An Autochthonous Human Case of Fasciolopsiasis in Nepal.

Fasciolopsiasis is rarely known as the parasitic disease in Nepal. Herein, we report a case of fasciolopsiasis in a 22-year-old man who was admitted in the hospital with abdominal pain, distension and loss of appetite for a month. He had previously diagnosed with acute viral hepatitis but, his abdominal pain was not resolving despite improvement in his liver function and general condition. During endoscopy an adult digenean worm was seen in the first part of the duodenum. After isolation, the worm was identified morphologically as Fasciolopsis buski. Microscogic examination of the patient's stool revealed eggs with a morphology consistent with F. buski. Eggs were yellow-brown, ellipsoidal, unembmbryonated, operculated, filled with yolk cells, with thin shell and ranging 118-130 µm in length and 60-69 µm in width. The abdominal pain of the patient was resolved after treatment with praziquantel. By the present study, it was confirmed for the first time that fasciolopsiasis is indigenously transmitted in Nepal. Accordingly, the epidemiological studies in humans and reservoir host animals should be performed intensively in near future.

Multi level ecological fitting: indirect life cycles are not a barrier to host switching and invasion.

Many invasive species are able to escape from coevolved enemies and thus enjoy a competitive advantage over native species. However, during the invasion phase, non-native species must overcome many ecological and/or physiological hurdles before they become established and spread in their new habitats. This may explain why most introduced species either fail to establish or remain as rare interstitials in their new ranges. Studies focusing on invasive species have been based on plants or animals where establishment requires the possession of preadapted traits from their native ranges that enables them to establish and spread in their new habitats. The possession of preadapted traits that facilitate the exploitation of novel resources or to colonize novel habitats is known as 'ecological fitting'. Some species have evolved traits and life histories that reflect highly intimate associations with very specific types of habitats or niches. For these species, their phenological windows are narrow, and thus the ability to colonize non-native habitats requires that a number of conditions need to be met in accordance with their more specialized life histories. Some of the strongest examples of more complex ecological fitting involve invasive parasites that require different animal hosts to complete their life cycles. For instance, the giant liver fluke, Fascioloides magna, is a major parasite of several species of ungulates in North America. The species exhibits a life cycle whereby newly hatched larvae must find suitable intermediate hosts (freshwater snails) and mature larvae, definitive hosts (ungulates). Intermediate and definitive host ranges of F. magna in its native range are low in number, yet this parasite has been successfully introduced into Europe where it has become a parasite of native European snails and deer. We discuss how the ability of these parasites to overcome multiple ecophysiological barriers represents an excellent example of 'multiple-level ecological fitting'.

Landscape influence on spatial patterns of meningeal worm and liver fluke infection in white-tailed deer.

Parasites that primarily infect white-tailed deer (Odocoileus virginianus), such as liver flukes (Fascioloides magna) and meningeal worm (Parelaphostrongylus tenuis), can cause morbidity and mortality when incidentally infecting moose (Alces alces). Ecological factors are expected to influence spatial variation in infection risk by affecting the survival of free-living life stages outside the host and the abundance of intermediate gastropod hosts. Here, we investigate how ecology influenced the fine-scale distribution of these parasites in deer in Voyageurs National Park, Minnesota. Deer pellet groups (N = 295) were sampled for the presence of P. tenuis larvae and F. magna eggs. We found that deer were significantly more likely to be infected with P. tenuis in habitats with less upland deciduous forest and more upland mixed conifer forest and shrub, a pattern that mirrored microhabitat differences in gastropod abundances. Deer were also more likely to be infected with F. magna in areas with more marshland, specifically rooted-floating aquatic marshes (RFAMs). The environment played a larger role than deer density in determining spatial patterns of infection for both parasites, highlighting the importance of considering ecological factors on all stages of a parasite's life cycle in order to understand its occurrence within the definitive host.

Differences in the compatibility of infection between the liver flukes Fascioloides magna and Fasciola hepatica in a Colombian population of the snail Galba sp.

