Fasciola hepatica and Paramphistomum daubneyi: vertical distribution of metacercariae on plants under natural conditions.

Four experiments on the metacercariae of Fasciola hepatica and Paramphistomum daubneyi were carried out under natural conditions in order to study their vertical location on submerged plants and to determine whether simultaneous cercarial shedding of both digenea causes changes in the distributions of the metacercariae. These experiments were performed in experimental boxes, each containing six tufts of rushes. Most metacercariae (73.0%) of F. hepatica were found along the walls of boxes, while 81.5% of P. daubneyi metacercariae were found on rush stems. In the case of snails infected by either of the two digenea, 80.1% of F. hepatica cercariae encysted on submerged parts of rushes and of the box walls near the water surface (to a depth of 1 cm), whereas 73.0% of P. daubneyi metacercariae were found in the lower sections (from -4 to -7 cm). If snails dually infected with P. daubneyi and F. hepatica were used, the vertical distributions of the metacercariae were significantly different from those found for snails infected by either of the two digenea. If snails having 42-day old infections with F. hepatica and other snails with 70-day old infections with P. daubneyi were simultaneously introduced into the boxes, the frequency of F. hepatica cysts was significantly lower in the section located under the water surface (29% only), while the frequencies of metacercariae in the lower sections (from -1 to -5 cm) were increased. Some significant changes were also observed for the metacercariae of P. daubneyi. The disturbance noted in the vertical distributions of F. hepatica and P. daubneyi metacercariae suggest that the encystment of F. hepatica cercariae can be disturbed by the simultaneous encystment of P. daubneyi cercariae, or conversely.

The use of tetraphyll as food for snails increases the intensity of cercarial shedding in Galba truncatula infected with Fasciola hepatica.

Experimental infections of Galba truncatula with Fasciola hepatica were carried out under laboratory conditions to determine why the use of Tetraphyll, when provided every 3 days as food for snails, stimulated the intensity of cercarial shedding. In snails raised on cos lettuce and Tetraphyll, the number of metacercariae was significantly higher than that recorded in controls on lettuce only. In the former group, numerous metacercariae were counted on day 1 of every 3-day period (when snails fed on Tetraphyll), while the numbers of these larvae strongly decreased on day 2 and were very low on day 3. The repetition of this experiment using microalgae or modified Boray's diet every 3 days as food for snails gave the same results. However, the number of metacercariae recorded on day 1 of every 3-day period was significantly higher in snails raised on lettuce and Tetraphyll than in those reared on lettuce and algae (or Boray's diet). According to the authors, the first contact of an infected snail with Tetraphyll induces a faster differentiation of cercariae within the rediae and the accumulation of many free cercariae in the snail's hemocoel, so that a second contact of the snail with this food 3 days after allows free cercariae to complete their glycogen and fat reserves and to rapidly exit from the snail. However, a temporary immobilization of the infected snail by Tetraphyll, or rather its degradation products, and a massed exit of cercariae cannot be excluded.

Fasciola hepatica and Paramphistomum daubneyi: field observations on the transport and outcome of floating metacercariae in running water.

Experimental investigations in eight open drainage ditches and furrows from central France were carried out to analyse the dispersal of floating metacercariae of two digenean species by running water and to determine the outcome of larvae which settled on Nasturtium officinale (watercress). The frequencies of larvae found after their transport by water ranged from 33% to 49.7%, thus indicating that more than half of the metacercariae used in this experiment had fallen to the bottom of the water during this transport. The nature of the site (furrow, or ditch supplied by a spring) had a significant effect on the distribution of floating larvae, while the digenean species had no effect. Low percentages of metacercariae on watercress were noted in furrows (3.5-4.3% of larvae) and ditches (0.8-1.3%). When the watercress grew, most larvae that had settled on leaves and stems died but there were always several living metacercariae on this plant (0.7-1.5% of larvae for Fasciola hepatica and 0.2-0.5% for Paramphistomum daubneyi). The weak buoyancy of these floating cysts on running water limited their dispersal and, consequently, led to a real diminution of risks incurred by definitive hosts towards these metacercariae.

