Studies on resistance in snails: Interference by nonirradiated echinostome larvae with natural resistance to Schistosoma mansoni in Biomphalaria glabrata

Most of the genetically selected juvenile Biomphalaria glabrata snails, normally strongly resistant to Schistosoma mansoni, lost their juvenile resistance to this parasite when other trematodes were concurrently present in the snail. Three echinostome species all were able to reduce this genetically controlled juvenile resistance: Echinostoma lindoense, E. paraensei, and e. liei. Subsequently, adult resistance to S. mansoni, clearly present in control snails of the same age and strain that were not doubly infected, failed to develop in most of the snails that also harbored echinostomes. Other snails, selected for resistance as adults to S. mansoni, also usually became susceptible to this parasite following infection with E. paraensei. The capacity of E. paraensei to interfere with the snails' resistance to S. mansoni was greater than that of E. lindoense. Destruction by predation of primary sporocysts of S. mansoni by echinostome rediae prevented completion of development of the S. mansoni infections. In a number of snails all primary S. mansoni sporocysts were consumed before secondary sporocysts could be formed. In most experimental snails, however, some of the schistosomes survived, often as a small number of degenerated secondary S. mansoni sporocysts. The capability of flukes to interfere with the natural defense of snails may be an important phenomenon whereby trematode species survive in their snail hosts.     

Acid phosphatase in granulocytic capsules formed in strains of Biomphalaria glabrata totally and partially resistant to Schistosoma mansoni

Cheng T. C. and Garrabrant T. A. 1977. Acid phosphatase in granulocytic capsules formed in strains of Biomphalaria glabrata totally and partially resistant to Schistosoma mansoni. International Journal for Parasitology7: 467–472. Acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase) has been demonstrated cytochemically in isolated granulocytes from the hemolymph of three strains of Biomphalaria glabrata. This enzyme was not detected in hyalinocytes. By employing acid phosphatase as a marker, it was determined that the cells comprising the capsule surrounding Schislosoma mansoni mother sporocysts in a totally and partially resistant strain of B. glabrata are granulocytes.The process of encapsulation of S. mansoni mother sporocysts in resistant B. glabrata was traced for 72 h post-penetration by miracidia and has been ascertained to involve two stages: (1) enlargement of the granuloma around intact sporocysts, followed by (2) disintegration of the parasite and a decrease in the size of the granuloma. There is an increase in the level of acid phosphatase activity within granulocytes comprising the granuloma during the second stage.Host cellular responses to S. mansoni mother sporocysts does not occur in susceptible snails.     

Schistosoma mansoni: Agglutination of sporocysts,and formation of gels on miracidia transforming in plasma of Biomphalaria glabrata

The resistance or susceptibility of Biomphalaria glabrata strains to strains of Schistosoma mansoni, the human blood fluke, are evidenced by the responses of snail hemocytes to sporocysts of the schistosome, both in vivo and in vitro. It is now reported that living sporocysts of the PR1 strain of S. mansoni agglutinate in the plasma of all tested strains of B. glabrata, in contrast to fixed sporocysts which agglutinate only in plasma from resistant snail strains. The agglutinating activity in resistant plasmas is not divalent cation dependent, and was not inhibited by the 26 carbohydrates and four amino acids tested. In addition, the observation that gelatinous deposits develop on transforming miracidia-sporocysts in B. glabrata plasmas is also reported. Both the agglutination and gel-formation phenomena may facilitate recognition of, and attacks on, sporocysts, thereby contributing to susceptibility and resistance in this host-parasite system.     

An ultrastructural study on ventricular encapsulation reactions in Biomphalaria glabrata exposed to irradiated echinostome parasites

Jeong K. H., Lie K. J. and Heyneman D. 1984. An ultrastructural study on ventricular encapsulation reactions in Biomphalaria glabrata exposed to irradiated echinostome parasites. International Journal for Parasitology14: 127–133. Encapsulation reactions around sporocysts of Echinostoma lindoense and E. paraensei have been studied in the ventricle of Biomphalaria glabrata using ultrastructural procedures. Amebocytes and ameboblasts migrate from the amebocyte-producing organ to the ventricle and there surround irradiated echinostome larvae. Specialized junction sites were found that linked amebocytes in the three phases of capsule formation, migratory, flattening and phagocytic. Active engulfing of portions of the tegument by amebocytes was followed by phagocytosis of parasite internal cells and formation in amebocytes of residual bodies of indigestible material. Regression of encapsulation reactions in the ventricle was frequently associated with formation of sear tissue. Comparisons made between types of encapsulation reactions observed around echinostomes and schistosomes in B. glabrata reveal differences in speed of response, number of cells involved and size of amebocyte pseudopodia.     

