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.     

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.     

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.     

Chemosensitivity of Philophthalmus megalurus (Trematoda) miracidia

Miracidia of the eyefluke Philophthalmus megalurus were tested in phi-chambers to determine if they reacted similarly to chemicals found stimulative for miracidia of Philophthalmus gralli, a closely related species. Philophthalmus megalurus miracidia were less responsive than P. gralli to the dicarboxylic amino acids and showed a significantly positive response only to 10 mM glutamic acid. These P. megalurus miracidia were chemonegative to 10 mM HCl and H2SO4, chemicals to which P. gralli miracidia gave a significantly positive response. Ammonia and Mg2+ did not elicit any response from P. megalurus miracidia.     

Echinostoma caproni: identification of enolase in excretory/secretory products, molecular cloning, and functional expression

In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a theoretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specifically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule.     

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.     

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.     

Reaction of Philophthalmus rhionica (Trematoda, Philophthalmidae miracidia to light

The light response of miracidia was studied by means of several original methodics. The heterogeneity in the character of light response between various larvae of the same age population was determined in the course of experiments. It was shown that a part of miracidia possesses the strict positive phototaxis. Moreover, in their movement to the light the larvae seem to orientate themselves on the light intensity gradient. Not all the miracidia possess the positive phototaxis ("+" miracidia). Some of them ("-" miracidia) leave for the dark side of camera under the influence of direct ray of light. There are also larvae indifferent to the light conditions in thepopulation. They can move in different directions in spite of the light ray direction ("0" miracidia). The number of "+", "-" and "0" miracidia in the population is inconstant. The number of the negative phototactic larvae grows with age and respectively the number of "+" miracidia decreases. It is obvious the "+" miracidia can transform into "0" or "-" forms, while there is no opposite transformation. The reasons of differences in the movement character of "+" and "-" miracidia are under discussion.     

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.     

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.     

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.     

Chemical stimulation of Schistosoma mansoni miracidial activity.

The movements of S. mansoni miracidia immersed in various solutions of organic chemicals were recorded by dark ground photography. Reduction of miracidial speed was recorded for miracidia in solutions of fatty acids and ammonia. The speed reduction was concentration dependant and pH dependant, indicating in both cases that the un-ionized molecule was responsible. Ammonia at concentrations between 0.4 mM and 0.8 mM elicited an increased "rate of change of direction" by miracidia. No changes in miracidial movements were recorded for miracidia immersed in aqueous solutions of amino acids, sugars or of molluscan nitrogenous excretion products other than ammonia. A possible role for chemo-klinokinetic behaviour patterns in miracidial host location was formulated.     

The organization of germinal material and dynamics of mother sporocyst reproduction in the genus Echinostoma (Trematoda: Echinostomatidae)

The reproduction in the first parthenogenetic generation--mother sporocyst (MS) in two species of echinostomes (E. caproni, E. paraensei) is investigated. A group of densely packed cells, which noticeably differ from others, occupies the posterior part of the miracidium. They are characterized by large sizes and a large bubble-shaped nucleus with heterogeneous nucleolus and strong dispersed chromatin. The use of histological and electron microscopic methods has shown that with observed similarity these cells are classified in two tyoes and have a completely different origin. First of all, large secretory cells stand out. In E. caproni miracidia their number averages 6.8 +/- 0.2 and linear sizes is 10-12 microns. Secretory cells possess a large bubble-shaped nucleus. The caryoplasm looks optically empty because of strong dispersion of chromatin. A large nucleolus occupies a bit eccentric position. Eosinophilic cytoplasm contains poorly noticeable at light-optical level accumulation of small granules. The second group of cells is represented by typical germinal cells (GC). The number of GC does not exceed six. Their polymorphy is well above that of secretory cells. The sizes of GC vary from 5.4 to 9 microns. The largest cells (8.1-9 microns) occupy the front position and usually are located between secretory cells. Intensively basophilic cytoplasm surrounds bubble-shaped nucleus with a large nucleolus like border with uniforming thickness. The heterochromatin is evenly distributed over the caryoplasm. Its content of nuclei is more than that in nuclei of secretory cells. That is why they do not look optically empty. So, it is "mature" germinal cells. Four or five cells are located directly behind "mature" cells. Their sizes are gradually decrease towards the posterior of the miracidium (the diameter of the smallest cells reaches 5.4 microns). Nuclei with a centrally located nucleolus are characterized by larger amount and more condensation of the heterochromatin than those in "mature" cells. Meanwhile, they concern to nuclei of bubble-shaped type. In general, all cells of second type represent the primary germinal cells distinguished by the stage of their differentiation. Also, 2-3 undifferentiated cells occupy the most posterior part of the miracidium. Their sizes average 5.55 +/- 0.18 microns. The nucleus contains a lot of densely packed heterochromatin. On parasitic phase of MS development undifferentiated cells give rise to secondary GC. Electron microscopic data in details confirm the situation described above. The essentially similar results were received during the investigation of E. Paraensei miracidia. The differences are observed in parameterical characteristics of germinal material and in small variability of the extent of germinal material development. With E. paraensei, germinal material may be represented by not only GC and undifferentiated cells, but one germ as well. So, our investigation has shown that germinal material of echinostomes represents typical germinal mass. The germinal material condition does not change on parasitic phase of E. caproni MS development during the first day of post infection (PI). The activization of germinal material coincides in the time with the beginning of schizocoel formation in 2 Days PI. On the 3rd day of PI, the proliferation of undifferentiated cells begins and the first germs are free to float in enormous schizocoel. After 8 days of PI, MS release the first rediae. During the following 2-3 days the other rediae formed by primary GC left MS. The release of rediae derived from secondary GC was observed later. So, E. caproni MS give rise to 12-16 rediae which is much less than the number of GC formed in MS. The earlier release of the first E. paraensei rediae by MS is predetermined by the difference in the structure of germinal mass in E. paraensei miracidia. Therefore, Echinostomatidae is intermediate between two groups of trematodes. The first group has MS that completely realize reproductive function in the time of miracidial formation; but the second group includes higher trematodes characterized by the transfer of reproductive time on a parasitic phase of MS development. The question concerning to so-called "pedogenetic larvae of trematodes" is discussed.     

