A stock of infected snails for the mass breeding of cercariae and schistosomules

A routine to maintain infected snails Biomphalaria glabrata able to produce 1-2 million cercariae per week and a mean shedding of about 3,500 cercariae per snail was quantitatively described. To a monthly production of about 5.3 and 6.5 million cercariae during the years of 1982 and 1983 it was necessary to expose an average of 1,557 and 1,957 snails, respectively. The efficiency described was maintained by infecting 2,000 snails per month (infectivity index of about 60%). A maximal production of 6,000 cercariae/snail was obtained in the period between 56-70 days after the snail infection by miracidia. By this period, however, 90% of the infected snails were dead. In the summer of 1982-1983 it was observed an impairment on the daily cercarial harvests which was related to ambient temperature increase, and the presence of rotifers in the aquaria water. When the cercariae were transformed into schistosomula in vitro, a yield of 55% schistosomula was obtained, with 7% of tail contamination. Lyophilization of cercariae produced 50.9 +/- 63 g dry weight per 1,000 cercariae.     

Cercarial shedding of Echinostoma cinetorchis and experimental infection of the cercariae to several kinds of snails

The development of Echinostoma cinetorchis in several snail species reared in laboratory aquaria was observed. The eggs from adult flukes collected from the intestine of rats were cultivated to miracidia, and exposed to Hippeutis sp. snails. Observations were made for cercarial shedding from the exposed snails. The cercariae shed from the snails were again exposed to several species of fresh water snails in order to observe metacercarial formation in the snails and their infectivity to final hosts. The results obtained in this study were as follows: 1. Twenty miracidia were exposed to each snail of Hippeutis sp. About 58.3% of the above snails (7 out of 12) were dead before shedding the cercariae, and the remainder shed the cercariae for a period of 7 to 9 days before death. 2. Cercarial shedding from the infected snails started from the 25th day after the exposure to miracidia, and the total number of cercariae shed per snail was 684 in average (range; 482-904). 3. The size of rediae developed in the infected Hippeutis sp. snails was 1,242 x 214 microns in average, and the number of rediae per snail was 350 in average (range; 120-510). 4. About 40 to 50 cercariae shed from the Hippeutis sp. snails were each exposed to several species of snails reared in the laboratory. The metacercarial formation was confirmed by dissecting the infected snails, 12 to 16 days after the infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Influence of low temperatures on the cercarial shedding of Paramphistomum daubneyi from the snail Lymnaea truncatula

Lymnaeids in nature are subjected to temperature fluctuations that may have an influence on the shedding of cercariae. Thus, experimental infections of Lymnaea truncatula by Paramphistomum daubneyi were performed to determine whether a sudden fall in temperature--daily dipping of infected snails into spring water at 14-15 degrees C, or at 6-8 degrees C--followed by its increase at 20 degrees C in the subsequent hour, had an influence on the characteristics of snail infection and cercarial production. The immersion of infected snails in cold water during a short period delayed the first cercarial shedding, at day 66 on average, in the 6-8 degrees C group vs at day 57 in the 14-15 degrees C group, or at day 49 in the 20 degrees C group. The percentage of cercaria-shedding snails was greater in the 6-8 degrees C group than in the 14-15 degrees C and the 20 degrees C ones: 41.8% vs 17.3% and 7.1%, respectively. The total number of cercariae given by each infected snail was also higher in the 6-8 degrees C group. A fall in the temperature of water during daily change, followed by its increase at 20 degrees C within the subsequent hour, stimulated the cercarial shedding of P. daubneyi.     

Plagiorchis elegans (Digenea: Plagiorchiidae) infections in Stagnicola elodes (Pulmonata: Lymnaeidae): host susceptibility, growth, reproduction, mortality, and cercarial production.

Eggs of Plagiorchis elegans were readily ingested by Stagnicola elodes of all ages, but were more infective to immature than mature snails. Infection enhanced the growth of the host in a dose-dependent manner. The number of cercariae released by immature snails increased with the age of the snail host; mature snails yielded fewer cercariae. Heavily infected snails tended to die prematurely, thereby reducing their total production of cercariae to levels below those of more lightly infected individuals. Even light infections castrated the snail host. Snails that acquired the infection as juveniles never produced eggs. Actively reproducing snails ceased egg laying within days of infection and never recovered. All parasite effects on the growth and reproduction of the snail host first manifested themselves during the early stages of infection, long before the development of daughter sporocysts and cercariae, and are therefore attributable to the effects of mother sporocysts. The study provides insight into how this natural enemy of mosquito larvae may be established in natural snail populations by means of strategically timed introductions of parasite eggs, with a goal of maximizing cercarial production for the biological control of sympatric mosquito larvae.     

