Pharmacological and morphological characteristics of the muscular system of the giant liver fluke (Fascioloides magna - Bassi 1875).

Motility is required for feeding, reproduction and maintenance of the fluke in the host's liver. According to that, the neuromuscular system can be an attractive drugable target for chemotherapy. Musculature of the Fascioloides magna is organized into three layers, an outer circular layer, beneath this layer the longitudinal layer, and third, the oblique, or diagonal layer underlies the longitudinal layer. In our study, the administration of atropine or caffeine did not cause classic muscle contractions of F. magna muscle strips. However, the Electrical Field Stimulation (EFS) induced stable and repeatable contractions, which enabled us to examine their sensitivity to the various substances. Acetylcholine (ACh) (300 µM and 1 mM), caused only a slight relaxation, without affecting the amplitude of spontaneous contractions or the amplitude of contractions induced by EFS. Contrary to that, atropine (100 µM) caused a significant increase in the basal tone and an increase of EFS-induced contractions. If acetylcholine is an inhibitory neurotransmitter in trematodes, the described effects of atropine are achieved by the blockade of inhibitory neurotransmission. On the other hand, with respect to the process of excitation-contraction coupling, the plant alkaloid ryanodine (30 µM) significantly reduced the basal tone, as well as EFS-induced contractions of F. magna muscle strips. Ryanodine inhibited the potentiating effect of atropine on the basal tone and contractions caused by EFS, which indicates that the contractile effect of atropine is dependent on Ca(++) release from intracellular stores. Caffeine (500 µM) caused relaxation of fluke muscle strips and at the same time significantly enhanced the EFS-induced contractions. Both effects of caffeine can be explained by entry of extracellular Ca(++) into muscle cells. The muscle contractility of F. magna depends both on the entry of extracellular calcium, and calcium release from intracellular stores, which are under the control of RyRs. Our results also suggest that antitrematodal drugs could potentially be developed from substances with selective anti-cholinergic activity.

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'.

In Vitro Effects of Mucus from the Mantle of Compatible (Lymnaea elodes) and Incompatible (Helisoma trivolvis) Snail Hosts on Fascioloides magna Miracidia.

The epidermal mucus covering the surface of a snail represents an important barrier to trematode larvae attempting to penetrate the snail and may play a role in mediating snail-trematode compatibility. In this study, Facioloides magna miracidia were exposed to mucus harvested from a compatible snail host, Lymnaea elodes (palustris), and from an incompatible snail, Helisoma trivolvis . In vitro treatment of freshly hatched miracidia with snail-derived mucus exerted dramatically different effects on larvae depending on snail species. At the lowest dilution of mucus tested (1:3) mean damage rates (tegumental damage and/or larval lysis and death) were as high as 100% for miracidia exposed to H. trivolvis mucus, while none of F. magna miracidia were damaged in L. elodes mucus. A dilution series for each snail species, and treatments with heat and proteinase K were performed to characterize the component(s) of mucus inducing the observed morphological changes. The damaging effects of H. trivolvis mucus were concentration dependent and completely abrogated by heat (65 C, 30 min) and proteinase treatment, strongly implicating a heat-labile protein(s) in mucus as the active cytotoxic agent(s). In contrast to our prediction that miracidial contact with mucus of compatible L. elodes would trigger larval transformation, mucus from either snail species tested exhibited little to no activity. Overall these data demonstrate the presence of a potent cytotoxic protein-like factor in the mucus of F. magna -incompatible H. trivolvis , and its absence in the mucus of the compatible snail, L. elodes . This finding supports the notion that the epidermal mucus layer may be serving as an important determinant of larval trematode-snail compatibility.

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.

Lymnaea cubensis, an experimental intermediate host for Fascioloides magna.

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.

Recovery of Fascioloides magna (Digenea) population in spite of treatment programme? Screening of Galba truncatula (Gastropoda, Lymnaeidae) from Lower Austria.

