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.     

The invasion of Biomphalaria pfeifferi by Schistosoma mansoni miracidia and the development of daughter sporocysts

Laboratory experiments have been carried out to determine the susceptibility of Gezira Biomphalaria pfeifferi snails to S. mansoni miracidia and the relationship between miracidia and daughter sporocyst production at the 10–17 day development stage. The relationship between snail numbers, miracidia numbers and water volume has also been studied. Two non susceptible snails, Bulinus truncatus and Cleopatra bulimoides, both of which occur naturally in Gezira canals, were tested to see if they act as decoys for S. mansoni miracidia.The results showed that the B. pfeifferi are 100% susceptible to S. mansoni invasion, at least to the daughter sporocyst development stage. The more miracidia that penetrated the more daughter sporocysts were produced, however individual variation and overlap were great. When one miracidium was released to find one snail it succeeded in low water volumes (5 m, 50 ml), but failed in 5 litres. When 100 miracidia were released mortality of snails was high suggesting superinfection particularly when only one or five snails were available. Among survivors daughter sporocyst counts were very high. Cleopatra and Bulinus snails do have a decoy effect when present in large numbers. In their presence the number of infected snails was marginally reduced and the number of daughter sporocysts greatly reduced. However, if superinfection is reduced by decoy effect, it is conceivable that Biomphalaria may be protected by decoy snails in circumstances where miracidia counts are high.     

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 ($\text{S}\text{C}\text{= 0.81}$).     

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.     

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.     

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.     

Alterations in total serum proteins and protein fractions in Biomphalaria glabrata parasitized by Schistosoma mansoni

Samples of serum from healthy Biomphalaria glabrata and from those infected with the larval stages of Schistosoma mansoni were subjected to quantitative electrophoretic analysis and total protein determinations. In the case of the infected snails, samples of serum were taken at 14 time intervals post-exposure to 5 miracidia ranging from 1 hr to 70 days.A total of 34 serum protein fractions have been identified in the hemolymph of uninfacted B. glabrata, although all these fractions usually do not occur in every snail. In infected snails, there is a gradual reduction in the staining intensity of the serum protein fractions until day 35 post-exposure to miracidia, and this is correlated with a reduction in the total serum protein concentration. By day 70 post-exposure to the parasite, the total protein concentration had declined to one-third of that in uninfected snails.Although the decrease in the amount of serum protein fractions as a result of parasitization by S. mansoni is generally nonselective; i.e., almost all the fractions are reduced, three fractions, 7, 9, and 10, do not appear to be reduced in quantity. These remain essentially unaltered in their intensity to staining for up to 70 days, the duration of this experiment.It is postulated that the reduction of serum protein is due to utilization by sporocysts of S. mansoni.     

Field studies on the bionomics of the free-living stages of St. Lucian Schistosoma mansoni.

Densities of Schistosoma mansoni miracidia and cercariae in natural habitats in three St. Lucian valleys were monitored over a 3-year period by exposure of sentinel snails, Biomphalaria glabrata, and a cercariometric technique, supplemented by sampling of field snails. Separate measures for control of S. mansoni transmission were under evaluation in two of the valleys. Sentinel snails became infected sporadically and their infection rates per valley ranged from 0·12% to 4·99%. S. mansoni miracidial inoculation rates ranged from 1 to over 4 per infected sentinel snail. Combined rainfall of more than 3 in on the day before any day of sentinel snail exposure interfered with miracidium-snail interaction. Densities of S. mansoni cercariae ranged from 0·05 to 21 per litre of water sampled. The number of cercariae detected in a habitat by cercariometry was directly proportional to the number of infected field snails. Sentinel snail infection rates exhibited a downward and an upward trend, respectively, in the controlled and uncontrolled areas, although the changes were not significant statistically.     

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.     

Purification and binding properties of hemagglutinin from Biomphalaria glabrata.

A hemagglutinin has been purified from Biomphalaria glabrata (PR-B) hemolymph, albumin glands, and egg masses using affinity chromatography with Sephadex gels. The purified material from any of the sources above demonstrated identical immunological properties during immunoelectrophoresis or immunodiffusion, and similar serological specificity for human A₁ erythrocytes and to a lesser extent A₂ erythrocytes. Hemagglutinin was able to bind in vitro to the tegumental surface of cultured Schistosoma mansoni sporocysts, cercariae, and miracidia. Sporocysts dissected from infected snails and shed cercariae were already found to have hemagglutinin on their tegumental surface as demonstrated by immunofluorescence. It is postulated that hemagglutinin binding to the surface of larval helminths may “mask” them from being recognized by the snail host's cellular defense system.     

