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The secreted cathepsin L-like proteinases of the trematode, Fasciola hepatica, contain 3-hydroxyproline residues.

The cysteine proteinases synthesized by the adult stage of the trematode Fasciola hepatica were found to be a very heterogeneous group of proteins as demonstrated by one- and two-dimensional gel analyses. N-terminal amino acid sequencing indicated the presence of at least two distinct gene products among the secreted cysteine proteinases. Enzymic studies and peptide sequence analysis of the excreted/secreted cysteine proteinases suggested a close relationship to the plant thiol cathepsins and the mammalian cathepsin L subfamily. The cloning of a representative cDNA for a putative Fasciola cathepsin confirmed similarities to the cathepsin L subfamily but revealed low identity with the cathepsin-like proteinases of the related trematode, Schistosoma, nematode cathepsins and the mammalian cathepsin B subfamily. Furthermore, peptide and protein sequencing revealed the modification of certain highly conserved prolines to unusual 3-hydroxyproline derivatives. This is the first report of modified prolines in any proteinase. This finding, as well as the high activities of these cathepsins at neutral to alkaline pH values, raises a number of questions as to the physiological function of these thiol cathepsins and their interaction with host tissues.

Amino Acid Sequence↗

[Seasonal variations of Fasciola hepatica infection in goats in the area of Haouz (Morocco)].

The prevalence and level of infection with Fasciola hepatica were assessed monthly over a 12-month period in goats slaughtered in the Haouz area, in correlation with the dynamics of Lymnaea truncatula populations. The infection rate was high in winter (23.8%) in January), and summer (17.1% in August), and relatively low in spring and at the beginning of autumn. The infected animals had a fluke burden range of 1-48; a mean of 13 was recorded in July. Lymnaeid snails were observed in their habitat throughout the year, but were more abundant during the hot months (July and August). Snails infected with F hepatica were found in February, June, July and August; their infection rate did not exceed 3%. These data enabled us to hypothesize that the main periods for transmission of fascioliasis were spring and fall; however, potential infections during other periods of the year were possible. The susceptibility of goats to infection with F hepatica appears to be similar to that reported in sheep.

Animals↗

Comparative efficacy of clorsulon and albendazole against Fasciola hepatica in cattle.

In a dosage-confirmation trial, anthelmintic activities of clorsulon and albendazole against Fasciola hepatica were evaluated and compared. Twenty-eight cattle (8 to 12 months old) with natural F hepatica infections were randomly allotted to 4 groups of 7 cattle each: group 1, no treatment (controls); group 2, clorsulon suspension given orally at 3.5 mg/kg of body weight; group 3, clorsulon suspension given orally at 7 mg/kg; and group 4, albendazole paste given orally at 10 mg/kg. At necropsies, performed 7 and 8 days after treatment, control cattle harbored a geometric mean of 133.2 F hepatica, 16.0 of which were immature. Clorsulon administered at 3.5 mg/kg or 7 mg/kg resulted in greater than 99% removal of F hepatica, including immatures. Albendazole treatment resulted in a 76% overall reduction in F hepatica, including a 91% reduction of immatures. Fascioloides magna also were found in the cattle, but neither clorsulon nor albendazole caused significant reductions of the parasite. Adverse reactions to the 2 drugs were not observed.

Albendazole↗

A histochemical study of the rediae and cercariae of Fasciola hepatica.

Histochemical studies have revealed hydrolytic enzyme activity in the redial tegument and caecal epithelium of Fasciola hepatica. A number of the hydrolases are known to be lysosomal marker enzymes and it is thought that they may be involved in intracellular digestion. The larval stages have also been shown to be reactive for succinate dehydrogenase and phosphorylase, but not for glucose-6-phosphatase.

Acid Phosphatase↗

PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails.