Variability in the prevalence of infection and cercarial production in Galba truncatula raised on a high-quality diet.

Two experiments using seven populations of Galba truncatula were carried out to analyse the effect of food quality (cos lettuce only, or cos lettuce+Tetraphyll) on the characteristics of infections: (1) in a single population of G. truncatula infected by one of three digenea (first experiment), and (2) in seven populations of G. truncatula differing in their susceptibility to Fasciola hepatica miracidia (second experiment). In most groups, food quality did not have a significant effect on the survival of snails. The prevalence of infection in five populations was significantly higher in snails raised on lettuce+Tetraphyll (first and second experiments), whereas it was close to those noted in lettuce only-reared groups in the last two populations (second experiment). Despite the higher growth of cercariae-shedding snails when raised on the mixed diet, no significant differences were noted. Significant effects of parasite species (first experiment) and of snail population (second experiment) on the life-spans of cercariae-shedding snails were noted, whereas food quality did not influence this parameter. Except for a single snail population, cercarial production in groups raised on lettuce+Tetraphyll was significantly higher than that in groups on lettuce. The variability noted in the prevalence of snail infections and in the intensity of cercarial shedding might be explained by differences in the susceptibility of snail populations to F. hepatica infections, and/or by the fact that Tetraphyll would not have the same appetency for all populations of G. truncatula.

Unusual snail species involved in the transmission of Fasciola hepatica in watercress beds in central France.

Four freshwater pulmonate species (Lymnaea ovata, L. stagnalis, Physa acuta, Planorbis leucostoma) were living in several watercress beds known for their relationships with human cases of fasciolosis, whereas L. truncatula was never found. The aims of these studies were to determine the prevalence of natural infections with Fasciola hepatica in snails and to verify if these species might ensure the full larval development of this trematode (with cercarial shedding) when they were experimentally subjected to F. hepatica only, or to co-infections with an other trematode species. Investigations were so carried out in six snail populations living in watercress beds (including three for P. acuta) and in four others originating from three brooks or a pond (as controls). Snails naturally infected with F. hepatica were found in two watercress beds inhabited by L. ovata (prevalence of infection: 1.4%) and P. leucostoma (0.1%), respectively. The L. ovata from the watercress bed could be infected at a higher size than those from the control population and the prevalence of this infection was greater in the bed population. Similar findings were noted for L. stagnalis. Despite single or dual infections, the results obtained with the four populations of P. acuta were unsuccessful. In contrast, the co-infections of young P. leucostoma with Paramphistomum daubneyi and F. hepatica resulted in the shedding of some F. hepatica cercariae. According to the authors, the occurrence of fasciolosis in these watercress beds would be the consequence of frequent natural encounters between parasite and snails (L. ovata, L. stagnalis), or of co-infections with P. daubneyi and F. hepatica (P. leucostoma). In watercress beds only colonized by P. acuta, a lymnaeid species would have ensured the larval development of F. hepatica but it would have been eliminated by P. acuta, as this last species was known to be invasive and could colonize open drainage ditches on siliceous soil.

The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection.

Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6-8 degrees C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93-96% vs 48-50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates.

The definitive and intermediate hosts of Fasciola hepatica in the natural watercress beds in central France.

Field investigations were carried out over a two-year period in 52 natural watercress beds located in the Limousin region of central France to list the mammal and bird species that frequented these sites. This enabled detection of the definitive hosts of Fasciola hepatica and determination of the prevalence of natural infection in snails. A total of 13 mammal and five bird species were listed in these watercress beds. Adult flukes were found in Lepus capensis (39.2%), Oryctolagus cuniculus (42.0%), and Sylvilagus floridanus (25.0%). No infection with F. hepatica was noted in the five species of rodents studied. Snails infected with F. hepatica were found in 14 watercress beds. The global prevalence of natural infection was 1.1% in Lymnaea truncatula and 0.3% in L. glabra. Among the other trematode larval forms detected, the most frequent was Haplometra cylindracea (0.5%). In the Limousin region, the presence of hares and rabbits in watercress beds ensured the continuation of the F. hepatica life cycle and permitted the subsequent infection of humans when this wild watercress was eaten.