Experimental infections of Galba sp. (origin, Colombia) with allopatric isolates of Fasciola hepatica from France or Fascioloides magna from the Czech Republic were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of either parasite. In snails exposed to F. hepatica, 7 of 400 snails harboured several rediae and only two snails contained a small number of free cercariae on day 50 post-exposure. In contrast, the intensity of F. magna infection in Galba sp. progressively increased from the F1 to F5 generations. Spontaneous cercarial shedding of F. magna occurred in 7 of 100 Galba sp. belonging to the F5 generation and the number of shed cercariae did not differ significantly from that noted in control Galba truncatula of French origin. Galba sp. from Colombia can be added to the list of potential intermediate hosts for F. magna.

Growth rate of the intermediate snail host Galba truncatula influences redial development of the trematode Fascioloides magna.

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.

Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna.

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.

Lymnaea fuscus (Pfeiffer, 1821) as a potential intermediate host of Fascioloides magna in Europe.

Experimental infections of two different populations of Lymnaea fuscus in France and Sweden, with a Czech isolate of Fascioloides magna were carried out to determine if this lymnaeid species enables parasite larval development. Species identification of both snail populations was performed using the morphology of the copulatory organ, and also confirmed by sequencing of the internal transcribed spacer 2 (ITS2) region of the snail genomic rDNA. Only juvenile snails measuring less than 3mm (1-3 weeks of age) were successfully infected (the viable cercariae were recorded) and infection prevalence decreased with age, as documented by increased shell height. In both French and Swedish L. fuscus populations, prevalence ranged between 1.1% and 58.8%. The mean number of metacercariae obtained from cercariae-shedding snails was 13.7 (±11.4), while the total cercarial production noted in snails dissected at day 85 post-exposure was 147.5 (±56.6). Compared to uninfected control snails, we observed reduced growth of infected snails. Despite age-related resistance of snail to the parasite, and limited cercarial production in these experimentally infected snails, F. magna was still able to complete larval development in L. fuscus.

[Developmental characteristics of the causative agent of fasciolopsiasis and its epidemiology in Vietnam].

The authors consider the specific features of development ofa Fasciolopsis buski trematode in the environment and intermediate and final hosts. It has been ascertained that under the conditions of Vietnam, the eggs may develop in the temperature range of 20 to 34 degrees C, which takes 20 to 51 days. The eggs die at less than 12 degrees C. As a new intermediate host of the trematode, the authors have registered the mollusk Indoplanorbis exutus, the development of the parasite in which occurs within 32-37 days at 27-33 degrees C. Invasiveness of adolescaria reduces with age. The parasite develops to sexual maturity in the final host within 36-51 days, which is much less than the time mentioned in the literature. In Vietnam, the porcine parasite is encountered everywhere, but the animals are more infected in the northern provinces than in the south of the country. The human parasite is also detected throughout the country, but comparatively rarely and in small quantities.

Efficacy of clorsulon against Fascioloides magna infection in sheep.

In a study to evaluate the efficacy of clorsulon against Fascioloides magna infection in sheep, 12 ewes were inoculated orally with 100 metacercariae of F magna, and 6 were treated with clorsulon (15 mg/kg of body weight) 8 weeks after inoculation. The sheep were euthanatized 16 weeks after inoculation, flukes were recovered, and the liver and other tissues were subjectively scored for the severity of lesions (0 to 4+). The number of flukes recovered from the clorsulon-treated group (3.8 +/- 1.2 flukes) was significantly (P = 0.025) lower than the number of flukes recovered from the group of untreated controls (10.0 +/- 6.6 flukes). The severity of lesions was significantly (P = 0.004) reduced (45.9%) in the treated group (2.0 +/- 1.1), compared with that in the untreated controls (3.7 +/- 0.5). In the untreated group, 3 sheep died and 1 became moribund 14 to 16 weeks after inoculation. The data suggested that a single treatment with clorsulon at a dosage of 15 mg/kg 8 weeks after inoculation was not effective in preventing F magna infection in sheep, because the survival of only a few F magna is potentially fatal in sheep within 6 months after infection.

PCR-based determination of internal transcribed spacer (ITS) regions of ribosomal DNA of giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899.