Emergence of cercariae of Echinostoma caproni and Schistosoma mansoni from Biomphalaria glabrata under different laboratory conditions

Release of Echinostoma caproni cercariae and Schistosoma mansoni from experimentally infected Biomphalaria glabrata snails maintained under different laboratory conditions was studied. Infected snails were isolated individually for 1 h in Stender dishes containing 5 ml of artificial spring water and the number of cercariae released during this time was recorded. Of numerous conditions tested, the addition of lettuce, the use of water conditioned by B. glabrata snails and a temperature of 35 degrees C significantly increased the release of E. caproni cercariae. A significant increase in cercarial release of S. mansoni was seen only in cultures fed lettuce. A temperature of 12 degrees C caused a significant decrease in cercarial release of both E. caproni and S. mansoni. Increased snail activity associated with feeding behaviour was probably responsible for the enhanced cercarial sheds observed in this study.     

Schistosoma mansoni infections in neonatal Biomphalaria glabrata snails.

In areas endemic for schistosomiasis, the population dynamics of the snail intermediate hosts have a direct effect on parasite transmission. The present study focused on the potential for neonatal Biomphalaria glabrata snails to become infected with Schistosoma mansoni and to produce cercariae under various conditions. It was found that snails as small as 0.74 mm in shell diameter could survive miracidial penetration and could release cercariae when as small as 1.6 mm in diameter. Cercariae produced by small snails were equally infectious for mice when compared with those shed by larger snails. Likewise, histological examination of neonatally exposed snails revealed normally developing parasites at all stages of infection. It was found that in 2 snail populations expressing either high or low susceptibility to the parasite, peak susceptibility occurred at 25 days of age in both groups. Daily cercarial production for neonatally exposed snails was initially low but increased dramatically as the snails grew, eventually reaching values as high as 2,100 cercariae/snail/day. A moderate to high percentage of snails infected as neonates was eventually capable of simultaneously producing both eggs and cercariae. These studies emphasize the potential importance of neonatal and preadult snails in helping to maintain foci of S. mansoni infection in endemic areas.     

Clonal diversity of the marine trematode Maritrema novaezealandensis within intermediate hosts: the molecular ecology of parasite life cycles

We quantified the clonal diversity of the New Zealand marine trematode Maritrema novaezealandensis (n = 1250) within Zeacumantus subcarinatus snail (n = 25) and Macrophthalmus hirtipes crab (n = 25) intermediate hosts using four to six microsatellite loci, and investigated the potential biological and physical factors responsible for the observed genetic patterns. Individual snails harboured one to five trematode genotypes and 48% of snails were infected by multiple parasite genotypes. Overall, the number of parasite genotypes did not increase with snail size, but was highest in intermediate-sized snails. Significantly larger numbers of parasite genotypes were detected in crabs (relative to snails; P < 0.001), with 16-25 genotypes recovered from individual crabs. Although crabs are typically infected by small numbers of cercariae sourced from many snails, they are occasionally infected by large numbers of cercariae sourced from single snails. The latter cases explain the significant genetic differentiation of trematode populations detected among their crab hosts (F(ST) = 0.009, P < 0.001). Our results suggest that the timing of infection and/or intraspecific competition among parasite clones within snails determine(s) the diversity of parasite clones that snails harbour. The presence of a large number of infected snails and tidal mixing of cercariae prior to infection results in crabs potentially harbouring hundreds of parasite genotypes despite the crabs' territorial behaviour.     

Schistosoma mansoni: influence of Biomphalaria glabrata size on susceptibility to infection and resultant cercarial production

Biomphalaria glabrata snails of the same age, but different sizes, were used to determine size-related susceptibility to Schistosoma mansoni miracidial infection and the influence of snail size on total cercarial production. Snails with shell diameters from less than 5 to greater than 17 mm were individually exposed to one or several miracidia, depending on the experiment. In snails exposed to multiple numbers of miracidia, the percentage of snails which developed patent infections was lower in snails with larger shell sizes. This was also reflected by fewer primary sporocysts per infected snail found in tissues of the larger snails. Upon determining cercarial production in these groups over a 1-month period there were no statistical differences between any groups in the numbers of cercariae produced per snail. However, upon determining the number of successful primary sporocysts found in cohort snails of each size group, cercarial production increased as a function of the number of successful primary sporocysts. This was verified by examining cercarial production in various size snails with known monomiracidial infections. Our data therefore confirm and extend earlier work using snails infected with unknown numbers of miracidia and clearly show that total S. mansoni cercarial development and decreased susceptibility of snails is a direct reflection of snail size and not necessarily age of the snail.     

