Fasciola hepatica: motility response to fasciolicides in vitro

The effects of a wide range of fasciolicides on the in vitro motility of Fasciola hepatica have been determined by means of an isometric transducer system. Carbon tetrachloride and diamphenethide do not affect movement at concentrations up to 500 and 100 micrograms/ml, respectively; at 1000 micrograms/ml, however, carbon tetrachloride induces a rapid tonic paralysis. Brotianide and the deacetylated metabolite of diamphenethide cause a rapid flaccid paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. In contrast, the effect of MK-401 is a long-term one, a flaccid paralysis occurring after 20 hr only at 200 micrograms/ml. Praziquantel also produces a flaccid paralysis of the fluke, but this follows an initial increase, then decrease in muscle tone. The effect is rapid at 500 micrograms/ml, but long-term at 100 and 200 micrograms/ml; at these lower concentrations there is also a stimulation of activity. Oxyclozanide , rafoxanide, niclofolan , bithionol, and hexacholorophene induce a rapid spastic paralysis of the fluke at concentrations of 1.0 micrograms/ml and above. Both phasic and tonic components are evident in the response at concentrations of 1.0 micrograms/ml and below; the phasic component disappears at higher concentrations. Nitroxynil produces a similar effect, evident at higher concentrations. Among the benzimidazoles, mebendazole, oxfendazole, and albendazole sulphoxide cause a suppression of motility, whilst thiabendazole and albendazole produce a stimulation of movement. The effects are not rapid, however, for only mebendazole at 500 micrograms/ml causes total inactivity of the fluke within a 12-hr period. Possible explanations for these effects on fluke motility are discussed.     

Fasciola hepatica: a technique for monitoring in vitro motility

An apparatus utilizing a force and displacement transducer is described for the direct and long-term recording of the motility in vitro of Fasciola hepatica. Normal movement is typically rhythmical, with bursts of more powerful contractions alternating with periods of lesser activity. Such rhythms and the overall level of activity are maintained for more than 30 hr. The fluke remains active for much longer periods of time: recordings of fluke movements have been made for up to 4 days. Potential damage to the fluke caused by the attachment system within the recording apparatus has been determined by the Evans' Blue Technique and scanning electron microscopy. It is restricted to the attachment sites, and does not spread to other parts of the body over the 30-hr normal activity period. Transmission electron microscope studies have shown that the tegument retains its structural and functional integrity over this period of time. There are advantages of the recording apparatus over previous kymographic methods for studying fluke motility.     

Fasciola hepatica: the effects of neuropharmacological agents upon in vitro motility

The effects of a wide range of neuropharmacological agents on the motility in vitro of Fasciola hepatica have been determined using an isometric transducer system. The neuromuscular blocking agents tubocurarine and decamethonium cause a long-term stimulation of the basal activity of the fluke. Acetylcholine causes an inhibition of activity. This effect is mimicked by the cholinergic agonists carbachol and nicotine, antagonised by the cholinergic blocking agents atropine and mecamylamine, and potentiated by eserine, a cholinesterase inhibitor. With nicotine and atropine the effects are accompanied by an increase in muscle tone at a concentration of 1 X 10(-2) M. Noradrenaline and adrenaline also cause some inhibition of activity, an effect antagonised by guanethidine, which blocks the release of noradrenaline. In contrast, dopamine stimulates fluke motility, whilst its antagonist dihydroergotamine causes an inhibition of activity. The monoamine oxidase inhibitors iproniazid and p-chloromercuribenzoic acid induce a stimulation of activity; with the latter there is an increase in muscle tone at a concentration of 1 X 10(-3) M. The amine depleting agents chloroamphetamine and reserpine, and the monoamine uptake inhibitors desipramine and nortriptyline produce an inhibition of fluke activity, as does the serotonin uptake inhibitor fluoxetine. High concentrations of chloroamphetamine (1 X 10(-2) M) and the uptake inhibitors (1 X 10(-3) M and above) also induce an increase in muscle tone. Serotonin causes a marked stimulation of motility. The pharmacological evidence is consistent with a neurotransmitter role of acetylcholine (inhibitory), dopamine (excitatory), and noradrenaline (inhibitory). The status of serotonin is discussed.     