Fasciolosis, caused by the trematode Fasciola hepatica, is a zoonosis of economic importance in livestock that is emerging as a chronic disease in humans. The intermediate hosts are lymnaeid snails, in which diagnosis of infection is traditionally based on cercarial shedding, tissue sectioning and crushing. We developed a PCR assay for the sensitive and specific detection of F. hepatica in field-collected Lymnaea sp. snails. A primer pair was designed to amplify a 405 bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica. The PCR assay showed a limit of detection of 10 pg of genomic F. hepatica DNA. No cross-reactions were observed with samples from other related trematode species or from the snail hosts Lymnaea columella and Lymnaea viatrix. DNA sequencing of the amplicon showed 100% homology with F. hepatica, and 75-89% homology with other trematodes on regions that did not include the entire set of primers. Two samples from Argentina were analysed. For snails in sample 1 (n = 240), identified as L. columella, the infection rate was 17.5 and 51.3% by direct examination and PCR, respectively. For snails in sample 2 (n = 34), identified as L. viatrix, the infection rate was 2.9 and 61.8% by direct examination and PCR, respectively. Differences in infection rates between these diagnosis methods were significant for both samples. Our PCR technique showed to be effective for detecting specific F. hepatica infections of low intensity in the intermediate host, and hence it could be used to study the epidemiological situation in a given area, as well as to assess host suitability for the parasite.

Animals↗

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.

Animals↗

Suitability of six lymnaeid snails for infection with Fasciola hepatica.

The suitability of Fossaria (Bakerilymnaea) cubensis and Pseudosuccinea columella from Louisiana as intermediate hosts for Fasciola hepatica was compared to P. columella and Stagnicola elodes from Ann Arbor, MI, S. attenuata from Hidalgo, Mexico, Lymnaea gedrosiana from Iran and L. natalensis from Senegal. P. columella from LA was shown to be a more suitable host (51.3% became infected) than F. (B.) cubensis (15.2% and 26.4% of two populations became infected). The infection rate for P. columella from MI, was 50% and for L. gedrosiana was 32.5%, whereas L. natalensis, S. elodes and S. attenuata were refractory. F. (B.) cubensis and P. columella have some degree of suitability as intermediate hosts for F. hepatica under laboratory conditions, but field observations of their habitat in southern LA and characteristic management of cattle indicate that the former snail is more important as an intermediate host in this enzootic area. Tissue sections of suitable snails had few histopathological effects but physical damage caused by rediae was pronounced, mainly in the digestive gland and in the mantle.

Animals↗

Resistance to Fasciola hepatica in rats and rabbits following implantation of adult flukes contained in diffusion chambers.

Resistance to Fasciola hepatica could be elicited in rats by implanting mature flukes subcutaneously or intraperitoneally in diffusion chambers. A similar resistance occurred even when the implants were removed two weeks before challenge. This excluded the possibility that the continuing presence of the sensitising flukes was necessary for the maintenance of resistance and also showed that the resistance could be elicited by only two weeks exposure to diffusible immunogens emanating from the flukes.

Animals↗

Activity of tubercidin against immature Fasciola hepatica in mice.

HaM/ICR females mice (20 to 25 g), infected with from 3 to 5 Fasciola hepatica metacercariae per os, were treated with a single dose of thepurine nucleoside analog tubercidin (7-deaza-adenosine; Tu) 7, 14, 21, and 28 days postexposure.Tu was administered either by direct intravenous injection or intraerythrocytically. Most of the flukes were killed and host survival rates were markedly increased if treatment was started within 3 weeks postexposure. The minimal single intravenous dose of Tu that was maximally effective was between 10 and 20 mg/kg.

Animals↗

Observations on Fasciola hepatica in donkeys from Morocco.

Regular monthly examination of a total of 422 donkey livers revealed Fasciola hepatica infection throughout most of the year, with an annual prevalence of 5.9% (range 0-20%). The monthly mean number of worms per animal varied from 0 to 6.4 (annual average 1.6). Of the infected animals 40% had less than ten worms, 28% between ten and 40, 24% between 41 and 70, and 8% over 71. The maximum number of flukes recovered from a single animal was 87. There were two peaks of infection, one in July (from the spring snail infection) and the other in December-February (from the snail infection of late summer-autumn).

Animals↗

Sero-diagnosis of Fasciola hepatica infections in cattle.

An antigen was prepared from metabolic products which were produced by maintaining Fasciola hepatica adults in RPMI tissue culture medium for 24 h. The antigen compared favourably with a soluble extract of adult flukes when used in an enzyme-linked immunosorbent assay (ELISA). Experimentally infected calves were used to evaluate the sensitivity of the test and a survey from areas of both traditionally high and low incidence of the disease was carried out. Evidence is presented which suggests that this metabolic antigen could be used for the sero diagnosis of naturally occurring infections. In addition the use of a microcomputer to read, file and analyse the results enabled a very large number of samples to be processed daily.