During the past decade, Fascioloides magna, the large American liver fluke, has spread within free-living deer in wetlands of the Danube in Lower Austria. The aim of this study was to determine the current infection rates with F. magna and other digenean parasites in the intermediate host snail Galba truncatula from risk areas in Lower Austria. A total of 3444 G. truncatula were collected and examined microscopically for the presence of digenean trematodes. A set of randomly selected snails and isolated trematode stages were also investigated molecular biologically by PCR and sequencing. Digenean parasites were detected with a prevalence of 2.41% (1.83% Paramphistomoidea; 0.46% Echinostomatoidea; 0.09% Strigeida; 0.06% Plagiorchiida). F. magna was found with an overall prevalence of 0.23%, which may indicate a recovery of the parasite population in spite of an ongoing triclabendazole treatment programme. Moreover, high risk areas and a seasonality of infections were observed.

[Age structure and dynamics of abundance of the adult Trematode Opisthioglyphe ranae (Fasciolida, Plagiorchidae) hemipopulation in lake frog from the Saratov reservoir].

Age structure and dynamics of abundance of adult Opisthioglyphe ranae, a parasite of intestine of lake frog, are analyzed. In the period of activity of the host (May-October), the hemipopulation of O. ranae maritae is represented by two generations, which are situated in host lesser than one year and differ from each other in the rate of maturation.

Optimization of metacercarial production for three digenean species by the use of petri dishes for raising lettuce-fed Galba truncatula.

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.

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.

First studies on the susceptibility of Omphiscola glabra (Gastropoda: Lymnaeidae) from central France to Fascioloides magna.

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.

NO nerves in trematodes, too! NADPH-diaphorase activity in adult Fasciolopsis buski.

The free radical nitric oxide (NO) is a unique molecule with an avidity to react with other molecules and is known to function as a neuronal messenger. This nitrergic transmitter with diverse functions in signal transduction, being a gas, is not stored in synaptic vesicles but is generated in various neuronal cells by a family of nitric oxide synthases (NOSs). The NADPH-d histochemical reaction is regarded as a selective marker for NOS in the neuronal tissue. With histochemical detection of NADPH-d, the presence of NOS is demonstrated in the digenetic trematode, Fasciolopsis buski. Strong NADPH-d staining was observed in the neuronal cell bodies in the two cerebral ganglia, the brain commissure and the nerve fibers in the main nerve cords. NADPH-d staining was also detectable in the innervation of the pharynx, the cirrus sac and the ventral sucker besides being observable sporadically in the nerve tributaries in the general parenchyma. NO released by the whole worm kept in PBS at 37 degrees C could also be measured biochemically. The NOS activity was assayed in the whole worm homogenate and also in the tissue homogenate containing only the anterior pre-acetabular part of the parasite body. The presence of NOS in this digenean parasite confirms that a nitrergic innervation occurs in the trematode group also as in other groups of exclusively parasitic helminths and that NO represents an old signal molecule in evolutionary scale.

[Bionomy, occurrence and importance of the giant liver fluke (Fascioloides magna), an important parasite of free-living ruminants]. / Bionómia, výskyt a význam cicavice obrovskej (Fascioloides magna), závazného parazita vol'ne zijúcich prezúvavcov.

The paper summarizes the most important knowledge of the giant liver fluke of ruminants, Fascioloides magna, with respect to its occurrence throughout the Northern Hemisphere, its fenotypic and genetic discriminative characteristics, its pathogenicity in various obligatory and facultative hosts and possible prophylaxis and medical control. The paper emphasizes the fact that new natural foci with heavy F. magna infections of deer regularly occur in Europe.

Mule deer (Odocoileus hemionus) and elk (Cervus elaphus) as experimental definitive hosts for Fascioloides magna.

In August 1992, six mule deer (Odocoileus hemionus hemionus) fawns and four elk (Cervus elaphus) calves (n = 2) or yearlings (n = 2) each were inoculated orally with 50, 250, or 2,000 metacercariae of the liver fluke Fascioloides magna to evaluate their potential to serve as definitive hosts. Animals were maintained for up to 403 days. Three mule deer each inoculated with 50 metacercariae survived the infection and shed eggs in feces; thus mule deer can function as definitive hosts for F. magna. The other three mule deer inoculated with 50 (n = 1) or 250 (n = 2) metacercariae died from fluke infection on days 91, 150, and 162 days postinoculation, respectively, and only immature F. magna were recovered. One elk calf inoculated with 2,000 metacercariae died from fluke infection 44 days after inoculation. The remaining three elk, each inoculated with 250 metacercariae, survived infection, and two of the three shed eggs in feces. The third elk contained only one immature F. magna at necropsy. The prepatent period in mule deer and elk was approximately 6 to 7 months.