Metacercarial aggregation in Digenea (Fasciola hepatica and Paramphistomum daubneyi): environmental or species determinism?

Metacercarial aggregation of Fasciola hepatica and Paramphistomum daubneyi was studied under experimental conditions to determine if the formation of these aggregates was influenced by environmental factors, or it was a characteristic of trematode species. This process was studied using the confinement of infected snails on the bottom of Petri dishes (diameter, 14 cm) for 3 days. The formation of metacercarial aggregates of F. hepatica was not significantly modified by environmental factors such as intensity and duration of lighting, quality and volume of water. Metacercariae of F. hepatica were more numerous on the Petri dish walls and 63.9% of them constituted aggregates. In contrast, most metacercariae of P. daubneyi were found on the Petri dish bottoms and 78.3% of them were isolated or in groups of two metacercariae each. The mean number of metacercariae per aggregate ranged from 6.7 to 12.2 in the case of F. hepatica, and from 2.7 to 4.5 in the case of P. daubneyi. However, these mean numbers were independent of the site of cercarial attachment. The tendency of cercariae to form metacercarial aggregations was a characteristic of F. hepatica and was species determined.

A field study of natural infections in three freshwater snails with Fasciola hepatica and/or Paramphistomum daubneyi in central France.

Natural infections of three freshwater snails with Fasciola hepatica and/or Paramphistomum daubneyi were studied during two periods in 1996 and 1997 (June-July and September-October) on 18 farms located in the departments of Vienne and Haute Vienne (central France), and known for low prevalences of F. hepatica infections in ruminants. A total of 1573 Lymnaea glabra and 1421 L. truncatula 6 mm high or more were collected in the meadows of 13 farms and dissected under laboratory conditions. Snails with single or concurrent infections of F. hepatica and/or

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.

Prevalence of Haplometra cylindracea infection in three species of Lymnaea snails in central France.

Field investigations were performed over a 2- or 3-year period at five sites in central France to determine the prevalence of Haplometra cylindracea infection in three species of adult Lymnaea, and to compare it with that of Fasciola hepatica infection recorded in the same snails. L. glabra, L. palustris, and L. truncatula harboured Haplometra larval forms but the prevalence of this infection varied according to the site and snail species studied. The prevalences in May ranged over 0-66.9% in L. glabra, 3-7.7% in L. palustris, and 0-31.5% in L. truncatula. The mean intensity of H. cylindracea infection in May did not show any significant variation, whatever the site and species studied. The prevalence of F. hepatica infection was significantly lower in snails collected in sites known for H. cylindracea infection than in those originating from nearby meadows (0-1% instead of 1-5%). H. cylindracea could develop in some Lymnaea species and its occurrence had an impact in snails by decreasing the prevalence of F. hepatica infection.

Allopatric combination of Fasciola hepatica and Lymnaea truncatula is more efficient than sympatric ones.

Parasites are capable of rapid evolutionary changes relative to their hosts, due to short life cycle, short generation time, and high fecundity. The direction of the evolution of parasite virulence can be studied in cross-transfer experiments, combining hosts and parasites from different localities, and comparing the outcome of established (sympatric and potentially locally adapted) and novel (allopatric) combinations of hosts and parasites. We aimed to compare the compatibility with snails hosts, the infectivity of metacercariae in rabbits and rats, and the fitness among different combinations (French-FF and Spanish-SS sympatries and allopatry-FS). The first isolate of Fasciola hepatica and its corresponding intermediate host, Lymnaea truncatula originated from Lugo's northwestern Spain. The second isolate of parasite and snail was collected in the Limoges area in central France. The Spanish snails were more susceptible to their sympatric trematode than the French snails. The Spanish flukes were more infective to intermediate hosts (snails) than the French flukes, but subsequent definitive hosts (rats or rabbits) infections remained similar. The estimated fitness was low in sympatric infections and highly similar (from 4.7 to 5.3). The fitness similarity corresponds, however, to different variations in life-history traits that could represent different strategies among the host-parasite local combinations. The infection rate in snails, metacercarial productivity, metacercarial infectivity, and the estimated fitness were better for allopatric combination (FS). The susceptibility data showed a higher efficiency of flukes in the allopatric snail population than in their local snail population. However, our results were obtained after one generation and from a single isolate and it remains to be determined if all allopatric fluke-snail isolates may present a better fitness. Nevertheless our results indicate that introduction of liver fluke-infected cattle should be monitored carefully, as it could result in the introduction of more efficient parasites.

