The susceptibility of Lymnaea fuscus to experimental infection with Fasciola hepatica

Three experiments on the infection of Lymnaea fuscus with Fasciola hepatica were carried out to determine if successful infections and maturation of the parasite were dependent on the size of snails at miracidial exposure. The first experiment was performed using 1-4-mm-high snails from 2 populations of L. fuscus and 1 population of Lymnaea palustris. In these snails each subjected to a single bimiracidial exposure, the prevalence of F. hepatica infection at day 35 postexposure ranged from 20.3% to 46.2% in snails measuring 1 mm in height at exposure; it was lower in the 2-mm snails and was 0 in higher size classes. The second experiment was performed by subjecting 1- and 4-mm L. fuscus to 1, 2, and 3 bimiracidial exposures. The prevalence of F. hepatica infection at day 35 postexposure was maximum in the 1-mm snails exposed once to miracidia and decreased with increasing number of exposures. The results were negative in 4-mm snails. Cercarial shedding of F. hepatica was studied in the third experiment using 1- and 2-mm L. fuscus each subjected to a single bimiracidial exposure. The total number of cercariae released from these snails was less than 50. From these results, it can be concluded that L. fuscus showed a partial resistance to F. hepatica infection due to snail age.     

Development of Fasciola hepatica in Lymnaea columella infected with miracidia derived from cattle and marmoset infections

The development of Fasciola hepatica from two species of definitive hosts, i.e. cattle (Bos taurus) and a marmoset (Callithrix penicillata) in the snail Lymnaea columella was determined based on the production of rediae and cercariae and snail survival rate. More rediae and cercariae at 60-74 days post-infection were produced by snails infected by cattle-derived miracidia (cattle group) than by those infected by marmoset-derived miracidia (marmoset group). Among the L. columella parasitized by the marmoset group, the survival rate and the percentage of positive snails were higher than among those parasitized by the cattle group. Eggs of F. hepatica released in cattle faeces were significantly bigger than those released in marmoset faeces. Miracidia originating from parasites that completed their development in cattle were more efficient in infecting the intermediate host. These results suggest that vertebrate-host origin influences the eggs produced by the parasite and the infection rates in the snail host L. columella.     

Larval productivity of Fasciola gigantica in two lymnaeid snails

Two groups of Galba truncatula and two groups of Lymnaea natalensis were experimentally infected with Fasciola gigantica to determine if snail species had an influence on the redial burden and cercarial shedding of this trematode when snails of both species were infected with the same isolate of miracidia. In the two groups used for the study of redial burden, the total number of free rediae was significantly higher at day 49 post-exposure in L. natalensis than in G. truncatula. In the groups used for cercarial shedding, the life-span of cercaria-shedding snails and those of infected snails which died without cercarial emission, and the duration of the prepatent period were significantly longer in L. natalensis than those noted in G. truncatula. However, the mean numbers of shed cercariae did not significantly differ and showed no differences in their daily distribution throughout the shedding period. These results demonstrate that G. truncatula might be the principal intermediate host of F. gigantica in Egypt, at least in the areas where this lymnaeid species lives.     

Prevalence and intensity of infections in the lymnaeid snail Omphiscola glabra experimentally infected with Fasciola hepatica, Fascioloides magna and Paramphistomum daubneyi

Single and double infections of juvenile Omphiscola glabra (Gastropoda: Lymnaeidae) with Paramphistomum daubneyi and/or Fasciola hepatica were carried out to determine the redial burden and cercarial production in snails dissected at day 60 or at day 75 post-exposure (p.e.) in the laboratory at 20 degrees C. The results were compared with those obtained with single-miracidium infections by Fascioloides magna. Compared to F. hepatica, low values were noted at day 75 p.e. for the prevalence of snail infections with P. daubneyi (4.6-8.3% instead of 23.6-25.9%), the total number of free rediae (10.7-17.9 per snail instead of 26.3-34.7), and that of free cercariae (112.8-136.9 per snail instead of 177.8-248.5). Despite a greater number of free rediae at day 75 p.e. (36.2-45.6 per snail), the prevalences of snail infections with F. magna and cercarial production were similar to those noted for F. hepatica. The results concerning F. hepatica and P. daubneyi might partly be explained by a progressive adaptation of O. glabra to sustain the larval development of these digeneans over the years, as this snail is a natural intermediate host of F. hepatica and P. daubneyi in central France since 1995. Compared with the high number of fully-grown rediae of F. magna in O. glabra, cercarial production seemed limited and this might be explained by the presence of high numbers of rediae which reduced the avaibility of nutrients for cercarial differentiation within the snail.     