Fasciola hepatica: site of resistance to reinfection in cattle

The effective site of resistance to reinfection of cattle with Fasciola hepatica was examined by recovery of challenge flukes from either the liver or body cavity. Calves infected 18 or 26 weeks previously with F. hepatica showed levels of resistance to reinfection of 56 and 94%, respectively, as assessed by recovery of flukes from the liver 15-16 weeks after challenge. Plasma glutamate dehydrogenase (EC 1.4.1.3; GLDH) enzyme activity estimations revealed only a marginal increase in these latter resistant calves compared with previously naive controls, indicating minimal liver damage as a result of migrating flukes. By comparison, when immature challenge flukes were recovered from the body cavity 4 or 14 days after infection of corresponding previously infected or naive calves, there was no significant difference in numbers. It appears, therefore, that, in cattle, resistance mechanisms are effective against challenge flukes at or soon after penetration of the liver.     

Fasciola hepatica: immunisation of rats by intraperitoneal injection of adult fluke antigen in Freund's adjuvant

Intraperitoneal injections of rats with freeze-dried, adult Fasciola hepatica material, which had been resuspended in phosphate-buffered saline and emulsified in Freund's incomplete adjuvant, reduced fluke burdens by 48 to 81% following oral infection. The addition of Bordetella pertussis to the adjuvant antigen emulsion enhanced the protection slightly (but not to a statistically significant degree); fluke antigens with B. pertussiss alone induced no protection.     

The pattern of mortality in mice experimentally infected with Fasciola hepatica

The extent and pattern of mortality of mice exposed to low levels of F. hepatica were determined for a 56-day period. Mice were exposed to 1, 2, 3, 4 or 6 metacercariae per mouse with emphasis placed on one cyst infections. Eighty per cent of all deaths occurred between days 27 and 35 post exposure with the mean death day for all being from 31 to 34 days postexposure. The mortality followed a statistically predictable pattern with the use of one-cyst infections as an extrapolation point. As the dose increased, the percentage mortality increased as a simple multiple of probabilities indicating the absence of synergism or antagonism in producing death when more than one parasite was present. Relatively low levels of infection in mice reliably produced high mortality within a limited time period and, thus, mortality could be used as a parameter of infection for studies with a variety of aims.     

Schistosoma mansoni: passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.     

Fasciola hepatica: Migration of newly excysted juveniles in resistant rats

Using transmission and scanning electron microscopy, the early migration of juvenile Fasciola hepatica was examined in naive and resistant rats. In naive rats, the migration of flukes to the peritoneal cavity was uneventful. In resistant rats, flukes were rapidly coated with antibody whilst still in the gut lumen and a proportion of the flukes were unable to penetrate the intestinal wall. Those that did penetrate were unharmed as they crossed the gut wall, but on entering the peritoneal cavity they were coated with antibody and host cells including eosinophils, neutrophils, macrophages, and mast cells. Eosinophils were seen degranulating onto the fluke surface, and this appeared to result in the erosion of the tegumental syncytium.     

Fasciola hepatica: increase of glycogen phosphorylase activity due to prostaglandins

Both prostaglandin E1 (PGE1) and prostaglandin F2 alpha (PGF2 alpha) stimulate the glycogen phosphorylase (EC 2.4.1.1.) activity of Fasciola hepatica. Whole or sliced parasites were incubated with PGE1 (2.8 X 10(-7) and 2.8 X 10(-5) M) and PGF2 alpha (2.1 X 10(-7) and 2.1 X 10(-5) M) and enzyme activity was measured in homogenates prepared immediately following the incubation. No substantially different effect was noted between the two assayed doses of prostaglandins. Prostaglandins appeared to be less effective in sliced parasites.     

Fasciola hepatica: basal infolds and associated vacuoles of the tegument.

The tegument of Fasciola hepatica has been shown to contain long invaginations of its basal plasma membrane, hereafter called basal infolds. Associated with the membranes of the infolds, and with the apical and basal plasma membranes, is a Na⁺ K⁺ ATPase (EC 3.6.1.3). Furthermore, polymorphic masses of acid mucopolysaccharide lie close to or against the sides of the infolds and the basal plasma membrane and also fill cytoplasmic tubules which connect the tegument with the tegumental cells. Fixation and incubation of flukes in hypertonic and hypotonic media have shown that the infolds respond to changes in external osmolarity by collapsing in the former and swelling in the latter. This is not simply a passive response, however, since the infolds return to near normal configuration and morphology within 1 hr in either hypertonic or hypotonic media, even though the whole fluke may be shrunken or turgid depending on medium osmolarity. The tegument, therefore, has many of the characteristics of a transporting epithelium. A theory outlining the possible mode of operation of the tegument as a transporting epithelium is proposed on the basis of the present structural, chemical, and physiological findings combined with current ideas of the role and functioning of standing gradients, and forward and backward channel systems in other transporting epithelia.     

