Search

Single-miracidium infections of Lymnaea cubensis (Pfeiffer) from Guadeloupe with the giant liver fluke Fascioloides magna (Bassi, 1875) (Digenea) were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of the parasite. Controls were constituted by a French population of Galba truncatula (Miller) (a single generation) infected according to the same protocol. It was recorded that prevalence and intensity of F. magna infection in L. cubensis progressively increased from F1 to F5 generations. Cercarial shedding of F. magna was noted only within F5 generation of L. cubensis. However, most measured parameters of infection in this species were significantly lower than those noted for G. truncatula and most L. cubensis died after a single shedding wave. Despite this, L. cubensis can be added to the list of potential intermediate hosts of F. magna.

Experimental infections of Egyptian Pseudosuccinea columella with one or two miracidia of Fasciola hepatica per snail were carried out to determine the developmental pattern (normal or abnormal) of redial generations and specify the number of free rediae developing in snails according to their generation. Controls were constituted by a French population of Galba truncatula infected according to the same protocol. Most infected P. columella showed a normal development of redial generations (96.2–98.1 vs 75.5–85.7 % for G. truncatula). In each redial category, free rediae were more numerous in P. columella than in G. truncatula, and their number were also greater in the two-miracidia groups than in single-miracidium infections for each lymnaeid considered separately. This increase in redial production was mainly due to the number of first mother (R1a) rediae producing daughter rediae only: 2 per P. columella (vs one redia in G. truncatula) in single-miracidium groups and 3.1 (vs 1.9) in the two-miracidia groups. In P. columella, the mean total number of free rediae developing in single-miracidium and bimiracidial infections was 77.2 and 117.6, respectively (instead of 33.5 and 52.1 rediae in G. truncatula). The number of F. hepatica rediae present in P. columella was related to the number of fully grown sporocysts and the quantity of R1a rediae which developed into the snail body.

International audience; The severity of toxoplasmic infection depends mainly on the immune status of the host, but also on the Toxoplasma gondii strains, which differ by their virulence profile. The relationship between the human host and T. gondii has not yet been elucidated because few studies have been conducted on human models. The immune mechanisms involved in the persistence of T. gondii in the brains of immunocompetent subjects and during the reactivation of latent infections are still unclear. In this study, we analyzed the kinetics of immune mediators in human nervous cells in vitro, infected with two strains of T. gondii. Human neuroblast cell line (SH SY5Y), microglial (CMH5) and endothelial cells (Hbmec) were infected separately by RH (type I) or PRU (type II) strains for 8 h, 14 h, 24 h and 48 h (ratio 1 cell: 2 tachyzoites). Pro-inflammatory protein expression was different between the two strains and among different human nervous cells. The cytokines IL-6, IL-8 and the chemokines MCP-1 and GROα, and SERPIN E1 were significantly increased in CMH5 and SH SY5Y at 24 h pi. At this point of infection, the parasite burden declined in microglial cells and neurons, but remained high in endothelial cells. This differential effect on the early parasite multiplication may be correlated with a higher production of immune mediators by neurons and microglial cells compared to endothelial cells. Regarding strain differences, PRU strain, but not RH strain, stimulates all cells to produce pro-inflammatory growth factors, G-CSF and GM-CSF. These proteins could increase the inflammatory effect of this type II strain. These results suggest that the different protein expression profiles depend on the parasitic strain and on the human nervous cell type, and that this could be at the origin of diverse brain lesions caused by T. gondii.

Experimental infections of Lymaea truncatula, using two susceptible snail populations (Berneuil, or Migné, central France) and four isolates of Fasciola hepatica miracidia differing in their mammalian host of origin (cattle, nutrias, rabbits, or sheep), were performed under laboratory conditions to determine whether the host of origin had an effect on the daily production of cercariae. Snails were each subjected to bimiracidial exposures and were then reared under semi-natural conditions (a constant temperature of 20 degrees C and natural photoperiod). Significantly lower values were noted in the rabbit groups for survival rates at day 30 post-exposure, as well as for prevalences of infection, snail growth. duration of shedding period, and the total numbers of cercariae these snails shed. The total number of cercariae shed by both nutria groups was significantly higher than those recorded in the six other infected groups. In the cattle, rabbit, and sheep (Berneuil only) groups, the peaks in the daily distribution of cercariae occurred between day 2 and day 4 after the first shedding, and the number of cercaria-shedding snails decreased with increasing number of shedding waves. In contrast, in the three other groups, the peaks were only observed between days 20 and 45. Snails shedding their cercariae during nine or more waves were numerous in these last groups. No infradian-type rhythm in the daily distribution of cercarial numbers over the shedding period was noted for any snail group. The highest production of F. hepatica cercariae in both nutria groups would be a consequence of a higher success rate of miracidia when they infected an allopatric population of snails. The absence of an infradian-type rhythm in the distribution of daily cercarial numbers in the eight groups suggests that this rhythm, if it occurs, would only be influenced by temperature and thus be limited to periods with optimal conditions for cercarial shedding.

Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rDNA, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna.

