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.     

Mitochondrial DNA polymorphism of a triploid form of Fasciola in Japan

Mitochondrial DNA polymorphism was characterized in a triploid form of Fasciola found in Japan in comparison with F. hepatica, F. gigantica and Korean Fasciola worm. Seventy worms of Fasciola from Japan, three of F. hepatica from Uruguay and Australia, two of F. gigantica from Thailand and one of Fasciola from Korea were used in the study. Mitochondrial DNA polymorphism was detected by restriction fragment length polymorphism (RFLP) using eight restriction enzymes, BamH I, Bgl II, Dra I, EcoR I, EcoR V, Hind III, Mfl I and Sca I. Three different types (types 1, 2 and 3) were detected from 76 Fasciola worms used in the study. Eight of 70 Japanese worms were categorized in type 2 (F. gigantica type), and the remaining 62 were in type 3 (F. hepatica type).     

Characterization of Fasciola samples by ITS of rDNA sequences revealed the existence of Fasciola hepatica and Fasciola gigantica in Yunnan Province, China

On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.     

Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam

The two species common of liver fluke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these species, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver flukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoonotic disease. Specimens examined came from domestic stock (cattle and buffalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.     

Effect of concurrent infection with Muellerius capillaris on the development of redial generations of Fasciola hepatica in Lymnaea truncatula.

The rediae of Fasciola hepatica were counted according to generation in adult and juvenile Lymnaea truncatula following single infection with Fasciola hepatica, double infection with F. hepatica and then Muellerius capillaris, or double infection with M. capillaris and F. hepatica. The rediae found in double infections were essentially first generation and an early cohort from the second generation. The following differences were observed in adult snails which underwent double infection when compared to corresponding single infections: i) dependent rediae were almost completely absent; ii) degenerating independent rediae were found in identical or decreased numbers; iii) living independent rediae of the first generation were decreased in number, while those of the second generation had variable decreased numbers. The results were similar in juvenile snails with double infections, except that the numbers of degenerating independent rediae were higher than those found in corresponding single infections and the numbers of rediae of the second generation were increased. The order of exposure in double infections had no influence on the number and maturation of fasciolid rediae.     

Fascioliasis and other plant-borne trematode zoonoses

Fascioliasis and other food-borne trematodiases are included in the list of important helminthiases with a great impact on human development. Six plant-borne trematode species have been found to affect humans: Fasciola hepatica, Fasciola gigantica and Fasciolopsis buski (Fasciolidae), Gastrodiscoides hominis (Gastrodiscidae), Watsonius watsoni and Fischoederius elongatus (Paramphistomidae). Whereas F. hepatica and F. gigantica are hepatic, the other four species are intestinal parasites. The fasciolids and the gastrodiscid cause important zoonoses distributed throughout many countries, while W. watsoni and F. elongatus have been only accidentally detected in humans. Present climate and global changes appear to increasingly affect snail-borne helminthiases, which are strongly dependent on environmental factors. Fascioliasis is a good example of an emerging/re-emerging parasitic disease in many countries as a consequence of many phenomena related to environmental changes as well as man-made modifications. The ability of F. hepatica to spread is related to its capacity to colonise and adapt to new hosts and environments, even at the extreme inhospitality of very high altitude. Moreover, the spread of F. hepatica from its original European range to other continents is related to the geographic expansion of its original European lymnaeid intermediate host species Galba truncatula, the American species Pseudosuccinea columella, and its adaptation to other lymnaeid species authochthonous in the newly colonised areas. Although fasciolopsiasis and gastrodiscoidiasis can be controlled along with other food-borne parasitoses, fasciolopsiasis still remains a public health problem in many endemic areas despite sustained WHO control programmes. Fasciolopsiasis has become a re-emerging infection in recent years and gastrodiscoidiasis, initially supposed to be restricted to Asian countries, is now being reported in African countries.     

Molecular identification of Fasciola spp. (Digenea: Platyhelminthes) in cattle from Vietnam

Fasciola spp. were collected from naturally infected cattle at a local abattoir of Khanh Hoa province, Vietnam, for morphological and genetic investigations. Microscopic examination detected no sperm cells in the seminal vesicles, suggesting a parthenogenetic reproduction of the flukes. Analyses of sequences from the first and second internal transcribed spacers (ITS1 and ITS2) of the ribosomal RNA revealed that 13 out of 16 isolates were of Fasciola gigantica type, whereas three isolates presented a hybrid sequence from F. gigantica and Fasciola hepatica. Interestingly, all the mitochondrial sequences (partial COI and NDI) were of F. gigantica type, suggesting that the maternal lineage of the hybrid form is from F. gigantica. No intra-sequence variation was detected.     

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.     

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     

The liver flukes Fasciola gigantica and Fasciola hepatica express the leucocyte cluster of differentiation marker CD77 (globotriaosylceramide) in their tegument

Glycosphingolipids from the parasitic liver flukes Fasciola gigantica and Fasciola hepatica were isolated and their carbohydrate moieties were structurally analysed by methylation analysis, exoglycosidase treatment, on-target exoglycosidase cleavage and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. For both liver fluke species, the ceramide monohexosides Gal1-ceramide and Glc1-ceramide were found in relative amounts of 1.0 to 0.1, respectively. From F. gigantica, the ceramide dihexoside was isolated in sufficient amounts to be structurally determined as lactosylceramide, Gal beta4-Glc1-ceramide, while for both liver fluke species the ceramide trihexoside was shown to be Gal alpha4Gal beta4-Glc1-ceramide, which is designated as either globotriaosylceramide, Pk-blood group antigen or CD77 leucocyte cluster of differentiation antigen. To our knowledge, this is the first report on the expression of globo-series glycosphingolipids in non-mammalian species. Ceramide analysis of ceramide monohexosides yielded as major components octadecanoic and 2-hydroxyoctadecanoic fatty acids together with C18- and C20-phytosphingosines. By the use of an anti-CD77 monoclonal antibody and the Escherichia coli Shiga toxin B1 subunit, globotriaosylceramide could be immunolocalised to the tegument of F. hepatica cryosections. The sharing of CD77 between liver flukes and their mammalian hosts fits in with the concept of molecular mimicry, which is closely parallel to the established imitation of host CD15 (Lewis X) displayed by the blood fluke Schistosoma mansoni.     