Fasciolopsis buski, the zoonotic intestinal flukes of pigs in South and Southeast Asia, is commonly prevalent in regions across Northeast India. Populations of the fluke collected from different parts of the region exhibit variations in morphology. The main objective of our study was to provide molecular characterization of the parasite so as to supplement morphological criteria, using ribosomal DNA cluster (rDNA), which is flanked by more conserved internal transcribed spacer (ITS) regions. We describe herein the ITS sequences of the parasite collected from swine hosts of Assam region. The ITS sequences of both egg and adult origins were found to be identical in length and composition. Phylogenetically, F. buski resembles closely the other members of family Fasciolidae, showing significant expectation value in the alignment. The results corroborate that the ITS sequences are not stage specific and are conserved through different stages of development of the fluke, and thus could be used as species markers.

Prevalence and intensity of infections in the lymnaeid snail Omphiscola glabra experimentally infected with Fasciola hepatica, Fascioloides magna and Paramphistomum daubneyi.

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 degrees 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.

Cercarial production of Fascioloides magna in the snail Galba truncatula (Gastropoda: Lymnaeidae).

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.

Scanning electron microscopy and chemical excystation of Fascioloides magna (Trematoda) metacercariae.

Scanning electron microscopy was used to study encysted metacercariae and newly excysted juveniles of Fascioloides magna. The outer cyst was rough, coarse and discontinuous in the ventral aspect; the inner cyst was smooth. The newly excysted metacercaria was plump and contained numerous tegumentary spines; large dome-shaped papillae were prominent around the oral sucker and on the rim of the acetabulum. Encysted metacercariae with outer cysts were excysted in an alkaline bile salts-trypsin medium at an elevated temperature in the absence of acid saline or acid pepsin pretreatment. Pretreatment in acid saline slightly decreased subsequent excystation, while pretreatment in acid pepsin slightly enhanced subsequent excystation in the alkaline bile salts-trypsin medium.

Miracidial penetration in Fascioloides magna (Trematoda).

The penetration of the miracidium of Fascioloides magna into the snail host Fossaria bulimoides is followed by examining the process at certain, timed intervals during the course of penetration. The cilia are lost early, but the epithelial cells may detach or they may be carried into the snail body. The apical gland releases vesicles which lose their outer coat in the snail tissue; similarly the vesiculated gland cells release vesicles which lose their coating in the snail tissue where there is a concomitant destruction of snail collagen fibers. Evidence is presented to support the concept of both a mechanical and an enzymatic penetration of snail tissues. Both transmission and scanning electron micrographs are used.

Biomphalaria glabrata embryonic (Bge) cell line supports in vitro miracidial transformation and early larval development of the deer liver fluke, Fascioloides magna.

A Bge cell co-culture system, previously shown to support the in vitro production of daughter sporocysts from mother sporocysts of Schistosoma mansoni and S. japonicum, has proven capable of supporting the in vitro development of intramolluscan stages of the deer liver fluke, Fascioloides magna. Miracidia commenced transforming within 4 h of incubation with Bge cells, and had completely shed their epidermal plates within 18-24 h. Redial stages were visible inside in vitro-transformed mother sporocysts after 12-16 days of co-culture with Bge cells, and emerged as fully-developed larvae starting at 14-20 days post-cultivation. Rediae survived over 60 days of in vitro culture, and reached a maximum size of 150-170 microns. Although particulate material was visible in their cecae, rediae were not observed to actively feed on Bge cells. Bge cells did not attach to or encapsulate larval stages at any time throughout the incubation period. Unlike Schistosoma spp., in which a high percentage of miracidia spontaneously shed their ciliated epidermal plates and transformed into mother sporocysts in Chernin's balanced saline solution (CBSS), transformation of F. magna was dependent on Bge cell products. Less than 5% of F. magna miracidia transformed spontaneously in either CBSS or Bge medium with 10% fetal bovine serum (complete or C-Bge). However, incubation of miracidia in either Bge cell-conditioned C-Bge medium or a greater than 30 kDa fraction concentrated from conditioned CBSS increased transformation rates to 67 and 83%, respectively. This secretory Bge cell factor(s) appeared to be protein in nature since its activity was completely abrogated by heat or proteinase K treatments. Overall, these results demonstrate that Bge cells are required for stimulating in vitro miracidial transformation and supporting early larval development of a fasciolid trematode under culture conditions. This is the first report of in vitro development of rediae from miracidia for a digenetic trematode.