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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.

Animals↗

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.

Aldehydes↗

Estimating the true prevalence of Fasciola hepatica in cattle slaughtered in Switzerland in the absence of an absolute diagnostic test.

A survey of 1,331 cattle presented for slaughter at two abattoirs in Switzerland was used to estimate the true prevalence of Fasciola hepatica infection and the diagnostic parameters of visual meat inspection, coproscopy after sedimentation technique, a commercial ELISA test for specific antibody detection in serum and the post mortem microscopic detection of eggs in bile. Faeces, blood and the gall bladder were taken from most cattle presented for slaughter. In addition, livers that were rejected by the meat inspectors were also dissected to examine for the presence of liver fluke. Bayesian techniques (Markov Chain-Monte Carlo) were used to estimate the diagnostic parameters of each of these procedures and the true prevalence of bovine fasciolosis. The true prevalence of F. hepatica infection was estimated at 18.0% (95% credible intervals 15.9-20.3%). The diagnostic sensitivity of coproscopy, bile examination, antibody ELISA and meat inspection were estimated at 69.0% (57.3-79.7%), 93.4% (88.0-97.5%), 91.7% (87.2-95.2%) and 63.2% (55.6-70.6%), respectively. The diagnostic specificity of the ELISA test was estimated at 93.7% (91.7-95.2%). These results demonstrate that the prevalence of bovine fasciolosis is higher than previously thought due to the low sensitivity of meat inspection. They also demonstrate that traditional coproscopy can be very efficient if there is repeated sampling, resulting in sensitivity of approximately 92%.

Abattoirs↗

Measurement of triclabendazole and its metabolites in liver flukes: method development and full validation.

The trematode parasite Fasciola hepatica is still the cause of considerable loss in livestock production all over the world. The knowledge of the pharmacological properties of the available flukicidal drugs is critical to control this parasite. Triclabendazole is an halogenated benzimidazole anthelmintic with potent flukicidal activity. A simple reversed-phase high-performance liquid chromatographic analytical method has been developed, validated and applied for the quantitative determination of the flukicidal drug triclabendazole and its sulpho- and hydroxy-metabolites in the liver fluke, F. hepatica. Triclabendazole, triclabendazole sulphoxide, triclabendazole sulphone, hydroxy-triclabendazole, hydroxy-triclabendazole sulphoxide and hydroxy-triclabendazole sulphone were measured simultaneously in this trematode parasite. Linearity, resolution, precision, accuracy, recovery, limits of detection and quantification of the method were determined. Drug extraction from the parasite's tissue homogenate was effectively performed using liquid extraction (acetonitrile), avoiding clean up by solid phase extraction, prior to analysis by reversed-phase high-performance liquid chromatography. The resolution of all the halogenated benzimidazole thiol molecules assayed was obtained on a C(18) reversed-phase (5 microm, 250 mm x 4.6 mm) column using acetonitrile and ammonium acetate as the mobile phase and ultraviolet detection at 300 nm. Regression analyses were linear over the concentration range examined (from 0.272 to 16.331 nmol/100 mg trematode protein) and the correlation coefficients of the calibration curves ranged between 0.996 and 1.000. The calculated limits of detection of the proposed method for the parent drug and its metabolites ranged between 0.007 and 0.079 nmol/100 mg trematode protein. The extraction efficiency for the different analytes from the parasite material was greater than 71%. The results obtained indicated that the developed chromatographic method was selective, accurate and easy to reproduce. The developed procedure was successfully applied to quantify triclabendazole/metabolites in F. hepatica incubated under ex vivo conditions, demonstrating to be efficient for the determination of the most extensively used flukicidal drug available for veterinary medicine, and its metabolites. The analytical method described here is an useful tool for the measurement of this flukicidal compound in different studies addresses to evaluate drug influx/eflux and metabolism in its main target parasite.

Animals↗

Clinical and biological abnormalities in patients after fasciolosis treatment.

