norvegicus causes changes in the hepatic function, possibly resulting from the cholestasis produced by the partial obstruction of the bile duct by the helminths.

Counter-regulatory anti-parasite cytokine responses during concurrent Plasmodium yoelii and intestinal helminth infections in mice

Malaria and helminth infections are two of the most prevalent parasitic diseases globally. While concomitant infection is common, mechanisms contributing to altered disease outcomes during co-infection remain poorly defined. We have previously reported exacerbation of normally non-lethal Plasmodium yoelii malaria in BALB/c mice chronically infected with the intestinal trematode Echinostoma caproni. The goal of the present studies was to determine the effect of helminth infection on IFN-γ and other key cytokines during malaria co-infection in the P. yoelii – E. caproni and P. yoelii – Heligmosomoides polygyrus model systems. Polyclonally stimulated spleen cells from both E. caproni - and H. polygyrus -infected mice produced significantly lower amounts of IFN-γ during P. yoelii co-infection than malaria-only infected mice. Furthermore, the magnitude of IFN-γ suppression was correlated with the relative amounts of IL-4 induced by these helminths ( E. caproni = low; H. polygyrus = high), but not IL-10. Concurrent malaria infection also suppressed helminth-associated IL-4 responses, indicating that immunologic counter-regulation occurs during co-infection with malaria and intestinal helminths.

Effects of diet on the lipid composition of Echinostoma caproni (Trematoda) in ICR mice.

High-performance thin-layer chromatography (HPTLC) was used to determine neutral lipids and phospholipids in the intestinal trematode Echinostoma caproni from experimentally infected ICR mice fed a high-fat diet (hen's egg yolk) as compared with worms from mice fed a standard laboratory diet. Worms were removed from the hosts at 2, 3, and 4 weeks postinfection (p.i.). Analysis by TLC-densitometry showed significantly greater amounts of triacylglycerols and free sterols at 2, 3, and 4 weeks p.i. in worms from mice on the high-fat diet as compared with worms from mice on the standard laboratory diet. Significantly greater amounts of phosphatidylcholine and phosphatidylethanolamine were found in worms from mice on the high-fat diet as compared with worms from those on the standard diet at 2 weeks p.i. but not at 3 and 4 weeks p.i. The results of this study suggest that the host diet influences the lipid content of E. caproni adults.

Zinc deficiency and host response to helminth infection: Echinostoma caproni infections in CBA mice.

The effect of zinc deficiency on the response of CBA mice to infection with the intestinal trematode Echinostoma caproni was examined. Young CBA mice were allocated to one of three dietary groups: a group fed a zinc deficient diet ad libitum, a control group pair fed a zinc sufficient diet and a control group fed a zinc sufficient diet ad libitum. The mice on the zinc deficient diet gained significantly less weight than the pair fed controls. In primary infections with six E. caproni metacercariae followed over a period of 128 days, zinc deficiency delayed worm expulsion. In addition, zinc deficiency resulted in a prolonged IgM response, a delayed IgG response and an increased IgA response towards the end of the experiment. Resistance to challenge infection day 21 following a primary infection with 25 E. caproni metacercariae was slightly, but not significantly, affected by zinc deficiency.

Development, hatching, and infectivity of Echinostoma caproni (Trematoda) eggs, and histologic and histochemical observations on the miracidia.

Light microscopy studies were done on the eggs and miracidia of the intestinal trematode Echinostoma caproni. Eggs were obtained by homogenizing adult worms derived from ICR mice in artificial spring water (ASW). To study egg development and to obtain miracidia, eggs were incubated in ASW in either a light or a dark environment for 10 days at 28 degrees C. Egg development was characterized and staged at 2-day intervals from day 0 to day 10 postembryonation. No significant difference in development was seen in eggs maintained under conditions of light or darkness. Eggs maintained in darkness for 10 days and exposed to incandescent light produced a large synchronous hatch of miracidia within 3 h of exposure to light. As expected, miracidia used to expose Biomphalaria glabrata snails produced patent infections at 5-7 weeks postinfection (p.i.). Embryonated eggs aged from 0 to 10 days that had been fed to B. glabrata snails also produced infections within 4 weeks of infection. Miracidial longevity studies showed that 50 newly hatched miracidia maintained at either 4 degrees, 12 degrees, 22 degrees, or 38 degrees C lived for 6, 28, 14, and 5 h, respectively. Histology studies were done on whole miracidia stained in 0.01% neutral red and in Schneider's acetocarmine. Histochemistry studies of miracidia stained with alcian blue (pH 2.5) showed the presence of acid mucopolysaccharides in the epidermal plates; miracidia treated with periodic acid-Schiff plus 0.5% malt diastase showed the presence of glycogen in the body and in the epidermal/subepidermal region. Miracidia stained with Lillie's Oil Red O showed the presence of sparse neutral fat droplets in the body.