Fasciola hepatica: The effect of challenge by the subcutaneous or intraperitoneal route with living adult flukes in sensitized rats

When rats, sensitized either by subcutaneous implantation of adult F. hepatica or by a normal oral infection with F. hepatica metacercariae, were challenged by implanting adult flukes in the peritoneal cavity, 23% of these flukes were killed in rats sensitized by subcutaneous implantation and 71% in the rats sensitized by the oral route. In contrast neither of these sensitization routes were effective against subcutaneous challenge with adult fluke. Histological evidence suggested that about half of the dead flukes found were killed shortly after transfer and these flukes were surrounded with mononuclear cells. The remaining dead flukes appear to have died after becoming surrounded with a cyst. These latter flukes were surrounded by neutrophils and this cell type was very prominent in the cysts of sensitized rats.     

Discovery and validation of serum biomarkers expressed over the first twelve weeks of Fasciola hepatica infection in sheep

Serum biomarkers associated with Fasciola hepatica infection of Corriedale sheep were analysed during the first 12 weeks of infection using surface-enhanced laser desorption ionisation time of flight mass spectrometry (SELDI-TOF MS). In the discovery phase of analysis, pooled sera collected at week 0 and at each week p.i. to week 12 were fractionated by anion-exchange chromatography and the protein mass fingerprints obtained in individual fractions were in the M/z range 1.5-150 kDa. A total of 2302 protein clusters (peaks) were identified that varied between time-points following infection with peaks increasing or decreasing in intensity, or showing transient variation in intensity, during the 12 weeks of parasite challenge. In the validation phase, candidate biomarkers in sera of individual sheep at weeks 3 and 9 p.i. were analysed, identifying 100 protein peaks, many of which are small peptides <10 kDa in size: 54% of these peaks were up-regulated in intensity at week 3 or 9 p.i. Twenty-six biomarkers were chosen for further study, ranging in size from 1832 to 89,823 Da: six biomarkers were up-regulated at weeks 3 and 9 p.i., 16 biomarkers were up-regulated only at week 9 p.i. and four biomarkers were down-regulated at week 9 p.i. Two biomarkers up-regulated at week 9 were identified as transferrin (77.2 kDa) and Apolipoprotein A-IV (44.3 kDa), respectively. The results show that the interaction between the host and F. hepatica is complex, with changes in biomarker patterns beginning within 3 weeks of infection and either persisting to weeks 9-12 or showing transient changes during infection. Identification of biomarkers expressed during ovine fasciolosis may provide insights into mechanisms of pathogenesis and immunity to Fasciola and may assist in the rational development and delivery of vaccines.     

Fasciola hepatica: effect of 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide on glycolysis in vitro

In vitro, 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide, a potent fasciolicide, causes a potent concentration-dependent inhibition of glucose uptake by mature Fasciola hepatica. In F. hepatica treated with the disulfonamide and then fed [U-¹⁴C]glucose, there was a 60% inhibition of glucose utilization and a corresponding inhibition of acetate and propionate formation. Treated fluke parasites possessed much lower levels of adenosine triphosphate, phosphoenolpyruvate, glucose 6-phosphate, and fructose 6-phosphate than untreated parasites and contained higher levels of glycerol and the free sugars fructose and mannose. Direct measurement of the effect of the disulfonamide on the glycolytic enzymes of F. hepatica demonstrated that 3-phosphoglycerate kinase (EC 2.7.2.3) and phosphoglyceromutase (EC 2.7.5.3) were inhibited. It is therefore suggested that the fasciolicidal activity of 4-amino-6-trichloroethenyl-1, 3-benzenedisulfonamide is due to inhibition of the enzymes 3-phosphoglycerate kinase and phosphoglyceromutase which effectively blocks the Embden-Myerhof glycolytic pathway.     

Schistosoma mansoni: challenge attrition during the lung phase of migration in vaccinated and serum-protected rats

Serum harvested from Sprague-Dawley rats twice vaccinated with gamma-irradiated cercariae of Schistosoma mansoni is able to protect naive recipients against a challenge infection, but the challenge parasites are susceptible to immune elimination over a very short period of time. Thus, vaccinated rat serum protects recipients against a percutaneous cercarial challenge when transferred on Day +5 but not Day 0 and protects recipients against a tail vein challenge with 5-day-old lung worms when transferred on Days 0 or +1, but not Days +4 or +5. Rats challenged with lung worms via the tail vein and given serum on Day +3 exhibit approximately half the protection expressed by counterparts that received serum on Day 0. However, vaccinated rat serum does not protect naive recipients against a lung worm challenge introduced directly into the liver. These data indicate that immune elimination of challenge parasites in the vaccinated rat model is site-dependent rather than stage-dependent, and most probably occurs during the lung phase of parasite migration.     