Infectivity of a microsporidium of mosquitoes (Nosema algerae) to larval stages of Schistosoma mansoni in Biomphalaria glabrata

Lai P. F. and Canning E. U. 1980. Infectivity of a microsporidium of mosquitoes (Nosema algerae) to larval stages of Schistosoma mansoni in Biomphalaria glabrata. International Journal for Parasitology10: 293–301. Nosema algerae derived from a closed colony of Anopheles stephensi was fed to Biomphalaria glabrata infected with Schistosoma mansoni. Mother and daughter sporocysts became hyperinfected but the snail tissues remained free of the microsporidia except for rare small aggregates of spores. These lay close to the sites occupied by mother or daughter sporocysts and were probably liberated from them. Irrespective of dose, fewer snails contained infected sporocysts when spores were given at 7 days post-miracidial infection than when given at 14 days. These periods corresponded respectively to stages when mother sporocysts only or daughter sporocysts as well were present in the snails. Infection of the sporocysts began in the tegumental cells, spread to the brood chamber and ultimately to the cercariae themselves. Heavily infected sporocysts contained fewer developing embryos. Doses of 10⁶ and 10⁷ spores/snail caused significant depression of cercaria output when given at 14 days but not at 7 days.     

Schistosoma mansoni: Cytotoxicity of hemocytes from susceptible snail hosts for sporocysts in plasma from resistant Biomphalaria glabrata

Sporocysts of Schistosoma mansoni (PR1 strain) survive and grow in Biomphalaria glabrata PR albino strain snails, whereas they are encapsulated and die in B. glabrata 10R2 strain snails. These processes also occur in an in vitro system in which the only living cells are those of sporocysts and snail hemolymph. Hemocytes of the susceptible snail are normally not effective in damaging sporocysts. However, when the encounter occurred in the presence of cell-free plasma from resistant snails, previously impotent hemocytes severely damaged sporocysts in 24 hr. The cytotoxic capacity of resistant strain hemocytes was not altered by plasma from susceptible snails. Furthermore, it was retained even when plasma was replaced by culture medium free of snail components. The nature of the plasma factor(s) which facilitated damage by otherwise impotent hemocytes is discussed, and evidence is evaluated for the hypothesis that snail resistance is dependent upon the specificity of cytophilic factors present both in the plasma and on the hemocyte plasma membranes.     

Resistance to schistosome infection in Biomphalaria glabrata induced by gamma radiation

Two strains of Biomphalaria glabrata were studied with respect to the effects of ionizing radiation on their susceptibility to Schistosoma mansoni infection. Gamma radiation at levels of 3.5 and 5 krad did not induce susceptibility in the resistant S-3 strain, but was found to initiate resistance in the susceptible PR-1 strain. In an attempt to understand the induced resistance in irradiated snails, histopathologic examinations and analyses of snail hemolymph were performed. Results indicated that miracidia invading irradiated snails were quickly surrounded and encapsulated by amoebocytes. Similarly, alterations in the hemolymph of irradiated snails suggested that radiation induced aging. It is suggested that radiation-altered snails may be of value in studying the defense mechanisms of these organisms.     

Acquired resistance in snails. Induction of resistance to Schistosoma mansoni in Biomphalaria glabrata

Acquired resistance to Schistosoma mansoni PR-I strain has been induced in Biomphalaria glabrata 442132 strain by infecting the snails with irradiated homologous miracidia. Present and previous results support the hypothesis that acquired resistance to trematodes in snails is an enhancement of the host's natural resistance to the parasite.     

Ribeiroia marini: Irradiated miracidia and induction of acquired resistance in Biomphalaria glabrata

Biomphalaria glabrata snails sensitized by exposure to X-irradiated miracidia of the trematode, Ribeiroia marini, acquired resistance to challenge with nonirradiated R. marini miracidia. Resistance was acquired within 1 day of sensitization; was strongest at 1 week, when infection rates of sensitized snails were 15% of the controls (i.e., $\text{S}\text{C}\text{= 0.15}$); and persisted for at least 3 weeks. By 30 days the difference between the infection rates of sensitized and control snails was no longer statistically significant. As in previous studies with echinostomes, acquired resistance to R. marini was characterized histologically by the destruction of irradiated sporocysts by host amoebocytes. Following destruction of all irradiated sporocysts, snails became resistant and encapsulated and destroyed nonirradiated challenge sporocysts within 1 day postchallenge. Associated with sporocyst destruction was an enlargement of the amoebocyte-producing organ, which showed intense mitotic activity. A proportion of the nonirradiated challenge sporocysts were also destroyed in most nonsensitized control snails, which consequently had a temporarily enlarged amoebocyte-producing organ. In contrast to acquired resistance reported to echinotomes, which is quite specific, acquired resistance to R. marini was associated with nonsusceptibility to both Echinostoma paraensei ($\text{S}\text{C}\text{= 0.19}$) and Schistosoma mansoni ($\text{S}\text{C}\text{= 0.81}$).     