14C uptake by Schistosoma mansoni from Biomphalaria glabrata exposed to 14C-glucose

Exposure of Biomphalaria glabrata infected with Schistosoma mansoni to ¹⁴C-glucose results in a greater uptake of original total snail label by the parasitized digestive gland-gonad, site of the developing daughter sporocysts and cercariae, than by the digestive gland-gonad of control animals. As a consequence of this greater uptake by the infected digestive gland-gonad, the albumen gland and remainder of the carcass of parasitized snails receive less label than do those areas in normal snails. Emergence of cercariae from the snail and daughter sporocyst mass account for a diversion of 12.6% of original total label from the infected snail itself. This diversion of label from the snail to the parasite may explain carbohydrate depletion in parasitized snails.     

Ribeiroia marini: pathogenesis and larval trematode antagonism in the snail, Biomphalaria glabrata

Larval trematode antagonism between Ribeiroia marini and Schistosoma mansoni was studied in the snail Biomphalaria glabrata. A laboratory-raised Puerto Rican strain of B. glabrata was exposed to single and double infections with given numbers of: (1) embryonated eggs of R. marini from laboratory rats, and (2) miracidia of S. mansoni from mice. Snails were maintained in outside environmental tanks in San Juan, Puerto Rico and larval trematode interactions were examined in a series of five experiments. Snails of all sizes were highly susceptible to single infections with R. marini. Rediae and cercariae caused extensive damage to the digestive gland and ovotestis resulting in premature death of snails. Heavily infected snails were castrated and stopped laying eggs. Snails infected first with S. mansoni were only partly susceptible to superinfection with R. marini given on Day 23. In a reverse experiment, snails infected first with R. marini were only partly susceptible to a second infection with S. mansoni given on Day 23. In simultaneous exposures, snails developed double infections (22%) with R. marini dominant and S. mansoni sporocyst and cercaria production reduced. While R. marini is not a strong direct antagonist against established S. mansoni infections, it has several attributes as a possible biological control agent: hardy eggs easily produced in rats; high infectivity to snails of all ages; and ability to castrate and prematurely kill B. glabrata. The R. marini-rat system described here provides a convenient laboratory and field model for the study of intrasnail trematode antagonism and biological control.     

Tissue responses exhibited by Biomphalaria alexandrina snails from different Egyptian localities following Schistosoma mansoni exposure

Snails’ susceptibilities to infection with Schistosoma mansoni were determined through observation of infection rates, total cercarial production and tissue responses of the first generation (F1) of Biomphalaria alexandrina snails, originally collected from different Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) and responses were compared between groups. The emergence of cercariae for a 3-month period and the calculation of survival and infection rates, in control (Schistosome Biological Supply Center; SBSC) and infected snails were evaluated. SBSC and Giza snails showed greater susceptibilities to infection and lower mortality rates. In addition, at 6 and 72 h post-exposure to miracidia all the snail groups showed no difference in the anatomical locations of sporocysts. The larvae were found in the head-foot, the mantle collar and the tentacles of the snails. Sporocysts showed normal development with low tissue reactions in SBSC and Giza snail groups infected with S. mansoni miracidia (SBSC). However, in Fayoum, Kafr El-Sheikh, Ismailia and Damietta snail groups, variable tissue responses were observed in which numerous hemocytes made direct contact with S. mansoni larvae forming capsules. The results suggested that, different responses of B. alexandrina snail’s hemocytes towards S. mansoni are related to the degree of susceptibility of these snails. So this is important in planning the strategy of schistosomiasis control.     

Intermediate host specificity in Schistosoma mansoni

Miracidia of Schistosoma mansoni penetrate into many kinds of snails, but development of normal sporocysts takes place only in certain species of Biomphalaria. Different populations of this snail vary greatly in laboratory infection rates with S. mansoni originating from diverse geographic localities. Cross-exposure experiments show that compatibility factors exist in both snails and parasites. Susceptibility of stocks of Biomphalaria to particular strains of S. mansoni is genetically determined and may be modified by selection in the laboratory. In a compatible snail, the sporocyst develops without host tissue reaction; in incompatible snails the early larvae are rapidly surrounded by amebocytes and fibroblasts, and destroyed. This reaction resembles the generalized host cellular response elicited by any foreign body. An individual snail exposed to many miracidia may have both developing and encapsulated sporocysts side by side within its tissues. The weight of current evidence suggests that elicitation or absence of this cellular response resides in the recognition or nonrecognition of the sporocyst as a foreign body. The sporocyst tegument surface, which forms within a few hours after miracidial penetration, may have a molecular conformation identical with that of the snail, or may be able to bind specific host molecules, so that detection and subsequent encapsulation by host cells are averted. Presuming genetic determination of the sporocyst surface structure and of the host cell detection capability, differing infection rates would result from the particular frequencies of relevant genes in the populations concerned.     