Liver and intestinal flukes.

A number of trematodes besides schistosomes parasitize humans and domesticated animals. Although they do not have as great a public health impact as schistosomiasis, they are prevalent in Southeast Asia as well as among the greater than 1 million immigrants from this region to North America. The human biliary flukes include C. sinensis, O. viverrini, and O. felineus. These chronic infections are often asymptomatic but over time may cause biliary thickening, cholangitis, and a predisposition to cholangiocarcinoma. Zoonotic trematode infections include the sheep liver fluke F. hepatica and the intestinal flukes Fasciolopsis, Echinostoma, Heterophyes, and Metagonimus.

Distribution of potential intermediate hosts for Fasciola hepatica and Fascioloides magna in Montana, USA.

A collection of lymnaeid snails in Montana was made over a 3 year period, in conjunction with a state-wide survey of the distribution of liver flukes in Montana. Collection areas were selected based on reports of infected cattle, sheep or wildlife, and with the intent of covering all geographic regions of the state. Snails were found at all 97 of the locations chosen for collections, with lymnaeids collected at 71 of the locations. The 97 sites were located in 28 of Montana's 56 counties. Nine lymnaeid species were collected, five of which have been reported either as natural or experimental intermediate hosts for Fasciola hepatica or Fascioloides magna. The two snail species most widely distributed over the areas enzootic for the flukes were Lymnaea modicella and Lymanaea caperata.

Hepatic and pulmonary pathology of experimental Fascioloides magna infection in guinea pigs.

The guinea pig was used to study the pathology of Fascioloides magna, an important pathogen for sheep. Although flukes migrated freely through various tissues in infected guinea pigs, the most serious lesions occurred in the liver and lungs. The sequential development of lesions indicated that flukes first invaded the quadrate lobe of the liver and subsequently migrated to other liver lobes and tissues. Six weeks post-infection, there was a marked drop in the recovery of flukes from the liver along with a dramatic increase in pulmonary involvement. Much of the hepatic and pulmonary pathology in infected animals was secondary to extensive vascular lesions caused by migrating flukes. In the liver, vascular lesions predominantly involved the portal and hepatic veins. Thrombophlebitis and locally extensive necrosis, resembling infarction, were observed. Vascular lesions in the lungs occurred in the pulmonary arteries leading to thrombosis and haemorrhagic infarction. Discovery of a fluke in a pulmonary artery, along with the pattern of hepatic and pulmonary lesions, suggested that flukes probably used the cardiovascular system as a pathway for dissemination. Death in fluke-infested guinea pigs was most often associated with severe pulmonary lesions. The nature and distribution of fluke-induced lesions observed in this study demonstrate that the guinea pig is a suitable animal model for Fascioloides magna infection in sheep.

[Studies on ecology of Fasciolopsis buski and control strategy of fasciolopsiasis].

The development of eggs of Fasciolopsis buski requires oxygen and the eggs cannot survive anaerobic conditions. The eggs have some resistance to low temperature and can be maintained at 4 degrees C for 3 to 4 months; however, the eggs are killed at 50 degrees C in four hours. The presence of salts can influence the development time of the eggs and reduce their hatching rate. Encysted cercariae exist not only on aquatic plants, but also on the surface of the water. The number of encysted cercariae floating on the water surface is about 3.6% of that of the total encysted cercariae. By inquiring into the case history we found that 10.3-12.8% of the patients and 35.1-40% of the infested pigs were possibly infected by drinking water contaminated with encysted cercariae. The authors suggest the use of fermented silage to feed pigs instead of fresh aquatic green fodders to prevent infection in the animals. In addition, aquatic plants such as water chestnut should be boiled for 1 to 2 minutes before eating to kill the encysted cercariae on the plants.