Schistosoma mansoni and Biomphalaria glabrata share epitopes: antibodies to sporocysts bind host snail hemocytes

Using an independent protocol, we have confirmed that sporocysts of the human blood fluke, Schistosoma mansoni, synthesize antigens which stimulate rabbit antibody activity to epitopes on infermediate snail host hemocytes. This molecular mimicry may aid S. mansoni to escape the innate immune system of this host, Biomphalaria glabrata.     

Schistosome sporocyst-killing Amoebae isolated from Biomphalaria glabrata.

Explants and swabs from the pericardium and mantle of three strains of Biomphalaria glabrata, two of them resistant to infection with Schistosoma mansoni, have yielded small amoebae, 3–5μm in diameter, in culture. These amoebae have been grown axenically through > 50 passages to date. The amoebae form cysts in dense cultures. When mixed with S. mansoni mother sporocysts in vitro, the amoebae adhere to and kill the trematodes within several hours. For 1–2 days thereafter, the amoebae proliferate rapidly at a generation time of about 5 hr, then return to normal growth. Sonically disrupted sporocysts also induce proliferation. Live sporocysts do not attract the amoebae or emit soluble substances which influence amoebal growth. Amoebae also adhered to and killed S. mansoni daughter sporocysts and cells derived from B. glabrata embryos; however, they did not harm S. mansoni cercariae or rediae of other trematode species. The proportion of mantle explants yielding amoebae was significantly higher (P<0.05) in one of the resistant snail strains than in the susceptible strain; however, whether amoebae contribute to snail resistance is unknown. Exposure of snails to S. mansoni miracidia did not influence the proportion of snails yielding amoebae.     

Distribution and variation of hemagglutinating activity in the hemolymph of Biomphalaria glabrata

A sensitive hemagglutination assay utilizing glutaraldehyde-fixed trypsinized calf erythrocytes (GTC) is described to test for agglutinin levels in hemolymph and albumen gland extracts from nine populations of Biomphalaria glabrata, and from B. straminea and B. obstructa. High levels of GTC-reactive hemagglutinin were found in all snail populations. There was no correlation between hemagglutinin titer and innate resistance of B. glabrata strains to Schistosoma mansoni. However, an increase in hemagglutinin titer occurs in B. glabrata M-RLc snails infected with Echinostoma lindoense and in snails sensitized and reexposed to this parasite.     

Hemocytes of Biomphalaria glabrata: Factors affecting variability

Variability in the hemocyte number of two geographic strains of Biomphalaria glabrata was studied. In each strain a logarithmic increase in hemocyte number associated with increasing shell size was observed. A two fold increase in circulating hemocytes occurred 2 hr following the exposure of a susceptible strain of B. glabrata to miracidia of Schistosoma mansoni. The hemocyte number was dependent on the temperatures at which the snails were maintained.     

Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata

Lie K. J., Jeong K. H. and Heyneman D. 1980. Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata. Intemational Journal for Parasitology10: 183–188. More than 85% of echinostome-infected albino B. glabrata laboratory strain snails develop miracidia-immobilizing substance(s) (MIS) in the hemolymph, while less than 5% of control uninfected snails show this ability. Snails infected with Echinostoma lindoense show a strong miracidial immobilizing test (MIT) when homologous miracidia are exposed to the hemolymph and a moderate response when E. liei and Paryphostomum segregatum miracidia are used. Infection with E. paraensei results in a high level of hemolymph MIS with E. lindoense miracidia, a moderate one with P. segregatum miracidia, and a weak one when hemolymph is tested against E. liei as well as the homologous E. paraensei miracidia. Infection with E. liei induces a strong MIT with E. lindoense miracidia whereas only a moderate one was observed when using homologous or P. segregatum miracidia. Infection with P. segregatum gives a moderate MIT reaction to miracidia of the homologous species, as well as to E. lindoense and E. liei, and only a weak response to E. paraensei miracidia. Infection with S. mansoni fails to induce hemolymph that shows MIS to any of the parasites tested. Production of hemolymph MIS is temporary. It begins one day postexposure, reaches its maximum 10–14 days postexposure, and declines to the preinfection level several weeks later. Infection of snails with irradiated parasites also results in a temporary production of hemolymph MIS.Uninfected snails show a tissue-extract MIS, which is especially strong when digestive gland extracts are used. However, these snails give little or no evidence of a hemolymph MIS.     

