Fasciola hepatica: the redial generations in juvenile Pseudosuccinea columella.

Juvenile Pseudosuccinea columella, measuring 1 or 2 mm in height, were subjected to single-miracidium infections with Fasciola hepatica to determine the developmental pattern of redial generations and count free and live rediae according to their generation. Controls were constituted of juvenile Galba truncatula infected according to the same protocol. In the four groups, redial counts were performed in snails dissected every week from day 7 to day 49 post-exposure at 20 °C. Most infected snails showed a normal development of redial generations, whatever the lymnaeid species. In P. columella, the total number of live rediae on day 49 was 24.6 and 34.6 per infected snail in the 1 and 2-mm groups, respectively (instead of 11.5 and 18.8 rediae in the corresponding groups of G. truncatula). A single mother redia (R1a) producing only daughter rediae of the second generation was noted in each snail of three groups, while the 2-mm P. columella showed the presence of a single (43 snails/71) or two (28/71) R1a redia(e) within their bodies. The mean number of other mother rediae and that of daughter rediae of the second generation were low in the 1 and 2-mm groups of both lymnaeids. Besides, there was a delay in redial development. The development of two live R1a rediae in several P. columella from the 2-mm group needs to verify if this process would be specific to P. columella or would occur in other lymnaeid species known for their good susceptibility to the digenean and their larger size to allow harbouring rediae.

Role of the lymnaeid snail Pseudosuccinea columella in the transmission of the liver fluke Fasciola hepatica in Egypt.

Experimental infections of three Egyptian Pseudosuccinea columella populations with sympatric miracidia of Fasciola sp., coming from cattle- or sheep-collected eggs, were carried out to determine the capacity of this lymnaeid to support larval development of the parasite. Using microsatellite markers, the isolates of Egyptian miracidia were identified as Fasciola hepatica. Apart from being independent of snail origin, prevalences ranging from 60.4 to 75.5% in snails infected with five miracidia of F. hepatica were significantly higher than values of 30.4 to 42.2% in snails with bi-miracidial infections. The number of metacercariae ranged from 243 to 472 per cercarial-shedding snail and was independent of snail origin, parasite origin and miracidial dose used for infection. If P. columella was subjected to two successive bi-miracidial infections with F. hepatica, prevalence of infection was 63.3%, with a mean of 311 metacercariae per snail. These values were clearly greater than those already reported for Radix natalensis infected with the same parasite and the same protocol. Successful experimental infection of P. columella with F. hepatica suggests that this lymnaeid snail is an important intermediate host for the transmission of fascioliasis in Egypt.

Fasciola hepatica: development of redial generations in experimental infections of Pseudosuccinea columella.

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.

Variations in local adaptation of allopatric Fasciola hepatica to French Galba truncatula in relation to parasite origin.

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.

Molecular identification of Fasciola spp. (Digenea: Fasciolidae) in Egypt.

A total of 134 Egyptian liver flukes were collected from different definitive hosts (cattle, sheep, and buffaloes) to identify them via the use of PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS1). Specimens of F. hepatica from France, as well as F. gigantica from Cameroon were included in the study for comparison. PCR products of ITS1 were subjected for digestion by RsaI restriction enzyme and visualized on agarose gel. According to RFLP pattern, Egyptian flukes were allocated into two categories. The first was identical to that of French hepatica flukes to have a pattern of 360, 100, and 60 (bp) band size, whereas the second resembled to that of Cameroonian gigantica worms to have a profile of 360, 170, and 60 bp in size. Results of RFLP analysis were confirmed by sequence analysis of representative ITS1 amplicons. No hybrid forms were detected in the present study. Taken together, this study concluded that both species of Fasciola are present in Egypt, whereas the hybrid form may be not very common.

The development of rediae of Fasciola hepatica in Radix natalensis subjected twice to bimiracidial exposures.

Experimental infections of Egyptian Radix natalensis with a French isolate of Fasciola 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 in R. 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.

Radix natalensis (Gastropoda: Lymnaeidae), a potential intermediate host of Fasciola hepatica in Egypt.

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.

Fasciola gigantica: larval productivity of sheep-derived miracidia in Radix natalensis and Galba truncatula.

Experimental infections of Egyptian Radix natalensis and French Galba truncatula with miracidia of Fasciola gigantica of sheep were carried out to determine the larval productivity of this parasite. Rediae and cercariae were thus counted in snails dissected at regular intervals from day 21 to day 49 postexposure (p.e.) at 24 degrees C, while cercarial shedding was studied in other two groups of snails after day 30 p.e. At day 49, the total number of free rediae and that of cercariae-containing rediae in R. natalensis (shell height, 10.0 mm) were 71.5 and 44, respectively, whereas mean values in G. truncatula (shell height, 5.7 mm) were 57.3 and 33 rediae, respectively. The life span of cercaria-shedding snails, the prepatent period, the patent period, and the total number of cercariae shed showed insignificant differences between both snail species. Compared to the data already obtained with a cattle isolate of parasite, the number of live rediae was significantly greater in G. truncatula and significantly lower in R. natalensis when exposed to sheep-originating miracidia. In cercariae, the differences between cattle- and sheep-derived infections were insignificant, whatever snail species. The results may be explained by the existence of an interpopulation of snail infection with F. gigantica, probably due to variations in frequency of natural encounters between this snail population and the parasite isolate. However, the better production of rediae and cercariae in G. truncatula might be due to the origin of snails used for this study because allopatric snails produced more larvae than sympatric congeners when they are subjected to experimental infections.