Snail host finding by Fasciola hepatica and Trichobilharzia ocellata: compound analysis of "miracidia-attracting glycoproteins"

The glycoconjugates from snail-conditioned water of Lymnaea truncatula and L. stagnalis which elicit typical host finding behavior in miracidia of Fasciola hepatica and Trichobilharzia ocellata were separated by anion-exchange chromatography and a two-step size-exclusion chromatography. We obtained fractions attractive for the parasites with MW of about 10(6) Da in both snail species. These fractions still contained species-specific information since miracidia responded only to molecules from their respective host snail. Analysis of the amino acid composition from the protein backbone revealed a similar composition in the effective fractions of both snails. Amounts of serine and threonine were higher than 30 mol %, which is typical for mucin-type glycoproteins. The carbohydrate moieties consisted mainly of galactose and fucose, but nine different other monosaccharides also were identified in smaller amounts. The heterogeneity of the molecules was also confirmed by the binding of six different lectins. Because of these characteristics, the effective molecules were termed "miracidia-attracting glycoproteins" (MAGs). MAGs may play an important role for parasite transmission, as they may increase the chance of an encounter between parasite and host and enable the miracidia to discriminate between their specific intermediate host and other unsuitable snail species.     

A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.     

Larval development of Fasciola hepatica in experimental infections: variations with populations of Lymnaea truncatula

A retrospective study was undertaken on 70 French populations of Lymnaea truncatula experimentally infected with Fasciola hepatica to determine whether or not susceptibility of snails to infection influenced redial and cercarial production. Results were compared with those obtained from two control populations, known for prevalences higher than 60% when experimentally infected with F. hepatica. In the 70 other populations examined, the prevalences ranged from 2 to 75%. In 55 of these populations, where the prevalence was more than 20%, a high proportion (50.1-56.8%) of snails died after cercarial shedding, whereas in the other groups (non-shedding snails with the most differentiated larvae being free cercariae, rediae containing cercariae, immature rediae, or sporocysts, respectively), snail death was significantly less. In 11 populations, where the prevalence values were 5-19%, only 14% of snails died after cercarial shedding, whereas snails with free cercariae, rediae with cercariae, or immature rediae showed significant increases in snail mortality. In the remaining four snail populations, with prevalences of less than 5%, the most differentiated larval forms were only immature rediae and/or sporocysts. Overall, the number of rediae containing cercariae significantly decreased with decreasing prevalence values. The low prevalence of experimental infection in several populations of snails might be explained by the occurrence of natural infections with miracidia originating from a mammalian host other than cattle, and/or by genetic variability in the susceptibility of snails to infection.     

Redial growth and cercarial productivity of Fasciola hepatica in three species of young lymnaeid snails

Experimental infections of 1-mm high snails using three populations of Lymnaea (L. glabra, L. ovata and L. truncatula) and a cattle strain of Fasciola hepatica miracidia were carried out under laboratory conditions to determine if the snail species had an effect on the number of free rediae, their growth, and cercarial productivity in relation to each redial category (R1a, R1b, R2a, or R2b/R3a). The total number of rediae ranged from 6.4 to 7.5 per snail. The mean body length of rediae varied from 1-1.2 mm (R1a) to 0.3-0.4 mm (R2b/R3a). The width of the intrapharyngeal lumen also varied from 26.0-38.8 microm to 3.0-4.2 microm, respectively. The redial category had a significant effect on both measurements, whereas snail species only had a significant influence on body length. The mean number of cercariae produced by all living rediae at day 49 post-exposure ranged from 63.0 in L. glabra to 87.2 in L. truncatula. In L. ovata and L. truncatula, 55.8% and 58.6% of cercariae, respectively, were produced by R2a rediae, whereas 53.9% of cercariae in L. glabra were formed by the R1b rediae. When young snails were infected with F. hepatica, the species of snail had an effect on the number of living rediae, their length and their cercarial productivity.     