Echinostoma liei miracidia and Biomphalariaglabrata snails: Effect of egg age,habitat heterogeneity,water quality and volume on infectivity

Infectivity of Echinostoma liei miracidia to NIH albino Biomphalaria glabrata declines significantly from 62% with eggs incubated for 10–24 days to 3% for eggs incubated for 30–42 days. In mass exposures of 25 snails to 125 miracidia in 1 liter of water infectivity was high (54–66 %) and not affected by the presence of lettuce, plastic sheets, chalk, detritus or snail-conditioned water. In distilled water or snail-conditioned water the proportion of infected snails exposed singly to five miracidia per snail in 5 ml was not significantly different from the results of mass exposures of 25 snails in 1 liter to the same snail: miracidia ratio. Some evidence is presented suggesting that infected snails are less likely to suffer mortality than uninfected snails during the first 7–10 days post-exposure.The results suggest that Echinostoma liei miracidial searching efficiency is robust in volumes of at least 1 liter and in a heterogeneous habitat. These aspects enhance the competitive potential of echinostomes as possible biological control agents for Schistosoma mansoni.     

Schistosoma mansoni: emergence of progeny-daughter sporocysts in monoxenic culture

Daughter sporocysts of Schistosoma mansoni released from mother sporocysts from Biomphalaria glabrata were cultured monoxenically for 21 days at 27.4 C in a medium consisting of a balanced salt-sugar solution supplemented with lactalbumin hydrolysate, inactivated fetal calf serum, serum Fraction A, and egg ultrafiltrate. The sporocysts were retained in transparent porous membranes so that their germinal development could be observed without interference from the underlying cells of Aedcs albopictus tissue cultures; the membrane permitted interchange of medium between sporocysts and cell cultures. The greatest increase in length, fivefold, occurred by 18 days. Progeny-daughters began to emerge at 9 days.The effects of pH, osmolality, and antibiotics were evaluated in axenizing solutions. Optimal conditions were a pH range of 6.5–7.6 and 113–153 mOsm. Antibiotics were not toxic.     

Effect of exposure to Echinostoma itliei miracidia on growth and survival of young Biomphalaria glabrata snails

Kuris A. M. 1980. Effect of exposure to Echinostoma liei miracidia on growth and survival of young Biomphalaria glabrata snails. International Journal for Parasitology10: 303–308. Exposure to miracidia of Echinostoma liei resulted in increased mortality and reduced growth of 1–2 mm albino Biomphalaria glabrata snails whether or not the snails became infected. Growth rates for infected and exposed but uninfected snails were significantly more variable than growth rates of unexposed snails. Retarded growth and increased mortality were detected as rapidly as seven to nine days after exposure. Neither growth nor survivorship of 4–6 mm snails was altered upon exposure to or infection by E. liei.     

Biomphalaria species in Alexandria water channels

Of the several species of Biomphalaria snails worldwide that serve as the intermediate host for Schistosoma mansoni, Biomphalaria alexandrina is a species that is indigenous to Egypt. Recently, there has been much debate concerning the presence of Biomphalaria glabrata and the hybrid of the species with Biomphalaria alexandrina. Due to this debate, the absence of a clear explanation for the presence of B. glabrata in Egyptian water channels and the probability that they may be reintroduced, we conducted this field study to identify Biomphalaria species present in Alexandria water channels. Laboratory-adapted susceptible snails to Schistosoma mansoni of the following species were used as a reference; Biomphalaria alexandrina, Biomphalaria glabrata and their hybrid. These snails were used to perpetuate the Schistosoma life cycle at the Theodor Bilharz Research Institute (TBRI), Cairo, Egypt. Morphological and molecular studies were conducted on these reference snails as well as on the first generation of Biomphalaria snails from two areas in the Alexandria governorate. The morphological study included both external shell morphology and internal anatomy of the renal ridge. The molecular study used a species-specific PCR technique.The results demonstrated that there was an absence of Biomphalaria glabrata and the hybrid from Alexandria water channels. Moreover, the susceptibility patterns of these reference snails were studied by measuring the different parasitological parameters. It was found that Biomphalaria glabrata and the hybrid were significantly more susceptible than Biomphalaria alexandrina to the Egyptian strain of Schistosoma mansoni. The results demonstrated that if Biomphalaria glabrata was reintroduced and adapted to the local environment in Egypt, it would have important epidemiologic impacts that would have a serious effect on the health of Egyptian people.