Bimiracidial infections of Lymnaea ovata with Fasciola hepatica were performed under laboratory conditions to determine the susceptibility of snails from six French populations to trematode infection. In five populations of L. ovata the prevalence of infection in the 1-mm groups ranged between 2.7% and 43.7% at day 35 postexposure; it decreased in the 2-mm snails and was zero in larger groups. In the snails from Thenay (periodically polluted brook) the prevalence of F. hepatica infection decreased from the 1-mm group to the 8-mm group (from 23.9% to 1.0%) and was zero in the 10-mm L. ovata. The total number of cercariae shed per snail was 18.3 in the 1-mm group, increasing to 117 in the 8-mm group. The latter findings could be interpreted as a consequence of periodic pollution in the brook of Thenay; pollution might disrupt the defense system of L. ovata and facilitate the subsequent larval development of F. hepatica.

International audience; Allopatric infections of French Galba truncatula with an Argentinean isolate of Fasciola hepatica were carried out to determine the infectivity of foreign miracidia in three snail populations differing by their susceptibility to French miracidia (two highly and one poorly susceptible populations). Sympatric infections of G. truncatula with French miracidia were used as controls. Compared to sympatric infections of G. truncatula, snail survival at day 30 post-infection in allopatric groups was significantly lower in a highly susceptible population and significantly greater in the other two. Prevalence in snails infected with the allopatric isolate was significantly lower (16.4-34.5 % instead of 58.6-72.1 %), whereas their patent period was significantly longer (a mean of 69.9-85.9 days instead of 6.4-20.7 days). The mean number of metacercariae was also higher in allopatric groups (236.5-897.3 per cercariae-shedding snail instead of 70.7-222.1). Owing to longer patent periods, the Argentinean isolate of F. hepatica was less pathogenic for these snails. The lower prevalence of infection, the longer patent period and the higher number of metacercariae noted in allopatric groups might be the consequence of an adaptive mechanism used by this digenean introduced to the New World to infect new populations of unusual intermediate hosts.

A total of 850 pre-adult Galba truncatula (shell height, 4 mm), originating from four French snail populations differing in shell height at the adult stage (from 6.5 to 12 mm), were individually subjected at 20°C to single-miracidium infections with Fascioloides magna. At day 75 post-exposure, the surviving snails were dissected, and rediae and cercariae were counted. Snail groups differed in shell growth during the experiment: from 1.8 ± 0.4 mm in group A up to 4.0 ± 1.1 mm in group D. The prevalence of F. magna infection, the numbers of free rediae and cercariae significantly increased together with increasing growth of infected snails during the experiment. Group A produced 1–6 first-generation rediae per snail and the mean daughter redia production ranged from 7.5 second-generation rediae (when a single first generation per snail developed) to 2.3 (6 first-generation rediae per snail). In contrast, up to ten first-generation rediae were noted in group D, and each mother redia gave daughter rediae with averages ranging from 1.5 (ten first-generation rediae per snail) to 13 (a single first generation per snail). In conclusion, the development of F. magna in G. truncatula exhibited both inter- and intrapopulation variability, where the development of rediae and cercariae was positively correlated with snail growth.

Two French populations of Galba truncatula were subjected to experimental infections with Egyptian and French isolates of Fasciola sp. miracidia, originating from cattle and sheep, to compare characteristics of snail infections in allopatric and sympatric groups. All sampled Egyptian isolates were identified as Fasciola hepatica using microsatellite markers. Compared to snails infected with French miracidia, snail survival at day 30 post-exposure was significantly greater in the Egyptian groups, while prevalence of infection was significantly lower (in an Egyptian group infected with cattle-derived miracidia) or did not show any significant differences in the other three cases. The total number of metacercariae was significantly higher in the four Egyptian groups. However, snail population and the mammalian origin of F. hepatica had also a significant effect on this parameter. The dissection of snail cadavers showed a significantly higher number of free rediae in the Egyptian groups, even if snail population also had a significant effect on the redial burden. Both Egyptian isolates of F. hepatica could easily develop in French snails, causing a low mortality in snails and inducing a metacercarial production higher than that noted in sympatric infections. However, the mammalian origin of F. hepatica eggs and the quality of snail populations as intermediate hosts had to be taken into account for studying local adaptation in reason of their effects on this process.

International audience; Slaughterhouse samples were analysed over a two-year period (September 2010-August 2012) in Jijel (northeastern Algeria) in order to determine seasonal variations in the prevalence and intensity of bovine paramphistomosis in a Mediterranean climate and identify paramphistome species using molecular biology. In spring and summer, significantly higher prevalences and lower parasite burdens were noted in bull calves, thus indicating an effect of season on these parameters. In contrast, the differences among seasonal prevalences or among seasonal parasite burdens were not significant in the case of old cows. Eleven adult worms from the slaughterhouses of Jijel and three neighbouring departments (Constantine, El Tarf and Setif) were analysed using molecular markers for species identification. Two different species, Calicophoron daubneyi and C. microbothrium, were found. The presence of these two paramphistomids raises the question of their respective frequency in the definitive host and local intermediate hosts.