Extremely low frequency magnetic field and the hatching rate of Fasciola hepatica eggs, the fecundity and survival of liver fluke-infected snail, Lymnaea truncatula

Eggs of Fasciola hepatica were exposed for 10 days to extremely low frequency magnetic field (ELFMF) at the frequency of 50 Hz and density of 2 mT (rms). The results show an accelerated hatching of F. hepatica eggs in relation to control (non-exposed) group. The host snails, Lymnaea truncatula, were divided into three groups; those of groups I and II were infected with the miracidia of F. hepatica hatched from control egg culture, whereas those of group III were infected with miracidia hatched from eggs affected by ELFMF. Thereafter, snails of groups II and III were exposed to ELFMF for 53 days, whereas those of group I were not exposed. At day 14 post infection, a significant decrease was observed in the number of cocoons laid by snails of group III, compared with control. Also, significant mortality in group III snails was observed 42 days post infection. The increased mortality and a lower number of cocoons laid by group III snails have probably resulted from enhanced stimulation of metacercarial parthenogenetic reproduction in consequence of infecting the molluscs with miracidia reared under ELFMF.     

Infectivity of Echinostoma friedi miracidia to different snail species under experimental conditions

The infectivity of Echinostoma friedi (Trematoda: Echinostomatidae) miracidia was studied experimentally in a range of laboratory-reared snails that coexist in the same natural locality, namely Radix peregra, Lymnaea fuscus, L. truncatula (Lymnaeidae), Gyraulus chinensis, Helisoma duryi (Planorbidae) and Physella acuta (Physidae), and snails from different geographical origins acting naturally or experimentally as intermediate hosts of Schistosoma spp., namely Planorbarius metidjensis (from Málaga, Spain), Biomphalaria glabrata (Guadeloupe), B. alexandrina (Egypt) (Planorbidae), Bulinus cernicus (Mauritius), B. globosus (Zambia), B. natalensis (South Africa) and B. truncatus (Niger) (Bulinidae). Six species of snails were found to be susceptible, with the rate of infection ranging from 0 to 36.7%. The highest infection was detected in R. peregra. The low host specificity of E. friedi might have an epidemiological significance as a requisite for a recent establishment in a new geographical area.     

Exposure to Fasciola hepatica miracidia increases the sensitivity of Lymnaea (Fossaria) humilis to high and low pH

Humidity and temperature have been considered important factors affecting the infectivity of Fasciola hepatica to its molluscan host. One hundred and thirty laboratory-reared Lymnaea humilis were exposed for 4 hr to the miracidia of F. hepatica over a pH range from 4.0 to 10.0, and their rates of survival were compared with 130 similarly treated but unexposed control snails. All control snails died within 24 hr at pH 4.0, but they showed better survival at pH 5.0-10.0. Their sensitivity to solutions with high and low pH, however, was increased if kept in the presence of F. hepatica miracidia. Snails exposed at pH 5.0 died within 24 hr, whereas most other pHs also affected survival such that by day 18 only those snails exposed at pH 7.2 remained alive. The increased sensitivity of the snails to pH could be explained by a damage-mediated release of parasite enzymes, because infectivity was highest at pHs associated with the lowest host mortality.     

The experimental production of Fasciola hepatica metacercariae from three aquatic populations of Galba truncatula

Laboratory investigations on three aquatic populations of Galba truncatula, originating from the Peruvian Altiplano and French Massif Central, were carried out during three successive snail generations to determine if these populations might be successfully used for the metacercarial production of Fasciola hepatica under experimental conditions. High numbers of surviving snails at day 30 post-exposure (>70%), high prevalences of F. hepatica infections (>60%), and prolonged productions of cercariae for a mean period of 35 to 47 days were observed in the three populations, whatever the snail generation. In the Peruvian population, metacercariae of F. hepatica significantly decreased in numbers from a mean of 251 in the parent snails to 124 per snail in the F2 generation, whereas no significant variation was observed in the two French populations. As these aquatic snails rarely emerged out of water, the use of these populations for the commercial production of F. hepatica metacercariae was of great interest, because the daily time spent watching the breeding boxes of snails was clearly shorter, thereby reducing the cost of producing metacercariae compared with using amphibious snails reared with romaine lettuce.     

