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In Greece, Fasciola hepatica, the sheep liver fluke, is common in sheep but only three human cases of fascioliasis have been reported. An epidemiological study was conducted in central Crete which proved to be a focus of F. hepatica. Sheep and snails were found positive for this parasite, but none of the 205 persons tested serologically.
A 36 year old man was diagnosed with eosinophilia and elevated liver-enzyme levels in a preoperative routine laboratory testing. In the course of the evaluation we found high levels of Fasciola hepatica specific antibodies which lead to an ERC with biliary aspiration after choleretic stimulation. The bile showed Fasciola eggs and subsequently we recovered a parasite from the bile duct. Abdominal CT- and MRI-Scans showed lesions of the liver consistent with hepatic fascioliasis. Focussed patient's history revealed ingestion of contaminated watercress in Turkey as the most likely source of infection. For patients with elevated liver-enzymes of unknown etiology with eosinophilia or in combination with a congruent patient's history even without eosinophilia serologic studies of Fasciola hepatica seem to be advisable. We suggest an ERC with biliary aspiration to prove the diagnosis in case of positive results, if the prepatency period of 4 months after the ingestion is over. Fasciola hepatica is rarely diagnosed in Germany probably due to the lack of awareness of the disease.
Cathepsin L proteinases (CL1 and CL2), the major components of Fasciola hepatica excretion/secretion products (E/S) are considered potential antigens of a vaccine against fascioliasis. The humoral response elicited by CL1 and CL2 in rats either immunized with the enzymes or infected with F. hepatica has been analysed, examining specific IgE and IgG subclass dynamics. The experiment was continued for 10 weeks and peripheral blood eosinophilia was also determined. Infected rats presented peaks of eosinophilia at weeks 3 and 7 post-infection, while those immunized with CL1 and CL2 were no different from controls. Total IgE in infected rats increased up to week 5, reaching 30 microg(-1) in some cases, then decreased slowly and rising again towards the end of the experiment. Determination of specific IgE, carried out in sera previously absorbed with Protein G-Sepharose, reached a peak in infected rats between weeks 2 and 5, depending on the individual. In immunized rats both total and specific IgE levels remained around the pre-immunization values. With regard to the IgG subclass responses to E/S products, in infected rats IgG1 predominated over IgG2a, and the reverse was true in rats immunized with CL1 and CL2 and tested against the respective antigens. In all cases an increase in IgG1 and IgG2a antibody titres was seen, with maximum levels being reached later (weeks 6-7) in infected rats than in immunized ones (weeks 4-5). No IgG2b or IgG2c responses were detected in any of the groups studied.
Knowledge of the morphological phenotypes of the liver fluke Fasciola hepatica (Trematoda: Digenea) is analysed. The influence of parasite age on its dimensions, the adult fluke growth model, variation in a biometric variable versus time, and variation in a biometric variable versus another biometric variable (allometric model) are revised. The most useful allometric model appears to be (y2 m-y2)/y2=c [(y1 m-y1)/y1](b), where y1=body area or body length, y2=one of the measurements analysed, y1 m, y2 m=maximum values towards which y1 and y2, respectively, tend, and c, b=constants. A method based on material standardization, the measurement proposal and allometric analysis is detailed. A computer image analysis system (CIAS), which includes a colour video-camera connected to a stereomicroscope (for adult studies) and a microscope (for egg studies), facilitates the processing of digital imaging. Examples of its application for the analysis of the influence of different factors on the liver fluke phenotype are shown using material from the Northern Bolivian Altiplano, where human and domestic animal fascioliasis is caused by F. hepatica only. Comparisons between the development of livestock fluke populations from highlands and lowlands are discussed and the relationships between host species and liver fluke morphometric patterns is analysed.
To estimate prevalence of amphistomiasis and its association with bovine Fasciola gigantica infections in Zambia, 709 faeces of cattle presented for slaughter from three major cattle rearing areas of Central, Southern and Western provinces were analysed. The prevalence rate of amphistomes was 51.6%. Egg counts per gram (EPG) of faeces ranged from 0 to 385 with a mean (+/- SEM) of 11.96 +/- 1.07. The origin of the cattle had a significant influence (P < 0.001) on the prevalence rate. Fasciola gigantica infections accounted for 46.7% of the cattle examined. The mean EPG count (+/- SEM) was 6.3 +/- 0.66 with a range of 0 to 223. A total of 34.6% were mixed infections while single Fasciola and amphistome infections represented 12.1% and 17.1%, respectively. Significantly more cattle (63.8%) were infected with either single or both trematode infections (P < 0.0001) than not. Mixed trematode infections were highest in the Southern province (80.0%) while the lowest were recorded in Central province (16.3%). A positive correlation (r(2) = 0.0428) was present in mixed infections. In order to minimize losses, epidemiology and cross-resistance of amphistomiasis and fascioliasis should be studied in different ecological regions of Zambia to formulate efficient control programmes.