Effects of UVB on interactions between Schistosoma mansoni and Biomphalaria glabrata

Ultraviolet B (UVB, 280-315 nm) radiation is detrimental to both of larvae of the digenetic trematode Schistosoma mansoni and its snail intermediate host, Biomphalaria glabrata. We explored effects of UVB on three aspects of the interaction between host and parasite: survival of infected snails, innate susceptibility and resistance of snails to infection, and acquired resistance induced by irradiated miracidia. Snails infected for 1 week showed significantly lower survival than uninfected snails following irradiation with a range of UVB intensities. In contrast to known immunomodulatory effects in vertebrates, an effect of UVB on susceptibility or resistance of snails to infection could not be conclusively demonstrated. Finally, exposure of susceptible snails to UVB-irradiated miracidia failed to induce resistance to a subsequent challenge with nonirradiated miracidia, a result similar to that reported previously with ionizing radiation.     

Physiologic studies of snail-schistosome interactions and potential for improvement of in vitro culture of schistosomes

Summary Despite extensive efforts to develop suitable media for rearing the intramolluscan stages of schistosomes, successful in vitro culture of these parasites remains elusive. Recent³¹P NMR studies demonstrated that the levels of free phospholipids, particularly phosphatidylcholine, in the digestive gland of the snail,Biomphalaria glabrata, were dramatically reduced when the host was infected withSchistosoma mansoni. It was speculated that absorption of host phosphatides may be an important source of membrane phospholipid precursors and fatty acids for developing sporocysts and cercariae. During the present investigations,B. glabrata was maintained on a high fat diet of egg yolk, and the lipid composition of control uninfected and infected snails examined by³¹P and¹³C NMR. In addition, the levels of host hemolymph metabolites, including glucose and urea, considered as indicators of parasite nutrient uptake, were monitored. The lipid level of snails fed egg yolk was greatly increased, and hosts developed patent infections in approximately half the time of infected snails maintained on lettuce. The composition of the free phospholipids accumulated in the tissues ofB. glabrata fed egg yolk were the same as those previously reported in the cercarial stage ofS. mansoni. Moreover, the fatty acids ofS. mansoni and those reported here in the neutral lipids and free phosphatides in the host tissues were similar. Uninfected snails maintained on lettuce had higher hemolymph levels of glucose than those reared on egg yolk, and infected hosts on egg yolk had significantly lower levels of hemolymph urea. β-hydroxybutyrate was the principal hemolymph metabolite in snails fed egg yolk, but was not detected in snails maintained on lettuce. The level of β-hydroxybutyrate in the hemolymph of snails on egg yolk was significantly reduced by infection. The results indicated that the pattern of host hemolymph nutrient utilization by larval schistosomes may be markedly altered by host diet, and it was concluded that host lipids may directly and indirectly be important nutrients for developing schistosomes. Future studies on in vitro culture of the intramolluscan stages of schistosomes should emphasize the potential role of lipids and attempt to define the nutritive value of those medium components that presently supply lipids in culture media, most notably serum.     

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.     

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.     

Schistosoma mansoni, NIH-Sm-PR-2 strain, in non-susceptible Biomphalaria glabrata: protection by Echinostoma paraensei

Sullivan J. T., Richards C. S., Lie K. J. and Heyneman D. 1981. Schistosoma mansoni, NIH-Sm-PR-2 strain, in non-susceptible Biomphalaria glabrata: Protection by Echinostoma paraensei. International journal for Parasitology11:481–484. Among seven inbred genetic stocks of Biomphalaria glabrata that are non-susceptible for the NIH-Sm-PR-2 strain of Schistosoma mansoni (PR-2), five stocks revert to nearly complete susceptibility when first infected with Echinostoma paraensei. These include both stocks in which PR-2 sporocysts are normally destroyed within 3–7 days, and stocks in which sporocysts often survive undeveloped for at least 3 weeks. Hence, these five stocks are resistant to but physiologically suitable for the development of PR-2. Of the two remaining stocks, one remains partly non-susceptible to PR-2, since less than 50 % of echinostome-infected snails revert to susceptibility, while the other stock remains completely non-susceptible to PR-2 following echinostome infection, due perhaps to a high level of residual resistance and/or unsuitability.