Comparison of humoral response in sheep to Fasciola hepatica and Fasciola gigantica experimental infection

Humoral response of sheep to F. gigantica was compared with the well known humoral response to F. hepatica, in order to explain the difference of susceptibility of sheep to these two parasites. In this work, a lesser susceptibility of sheep to F. gigantica than to F. hepatica infection was confirmed. Humoral response to F. hepatica infection is similar to that previously described by several authors. IgG level of F. gigantica infected sheep increased from week 2 post-infection (2WPI) and displayed a peak at 13WPI. F. gigantica excretory-secretory products (FgESP) analyzed by SDS-PAGE showed at least 31 bands from 12.0 to 127.6 kDa in FgESP. Western blot indicated that F. gigantica infected sheep sera recognized, in FgESP, at least 30 antigens from 7.8 to 119.2 kDa of which 12 major bands recognized after OWPI. In FhESP and FgESP, F. hepatica infected sheep serum reacted only with the lower molecular mass antigens, while F. gigantica infected sheep serum reacted with the lower and the higher molecular mass antigens. These differences of antigenic recognition might be associated with the difference of susceptibility of sheep. Further investigation must be done to study the mechanism of resistance between the sheep infected with F. hepatica or F. gigantica.     

Identification of Fasciola species isolated from Egypt based on sequence analysis of genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers

Fascioliasis has a negative impact on the farming industry in both developed and developing countries, rather than a public health challenge. This study was performed to identify Fasciola sp. from different definitive hosts (buffalo, cattle, and sheep) based on the molecular parameters and spermatogenesis. Ninety-one adult flukes were collected from livers of slaughtered animals at abattoirs in different prefectures in Egypt. Microscopic examination of the analyzed flukes showed many normal spermatozoa in the seminal vesicles (spermic), suggesting that they have the ability of spermatogenesis. This study showed that no parthenogenic Fasciola species occurred in Egypt. Molecular analysis was performed utilizing genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers. Whereas 16 animals proved to have infection with a single Fasciola species, 2 were infected with both F. hepatica and F. gigantica. The results indicated that sheep were prone to F. hepatica (8 out of 10 animals) more than F. gigantica infection. Sequences of ITS1 and ITS2 ribosomal region indicated that the flukes were categorized into 3 groups F. hepatica-type (47), F. gigantica-type (42) and 2 flukes possessed sequences of both types indicating an existence of different alleles at the same loci. Unique overlapping of T/C bases were detected in both ITS1 (Position 96) and ITS2 (Position 416). Based on results of mitochondrial gene markers (NDI and COI), flukes were classified into F. hepatica-type and F. gigantica-type. Extensive intra-sequence polymorphism was detected at both markers. NDI and COI sequences of Egyptian strain of F. gigantica showed pronounced diversity compared with relevant sequences at database.     

Fasciola hepatica and Fasciola gigantica: comparison of cellular response to experimental infection in sheep

Cellular responses to Fasciola gigantica and to Fasciola hepatica infection in sheep were compared. Eosinophil numbers increased more quickly and strongly in F. gigantica-infected sheep than in F. hepatica-infected sheep. In both groups, peripheral blood mononuclear cell (PBMC) proliferation in response to the parasitic excretory-secretory products (ESP) showed similar kinetics. Interferon-gamma (IFN-gamma) production by ESP-stimulated PBMC was early and showed similar kinetics in both groups. Interleukin-10 (IL-10) production by FhESP-stimulated PBMC was very high throughout infection even at 0 weeks post-infection (WPI) in F. hepatica-infected sheep, while in F. gigantica-infected sheep, IL-10 production by FgESP-stimulated PBMC increased between 1 and 4 WPI. IL-10 production in F. gigantica-infected sheep was significantly lower than in F. hepatica-infected sheep during infection. The lower susceptibility to F. gigantica infection in sheep could be explained by the more intense cellular response induced by the parasite and the weaker capacity of F. gigantica to evade the immune response.     

The structure and cytochemistry of the neurosecretory cells of Fasciola gigantica Cobbold and Fasciola hepatica L.

Histochemical studies of the nervous system of Fasciola gigantica and Fasciola hepatica were undertaken. Neurosecretory cells were detected by Gomori's aldehydefuchsin, Bargmann's chrome hematoxylin-phloxin, Mallory's triple stain, periodic acid-Schiff, Heidenhain's Azan and alcian blue after potassium permanganate oxidation. Two types of neurosecretory cells were recognized and designated as "A" and "B". Type "A" cells occurred in small numbers in the brain and subesophageal mass and type "B" cells ubiquitous in distribution. The reactions of these cells to the standard stains for neurosecretory substance generally, were less intense than the neurosecretory cells of other animals such as crustaceans and insects. The structure, organisation, distribution and cytochemistry of neurosecretory cells in Fasciola gigantica and Fasciola hepatica is discussed.     

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

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

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.