Two hundred Galba truncatula, originating from a French population, were subjected to single-miracidium infections with an allopatric isolate (origin, Argentina) of Fasciola hepatica. The control group was constituted by 100 snails coming from the same population and exposed to sympatric miracidia of F. hepatica according to the same protocol. Snail samples were collected bimonthly from both groups between day 14 and day 112 p.e. (at 20 C) and snail dissections were performed to count free rediae, intraredial morulae and free cercariae. Third and fourth generation rediae were significantly more numerous in the allopatric group, while the number of first generation rediae was significantly lower. In the sympatric group, the decrease in the number of intraredial morulae in the first, second and third redial generations was significantly faster. Free cercariae within the snail body were significantly more numerous in the sympatric than in the allopatric groups, whatever the date of snail dissection. The changes in redial development and cercarial production noted in the Argentinean group might be due to the evolution of South American flukes in a divergent way after the introduction of foreign infected ruminants in this continent from the 15th century., Facultad de Ciencias Veterinarias

International audience; Experimental infections of Galba truncatula (two populations) with Fasciola hepatica or Paramphistomum daubneyi were carried out to study the effect of water temperature changes (3 h at a mean of 12 °C every week) on cercarial shedding during the patent period. The results were compared with those of control snails infected according to the same protocol and always maintained at 20 °C. Compared to controls, a significant increase in the number of cercariae-shedding snails, a significantly longer patent period and significantly greater cercarial production were noted in temperature-challenged snails, regardless of the type of digenean infection. In contrast, the number of incompletely formed metacercariae was significantly higher in temperature-challenged snails than in controls. Incompletely formed metacercariae of F. hepatica consisted of cysts whose colour remained whitish after shedding (25.4% for temperature-challenged snails) or whose dome was flattened after encystment (74.6%). Those of P. daubneyi were totally dark brown or blackish after formation. These incomplete metacercariae might originate from young differentiating cercariae within the snail body (F. hepatica) or from cercariae which died just after encystment (P. daubneyi). The use of regular temperature changes for snails infected with F. hepatica or P. daubneyi must be monitored carefully during collection of metacercariae to select completely formed cysts for infecting definitive hosts.

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.

Several experiments on the breeding of trematode-infected Galba truncatula for obtaining and packaging Fasciola hepatica and Paramphistomum daubneyi metacercariae were carried out to determine the more convenient methods to use for commercial production of these infective stages. Compared to the breeding of infected snails in aquaterraria, the use of 14-cm Petri dishes allowed a greater prevalence of snail infection and a higher number of metacercariae. The production of these larvae was still 2.3–3.4 times greater if infected snails were dissected during the patent period. The aspiration of these metacercariae at the extremity of a Pasteur pipette significantly shortens the time necessary for their transfer from Petri dishes to Eppendorf tubes. Using 14-cm Petri dishes, snail dissection and metacercarial aspiration for their transfer strongly reduce the cost price for metacercarial production of the trematodes Fasciola hepatica and Paramphistomum daubneyi.

Allopatric and sympatric infections of Lymnaea neotropica and Lymnaea viatrix var. ventricosa with Argentinean and French isolates of Fasciola hepatica were carried out to determine the capacity of these snails to produce metacercariae and to verify if this capacity changed with snail generation. The same process was also made with a French population of Galba truncatula known to be highly susceptible to French isolates of the parasite. In each lymnaeid species separately considered, the survival rate at day 30 post-exposure and prevalence of F. hepatica infection in the group infected with Argentinean miracidia were significantly greater than those recorded in the corresponding French one. Compared to infected G. truncatula, both South American lymnaeids had longer patent periods and produced a higher number of metacercariae. The highest infections were noted with L. v. ventricosa. In the three snail species, metacercarial production was more important with the Argentinean isolate of miracidia than with the French one. If three successive generations of L. v. ventricosa are exposed to the same French isolate of miracidia, cercarial production significantly increased from parents to the F2 generation, while the other characteristics of infection only showed insignificant variations. L. neotropica and L. v. ventricosa are better intermediate hosts for French F. hepatica than local G. truncatula. The numerical increase of shed cercariae in the F1 and F2 generations of L. v. ventricosa demonstrates a rapid adaptation of this species to the French isolate of the parasite., Facultad de Ciencias Veterinarias, Centro de Diagnóstico e Investigaciones Veterinarias

PurposeTo evaluate the RARE (Réseau Action-Recherche sur l'Epilepsie) program, a model of managing and treating people with epilepsy (PWE) at a primary health-care level in rural areas of Mali, we assessed treatment efficacy and compliance of patients who underwent the first year follow-up.MethodsA network of rural general practitioners (GPs) settled in six rural districts of the regions of Koulikoro, Segou and Sikasso, was involved in the diagnosis, evaluation and monitoring of all the identified PWE and in the distribution of phenobarbital (PB). All the participants were included in a prospective database and followed-up by GPs at 4 months intervals during the first year. Seizure frequency, treatment doses and appearance of adverse events (AEs) were systematically recorded. Efficacy was evaluated in terms of reduction of seizures frequency while noncompliance in terms of time to study withdrawal for any cause.Key findings596 patients treated with PB were included in the analysis. Of these, 74.0% completed the first year follow-up. At the final visit, 59.6% were seizure-free: 31.0% for 12 months, 10.2% for 8 months and 18.4% for 4 months. Adults and patients with convulsive seizures were the most drug-resistant (pSignificanceThis study showed a good response and compliance to the treatment and allowed the identification of some factors associated with failure of management in a setting very near to clinical practice. Awareness campaigns are needed to assure a broader accessibility to treatment and to improve the compliance and continuity with treatment programs.