Local adaptation of the trematode Fasciola hepatica to the snail Galba truncatula

Experimental infections of six riverbank populations of Galba truncatula with Fasciola hepatica were carried out to determine if the poor susceptibility of these populations to this digenean might be due to the scarcity or the absence of natural encounters between these snails and the parasite. The first three populations originated from banks frequented by cattle in the past (riverbank group) whereas the three others were living on islet banks without any known contact with local ruminants (islet group). After their exposure, all snails were placed in their natural habitats from the end of October up to their collection at the beginning of April. Compared to the riverbank group, snails, which died without cercarial shedding clearly predominated in the islet group, while the other infected snails were few in number. Most of these last snails released their cercariae during a single shedding wave. In islet snails dissected after their death, the redial and cercarial burdens were significantly lower than those noted in riverbank G. truncatula. Snails living on these islet banks are thus able to sustain larval development of F. hepatica. The modifications noted in the characteristics of snail infection suggest the existence of an incomplete adaptation between these G. truncatula and the parasite, probably due to the absence of natural contact between host and parasite.     

Effect of the exposure to Fasciola hepatica (Trematoda: Digenea) on life history traits of Lymnaea cousini and Lymnaea columella (Gastropoda: Lymnaeidae)

The snails Lymnaea columella and Lymnaea cousini have both been reported as intermediate hosts of Fasciola hepatica in Colombia. The effect of the exposure to the parasite on survival, fecundity and size of these snails was evaluated by means of experimental infections and the life history traits of control and exposed groups were compared. Infection rates were 82.2 and 34% for L. columella and L. cousini, respectively. A reduction in fitness was observed in both species when exposed to the parasite: fecundity alone was reduced in L. columella whereas in L. cousini there was also a decline in survival rate. Unlike other studies, increased size was not observed in either species. On the contrary, a reduction in growth rate was observed in L. columella.     

Fasciola hepatica: effects on the antioxidative properties and lipid peroxidation of rat serum

Fasciola hepatica infection is accompanied by increased formation of reactive oxygen species. The aim of this study was to analyze antioxidative properties of rat serum in the course of fasciolosis. Wistar rats were infected per os with 30 metacercariae of F. hepatica. Activities of antioxidant enzymes and concentrations of non-enzymatic antioxidants in serum were determined at 4, 7, and 10 weeks post-infection (wpi). Activity of superoxide dismutase (Cu, Zn-SOD) significantly decreased (by 35% during the migratory phase, by 40 and 23% at 7 and 10 wpi, respectively), while glutathione reductase activity significantly increased (by 62, 65, and 41%, at 4, 7, and 10 wpi, respectively). No significant changes were found in the activity of glutathione peroxidase. Significant decreases in concentrations of reduced glutathione, vitamins C, E, and A were observed, particularly during the migratory phase of fasciolosis (at 4 wpi). These changes were accompanied by enhancement of lipid peroxidation processes as evidenced by increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Concentrations of MDA and 4-HNE at 4 wpi increased by 38% and by 59%. MDA increased by 51% at 7 wpi and by 79% at 10 wpi, while 4-HNE increased by 87 and 118%, respectively. The results indicate that fasciolosis is associated with enhanced oxidative reactions and reduced antioxidant defense capability of rat serum.     

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.     

Schistosoma mansoni: Effect of insulin and a low-molecular-weight fraction of serum on schistosomula in chemically defined media

Improved chemically defined media for the in vitro maintenance of Schistosoma mansoni schistosomula are described. Artificially transformed schistosomula could be maintained for 7–13 days in a mixture of equal volumes of RPMI 1640 and F-12 supplemented with 30 nM sodium selenite (DSM). Addition of 50 μg/ml insulin increased the survival time to 13–22 days. Insulin at concentrations lower than 25 μg/ml was not effective. Other proteins like hemoglobin, bovine serum albumin, human serum albumin, and lysozyme were also ineffective. A low-molecular-weight fraction from human serum that passes through an Amicon PM 10 filter (10K fraction) increased the survival time to 19–30 days. The schistosomula maintained under these conditions were actively motile for the above periods but did not grow to a significant extent and did not reach the closed-gut stage. However schistosomula maintained for 7 days in DSM or in DSM containing 50 μg/ml insulin and then transferred into DSM-serum (1:1) developed normally after an adaptation period. Insulin greatly increased the initial rate of development and the resistance of mechanically transformed schistosomula to antibodies and complement. Thus, in chemically defined synthetic medium (DSM) in the presence of 50 μg/ml insulin, schistosomula developed a resistance similar to that reached in the presence of 50% serum, but at a somewhat slower rate. On the other hand, in synthetic medium alone without insulin, both the development rate and the extent of resistance were much lower.