Echinostoma caproni: intestinal pathology in the golden hamster, a highly compatible host, and the Wistar rat, a less compatible host

The histopathological changes induced by Echinostoma caproni (Trematoda: Echinostomatidae) in a high (golden hamster) and a low compatible host (rat) were compared at 15 and 30 days post-infection. Infection of rats was characterized by a progressive increase in erosion of villi and elevated numbers of goblet cells, which could be related to the early expulsion of the parasite in a host of low compatibility. In contrast to rats, the number of goblet cell in E. caproni-infected hamsters was low, but increased numbers of neutrophils and mesenteric inflammatory cells were observed. This indicated that local inflammatory responses in hamsters were greater than in rats. An immunohistochemical study using polyclonal IgG anti-E. caproni excretory-secretory antigens demonstrated a greater level of passage of E. caproni antigens through the intestinal mucosa in hamsters than in rats, probably in relation to the greater inflammatory response. Our results indicate the fact that early inflammatory responses could be important for the establishment of E. caproni chronic infections in highly compatible hosts.     

Development of an antibody-based capture enzyme-linked immunosorbent assay for detecting Echinostoma caproni (Trematoda) in experimentally infected rats: kinetics of coproantigen excretion

The present study reports on the development of a coproantigen capture enzyme-linked immunosorbent assay (ELISA) for detecting Echinostoma caproni in experimentally infected rats. The capture ELISA was based on polyclonal rabbit antibodies that recognize excretory-secretory (ES) antigens. The detection limit of pure ES was 3 ng/ml in sample buffer and 60 ng/ml in fecal samples. The test was evaluated using a follow-up of 10 rats experimentally infected with 100 metacercariae of E. caproni, and the results were compared with those of other diagnostic methods such as parasitological examination and antibody titers determined by indirect ELISA. Coproantigens were detected in all the infected rats from the first day postinfection (DPI). The period of maximal coproantigen excretion was between 7 and 21 DPI. The values remained positive until 49-56 DPI, coinciding with the disappearance of the eggs in the stool samples of the infected rats. The kinetics of coproantigen detection were correlated with those of egg output. The present assay provides an alternative tool for the diagnosis of the echinostome infections. The proposed capture ELISA makes possible an earlier diagnosis than that provided by parasitological examination and indirect ELISA and also allows for the differentiation of past and current infections. Our results show that this assay can also be used to monitor the course of echinostome infections.     

Echinostoma caproni: kinetics of IgM, IgA and IgG subclasses in the serum and intestine of experimentally infected rats and mice

The kinetics of specific immunoglobulin M, A and IgG subclasses against Echinostoma caproni (Trematoda: Echinostomatidae) were analyzed in serum and intestinal fluid of two host species (Wistar rats and ICR mice) in which the course of the infection markedly differs. In rats, the worms were rapidly expelled, whereas E. caproni evokes in mice long-lasting infection. The pattern of antibody responses in both serum and intestinal samples was different in each host species. Serum responses in mice were characterized by significant increases of IgM, IgA, total IgG, IgG1 and IgG3, but not IgG2a. In contrast, serum responses in rats showed elevated levels of IgM, probably in relation to thymus-independent antigens, and slight increases of total IgG, IgG1 and IgG2a. At the intestinal level, increases of IgM and IgA levels were observed in mice. In regard to IgG subclasses, increases in both IgG1 and IgG2a were detected. Later decreases to normal values in IgG2a were also detected. In rats, only increases in total IgG and IgG2a were found. According to our results the development of long-lasting E. caproni infections in mice appears to be associated with a dominance of Th2 responses at the systemic level and balanced Th1/Th2 responses at the local level, characterized by initial increases in IgG1 and IgG2a levels. In contrast, the worm expulsion appears to be related to increases in local IgG2a levels.     

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.     

Kinetics of antibodies and antigens in serum of mice experimentally infected with Echinostoma caproni (Trematoda: Echinostomatidae)

The present study reports on the kinetics of antibodies and antigens in serum of mice experimentally infected with 75 metacercariae of Echinostoma caproni during the first 12 wk postinfection (wpi). Antibody titers in the serum of mice were determined by an indirect enzyme-linked immunosorbent assay (ELISA) using excretory/secretory (ES) antigens of E. caproni. The early detection of antibodies against ES antigens of E. caproni is feasible using indirect ELISA. Mice developed significant antibody responses at 2 wpi, and the values progressively increased until the end of the experiment. This may be related to the intestinal absorption of adult worm antigens that induces humoral responses. The presence of E. caproni circulating antigens was determined by a capture ELISA based on polyclonal rabbit antibodies against ES antigens of E. caproni. High levels of seroantigens in mice were detected by 1-2 wpi, probably because of the local inflammatory responses in mice induced by the adult worms. A drop in circulating antigen levels was observed at 9 wpi, which could reflect changes in the intestinal tissues over the course of the infection.     

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.     