International audience; Field investigations in 361 liver fluke infected cattle- or sheep-breeding farms on acid soil were carried out during thirty years in March and April to record indicator plants in relation to the category of site colonized by the intermediate host of liver fluke, the snail Galba truncatula. Seven types of snail zones and six species of indicator plants were recorded in the 7709 positive sites studied. The most frequent habitats were located at the peripheral extremities of open drainage furrows. Juncus acutiflorus, Juncus effusus, Glyceria fluitans, and Agrostis stolonifera were the indicator plants. Plant indicators were highly efficient (38-80% of variance explained) when used with the size of site area within a hydrographical zone. The identification of positive sites for G. truncatula is a key for controlling the intermediate host on pastures through biological control with predatory molluscs. The positive sites may be detected using a two-step method including first categorization of hydrographical zones and then, within a zone, use of one or several indicator plants (frequently J. acutiflorus).

Natural infections of Galba truncatula with Haplometra cylindracea were followed from 2001 to 2009 to determine if their characteristics were similar when snails came from water collections frequented by Bufo bufo or by frogs and newts for their egg-laying. Snail samples were collected from both types of sites to count shed cercariae for three days and also free cercariae when snails were dissected. In sites only frequented by B. bufo, cercarial shedding occurred earlier than in those colonized by frogs and newts (March instead of April-May). In contrast, the number of cercariae shed during three successive days was significantly higher in May. This variation in the dates of cercarial shedding might be due, either to a synchronism between cercaria-releasing snails and the presence of the definitive host (tadpoles) in water collections, or to an earlier infection of overwintering snails in autumn by H. cylindracea miracidia in the case of toad-frequented sites.

We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.

International audience; Experimental infections of Egyptian Radix natalensis with French miracidia of Fasciola hepatica were carried out to determine if this snail might act as an intermediate host in the life cycle of this digenean in Egypt. Single exposures of R. natalensis to miracidia (2/snail) and two successive exposures (a total of 4 miracidia/ snail) were performed using lymnaeids measuring 1 to 6 mm in height. Live larval forms of F. hepatica were noted in single- and double-exposed snails. In double exposures, a significant increase of snail survival on day 28 post-exposure (at 24 degrees C) and an decrease in prevalence were noted when the height of snails at exposure was increasing. Cercariae of F. hepatica were shed by these snails (90.7/snail) during a mean patent period of 24.3 days. All snails have released these cercariae during 2-13 waves of shedding. According to these results, R. natalensis can be considered a potential intermediate host of F. hepatica in Egypt.

Experimental infections of EgyptianRadix natalensiswith a French isolate ofFasciola hepatica(each snail was subjected twice to a bimiracidial exposure) were carried out to determine how many sporocysts grew in these snails and to study the developmental patterns of redial generations. Single-sporocyst infections were found in 69.3% (34/49) of infected snails, with equivalent numbers of normal and abnormal patterns. Snails with two- and three-sporocyst infections were 24.4% and 6.1%, respectively. In single- and two-sporocyst infections at days 42 and 56 post-exposure, the total redial burden was significantly higher in snails with a normal redial development. In two- and three-sporocyst infections, the overall maturity of rediae was delayed at days 42 and 56. The high frequency of abnormal patterns inR. natalensis(53.1% of all infected snails showed degeneration of a first mother redia) might be due to incomplete adaptation between the snail population and the parasite. The delayed redial maturity in two- and three-sporocyst infections can mainly be explained by the volume of the snail body, which would be insufficient to allow the simultaneous differentiation of most rediae over time.

Des expositions individuelles mono- et bimiracidiennes de Galba truncatula avec Fasciola hepatica (deux groupes) ou avec Paramphistomum daubneyi (deux groupes) ont ete realisees dans les conditions du laboratoire afin de comparer la production parasitaire chez des limnees qui emettent des cercaires (mollusques EC) par rapport a celle trouvee chez des individus parasites mais sans emission (limnees NEC). Les limnees vivantes et les cercaires independantes ont ete comptees chez les mollusques des deux categories apres leur dissection a intervalles reguliers (a 24°C). Dans les quatre groupes, les nombres de redies et de cercaires independantes notes au 75 eme jour post-exposition (F. hepatica) ou au 85 eme jour (P. daubneyi) sont significativement plus eleves chez les mollusques EC que chez les NEC. Chez ces derniers, le nombre de cercaires ne presente pas de variation significative du 45 eme au 75 eme jour (F. hepatica: les deux groupes) ou du 55 eme au 85 eme jour (P. daubneyi, infestations individuelles a un seul miracidium) alors qu'il decroit de maniere significative lorsque la duree de l'infestation augmente (P. daubneyi, infestations a deux miracidiums). Chez les limnees NEC, la presence de cercaires independantes trop nombreuses dans le corps du mollusque (celui-ci ne permet le developpement que d'un nombre donne de redies) bloquerait rapidement le developpement des redies et la differenciation intra-redienne des autres cercaires. La diminution numerique des cercaires de P. daubneyi chez les mollusques NEC (groupe a deux miracidiums) pourrait etre due a la lyse des nouvelles cercaires juste formees, probablement en raison d'un manque d'aliments disponibles pour celles-ci dans le corps du mollusque.