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.     

Effect of temperature on emergence, survival and infectivity of cercariae of the marine trematode Renicola roscovita (Digenea: Renicolidae)

Marine bivalves harbour a diversity of trematode parasites affecting population and community dynamics of their hosts. Although ecologically and economically important, factors influencing transmission between first (snail) and second (bivalve) intermediate hosts have rarely been studied in marine systems. In laboratory experiments, the effect of temperature (10, 15, 20, 25 degrees C) was investigated on (1) emergence from snails, (2) survival outside hosts and (3) infectivity in second intermediate hosts of cercariae of the trematode Renicola roscovita (Digenea: Renicolidae), a major parasite in North Sea bivalves. Emergence of cercariae peaked at 20 degrees C (2609 +/- 478 cercariae snail(-1) 120 h(-1)) and was considerably lower at 10 degrees C (80 +/- 79), 15 degrees C (747 +/- 384) and 25 degrees C (1141 +/- 334). Survival time decreased with increasing temperature, resulting in 50% mortality of the cercariae after 32.8 +/- 0.6 h (10 degrees C), 26.8 +/- 0.8 h (15 degrees C), 20.2 +/- 0.5 h (20 degrees C) and 16.6 +/- 0.3 h (25 degrees C ). Infectivity of R. roscovita cercariae in cockles Cerastoderma edule increased with increasing temperature and was highest at 25 degrees C (42.6 +/- 3.9%). However, mesocosm experiments with infected snails and cockle hosts in small aquaria, integrating cercarial emergence, survival and infectivity, showed highest infection of cockles at 20 degrees C (415 +/- 115 metacercariae host(-1)), indicating 20 degrees C to be the optimum temperature for transmission of this species. A field experiment showed metacercariae of R. roscovita to appear in C. edule with rising water temperature in April; highest infection rates were in August, when the water temperature reached 20 degrees C. Since another trematode species (Himasthla elongata; Digenea: Echinostomatidae) occurring at the experimental site showed a similar temporal pattern, trematode transmission to second intermediate bivalve hosts may peak during especially warm (> or = 20 degrees C) summers in the variable climate regime of the North Sea.     

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.     

Relationship between Snail Population Density and Infection Status of Snails and Fish with Zoonotic Trematodes in Vietnamese Carp Nurseries

Background

Fish-borne zoonotic trematodes (FZT) are a food safety and health concern in Vietnam. Humans and other final hosts acquire these parasites from eating raw or under-cooked fish with FZT metacercariae. Fish raised in ponds are exposed to cercariae shed by snail hosts that are common in fish farm ponds. Previous risk assessment on FZT transmission in the Red River Delta of Vietnam identified carp nursery ponds as major sites of transmission. In this study, we analyzed the association between snail population density and heterophyid trematode infection in snails with the rate of FZT transmission to juvenile fish raised in carp nurseries.

Methodology/Principal Findings

Snail population density and prevalence of trematode (Heterophyidae) infections were determined in 48 carp nurseries producing Rohu juveniles, (Labeo rohita) in the Red River Delta area. Fish samples were examined at 3, 6 and 9 weeks after the juvenile fish were introduced into the ponds. There was a significant positive correlation between prevalence of FZT metacercariae in juvenile fish and density of infected snails. Thus, the odds of infection in juvenile fish were 4.36 and 11.32 times higher for ponds with medium and high density of snails, respectively, compared to ponds where no infected snails were found. Further, the intensity of fish FZT infections increased with the density of infected snails. Interestingly, however, some ponds with no or few infected snails were collected also had high prevalence and intensity of FZT in juvenile fish. This may be due to immigration of cercariae into the pond from external water sources.

Conclusions/Significance

The total number and density of potential host snails and density of host snails infected with heterophyid trematodes in the aquaculture pond is a useful predictor for infections in juvenile fish, although infection levels in juvenile fish can occur despite low density or absence infected snails. This suggests that intervention programs to control FZT infection of fish should include not only intra-pond snail control, but also include water sources of allochthonous cercariae, i.e. canals supplying water to ponds as well as snail habitats outside the pond such as rice fields and surrounding ponds.     

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.     

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.     