The antibody response in serum, intestinal wall and intestinal lumen of NMRI mice infected with Echinostoma caproni

Groups of mice (NMRI) were infected with 0, 6 and 25 metacercariae of the intestinal trematode Echinostoma caproni. An enzyme linked immunosorbent assay (ELISA) was developed for measuring the specific IgM, IgG and IgA antibody responses in serum, small intestinal tissue and small intestinal lumen of the mice, by using a crude adult E. caproni antigen. In infected mice, significant levels of IgM were measured in the sera from day 14 after infection and of IgG and IgA from day 28 after infection. Early in the infection, the mean level of serum IgM was higher in 6-worm infections than in 25-worm infections. Late in the infection, higher mean levels of IgA were reached in the serum of mice with high than in those with low dose infections. The onset of appearance of antibodies in the tissue of the small intestine reflected the picture seen in the serum. For IgM, and to a lesser degree for IgG, the highest mean antibody levels appeared in the posterior sections of the small intestinal wall, where also the parasites were located. The mean level of IgA, however, was uniform throughout the length of the small intestinal tissue. High levels of specific IgA were detected in the lumen of the small intestine on day 28 after infection, especially in the anterior sections, where only few parasites were located. No specific IgM or IgG could be detected in the intestinal lumen on this day. The results are related to the intestinal location and the pattern of expulsion of E. caproni in the mouse host.

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 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.

Negative effect on early post-implantation pregnancy and progesterone levels in mice infected with the intestinal trematodeEchinostoma caproni

Infection in mice (BALB/cABom) with the intestinal trematode Echinostoma caproni had a negative influence on pregnancy. The effect of the infection set in immediately following implantation (day 5 of pregnancy) resulting in fewer foetuses being present in infected mice on day 9 than in the controls. Ovulation, fertilization of eggs, and implantation itself were obviously not impaired. The infected mice had significantly lower serum progesterone levels on day 5 of pregnancy than the non-infected controls. It is speculated that the progesterone levels in the infected female mice were too low to secure early post-implantation gestation.

Echinostoma caproni in rats: worm population dynamics and host blood eosinophilia during primary infections with 6, 25 and 50 metacercariae and resistance to secondary and superimposed infections

Hooded-Lister rats were inoculated with 6, 25, 50 or 100 metacercariae of the intestinal trematode Echinostoma caproni. Worm establishment and the pattern of egg excretion were followed during the course of primary infections with 6, 25 and 50 metacercariae. Peripheral blood eosinophilia was followed at all infection levels. After 1 month, worm recovery and faecal egg output showed a gradual decline with increasing duration of infection. High worm burdens were expelled later than smaller worm burdens, and egg output persisted longer in animals exhibiting a high initial egg output. The level of blood eosinophilia increased with increasing degree of infection and with the level of egg output. A marked concomitant resistance to superimposed infection was observed on the challenge of rats harbouring 21- and 49-day-old infections with 50 metacercariae. In addition, rats were partially resistant to secondary infection at challenge day 14 following anthelmintic removal of primary 7-day-old infections with 50 metacercariae and were completely resistant at challenge day 7 following elimination of a primary 14-day-old primary infection.

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

Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode. International Journal for Parasitology 19: 111–118. 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. SDSPAGE 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.

Echinostoma revolutum in mice; dynamics of the antibody attack to the surface of an intestinal trematode

Echinostoma revolutum in mice: Dynamics of the antibody attack to the surface of an intestinal trematode. International Journal for Parasitology 16: 475–482. Two weeks after infection of mice with the intestinal trematode Echinostoma revolutum, sera became positive for antibodies (IgG, IgM, IgA) to the surface of in vitro excysted juveniles, as tested by IFAT, coinciding with development of immunity to reinfection. Juveniles also bound C3 when incubated in normal serum in vitro. Antibody- or C3-covered juveniles were attacked by cells in an in vitro peritoneal-cell-adherence test, but antibodies, C3 or cells had no effect on the survival of juveniles in vitro after 24 h. In vivo excysted juveniles, recovered from the intestine of immune mice 2 h after reinfection with metacercariae, were covered with IgG and IgA, but IgM, C3 or cells were not observed on their surface. A high degree of specificity of the E. revolutum surface antibodies was observed, with no cross reactions to schistosomulae of Schistosoma mansoni or vice versa. IFAT with sections of 1–4-week-old flukes incubated in immune serum indicated, that antibody also bound to the surface of older flukes. Juveniles completely lost the surface-bound antibodies within 5 h of being transferred to medium, but reexposure to immune serum made them positive again. It is suggested, that shedding of surface-bound antibodies may be an adaptation of these flukes to withstand the hosts immune attack.