Experimental infections of pre-adult Galba truncatula (two populations) using one, two, three or five miracidia of Fasciola hepatica per snail were carried out to determine the total number of cercariae produced by snails when they were sacrificed at day 70 post-exposure (at 24°C). When the number of miracidia used for each snail increased at exposure, significant numerical augmentations of live rediae and of intraredial differentiating cercariae were noted. In contrast, only insignificant differences between mean numbers of free cercariae were found. The number of metacercariae produced by each snail (after shedding and/or after dissection) increased in infections with up to three miracidia per snail in each population and strongly decreased in infections with five miracidia per snail. Compared to the other types of infections, the delay of cercarial differentiation noted in the five-miracidia groups was probably due to the volume of the snail host, which would be too small to assure the complete development of five sporocysts.

International audience; Experimental infections of Galba truncatula with Paramphistomum daubneyi were carried out to determine at day 50 (at 24 degrees C) the numbers of sporocysts, which grew in infected snails via the count of first- and second-generation rediae. In snails individually exposed to one, two, three, four, or five miracidia, the numbers of first-generation rediae increased from the one-miracidium group to the five-miracidium snails (from a mean of 6.7 to 26.1), while second-generation rediae decreased in number (from 6.2 to 0.9, respectively). This scale of redial numbers was used to determine the number of sporocysts, which grew in naturally infected snails collected from sedimentary or acid soils between 1993 and 2006. In cercariae-containing snails, natural infections resulting from the development of one to five sporocysts were found in both samples of G. truncatula examined. The numbers of 3-, 4-, and 5-sporocyst infections were increasing over time since 1997, 2000, and 2003, respectively. The utility of such multiple-sporocyst infections is open to question, as the differentiation of second-generation rediae and that of procercariae were delayed and always limited. They might be interpreted as a consequence of a zoonosis, which has been spreading since 1990 in ruminants of central France.

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°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.

International audience; Experimental infections of Galba truncatula with Fasciola hepatica, Fascioloides magna, or Paramphistomum daubneyi were carried out at 20 degrees C to determine if the use of 14-cm petri dishes for breeding lettuce-fed snails enhanced the characteristics of snail infections. Compared to infected snails raised in boxes up to day 30 post-exposure and later in individual 35-mm dishes, the survival of G. truncatula kept in 14-cm dishes and the shell height of cercariae-shedding snails during the first 45 days were higher, whatever the digenean species is. The consequence of such enhanced characteristics was a greater production of metacercariae in the case of F. hepatica (1.7 to 5.6 times higher) and P. daubneyi (2.3 times). In contrast, metacercariae of F. magna were few in number, whatever the method of snail breeding is, and this might be explained by a still incomplete adaptation between the parasite of Czech origin and the French population of G. truncatula. The use of these 14-cm dishes reduced the time necessary for snail maintenance and metacercaria collection, thus allowing a decrease in the cost price of these larvae for commercial production.

As large numbers of metacercariae of Fasciola hepatica are necessary for research, experimental infections of Galba truncatula and Pseudosuccinea columella with this digenean were carried out to determine the better intermediate host for metacercarial production and, consequently, the most profitable snail for decreasing the cost price of these larvae. Pre-adult snails (4 mm in shell height) originating from two populations per lymnaeid species were individually exposed to two or five miracidia, raised at 23 °C and followed for cercarial shedding up to their death. Compared to values noted in G. truncatula, the survival of P. columella on day 30 post-exposure was significantly greater, while the prevalence of F. hepatica infection was significantly lower. In the four P. columella groups, metacercarial production was significantly greater than that noted in the four groups of G. truncatula (347-453 per cercariae-shedding snail versus 163-275, respectively). Apart from one population of G. truncatula, the use of five miracidia per snail at exposure significantly increased the prevalence of F. hepatica in P. columella and the other population of G. truncatula, whereas it did not have any clear effect on the mean number of metacercariae. The use of P. columella for experimental infections with F. hepatica resulted in significantly higher metacercarial production than that noted with G. truncatula, in spite of a lower prevalence for the former lymnaeid. This finding allows for a significant decrease in the cost price of these larvae for commercial production.

International audience; Single-miracidium infections of Fascioloides magna in two populations of Galba truncatula were carried out under laboratory conditions to count free rediae and cercariae in snail cadavers just after death. Cercaria-shedding snails were in low numbers, and their shell height at day 60 p.e. was significantly greater than that of numerous infected snails that died without cercarial shedding. In snails that died between days 44 and 60 p.e. (at 20 degrees C), the numbers of second-generation rediae significantly increased with increasing shell heights of infected snails. First-generation rediae showed insignificant, quantitative variations, while scarce rediae of the third generation were only found in the highest snails. Cercariae were only produced by the second redial generation. In both groups of snails, free cercariae appeared from 6 mm of shell height, and their numbers increased in the upper classes up to 32.9 per snail. Metacercariae were only found from 9 mm of shell height and were in low numbers. The global cercarial production ranged from 163.5 to 210.0 in the highest classes of snail size from both groups and was limited, whereas the mean burdens of free rediae fluctuated from 39.5 to 43.9. The death of numerous infected snails without cercarial shedding might be explained by the presence of a very high number of second-generation rediae simultaneously growing within the body of these snails.