OBJECTIVE: The aim of this communication was to report clinical disorders and biological abnormalities in patients followed over a 3- to 18-year period after the last date of fasciolosis treatment. These observations were noted in 87 individuals (out of 711) during a 30-year epidemiological survey. RESULTS: We noted diffuse pain in the right hypochondrium, persisting 2 to 6 years after treatment (3.7% of persons), and weight loss appearing during treatment and persisting afterwards (2.1%). CONCLUSIONS: Our current state of knowledge leads us to asking whether pain in the right hypochondrium and weight loss would not be due to the presence of persistent hepatic lesions following fasciolosis treatment. Other investigations in patients after treated fasciolosis are necessary to confirm these abnormalities and determine their frequency.

Animals↗

An analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to its homologs in the phylum Platyhelminthes.

A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.

Amino Acid Sequence↗

Bayesian hierarchical modelling to enhance the epidemiological value of abattoir surveys for bovine fasciolosis.

Four classes of Bayesian hierarchical models were evaluated using an historical dataset from an abattoir survey for fasciolosis conducted in Victoria, Australia. The purpose of this analysis was to identify areas of high prevalence and to explain these in terms of environmental covariates. The simplest of the Bayesian models, with a single random effect, validated the use of smoothed maps for cartographic display when the sample sizes vary. The model was then extended to partition the random effect into spatially structured and unstructured components, thus allowing for spatial autocorrelation. Rainfall, irrigation, temperature-adjusted rainfall and a remotely sensed surrogate for rainfall, the normalised difference vegetation index (NDVI), were then introduced into the models as explanatory variables. The variable that best explained the observed distribution was irrigation. Associations between prevalence and both rainfall and NDVI that were significant in fixed effects models were shown to be due to spatial confounding. Nevertheless, provided they are used cautiously, confounded variables may be valid predictors for the prevalence of disease.

Abattoirs↗

A retrospective survey of liver fluke disease in livestock based on abattoir data in Shiraz, south of Iran.

Meat-inspection records in an abattoir located in the Fars province (southern part of Iran) from 20 March 1999 to 19 March 2004 were used to determine the prevalence and long-term trend of liver fluke disease in sheep, cattle and goats in the region. A total of 844,039 animals (cattle 131,716; sheep 577,090; goats 135,233) slaughtered in the 5-year period and overall 34,856 (4.1%) livers were condemned. Fasciolosis and dicrocoeliosis were responsible for 54 and 21% of total liver condemnations, respectively. The prevalence of liver condemnations due to fasciolosis was decreased from 3.89, 3.20 and 2.63% in 1999-2000 to 1.07, 0.59 and 0.24% in 2003-2004 for cattle, sheep and goats, respectively. The corresponding features for dicrocoeliosis were similar, declining from 1.47, 1.76 and 2.10% in 1999-2000 to 0.69, 0.34 and 0.25% in 2003-2004, respectively. Drought climatic conditions in conjunction with a greater awareness among farmers could be responsible factors.

Animals↗

Associations between anti-Fasciola hepatica antibody levels in bulk-tank milk samples and production parameters in dairy herds.

Our primary objective was to determine the relationships between Fasciola-specific antibody levels in bulk-tank milk and measures of productivity to estimate economic losses that are associated with Fasciola infections. A bulk-tank milk sample was collected in March 2004 from 1105 dairy herds in Flanders and the antibody levels against Fasciola hepatica (ODRf) and Ostertagia ostertagi (ODRo) were determined. The association of ODRf with four production parameters (milk yield, milk-protein %, milk-fat % and inter-calving interval) was assessed by multivariable linear-regression models. Production data were available for 463 out of the 1105 herds sampled. An increase in ODRf from the 25% quantile (0.428) to the 75% quantile (1.064) was associated with a decrease in the annual average milk yield of 0.7kg/(cowday) (P=0.002), with a decrease in the average milk-fat % of 0.06% (P<0.001) and with an increase of the mean inter-calving interval of 4.7 days (P=0.03). No significant relationship was found with the average milk-protein %. When the relationships of ODRf and ODRo with milk yield were tested simultaneously, we saw an additive rather than synergistic effect of concurrent infections.

Animals↗

Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test.