The enzyme-linked immunosorbent assay (ELISA) was evaluated for the diagnosis of naturally acquired Fasciola gigantica infection in cattle in comparison with conventional parasitological techniques. Using unfractionated whole worm extract of F. gigantica as the antigen, it was observed that there was a good correlation between ELISA positivity and positive diagnosis of fascioliasis by post mortem liver examination, bile egg sedimentation and faecal egg sedimentation. There was, however, a disparity between ELISA results and faecal egg flotation results.
Fasciola hepatica and F. gigantica are polymorphic liver flukes that show considerable overlap between species, and various protein separation techniques have been used as alternative means of differentiation. Acid and alkaline polyacrylamide gel electrophoresis (PAGE) show differences between F. hepatica and F. gigantica. Following SDS-PAGE, F. hepatica proteins are characterised by the presence of eight major peptide bands, with molecular weights estimated as 48, 45, 43.5, 37, 33, 29, 27 and 25.5 kDa. In contrast, F. gigantica shows only five major protein bands of 57.6, 54, 48, 29 and 27 kDa. Isoelectric focusing (IEF) demonstrates 17 bands from F. hepatica and 22 bands from F. gigantica between pH 3.5 and pH 10. Although many bands appear common to both species, some are species-specific. Six cases of human acute fascioliasis diagnosed clinically, haematologically and immunologically are also studied. Gel immunodiffusion and immunoelectrophoresis, using adult F. hepatica and F. gigantica antigens, are used to determine the species, and indicate that the antisera are more specific for F. hepatica.
Fascioliasis, caused by the liver fluke Fasciola hepatica, is an infection that occurs worldwide, although humans are accidental hosts. F. hepatica infection comprises two stages, hepatic and biliary, with different signs and symptoms. Stool examination and ELISA can be used for the initial diagnosis. Radiographic techniques, such as computerised tomography and ultrasonography, as well as magnetic resonance imaging, are used widely for confirmation and follow-up of the disease. Invasive techniques, such as percutaneous cholangiography, endoscopic retrograde cholangiography and liver biopsy, may aid in the diagnosis but are not essential. Triclabendazole is recommended as the first-line agent for the treatment of F. hepatica infection, with bithionol as an alternative.
Fasciola hepatica tegument antigens were isolated from intact worms in the cold by using Nonidet P-40. Proof of the tegumental nature of the antigens was shown by the peroxidase-antiperoxidase immunocytochemical technique at the light microscope level. The potential of F. hepatica tegument antigens for the immunodiagnosis of rabbit and human fascioliasis was shown by Ouchterlony immunodiffusion, although cross-reactivity was evident in one of six serum samples from patients infected with Schistosoma mansoni. A genus-specific Fasciola antigen was found in F. hepatica tegument. Finally, F. hepatica tegument contained antigens which protected mice from challenge infection with S. mansoni.
The economic effects of the common helminth parasites of sheep are described and the current methods of prophylaxis adopted by sheep farmers in the United Kingdom reviewed. More efficient prophylactic programmes are outlined and the economic benefit accruing from two programmes designed to control fascioliasis and parasitic gastroenteritis are presented in detail.
Condemnation of meat and offal in 65 selected abattoirs in England and Wales is reviewed. Returns cover about 14 per cent of animals slaughtered for food. In the decade under review, a marked reduction in losses caused by fascioliasis was noted in cattle and sheep, while other parasitic conditions show no decrease. Abscesses continue to be substantial cause of loss in cattle and pigs. Condemnation on account of bruising has increased.
Groups of eight Welsh Mountain sheep were dosed with diamphenethide at the rate of 70 mg/kg bodyweight at either one, four, six or eight weeks after artificial infection with approximately 300 Fasciola hepatica metacercariae. Comparisons were made with similarly infected but undosed sheep and with sheep which were neither infected nor dosed. The good clearance of flukes up to six weeks of age (above 97 per cent on pooled data) was reflected in the plasma concentrations of the accepted liver damage marker enzymes glutamate dehydrogenase and gamma-glutamyl transpeptidase. Highly significant correlations were demonstrated between the numbers of flukes recovered, the plasma levels of these enzymes and haemoglobin and plasma albumin values. At 70 mg/kg, diamphenethide was shown to be able to control F hepatica populations of up to six weeks of age. The systematic use of diamphenethide at this dose level at intervals of up to six weeks during the period of metacercarial challenge should prevent ovine fascioliasis.
Soluble polypeptides extracted from adult Fasciola sp. were analysed by SDS-PAGE, and their antigenicity was characterized by enzyme-linked immunotransfer blot (EITB) using sera from experimentally and naturally infected cattle. Polypeptides of adult Fasciola sp. with estimated molecular mass of 64-52 kDa, 38-28 kDa, 17 kDa, 15 kDa, 13 kDa and 12 kDa, were recognized by sera obtained in both early and late stages of infection. Furthermore, two polypeptides of more than 160 kDa were detected by sera only in the early stage of infection. The results of EITB using sera of dairy cows naturally infected with Fasciola sp. suggest that polypeptides of 64-52 kDa may be candidates as serodiagnostic antigen of bovine fascioliasis.