Potential of Biomphalaria tenagophila from Pampulha lake, Belo Horizonte, MG, as a host of Schistosoma mansoni

Biomphalaria tenagophila snails, from a population originally obtained from "Pampulha" lake, Belo Horizonte, Minas Gerais State, Brazil, were exposed to miracidia from four strains of Schistosoma mansoni: "LE" and "HK" from Belo Horizonte, "AL" from Alagoas and "SJ" from S?o José dos Campos, S?o Paulo. The "LE", "AL" and "SJ" strains are maintained in the laboratory and the "HK" strain was obtained from feces of a patient residing near to "Pampulha" lake. Infection rates were of 4% ("LE" strain), 6% ("HK" strain), 30% ("SJ" strain) and 40% ("AL" strain). These infection rates were similar to those obtained by others authors for B. tenagophila from Minas Gerais. Experimentally infected snails when compared to B. glabrata of the control group and B. tenagophila naturally infected in "Pampulha" lake shed similar number of cercariae (2000 cercariae/snail). The high density of B. tenagophila in the "Pampulha" lake, the number of cercariae shed by naturally infected snails, the great number of persons who use the water for fishing and swimming, and the water contamination with human feces, are favourable factors for growing the Schistosomiasis focus in the lake.     

Experimental double infection of Biomphalaria glabrata snails with Angiostrongylus cantonensis and Schistosoma mansoni

Two groups of Biomphalaria glabrata snails primarily infected with Angiostrongylus contonensis were secondarily exposed to infection with Schistosoma mansoni. To investigate any anatagonistic effect of the first infection on a superimposed one and to compare to singly and non-infected snails, a series of experiments was undertaken in which snails were individually exposed, variously, to 1,000 and 2,000 first-stage larvae of A. cantonensis and then to 5 and 10 miracidia of S. mansoni 1 day and 3 weeks later. Snails became infected with S. mansoni in both groups of snails with double infections and shed cercariae after the same incubation period as in the singly infected groups. The number of snails shedding cercariae simultaneously was similar in single and double infection groups during the first two weeks of shedding, after which this number decreased somewhat in doubly infected groups. Snails with double infection showed higher cumulative mortality rates than in snail groups with single infection with either A. cantonensis or S. mansoni. Therefore, initial infection of B. glabrata with A. cantonensis produced no inhibitory or retarding effect on subsequent infection of snails with S. mansoni.     

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.     

Effects of snail size on encystment of Echinostoma caproni in juvenile Biomphalaria glabrata (NMRI strain) and observations on the survival of infected snails

The effects of snail size on encystment of Echinostoma caproni cercariae in neonatal and juvenile Biomphalaria glabrata (NMRI strain) snails were studied. Encystment in neonatal (0.7-1.1 mm shell diameter) and juvenile (2-3 mm shell diameter) snails was compared 24 h post-infection (PI) following individual exposure of snails of each size to 1, 5, 10, 25 and 50 cercariae. Significantly more cysts were recovered from juveniles exposed to 1, 5, 10 and 50 cercariae than from neonatals with comparable exposure. Size of B. glabrata was a major factor in determining cyst burden in this planorbid. Survival of infected versus uninfected neonatals and juveniles was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 3, 6 and 7 days PI when compared to the uninfected controls. There was no significant decrease in the survival of juveniles exposed to 10 cercariae compared to uninfected controls at any time point. Snail size was a factor in mortality associated with echinostome cercarial penetration and encystment.     

Schistosoma mansoni: Long-term maintenance of clones by microsurgical transplantation of sporocysts

Schistosoma mansoni sporocysts originally derived from monomiracidially infected Biomphalaria glabrata snails were serially transplanted into the cephalopedal sinus of anesthetized snails by the microsurgical implantation of fragments of parasitized hepato-pancreas and ovotestis. Three to six passages each of five male and five female clones were maintained for as long as 2.0 years. Of the recipient snails which survived surgery, 87% released cercariae, usually beginning 5–7 weeks after surgery. The percentage of snails which released cercariae increased with successive passages. The mean survival time of surgically infected snails after cercarial emergence began was 9.2 ± 0.5 weeks, nearly the same as that of miracidially infected snails. Longevities of snails infected with male or female clones were similar. Recipient snail size and age did not influence cloning success. Beginning 5 weeks from the onset of cercarial emergence large numbers of cercariae (a mean of 3900/snail from male clones and 1300/snail from females) were obtained during each shedding period. These results clearly demonstrate that the microsurgical transplantation of sporocysts is a practical means of maintaining and expanding populations of genetically homogeneous schistosomes (clones).