Fasciola hepatica: characteristics of infection in Lymnaea truncatula in relation to the number of miracidia at exposure.

Experimental infections of Lymnaea truncatula by Fasciola hepatica were carried out in three snail populations to determine whether the number of miracidia used for each snail at exposure (1, 2, 5, 10, or 20 per snail) had any influence on the characteristics of Fasciola infection and metacercarial production. The number of miracidia had a significant influence on snail survival at day 30 postexposure and the frequency of infected L. truncatula that died without shedding (NCS snails). The frequency of NCS snails, the growth of cercaria-shedding snails throughout the experiment, the time between exposure and the first cercarial shedding, the duration of shedding, and the number of metacercariae were independent of the number of miracidia used for each snail. The highest metacercaria productivity for each miracidium was found in single-miracidium infections. Single-miracidium infections were the most effective, as the mean number of cercariae was the same as in other groups, whereas their survival rate was much higher.     

Local adaptation of the trematode Fasciola hepatica to the snail Galba truncatula

Experimental infections of six riverbank populations of Galba truncatula with Fasciola hepatica were carried out to determine if the poor susceptibility of these populations to this digenean might be due to the scarcity or the absence of natural encounters between these snails and the parasite. The first three populations originated from banks frequented by cattle in the past (riverbank group) whereas the three others were living on islet banks without any known contact with local ruminants (islet group). After their exposure, all snails were placed in their natural habitats from the end of October up to their collection at the beginning of April. Compared to the riverbank group, snails, which died without cercarial shedding clearly predominated in the islet group, while the other infected snails were few in number. Most of these last snails released their cercariae during a single shedding wave. In islet snails dissected after their death, the redial and cercarial burdens were significantly lower than those noted in riverbank G. truncatula. Snails living on these islet banks are thus able to sustain larval development of F. hepatica. The modifications noted in the characteristics of snail infection suggest the existence of an incomplete adaptation between these G. truncatula and the parasite, probably due to the absence of natural contact between host and parasite.     

Cercarial productivity of redial generations in single-miracidium infections of Lymnaea truncatula with Paramphistomum daubneyi or Fasciola hepatica

Single-miracidium infections of Lymnaea truncatula with Paramphistomum daubneyi or with Fasciola hepatica were carried out under laboratory conditions to count free rediae, their germinal embryos, and to determine the cercarial productivity of each redial generation. In snails infected by P. daubneyi, the cercariae were produced by the first (8.7 cercariae per redia) and second (8.9 per redia) generations. At day 63 post-exposure, they corresponded, respectively, to 53.9% and 46.1% of cercariae produced by all rediae. In snails infected by F. hepatica, the majority of cercariae were produced by the R2a group (18.2 cercariae per redia) and corresponded to 66.0% of cercariae produced all rediae. The cercariae produced by the other redial groups were more limited in number: 17.5 per redia in the R1b group (28.7%) and 2.0 per redia in the R2b/R3a group (5.3%). Cercarial productivity of P. daubneyi until day 63 post-exposure was more limited in number than that of F. hepatica: a total of 145 cercariae per snail versus 427 per snail.     

Comparative studies of the defense mechanism against Schistosoma japonicum of schistosome-susceptible and -resistant Oncomelania nosophora

The amphibious snail Oncomelania nosophora is an intermediate host of Schistosoma japonicum. Previously we reported that there are two strains of the snail, one resistant and one susceptible to a Mindoro, the Philippines, strain of S. japonicum. The resistant snails were collected from Nirasaki and susceptible snails from Kisarazu, Japan. To determine early cellular responses in the two snail strains, we examined histologic alterations in the snails for up to 18 h after the initial exposure to miracidia. In both strains, the penetrating miracidia were distributed in the foot, mantle, gills, heart, stomach, and kidney, and the mean number of penetrating miracidia was similar in both strains. After penetration, snail hemocytes migrated toward the larvae, and by 12 h after exposure, substantial numbers of penetrated larvae were surrounded and encapsulated by hemocytes. The percentage of larvae encapsulated by hemocytes during 12-18 h after the exposure was significantly higher in the resistant Nirasaki strain (60.9+/-19.8%) than in the susceptible Kisarazu strain (42.3+/-15.0%). In a few snails of the Nirasaki strain, all the larvae found were encapsulated by hemocytes. The differences in hemocyte responses between the two strains may explain the susceptibility of the snails to schistosome larvae.     