Host reactions in Biomphalaria glabrata to Schistosoma mansoni miracidia, involving variations in parasite strains, numbers and sequence of exposures

M-line Biomphalaria glabrata snails are susceptible to Puerto Rican (PR-1) strain of Schistosoma mansoni, but are resistant to a St. Lucian (LC-1) strain. 10-R2 B. glabrata snails are resistant to both strains of S. mansoni. When 10-R2 snails were exposed repeatedly to PR-1 S. mansoni miracidia for 5 consecutive days, all of the sporocysts were encapsulated and destroyed by the snails. Thirty-four per cent of sporocysts examined in M-line snails with similar exposures were also degraded. In double concurrent infections of M-line B. glabrata with [³H]leucine-labeled and unlabeled PR-1 and Lc-1 S. mansoni, the incompatible Lc-1 miracidia were selectively attacked and destroyed. This destruction occurred irrespective of the sequence of exposure of the 2 strains of miracidia, and whether or not the miracidia were labeled. Successful superinfection of M-line B. glabrata with homologous S. mansoni miracidia was obtained at least 4 days after the primary exposure to the miracidia.     

The responses of Philophthalmus gralli and P. megalurus miracidia to light, gravity and magnetic fields

Miracida of an eyefluke of birds, Philophthalmus gralli, which are positively geotactic, exhibited a positive north-seeking magnetotaxis when subjected to magnetic field strengths from 3 × 10^?4 to 2 × 10^?2 T. A closely-related species, P. megalurus, which is positively geotactic only in complete darkness, exhibited no magnetotaxis under similar conditions. A positive phototactic response overrode the north-seeking magnetotaxis when P. gralli miracidia were subjected to both stimuli in a competing system. No detector mechanisms for magnetic fields are known in miracidia of digenetic trematodes. The order of responsiveness for P. gralli miracidia then is: geotaxis > phototaxis > magnetotaxis.     

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.     

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.     

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.     

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.     

Snail odour-clouds: spreading and contribution to the transmission success of Trichobilharzia ocellata (Trematoda, Digenea) miracidia

Chemical communication among freshwater organisms is an adaptation to improve their coexistence. Here,we focus on the chemical cues secreted by the freshwater gastropod Lymnaea stagnalis, which are known to stimulate behavioural responses of Trichobilharzia ocellata (Plathelminthes, Digenea, Trematoda) miracidia. Such responses are commonly claimed to influence transmission positively, but in response to chemical cues miracidia randomly change their swimming direction. This kind of response does not necessarily increase transmission, because miracidia may be trapped at the periphery of very large snail odour-clouds, which may prevent them from approaching the snail. On the other hand, the odour clouds may be too small to improve host-localisation. To shed light on these scenarios, the spreading of molecules released around L. stagnalis (active space) was visualised by recording host-finding responses of T. ocellata miracidia when they approached snails. Behavioural responses of miracidia indicated the spreading of compounds forming an attractive active space only around the host-snail L. stagnalis, but not around sympatric non-host-snail species. The active space increased approximately linearly with the time the snail rested at the same spot and within 5 min it reached a volume of more than 30 times that of the snail. We also demonstrated in a large-scale experiment, that the active space of L. stagnalis significantly increases the transmission success of T. ocellata miracidia. Additionally, the microhabitat selection of T. ocellata miracidia was studied, demonstrating that peripheral locations near the water surface were preferred, which are also preferred sites of L. stagnalis. Improved chemoperception and microhabitat selection may have been a consequence of coevolution with snails and benefited miracidia, which became efficient transmissive stages. Electronic Supplementary Material Supplementary material is available for this article at