A quantitative approach to the experimental transmission success of Echinostoma friedi (Trematoda: Echinostomatidae) in rats

Using a range of parameters, the ability of rats (Rattus norvegicus) to successfully transmit Echinostoma friedi to the next host was examined under experimental conditions. The concept of Experimental Transmission Success (TM), defined as the number of hosts that become successfully infected after exposure to a number of infective stages produced by a previous host per unit of inoculation at which this latter host was exposed, was introduced. Using data for the egg output and miracidium hatching and infectivity, the TM permits us to estimate the ability of a particular definitive host species to successfully transmit a parasite species. This concept may be also useful to compare the transmission fitness of a parasite in different definitive host species. Moreover, variations of the Experimental Transmission Success over the course of the infection were calculated by the use of the Weekly Experimental Transmission Success (TMW). Overall, considering the complete duration of the experiment, the TM of E. friedi using rats as definitive hosts was 0.68 infected snails/metacercaria. However, positive values of the TMW were only obtained from 2 to 4 wk post-infection, with a maximum during the third wk post-infection. When comparing the TM values of E. friedi in rats with those calculated in hamsters on the basis of previously published data, E. friedi appears to be more appropriate to move through this portion of its life cycle when using hamsters (Mesocricetus auratus) as the final host than rats.     

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.     

Interspecific antagonism and virulence in hosts exposed to two parasite species

Co-infection of host organisms by multiple parasite species has evolutionary consequences for all participants in the symbiosis. In this study, we co-exposed aquatic-snails (Biomphalaria glabrata) to two of their trematode parasites, Schistosoma mansoni and Echinostoma caproni. In co-exposed snails, E. caproni prevalence was 63% compared to only 23% for S. mansoni. Co-exposed E. caproni-infected snails exhibited reduced fecundity, higher mortality, and higher parasite reproduction (higher virulence) compared to hosts exposed to echinostomes alone. Conversely, co-exposed S. mansoni-infected snails released fewer parasites and produced greater numbers of eggs compared to hosts exposed to S. mansoni alone. These results suggest that co-exposure not only influences the establishment (presence or absence) of particular parasite species, but also impacts host life history, parasite reproduction, and the virulence of the interaction.     

Humoral and cellular response to infection with Echinostoma revolutum in the golden hamster, Mesocricetus auratus

Laboratory hamsters (Mesocricetus auratus) were infected with Echinostoma revolutum (Trematoda). Immunoelectrophoretic studies of hamster serum showed no demonstrable antibody response to E. revolutum. Histopathologic examination of intestinal tissue of infected hamsters showed erosion of intestinal villi and lymphocytic infiltration as the primary host response. Spleens from infected hamsters were hyperplastic during the first 3 weeks of infection and atrophic from 4 to 8 weeks postinfection. Hamsters were unable to acquire a resistance to E. revolutum infection. Lack of resistance was demonstrated in hamsters where the parasite infection was no longer detected based on the absence of eggs in the faeces; these hamsters were then reinfected. Hamsters treated with the anthelmintic oxyclozanide were also reinfected with E. revolutum.     

Production and characterization of monoclonal antibodies against excretory/secretory products of adult Echinococcus granulosus, and their application to coproantigen detection

Two IgM murine monoclonal antibodies (MAbs), EgC1 and EgC3, were produced against the excretory/secretory (E/S) products of Echinococcus granulosus adult worms. Immunoblotting revealed that both predominantly recognized a 50 kDa antigen in the somatic extract and an 85 kDa component in the E/S products. Immunolocalization showed that both MAbs reacted with the tegument of the parasite, and additionally EgC3 reacted with parenchyma and the tegument lining the external surface of the reproductive organs. A coproantigen capture ELISA was developed using a rabbit polyclonal antibody against E/S products from adult tapeworms as catching antibodies, and each one of MAbs as detecting antibody. The assays detected seven out of eight (EgC1), and eight out of eight (EgC3) experimentally infected dogs (worm burdens ranging from 61 to 57,500), using heat-treated samples obtained at prepatent period, and none (n=8) of helminth-free samples. Time course analysis showed that, after a 12-25 days lag, coproantigen levels rose above cut off O.D. values and typically peaked around 30 days post-infection (DPI) at the end of the experiment. One dog experimentally infected with Taenia hydatigena metacestodes was slightly detected as positive at different time points after 30 DPI. Both MAbs showed a similar pattern of recognition, but T. hydatigena antigens were undetectable for a longer period, and reached lower O.D. values with EgC1. Interestingly, fecal samples from two experimentally infected dogs with Echinococcus multilocularis were not recognized by the EgC1 assay, suggesting a potential value as species-specific diagnostic tool.     