Aspects of the maintenance of the life cycle of Fasciola hepatica in Lymnaea columella in Minas Gerais,Brazil

Fascioliasis is a parasitic disease of domestic ruminants that occurs worldwide. The lymnaeid intermediate hosts of Fasciola hepatica include Lymnaea columella, which is widely distributed in Brazil. A colony of L. columella from Belo Horizonte, MG, was reared in our laboratory to be used in studies of the F. hepatica life cycle, the intermediate host-parasite relationship and development of an anti-helminthic vaccine. In the first experiment 1,180 snails were exposed to miracidia of F. hepatica eggs removed from the biliary tracts of cattle from the State of Rio Grande do Sul. In the second and third experiments the snails were exposed to miracidia that had emerged from F. hepatica eggs from Uruguay, maintained in rabbits. The rates of infection in the first, second and third experiments were 0, 42.1 and 0% respectively. Over 15,806 metacercariae were obtained and stored at 4 degrees C. Four rabbits weighing 1.5 kg each were infected with 32-44 metacercariae and two with 200. Three rabbits begin to eliminate eggs of the parasite in the feces from 84 days after infection onwards. The biological cycle of F. hepatica in L. columella and the rabbit was completed within 124 days.     

Austropeplea ollula (Pulmonata: Lymnaeidae): a new molluscan intermediate host of a human intestinal fluke, Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

Three freshwater snail species of the family Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula and Fossaria truncatula. Out of 3 lymnaeid snail species, A. ollula was naturally infected with the Echinostoma cinetorchis cercariae (infection rate = 0.7%). In the experiments with the laboratory-bred snails, F. truncatula as well as A. ollula was also susceptible to the E. cinetorchis miracidia with infection rates of 25% and 40%, respectively. All of three lymnaeid snail species exposed to the E. cinetorchis cercariae were infected with the E. cinetorchis metacercariae. It is evident that A. ollula acts as the first molluscan intermediate host of E. cinetorchis in Korea, and F. truncatula may be a possible candidate for the first intermediate host of this intestinal fluke. Also, three lymnaeid snail species targeted were experimentally infected with E. cinetorchis metacercariae.     

Fasciola hepatica: cercarial productivity of redial generations in long-surviving Galba truncatula

Bimiracidial infections of Galba truncatula with Fasciola hepatica were carried out to determine the effect of food quality on the frequency of 1- and 2-sporocyst infections, to analyse its impact on the developmental patterns (normal, or abnormal) of redial generations, and to verify its consequences on cercarial production. These investigations were performed in snails reared at 20 degrees C and provided with cos lettuce and commercial fish food (Tetraphyll) as a food source until their death. Double-sporocyst infections with normal development of redial generations were recorded in 43.9% of infected snails (out of 296). Single-sporocyst infections were noted in the other snails, with normal development of generations in 53.7% and abnormal development (the first mother redia early degenerated) in 2.4%. Four successive redial generations were found in long-surviving snails (more than 90 days). In both 1- and 2-sporocyst infections, showing normal development of generations, the daughter rediae, which exited from the first mother redia (R2a rediae), constituted the greater group of free rediae and produced the highest percentages of cercariae (46.2-48.2%). However, the development of these rediae inside the snail body was slower in 2-sporocyst infections than in 1-sporocyst infections. The numbers of rediae noted in subsequent generations (R2b/R3a and R3b/R4a rediae) were similar, whatever the number of full-grown sporocysts. The number of shed cercariae recorded in the 1- and 2-sporocyst infections did not significantly differ. When long-surviving snails died, 19.8-20.7% of cercariae produced by free rediae (essentially by R2b/R3a and R3b/R4a rediae) were still present in their bodies. The increased frequency of 2-sporocyst infections demonstrated that food quality had a significant effect on the redial burden of F. hepatica developing inside G. truncatula.     

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of >99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.