International audience; A retrospective study on a total of 669 Galba truncatula (three groups) experimentally infected with Fasciola hepatica was carried out to determine why 6- to 8-day interwave intervals, separating the successive waves of cercarial shedding, occurred with a regular pattern in some snails during the whole patent period. In the three groups of snails, the number of cercariae per shedding wave peaked at the second wave and subsequently decreased up to the fifth wave. The mean length of interwave intervals ranged from 6.8 to 7.8 days and only showed insignificant variations. The number of free cercariae recorded at the end of each interwave interval significantly decreased over the patent period. Similar findings were also noted for intraredial cercariae in the first redial generation and the first cohort of the second generation. By contrast, the number of intraredial cercariae significantly increased along the patent period from the second interwave interval. In the case of each interval separately considered, most numerical variations noted for free cercariae or for intraredial cercariae were insignificant. The periodicity of 6.8-7.8 days found for interwave intervals in the present study might correspond to the infradian-type rhythm already reported for the cercarial shedding of F. hepatica. However, snails showing such regular pattern in cercarial shedding along the patent period were few in number, and one may wonder about the reasons of such snails in the case of F. hepatica.

International audience; Experimental infections of Omphiscola glabra (preadult snails), originating from central France, to a Czech isolate of Fascioloides magna miracidia were carried out to determine if the local populations of O. glabra may ensure the larval development of this parasite and to compare these results with those noted for a natural snail host, Galba truncatula. The presence of experimentally infected snails was noted in the six populations of snails studied. However, only a few snails shed their cercariae (O. glabra 5.3 to 17.1%, G. truncatula 15.1% in the first population, and no shedding in the other). The shell heights of cercariae-shedding (CS) snails were significantly greater than those of other infected snails, for O. glabra as well as for G. truncatula. The number of metacercariae noted in each snail group was low and showed insignificant variations. When experimental infections of O. glabra were performed in relation to the shell height of snails (from 1 to 14 mm) at miracidial exposure, the prevalence of infected snails significantly decreased with increasing shell heights at exposure. However, the presence of CS snails was only noted from the 5-6 to the 9-10 mm groups, and the mean number of metacercariae per group ranged from 27 to 44.2. Despite the high infectivity of the Czech isolate of F. magna miracidia, there was an incomplete adaptation with the French G. truncatula and O. glabra used in this study, as the metacercarial production was low, and cercarial shedding only occurred for snails which showed a strong increase of their shell height during F. magna infections.

Field investigations in two lymnaeid habitats located along river banks were carried out over three years to study snail colonization in relation to water-level variations, to determine the number of annual generations, and to analyse the ability of G. truncatula to emerge from water. As these river banks were located 6.7 km upstream from a dam, water recedes each day, thus permitting the study of snails on banks for several hours per day. From March to December, snails followed seasonal variations of water-level. However, only a few G. truncatula were observed following the daily retreat of water. Two annual generations of G. truncatula, the first from the onset of July and the second in December, were noted in these habitats. When water receded each day, the mean distance covered by non-aestivating snails ranged from 25 to 44 cm. However, they escaped water rise by seeking refuge in still emerged zones of their habitats. From July to October, snails following each day water receding showed a significantly quicker growth than that of G. truncatula which only moved in the same zone of their habitat. Populations of G. truncatula living along these river banks would be completely adapted to their habitat. The escape of snails from water rise between July and October might correspond to a physiological reaction of survival when a too rapid increase of water-level occurs.

Field investigations on the control of Galba truncatula by Zonitoides nitidus were carried out over the past 30 years in the different types of lymnaeid habitats located in central France. When a layer of mowed vegetation was used to cover G. truncatula habitats at the end of June, the introduction of adult Z. nitidus (20/m2) eliminated lymnaeid populations after 2 years of control in habitats located in swampy meadows, around the heads of intermittent springs, in areas trampled by cattle, and along river or pond banks. In the case of wild watercress beds, 3 years were necessary. The best results (elimination of G. truncatula after a single year of control) were obtained using an association of snails (Z. nitidus + Oxychilus draparnaudi), or a mixed control (a first application of 0.1 mg/l CuCl2, followed 3 months later by the introduction of Z. nitidus). Recolonization of treated habitats by G. truncatula coming from downstream populations was noted 3 years following the last application of biological control. Apart from the regular introduction of Z. nitidus in several watercress beds, the use of this snail to control G. truncatula has not become generalized in cattle- and sheep-breeding farms. The reasons for this situation are probably the complexity of applying this control in the field by nonspecialists and the difficulty of selecting the period of snail control at the end of June due to frequent local rainfall at this time.

International audience; OBJECTIVE: The aim of this communication was to report clinical disorders and biological abnormalities in patients followed over a 3- to 18-year period after the last date of fasciolosis treatment. These observations were noted in 87 individuals (out of 711) during a 30-year epidemiological survey. RESULTS: We noted diffuse pain in the right hypochondrium, persisting 2 to 6 years after treatment (3.7% of persons), and weight loss appearing during treatment and persisting afterwards (2.1%). CONCLUSIONS: Our current state of knowledge leads us to asking whether pain in the right hypochondrium and weight loss would not be due to the presence of persistent hepatic lesions following fasciolosis treatment. Other investigations in patients after treated fasciolosis are necessary to confirm these abnormalities and determine their frequency.