An ELISA was developed for the detection of Fasciola hepatica antibody in serum of cattle. The assay was applied to sera from 258 naturally infected cattle, 256 non-infected cattle and six calves experimentally infected with F. hepatica. The diagnostic sensitivity and specificity of the ELISA test was 98% (95% confidence intervals, 96-100%) and 96% (95% confidence intervals, 93-98%) respectively at a cut-off value of 15% positivity. The results using sera from the experimentally infected calves showed that antibodies were first detected 2-4 weeks after infection. The ELISA test was also compared to the commercially available Bio-X bovine F. hepatica ELISA kit. A subset of 39 positive sera and 47 negative sera were selected from the samples used to evaluate the in-house test. The results indicated that the agreement between the two tests was almost perfect (k statistic=0.82).

Animals↗

Anthelmintic resistance--looking to the future: a UK perspective.

The full extent of anthelmintic resistance in nematodes of farm animals is not known. Resistance can be detected with a faecal egg count reduction test and two in vitro tests, the egg hatch and larval development tests. The sensitivity of these two in vitro tests can be increased by using discriminating doses rather than calculating LD50 values. Only benzimidazole resistance can be detected with PCR based tests because the molecular mechanisms of resistance to levamisole and the macrocyclic lactones remain unknown. Resistance detection is important because it enables the appropriate management strategies to be put in place. The development of resistance is delayed by keeping sufficient parasites in refugia (not exposed to anthelmintic), but the necessary management details have not yet been validated in the field. It is probably too late to use combination products to delay the development of resistance, except in cattle but quarantining animals to prevent introduction of resistant helminths onto a farm is important. Dilution of resistant worms with susceptible ones is only at the preliminary research stage and the application of non-chemical methods of control to delay resistance is not yet a practical option. Extensive research is required to manage resistance, especially in the control of resistance in Fasciola hepatica.

Animals↗

Humoral and cellular immune responses to Fasciola gigantica experimental infection in buffaloes.

Humoral and cellular immune responses to Fasciola gigantica experimental infection in buffaloes were studied. The results showed that 33.4+/-9.1% of the infection dose was recovered as adult flukes from infected animals at necropsy. Significant differences of weight gain between infected and non-infected buffaloes was observed at 4 MPI (months post-infection). Anti F. gigantica excretory-secretory products (FgESP)-IgG levels increased significantly from 3 WPI (weeks post-infection) and displayed a peak at 13 WPI. Western blot indicated that in FgESP six major bands of 11.5, 19.0, 23.4, 29.8, 47.5 and 53.2kDa were recognized by F. gigantica-infected buffaloes sera after 0 WPI. Eosinophil numbers increased significantly from 3 WPI in F. gigantica-infected buffaloes and displayed a peak at 8 WPI. Peripheral blood mononuclear cells (PBMC) proliferation induced by FgESP increased from 2 WPI with a peak at 5 WPI. IFNgamma secretion by FgESP-stimulated PBMC appeared early from 1 WPI with three peaks at 2, 5 and 8 WPI, respectively. IL-10 production was observed from 2 WPI with two peaks at 4 and 9 WPI, respectively. Our results suggested that buffaloes were highly susceptible to F. gigantica infection, and this susceptibility could be associated with the late and weak cellular immune response in the early phase of infection and the Th0-like response throughout the infection.

Animals↗

Effect of Fasciola gigantica infection on adrenal and thyroid glands of riverine buffaloes.

Effect of Fasciola gigantica infection on adrenal and thyroid glands was investigated using eight male, yearling Murrah buffaloes. The animals were randomly assigned to two groups of four buffaloes each (Group-A, infected; Group-B, non-infected control). Animals of Group-A were orally infected with 1000 F. gigantica viable metacercariae, keeping other four animals of Group-B as uninfected control. In the infected buffaloes, the clinical signs began appearing from 7th week postinfection (p.i.) and eggs were detected in the faeces between day 93 and 99 (95.5+/-1.25) postinfection (p.i.). The serum cortisol level, revealed a significant (P<0.05) rise during initial stage of the infection, followed by a continuous fall from 12th week onward. Peak cortisol level on 10th week (13.30+/-2.57ngml(-1)) was associated with eosinophilia (11.0+/-0.95%). However, non-infected controls maintained almost uniform cortisol levels (3.97+/-0.15-5.88+/-0.09ngml(-1)) throughout the period of the study. The pathological changes of adrenal glands were correlated with physiological dysfunction of the glands. The levels of T(3) and T(4) were significantly (P<0.05-0.01) low from 14th week onward and were synchronous with in situ migration, growth and development of F. gigantica. Significant reduction in the thyroid hormones was further supported by histopathological evidence of lymphocytic thyroiditis confirming hypothyroidism. A decrease in Hb, PCV, total erythrocyte counts and appearance of reticulocytes in the blood of the infected buffaloes suggested regenerative anemia, which could partly be due to hypothyroidism.