The role of light and gravity in the experimental transmission of Echinostoma caproni (Digenea: Echinostomatidae) cercariae to the second intermediate host, Biomphalaria glabrata (Gastropoda: Pulmonata)

Trematode cercariae inhabit predictable environments and respond to trigger cues with genetically fixed releaser responses when foraging for the upstream host. The effect of light and gravity on the transmission of Echinostoma caproni cercariae to Biomphalaria glabrata was investigated experimentally. Transmission chambers were constructed of clear polyvinyl chloride pipe. Snails were constrained within the chamber to prevent movement, while permitting the cercariae to swim freely. A trial consisted of 2 infected B. glabrata shedding E. caproni cercariae placed at the center of the chamber, with 5 uninfected B. glabrata placed 10 cm on either side (or above and below) of the shedding snails as sentinels. There was no significant difference in the prevalence of infection sentinel snails in either experiment (light vs. dark or top vs. bottom); however, mean intensity was significantly higher in sentinel snails in the dark portion of the chamber (42.5 vs. 10.4; P = 0.001) and the top of the transmission chamber (66.1 vs. 38.0; P = 0.0003). There was a high correlation between the number of metacercariae collected from sentinel snails and the total number of infective units (metacercariae + unsuccessful cercariae): r = 0.992 (light vs. dark) and r = 0.957 (top vs. bottom), respectively, at cercariae densities estimated from 22 to 3,304/L. The results suggest that cercariae of E. caproni exhibit negative photo- and geotaxis in searching for a second intermediate host. Stereotypical releaser responses to environmental trigger cues (light and gravity) allow E. caproni cercariae to exploit flexible strategies for completing the life cycle consistent with the broad range second intermediate and definitive hosts used by E. caproni cercariae and adults, respectively.     

Screening trematodes for novel intervention targets: a proteomic and immunological comparison of Schistosoma haematobium, Schistosoma bovis and Echinostoma caproni

With the current paucity of vaccine targets for parasitic diseases, particularly those in childhood, the aim of this study was to compare protein expression and immune cross-reactivity between the trematodes Schistosoma haematobium, S. bovis and Echinostoma caproni in the hope of identifying novel intervention targets. Native adult parasite proteins were separated by 2-dimensional gel electrophoresis and identified through electrospray ionisation tandem mass spectrometry to produce a reference gel. Proteins from differential gel electrophoresis analyses of the three parasite proteomes were compared and screened against sera from hamsters infected with S. haematobium and E. caproni following 2-dimensional Western blotting. Differential protein expression between the three species was observed with circa 5% of proteins from S. haematobium showing expression up-regulation compared to the other two species. There was 91% similarity between the proteomes of the two Schistosoma species and 81% and 78·6% similarity between S. haematobium and S. bovis versus E. caproni, respectively. Although there were some common cross-species antigens, species-species targets were revealed which, despite evolutionary homology, could be due to phenotypic plasticity arising from different host-parasite relationships. Nevertheless, this approach helps to identify novel intervention targets which could be used as broad-spectrum candidates for future use in human and veterinary vaccines.     

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.     

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.     

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.     

Echinostoma luisreyi n. sp. (Platyhelminthes: Digenea) by light and scanning electron microscopy

The entire life cycle of the Brazilian Echinostoma luisreyi n. sp., of the 37-collar spine E. revolutum group, has been observed under experimental conditions. The snail Physa marmorata serves as its first natural intermediate host. This species and the planorbid Biomplalaria glabrata act as experimental second intermediate hosts. The worm recovery rate was 51.3 and 0.6% for mice and hamsters, respectively, but the infection did not develop in the quail Coturnix coturnix. The natural vertebrate host is not known. The most important morphological character of the new species separating it from the other Echinostoma species studied is the oral corner spines that increase in size from the latero-oral to the ventro-oral regions. The ratio of the sizes of the smaller oral spines and the larger aboral ones was 1.7:1.0. Also, the excretory pore is radially wrinkled and dorsally subterminal. Images obtained by scanning electron microscopy confirmed the characters that differentiate the new species and the most closely related species.     

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.     

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.     

Germinal elements and their development in Echinostoma caproni and Echinostoma paraensei (Trematoda) miracidia.

Among the large cells located in the posterior of Echinostoma caproni and E. paraensei miracidia are secretory cells, germinal cells (GC), and undifferentiated cells. Secretory cells do not give rise to progeny, whereas GC do. Undifferentiated cells develop into GC that can also divide to produce embryos. Cleavage of GC of E. caproni occurs only after the parasite has entered the snail host and develops into a sporocyst. With E. paraensei, GC are larger than noted for E. caproni, and in 3 of 23 miracidia examined, germinal cell cleavage had occurred in the miracidium such that an embryo containing 20-25 blastomeres was present. Observations on the germinal elements of miracidia help to explain previous results showing that (1) E. paraensei sporocysts release mother rediae a few days earlier than do sporocysts of E. caproni, and that (2) a single mother redia is produced ahead of all others by sporocysts of E. paraensei, but not by sporocysts of E. caproni. The present study adds support to the concept that E. caproni and E. paraensei are distinct species.