A total of 59 natural watercress beds in the Limousin region (central France) was surveyed over a 15-year period (1990-2004) to detect the contamination of watercress by the metacercariae of Fasciola hepatica and to determine the presence of larval forms in the two species of lymnaeids which live in these waterholes in June and July. The number of beds contaminated with F. hepatica metacercariae varied over the years, and the burden of the larvae on plants was low: a mean of 2.6-6.3 per bed. The same variability was also noted for natural infections of Galba truncatula with F. hepatica, as the annual prevalences ranged from 1.2% to 2.4%. Natural infections of Omphiscola glabra with F. hepatica were only detected from 1996 and the annual prevalences subsequently increased up to 1.4-1.8% between 2001 and 2004. However, for both lymnaeids, the variations in these prevalences with year were insignificant. The contamination of these beds with F. hepatica over the past 15 years was similar to that recorded in the same sites between 1970 and 1986. The main changes were the appearance of another digenea, Paramphistomum daubneyi, in the beds, and the possibility for O. glabra to naturally sustain the larval development of F. hepatica.

As most natural watercress beds in central France are located upstream of the permanent habitats of two lymnaeid species, Galba truncatula and Omphiscola glabra, field investigations were made from 1999 to 2004 on 67 beds to determine why the contamination of watercress with Fasciola hepatica is irregular over time in these sites, while definitive hosts, especially lagomorphs, are regularly found infected around them. Snails are able to migrate upstream in winter and spring towards the beds, and a 4-year survey demonstrated the existence of annual variation in the colonization of these sites by snails. In the 45 beds irregularly contaminated with F. hepatica over time, 37.7–62.2%, according to the year, were not populated by lymnaeids, in spite of the presence of snail populations downstream. G. truncatula was found alone in 8.8–13.3% of sites and was the first colonizing snail in 24.3–33.3% when the two lymnaeid species successively settled in these waterholes. The colonizing ability of O. glabra was more limited, as it was observed alone in 2.2% of beds and was the first colonizing snail in only 2.2–20% of them. The distances covered by these snails significantly increased with increasing migration time. After migration, a few overwintering snails (3.8% of G. truncatula and 6.8% of O. glabra) are able to colonize the beds, but their numbers decreased when the distance of migration was greater. O. glabra migrated more quickly and reached more watercress beds than G. truncatula. However, this did not influence the natural infections of snails, which were more frequent in the F1 of G. truncatula. The variability observed in the colonization of beds by snails might explain the regular or irregular contamination of wild watercress by metacercariae of F. hepatica. One of the factors which may explain this variability is the occurrence of showers in spring, so that a few snails are able to reach watercress beds after their upstream migration.

A retrospective study on 1,211 snails naturally infected with Fasciola hepatica in central France was carried out to determine the numbers of full-grown sporocysts from which infections derived, to specify the developmental patterns of redial generations (normal or abnormal) and to count live and free rediae. In the department of Haute Vienne (siliceous subsoils), most snails showed single-sporocyst infections, with normal (46.3%) or abnormal (33.9%) development of redial generations. Two-sporocyst infections were scarcer (a total of 10.6%), while snail co-infections with F. hepatica and Paramphistomum daubneyi (8.0%) were found since 1996. In the department of Indre (calcareous subsoils), single-sporocyst infections showing normal development of rediae were the most numerous (58.0%), while the frequency of single-sporocyst infections with abnormal development was strongly decreased (4.3%). Two-sporocyst infections (with normal development of generations) and co-infections with F. hepatica and P. daubneyi slightly increased in frequency. The redial burdens found in snails collected from the department of Indre were significantly higher than those noted in snails originating from the department of Haute Vienne, whatever the type of snail infection; and these increases in numbers especially concerned the rediae of the second and subsequent generations. The results might be explained by the lower calcium ion content (7–23 mg/l) present in waters from the department of Haute Vienne which would induce a slower growth of infected snails (the shell height of adults scarcely reached 8 mm) and, consequently, would create barely favourable conditions for the development of the first rediae of the first generation within snails. However, the quality of the diet provided as food for snails and its influence on the development of redial generations cannot be excluded.

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.

Experimental infections of Galba truncatula with Fasciola gigantica or F. hepatica were carried out under laboratory conditions (20 degrees C) to determine the characteristics of rediae of both species via their morphometry and to find reliable measurements that might be efficiently used to discriminate between the rediae of both species of Fasciola. These results were compared to those of another snail: Radix natalensis, infected with either F. gigantica or F. hepatica under the same protocol. At day 28 post-exposure, abortive infections with F. hepatica were found in a group of R. natalensis. By contrast, live rediae were observed in the other three groups. The group of infected snails and the redial category significantly influenced the mean values of the seven measurements studied and those of three indices. Using the PSLD Fisher test, it was found that the index, distance from the anterior end of the body to the collar/length of the body, was an efficient means of distinguishing the rediae of F. hepatica from those of F. gigantica [second-appearing mother rediae (R1b) of the first generation, 0.14 instead of 0.22; daughter rediae (R2a) produced by the first mother rediae, 0.19 instead of 0.24]. Another index, distance from the anterior end of the body to the collar/diameter of the collar, could also be used to discriminate between rediae (R1b, 0.80 for F. hepatica instead of 1.09 for F. gigantica; R2a, 0.90 instead of 1.26, respectively). Compared to measurements recorded for the rediae of F. hepatica, rediae of F. gigantica can be characterized by the following measurements: the diameter of the pharyngeal lumen and the distance from the anterior end of the body to the collar for larvae developed in R. natalensis, and the length of the body and the distance from the posterior end of the body to lateral projections for those found in G. truncatula. The species of snail host and, consequently, its growth, as well as the species of Fasciola, had a significant influence on the morphometric characters of the redial stage.