Animals↗

Serum and bile antibody responses (IgG and IgA) during subclinical Fasciola hepatica infection in sheep.

The kinetics of serum and bile immunoglobulins (IgG and IgA) directed against Fasciola hepatica in the course of subclinical infection induced experimentally was investigated in sheep. Serum activities of glutamate dehydrogenase and gamma-glutamyltransferase were used as markers of the different fluke stages during infection and associated liver damage. Specific serum and bile immunoglobulins followed a similar kinetic pattern, increasing progressively from infection throughout the prepatent period and tended to decrease when adult flukes became established in the bile duct. IgA titres were lower than those of IgG. Specific IgG and IgA bile titres reached maximum values at 14 weeks postinfection that were considerably lower than the serum titres during the whole experimental period. The major bile immunoglobulins are probably derived directly from plasma. The immunoglobulin kinetic pattern could be related to changes in serum liver enzyme activities.

Animals↗

Evidence of phenotypic differences between resistant and susceptible isolates of Pseudosuccinea columella (Gastropoda: Lymnaeidae) to Fasciola hepatica (Trematoda: Digenea) in Cuba.

Isolates of Cuban Pseudosuccinea columella susceptible and non-susceptible to Fasciola hepatica miracidial infection were compared for cellular reaction to miracidial development, shell morphometrics, mantle pigmentation pattern, and egg laying behaviour. At 24 h post-exposure, non-susceptible snails showed an early cellular host reaction to encapsulate and phagocytise the miracidium, whereas viable transforming miracidia were observed in exposed susceptible snails, indicating that non-susceptibility resulted from resistance rather than physiological unsuitability. Susceptible isolates (more than 80% susceptible to infection) showed significantly higher values of the morphometric indices shell width/length and aperture width/length than resistant isolates (100% resistant). Mantle pigmentation pattern was analysed using Visilog software; the cumulative distances between mantle spots were plotted and compared between isolates, showing a clear distinction between groups. Egg-laying behaviour also showed significant differences with susceptible snails preferring to lay their eggs on the container's walls whereas resistant snails preferred to lay their eggs on the bottom of the container.

Animals↗

Antibody response in cattle, sheep and rats to infection with gamma-irradiated metacercariae of Fasciola hepatica.

Cattle, sheep and rats were infected orally with gamma-irradiated (4 krad) metacercariae of fasciola hepatica, or with normal metacercariae. The antibody response was monitored in each host to metacercarial tegument (T0), juvenile tegument (T1), adult tegument (T2) and gut antigens. The response was examined at weekly intervals for cattle and sheep throughout 15 weeks of infection and four weeks after infection in rats, using an indirect fluorescent antibody labelling technique. It was found that the irradiated metacercariae engendered a normal humoral response to T0, T1 and gut antigens in all three hosts although the antibody levels were somewhat reduced due to an early death or stunting of the flukes. T0 and T1 appeared to be antigenically similar. Antibodies against T2 appeared late in the animals infected with gamma-irradiated metacercariae and the titres attained were considerably lower than in the controls. the T2 antigen stimulus in the animals given gamma-irradiated metacercariae was probably provided by flukes which 'broke through' the developmental barrier imposed by irradiation and which were found alive at autopsy.

Animals↗

Aberrant energy-linked reactions in mitochondria isolated from the livers of rats infected with the liver fluke Fasciola hepatica.

The respiratory properties of mitochondria isolated from the livers of rats infected with the parasite Fasciola hepatica were examined. Oligomycin-sensitive ATPase activity was also examined during the acute stage (2-4 weeks post-infection). At 2,4 and 6 weeks post-infection, mitochondrial respiration in vitro (supported by site I and site II substrates) was completely uncoupled. Limited respiratory control had returned by 11 weeks post-infection, but complete recovery was not observed even at 21 weeks post-infection. At 4 weeks post-infection, uncoupled respiration (from all three energy-conserving sites) was also markedly attenuated (to the greatest extent with NADH-linked substrate). Except for pyruvate-supported respiration, this attenuation was not apparent at any other stage of the infection. The attenuation of pyruvate-supported respiration declined, but was still present, at 6 weeks post-infection. In addition to these perturbations in mitochondrial respiratory properties, mitochondrial ATPase activity at 4 weeks post-infection was insensitive to oligomycin, indicating a change in the structural integrity of the ATPase complex.

Adenosine Triphosphatases↗