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.

Prevalence of Paramphistomum daubneyi infection in cattle in central France.

Investigations at the slaughterhouse of Limoges (Central France) were performed in 1994-1996 to determine the prevalence of Paramphistomum daubneyi infection in cattle. In 1994/1995, higher prevalences of P. daubneyi were recorded in May, October, and January. In 1996, smaller variations were recorded, and prevalence in April and May was higher. The prevalence of P. daubneyi in summer was lower. Breed and age of cattle were not significantly associated to P. daubneyi infection, but females were significantly more infected than males. P daubneyi infection was related to that of Fasciola hepatica, but not to that of Dicrocoelium lanceolatum.

Paramphistomum daubneyi and Fasciola hepatica: influence of temperature changes on the shedding of cercariae from dually infected Lymnaea truncatula.

Dual infections of Lymnaea truncatula with Paramphistomum daubneyi and Fasciola hepatica were performed to determine whether temperature changes in snails (daily water change with spring water at 6 degrees-8 degrees C, which subsequently increased to room temperature at 20 degrees C) would influence snail infection and the production of cercariae by both trematodes. At day 30 post-exposure the surviving snails were individually placed in petri dishes to constitute two groups. Snails from the first group were maintained at a temperature of 20 degrees C, and the water in the petri dishes was changed daily. The protocol was identical for the second group of snails except that the water temperature was 6 degrees-8 degrees C when changed. The interval between exposure and the first shedding of cercariae in snails immersed in cold water for a short period was longer (67-69 days instead of 48-50 days in the 20 degrees C group). In both groups, snails infected only with F. hepatica or P. daubneyi or with both trematodes were detected. In snails infected only with F. hepatica the frequency of cercaria-shedding snails and the total number of metacercariae were significantly greater in the 20 degrees C group. Inversely, in snails infected only with P. daubneyi the frequency of cercaria-shedding snails and the number of metacercariae were significantly greater in the 6 degrees-8 degrees C group. In snails harboring both trematode larval forms, no significant difference in the frequencies of cercaria-shedding snails between the two groups was noted. Metacercariae of both trematodes were obtained from these snails. In the 20 degrees C group, F. hepatica metacercariae were more numerous, whereas in the 6 degrees-8 degrees C group the number of P. daubneyi metacercariae was greater. From these results it appears that greater activity of P. daubneyi cercariae occurs in snails subjected to daily temperature changes (from 6 degrees to 20 degrees C).

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.

Infection of Lymnaea truncatula and Lymnaea glabra by Fasciola hepatica and Paramphistomum daubneyi in farms of central France.

Natural infection of Lymnaea glabra and Lymnaea truncatula by Fasciola hepatica and/or Paramphistomum daubneyi was studied at two periods of risk (June-July and September-October) in 11 French farms known for their high prevalences of F. hepatica infection in ruminants. A total of 1,778 L. truncatula and 2,396 L. glabra measuring 6 mm or more in height were collected to determine the prevalence of natural infection with F. hepatica and P. daubneyi. The role of four factors, i.e. season, snail co-infection (F. hepatica-P. daubneyi), ruminant host and the existence of single or mixed intermediate host communities, was investigated. There were no differences in prevalences between the two risk periods. Co-infections in L. glabra were more frequent than expected. The prevalences of infection with F. hepatica or P. daubneyi in L. glabra were higher in farms rearing sheep than in farms rearing cattle. A similar finding was also noted for L. truncatula infected with P. daubneyi only. L. glabra was a much better intermediate host for F. hepatica and P. daubneyi when it was the only available snail, possibly indicating an adaptation of parasites to their less usual host in local conditions. The two trematodes preferably developed in L. truncatula rather than in L. glabra when both host species lived in the same places.