Snails from two populations highly susceptible to Fasciola hepatica and their F1 generations were subjected to individual bimiracidial exposures to determine if changes noted in infection parameters were due to an effect imposed on the snail by the parasite, or to some other effect such as the food used for the snails. Apart from the higher survival of unexposed parents at day 30 post exposure (p.e.) and their higher shell heights at day 45 p.e., the differences between the survival rates of exposed parents, prevalences of infections, and shell sizes were not significant. In the F1 snails born to previously infected parents, the prevalences of F. hepatica infection and cercarial production were significantly lower than those noted for the F1 born to unexposed parents. The survival of these snails and their shell growth did not show any significant variation. The F1 snails born to previously exposed snails would have developed a partial resistance against F. hepatica and this process would probably be maximal in the first 2 weeks of larval development inside the snail.

As larval forms of Fasciola hepatica have periodically been detected in Omphiscola glabra after their collection from watercress beds or from meadows since 1995, field investigations in 37 populations of O. glabra were carried from 1996 to 2002. This was done in order to determine the changes in prevalences and intensities of these natural infections with F. hepatica in relation to the type of snail habitat and the year of snail collection. Snails infected with F. hepatica were found in all samples made in swampy meadows and roadside ditches in all years. In fenced pools and walled gardens, snail infections were only found from 1998 and 1999 onwards, respectively. In the four types of habitats, the prevalences of F. hepatica infections increased slightly over time (0.8–2.1% for snails sampled in swampy meadows, for example) but this increase varied with the habitat. The mean shell heights of infected snails (6.2–7.8 mm) were similar whatever the type of habitat. The numbers of cercariae-containing rediae counted in snails sampled in swampy meadows, roadside ditches, and fenced pools significantly increased over time. Significant numerical variation between these redial burdens was also observed in relation to snail habitat. As the larval development of F. hepatica is facilitated by the presence of another trematode larval form (Paramphistomum daubneyi), the finding of some naturally infected O. glabra in watering places known to have no contact with domestic or wild large mammals might be explained by the development of P. daubneyi in small mammals such as lagomorphs. However, a progressive adaptation of F. hepatica miracidia to O. glabra over time, which would permit the infection of snails at sizes larger than 2 mm, could not be excluded.

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.

A retrospective study was carried out on the experimental infections of Lymnaea truncatula with Fasciola hepatica performed over the last 20 years to determine if the populations of snails living in highland or lowland countries had the same ability to sustain trematode larval development. The six highland populations originated from the Peruvian Altiplano (altitude 2,800 m), the French Alps (2,300 m), and the Massif Central (900-1,400 m), whereas the 13 lowland populations came from different sites located in central France (90-250 m). Bimiracidial infections of 4-mm-high snails were performed to study cercarial shedding and to quantify their redial burden. Compared to lowland populations, snail survival at day 30 post-exposure was significantly higher in the highland L. truncatula (57-75% compared to 31-45%) and their lifespan was greater (a mean of 87-96 days for cercaria-shedding snails compared to 64-77 days). The prevalences of F. hepatica infections, the numbers of free rediae within snail bodies, and the numbers of cercariae did not show any significant differences between highland and lowland snails although the numbers of cercariae were clearly higher in the Peruvian and three French highland populations of L. truncatula. The long survival times of highland snails under laboratory conditions might be an adaptation of these L. truncatula to the more extreme highland climate. The better ability of highland snails to sustain parasite larval development suggests that they would be better intermediate hosts in the life cycle of F. hepatica than lowland populations.

As abrupt changes in water temperature (thermal shock) triggered a significantly greater cercarial emergence of Fasciola hepatica from experimentally infected Galba truncatula, laboratory investigations were carried out to study the influence of light on cercarial emergence in snails subjected to a thermal shock every week (a mean of 12 °C for 3 h) during the patent period. Thermal shock for these temperature-challenged (TC) snails was carried out outdoors under artificial or natural light, or indoors under constant artificial light. Compared with the infected control snails always reared indoors at 20 °C, the number of cercariae in TC snails subjected to a thermal shock and natural light outdoors was significantly greater. The repetition of this experiment by subjecting TC snails to the same thermal shock indoors under an artificial light level ranging from 600 to 3000 lux did not show any significant difference among the numbers of cercariae in the different subgroups. A detailed analysis of the results noted in the TC snails subjected to natural light during the thermal shock demonstrated that the number of cercariae-releasing snails was significantly higher between 601 and 1200 lux and for the highest nebulosity values (7–8 octas, which corresponds to a sufficiently or completely overcast sky). Contrary to the intensity of artificial light, which did not influence cercarial emergence, the natural light level had a significant effect on this process when F. hepatica-infected snails were subjected to a regular thermal shock during the patent period.

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.