Update of fasciolosis-transmitting snails in Egypt (review and comment).

Several snail species may contribute in transmission of fasciolosis in Egypt. These molluscs show a variable sensibility to natural infections with Fasciola species. Radix natalensis is considered to be the essential intermediate host for F. gigantica based on field and experimental studies. Cercarial production from R. natalensis experimentally infected with F. gigantica is affected by the species of definitive host from which the eggs are obtained, as well as the different laboratory conditions. Another lymnaeid, Galba truncatula, may play a role in transmitting this parasite in Egypt, as it was found naturally infected with F. gigantica. Latter snail species, originated from France, was susceptible to experimental infections with Egyptian miracidia of F. gigantica and it had a cercarial production close to that of local R. natalensis. Two other snails, Pseudosuccinea columella and Biomphalaria alexandrina, were naturally found harboring larvae of Fasciola sp. At the level of intermediate hosts of F. gigantica, the conditions are thus favorable in Egypt to transmit fasciolosis which could also be caused by another fasciolid, F. hepatica, as the existance of this fluke was confirmed in Egypt.

The effect of clomiphene citrate and its Zu or En isomers on the reproductive system of the immature male rat.

The effect of clomiphene citrate (CC) and of its Zuclomiphene (ZuC) and Enclomiphene (EnC) isomers on the reproductive organs of immature male rats under different experimental conditions is reported. CC, ZuC, and EnC were administered daily to groups of either intact or castrated rats between the age of 21-44 d. This led to inhibition of weight increase of testis and accessory glands in the intact group. Spermatogenesis was arrested at the stage of primary spermatocyte following CC and ZuC treatment, and at the stage of young spermatids by EnC treatment. In the castrated group clomiphenes significantly stimulated weight increase of seminal vesicles (SV) compared with castrated control animals, but the former group were unable to achieve organ weight gain comparable to that in normal controls. Administration of human Chorionic Gonadotropin (hCG) together with CC or each of its isomers to intact animals, abolished the drug effect on spermatogenesis and on reproductive organ growth. Administration of CC, ZuC, and EnC together with testosterone to castrated animals, abolished the drug effect on growth inhibition of accessory glands. In intact treated rats LH and testosterone secretion were suppressed by all forms of clomiphenes. In the castrated group ZuC proved to be the most potent inhibitor of LH secretion. Therefore, it is inferred that ZuC and EnC have different potencies as far as their biological activity in the immature male rat is expressed.

Cercarial shedding from Galba truncatula infected with Fasciola gigantica of distinct geographic origins.

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.

Fasciola gigantica: larval productivity of three different miracidial isolates in the snail Lymnaea truncatula.

Bimiracidial infections of Lymnaea truncatula with three isolates of Fasciola gigantica, originating from China, Egypt and Madagascar, were carried out to determine the effect of geographic origin of the parasite on the larval productivity of redial generations. The prevalences of experimental infections in snails exposed to strains from Madagascar, China and Egypt were 20.8%, 60.0% and 80.0%, respectively. At day 49 post-exposure (p.e.), the total number of free rediae in snails infected with the Egyptian isolate was significantly higher than that recorded in the Madagascan group. On the other hand, at day 49 p.e., the majority of cercariae in the Chinese and Egyptian groups were produced by R2a rediae (70.6% and 66.6% of cercariae produced by all live rediae), while, in the Madagascan group, the cercariae were produced mainly by the first redial generation. Snails infected with the Egyptian isolate of miracidia developed more live rediae and, consequently, could produce a higher number of cercariae. As a result, L. truncatula snails were highly adapted to infections with the Egyptian and Chinese isolates of F. gigantica.

Larval productivity of Fasciola gigantica in two lymnaeid snails.

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

Fasciola hepatica and Fasciola gigantica: comparative morphometric studies on the redial stage of both species.

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.

Fasciola gigantica: the growth and larval productivity of redial generations in the snail Lymnaea truncatula.

Bimiracidial infections of French Lymnaea truncatula with a Madagascan isolate of Fasciola gigantica were carried out under laboratory conditions to study the growth of rediae and their larval productivity in relation to the different redial generations. The total numbers of rediae and their mean lengths significantly increased with the duration of infection until day 49 post-exposure (p.e.). Significant differences in the lengths between the different redial generations were noted. At day 49 p.e. (at 20 degrees C), the cercariae were produced by the first redial generation, while the productivity of other redial groups was delayed. This last finding shows a slow larval development of this Madagascan isolate of F. gigantica in this French population of L. truncatula.