Fasciola hepatica: an unusual development of redial generations in an isolate of Lymnaea truncatula.

Single-miracidium infections of Lymnaea truncatula by Fasciola hepatica were experimentally carried out to identify the redial generations of this trematode when the larval development was unusual (when the first-appearing mother redia, or R1a redia, died after its exit from the sporocyst). Four parameters were measured in the body and pharyngeal region at weekly intervals. At day 49 post-exposure at 20°C, the body of the second mother rediae (R1b) was significantly longer than that of the subsequent generations, R2a and R2b/R3a (a mean of 3.0 mm instead of 1.0 and 0.9 mm, respectively). The body was significantly wider in the R1b and R2a groups than in the R2b/R3a rediae. The pharyngeal lumen was significantly wider in the R1b group than in the R2a and R2b/R3a rediae (a mean of 48.6 µm instead of 10.8 and 3.3 µm at day 49). The thickness of the pharyngeal wall did not differ in the R1b and R2b/R3a groups, but was significantly lower in the R2a group (19.5 µm instead of 23.0-23.6 at day 49). There was better development of R1b and R2b/R3a rediae in the snails when the R1a redia died, compared with normal larval development (with a living R1a redia).

Morphological variability in Fasciola hepatica eggs in ruminants, rodents and lagomorphs.

The length and width of 1297 Fasciola hepatica eggs shed in cattle hosts, 337 in sheep and 199 in nutria, were measured from several parts of France. The data were compared with those obtained from other studies in Spain, France (where rats were also investigated), Germany and the Netherlands. One way analysis of variance and discriminant analysis were used to assess differences between host origins. The distribution of length and width of eggs were analysed using skewness and kurtosis Fisher coefficients. The eggs recovered from sheep, cattle, rodents and lagomorphs were different in size: the eggs found in rodents (length L x width W in microm: 8592) and lagomorphs (L x W in microm: 9100) were smaller than those found in sheep and cattle (L x W in microm: 10,000). These morphological differences in F. hepatica eggs were host-induced in rats (L x W in microM: 9709 in cattle to 8949 in rats) and rabbits (L x W in microm: 9709 in cattle to 8432 in rabbits). These differences in size of eggs might correspond to their being less able to develop into miracidia in less frequent hosts such as rodents and rabbits.

Unusual transmission of the liver fluke, Fasciola hepatica, by Lymnaea glabra or Planorbis leucostoma in France.

Cases of fasciolosis in ruminants have been recorded in several French farms in the absence of Lymnaea truncatula, which is considered the only snail intermediate host in western Europe. These farms harbored other species of freshwater snails in large numbers (Lymnaea glabra, Physa acuta, or Planorbis leucostoma) and, in many cases, had cattle or sheep infected by another trematode (Paramphistomum daubneyi). These other freshwater snails may serve as intermediate hosts for F. hepatica due to a coexisting infection with P. daubneyi. We have demonstrated that L. glabra, either infected with F. hepatica alone or coinfected by P. daubneyi, was capable of developing a F. hepatica infection. A broader range of L. glabra size classes (up to 10 mm in height) were susceptible to infection if simultaneously infected with P. daubneyi. Planorbis leucostoma can only serve as an intermediate host for F. hepatica, if infected with P. daubneyi. Lastly, P. acuta smaller than 4 mm cannot serve as an intermediate host. These results may explain, in part, the maintenance of low-level F. hepatica infections in the absence of the normal intermediate host, L. truncatula.