Adrenal Glands↗

Evaluation of relationship between repeat breeding and Fasciola hepatica and hydatid cyst infections in cows in Elazig district of eastern Turkey.

Sera samples from 89 dairy cows with repeat breeding and 111 healthy pregnant dairy cows (controls) from Elazig province of eastern Turkey were tested for presence of Fasciola hepatica and Hydatid cyst antibodies by use of an indirect enzyme linked immunosorbent assay (indirect-ELISA). One hundred and twenty one (60.5%) and 93 (46.5%) cows were found to be positive to F. hepatica and Hydatid cyst antibodies, respectively. Fifty two of 89 cows (58.4%) with a history of repeat breeding were seropositive to F. hepatica, and 43 of 89 (48.3%) were seropositive to Hydatid cyst antibodies. Whereas, seropositivity rates were 62.1% (69/11) for F. hepatica and 45% (50/111) for Hydatid cyst in healthy pregnant cows (control group). There were no statistically relationship between infected and control group (P>0.05). In conclusion, we could not detect any relationship between repeat breeding and F. hepatica and Hydatid cyst infections in cattle.

Animals↗

Fasciola hepatica in the human eye.

During intraocular surgery of a 44-year-old woman from Guilan Province in north Iran, a small flat parasite was removed from the anterior chamber angle of the left eye. According to morphological characterisation of the parasite, it was identified as Fasciola hepatica. The route of entry of the parasite was not identified. Meanwhile, stool and serology examinations of the patient for diagnosis of Fasciola infection were negative. In endemic areas of F. hepatica infection, ocular involvement should be considered in cases of uveitis, despite no other systemic involvement.

Adult↗

An outbreak of subacute fasciolosis in Soay sheep: ultrasonographic biochemical and histological studies.

A naturally occurring outbreak of subacute fasciolosis in a group of 17 seven-month-old Soay ram lambs was studied following the sudden death of two sheep. In addition to standard biochemical investigations, ultrasound examination of the liver and cranial abdominal cavity was undertaken. There was a significant positive linear correlation between liver weight and ultrasonographic determination of liver size (R=0.72, P<0.05). Ultrasonographic examination of the liver revealed multiple hyperechoic dots in the parenchyma giving a granular appearance to the hepatic texture in three sheep corresponding to the most advanced histopathological changes as determined by the size of the abscesses and their relatively mature fibrous capsules, and areas of hepatic necrosis. No distension of the bile duct system was noted nor was the gall bladder imaged. While serum concentrations of albumin, globulin and certain liver enzymes assisted in the diagnosis of subacute fasciolosis in sheep, only glutamate dehydrogenase, and gammaglutamyl transferase remained elevated four weeks after triclabendazole treatment.

Animals↗

Fasciola hepatica procathepsin L3 protein expressed by a baculovirus recombinant can partly protect rats against fasciolosis.

Fasciola hepatica juveniles express immunodominant cathepsin L proteins, which are mainly found in their immature, procathepsin form. A gene encoding such a procathepsin L (FheCL3) was expressed by a baculovirus recombinant and by Saccharomyces cerevisiae. The glycosylated FheCL3 proteins obtained by both systems were used in a vaccination/challenge experiment in rats. Both antigens evoked similar antibody responses, but only the baculovirus expressed FheCL3 caused a significant protection against the number of liver flukes (52% protection, P=0.01), whereas the S. cerevisiae expressed FheCL3 did not. In a second experiment in rats, deglycosylated versions of both antigens were used, but this did not improve their efficacies.

Amino Acid Sequence↗