Previous studies of ours have demonstrated that a recombinant protein (Fh15) related to fatty acid-binding proteins did not induce significant protection in rabbits challenged 2 or 4 wk postimmunization over nonimmunized controls. In the current study, rabbits were immunized with Fh15 and challenged with Fasciola hepatica metacercariae 12 and 20 wk later. In the current study in which longer lag periods for challenge infection after the second immunization were used, worm burden reductions compared to adjuvant controls were a significant 43% and 76%, respectively. Importantly, rabbits immunized with Fh15 had significant numbers of immature flukes, 66% in the 12-wk period and 84% in the 20-wk lag period as compared to controls. In addition, liver lesions were clearly diminished in the vaccinated rabbits. Enzyme-linked immunosorbent assay absorbance values showed that immunized rabbits developed high antibody levels to Fh15 from 8 wk after the first immunization and did not increase after challenge. These results suggest that a recombinant F. hepatica molecule related to fatty acid-binding proteins induces protective (worm burden reductions), anti-fecundity (immature flukes), and anti-pathology (less liver lesions) effects in rabbits and may serve as a model for the immunoprophylaxis of fascioliasis.
A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.
A member of the Fasciola hepatica saposinlike/NK-lysin protein family with lytic activity on human peripheral blood mononuclear cells and erythrocytes was recently described. The current study was designed to test the immunoprophylactic potential of this protein termed FhSAP-2 against infection with F. hepatica in rabbits. Two doses of 50 microg of recombinant FhSAP-2 (rFhSAP-2) emulsified in TiterMax were injected subcutaneously on the dorsal surface of 4 rabbits at 2-wk intervals. Four weeks after the second immunization, the rabbits were infected orally with 25 F. hepatica metacercariae. Four non-immunized-infected rabbits were used as controls. An enzyme-linked immunosorbent assay revealed high levels of antibodies to both rFhSAP-2 and F. hepatica excretory-secretory antigens by 2 wk after the first immunization, which were always significantly higher in immunized-infected rabbits than in control-infected rabbits. On the completion of the trial, vaccinated rabbits had 81.2% less flukes than controls. Moreover, F. hepatica egg counts in feces, as well as in bile collected from the gall bladders from vaccinated animals, were lower, 83.8 and 73%, respectively, compared with controls. The vaccinated rabbits also had significantly lower amounts of parasite antigen in stool and bile samples than controls. Last, evaluation of macroscopic liver lesions revealed that the rabbits vaccinated with rFhSAP-2 had milder lesions than the infected-control rabbits. These findings support the hypothesis that this novel rFhSAP-2 protein has immunoprophylactic potential against fascioliasis in rabbits including antifecundity and antipathology effects. This is the first report on experimental vaccination of rabbits against F. hepatica with a purified, defined, recombinant protein related to a member of the saposinlike protein family.
A 436-bp complementary DNA (cDNA) was isolated from an adult Fasciola hepatica cDNA expression library by screening with the serum from a rabbit infected with F. hepatica for 4 wk. The deduced amino acid sequence encoded by this cDNA is an 11.5-kDa polypeptide that has significant homology to F. hepatica NK-lysin protein, to several members of saposin-like or NK-lysin protein families, as well as 3 amoebapore precursors of Entamoeba histolytica. The most striking feature observed within this protein, denoted FhSAP-2, is the presence of 6 conserved cysteine residues arranged within 5 amphipathic alpha-helical domains and the presence of 7 hydrophobic residues in strictly conserved positions. Using enzyme-linked immunosorbent assay it was found that rFhSAP-2 is highly reactive with sera from rabbits infected with F. hepatica for 2-14 wk as well as with sera from humans with chronic fascioliasis. An anti-rFhSAP-2 rabbit antiserum reacted with F. hepatica excretory-secretory antigens by Western blot, revealing a major 11.5-kDa and 2 minor 46- and 67-kDa antigenic polypeptides. This suggests that FhSAP-2 may be an antigen released from cytoplasmic storage granules present within F. hepatica parasites. rFhSAP-2 also exhibits a strong lytic activity on human erythrocytes and peripheral blood mononuclear cells. This suggests that cell lysis could be 1 of the biological functions of this protein.
Rabbits infected with Fasciola hepatica develop precipitins to adult worm heomogenates, as observed by Ouchterlony double immunodiffusion (ID) and counterelectrophoresis (CEP). When they are successfully treated with a fasciolicidal drug such as rafoxanide at 5, 6, or 11 weeks of infection their precipitins drop dramatically by 2 weeks post-treatment, they are virtually negative by 4 weeks, and have no detectable precipitins by 5 or 6 weeks post-treatment. The results suggest that ID or CEP can be utilized to show chemotherapeutic success in rabbits infected with F. hepatica and warrant further studies as to their possible application in human fascioliasis.