Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Fasciola hepatica in milk

Antibodies against Fasciola hepatica were detected in serum and individual milk samples of dairy cattle using an ELISA. Percentage positivity (PP) values in milk samples were related to serum PP values and were not influenced by days into lactation. The correlation coefficient between serum and individual milk samples was highly significant (r=0.84, P<0.005). The correlation coefficient between herd seroprevalence and herd milk antibody prevalence was 0.96. The correlation coefficient between prevalence measured by faecal egg count and both seroprevalence and milk antibody prevalence within the herd was 0.87. The diagnostic sensitivity and specificity for milk were 92% (95% CI=89-96) and 88% (95% CI=85-91), respectively, when the serum test was considered as a gold standard. In conclusion, the level of antibody to F. hepatica in milk is significantly correlated with the antibody level in serum and this ELISA is suitable as a means of routine veterinary diagnosis of exposure to F. hepatica in cattle and an alternative to testing sera.     

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.     

Failure of a recombinant Schistosoma bovis-derived glutathione S-transferase to protect cattle against experimental Fasciola hepatica infection

The potential of a recombinant Schistosoma bovis 28-kDa glutathione S-transferase (rSb28GST) to protect cattle against Fasciola hepatica was tested in a vaccination trial. Thirty two calves were randomly divided into four groups of eight animals. Calves of the three vaccine groups received two intramuscular injections at 3 weeks interval, of 0.250mg rSb28GST in either aluminium hydroxide (Al(OH)(3)), Quil A, or PBS emulsified in an equal volume of Freund's complete adjuvant (FCA).Animals of the control group received injections of Al(OH)(3)/PBS only. All animals were challenged orally with a total of 360 metacercariae of F. hepatica, spread over 6 weeks.All groups of vaccinated animals produced measurable IgG antibody titers to rSb28GST after vaccination. Animals immunised with FCA adjuvanted vaccine had the highest and more durable antibody titers and only sera from this group recognised an approximately 24kDa protein band from F. hepatica, that is thought to be a F. hepatica GST. Despite a good antibody response differences in cumulative faecal egg output between the groups were not statistically significant. In addition, no significant difference was found between groups in terms of total worm numbers or percentage of immature flukes recovered at necropsy. In conclusion, the recombinant S. bovis 28kDa GST was not found to adequately protect cattle against experimental F. hepatica challenge, using either aluminium hydroxide, Quil A or FCA as adjuvant.     

Serological responses of cattle after treatment and during natural re-infection with Fasciola hepatica, as measured with a dot-ELISA system

The dot-ELISA reaction was used to study the dynamics of IgG titers in cattle naturally infected with Fasciola hepatica after anthelmintic treatment and during reinfection. Excretion/secretion products (ES) of the parasite were used as antigens for the dot-ELISA. IgG antibodies were no longer detectable by dot-ELISA, 4-6 months after nine animals received the first of three weekly doses of triclabendazole (15 mg kg(-1)) and were then maintained on a pasture free of F. hepatica metacercariae. Six fluke-free cattle began shedding F. hepatica eggs 3-6 months after grazing a pasture contaminated with metacercariae of the parasite. A detectable increase in dot-ELISA IgG antibody levels was observed 2-4 weeks after natural reinfection by grazing a similar pasture contaminated with F. hepatica metacercariae. The usefulness of the dot-ELISA system to diagnose chronic infection by serology is complicated by previous treatment against the parasite. It is concluded that the ES antigens can be useful to detect early infection of cattle with F. hepatica in a dot-ELISA system     

Relationships between host species and morphometric patterns in Fasciola hepatica adults and eggs from the northern Bolivian Altiplano hyperendemic region.

The highest prevalences and intensities of human fasciolosis by Fasciola hepatica are found in the northern Bolivian Altiplano, where sheep and cattle are the main reservoir host species and pigs and donkeys the secondary ones. Morphometric comparisons of many linear measurements, areas and ratios of F. hepatica adults (from sheep, cattle and pigs) and eggs (from sheep, cattle, pigs and donkeys) in natural liver fluke populations of the Bolivian Altiplano, as well as of F. hepatica adults and eggs experimentally obtained in Wistar rats infected with Altiplanic sheep, cattle and pig isolates, were made using computer image analysis and an allometric model. Although morphometric values of adult flukes from natural populations of sheep, cattle, and pigs showed great overlap, there were clear differences in allometric growth. The allometries analyzed were: body area (BA) versus body length (BL), BA versus body width (BW), BA versus perimeter (Pe), BA versus distance between posterior end of body and ventral sucker (P-VS), BL versus BW, BL versus Pe, and BL versus P-VS. These allometries show a good fit in the seven pairs of variables in all the populations examined. Comparative statistical analysis of the allometries shows that fluke adult populations from sheep, cattle and pigs significantly differ in BL versus BW and BL versus P-VS functions. Statistical analysis of F. hepatica egg size shows characteristic morphometric traits in each definitive host species. In experimentally infected rats, fluke adult allometry and egg morphometry do not vary depending on the Altiplanic definitive host species isolate. Our study reveals that the definitive host species decisively influences the size of F. hepatica adults and eggs, and these influences do not persist in a rodent definitive host model.     

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.     

The efficacy of formulations of triclabendazole and ivermectin in combination against liver fluke (Fasciola hepatica) and gastro-intestinal nematodes in cattle and sheep and sucking lice species in cattle

OBJECTIVE: To assess the efficacy of two formulations of triclabendazole and ivermectin in combination against liver fluke (Fasciola hepatica), gastro-intestinal nematodes and sucking louse species in cattle and sheep. PROCEDURE: A study of 540 cattle and 428 sheep at 18 sites throughout Victoria and New South Wales was undertaken. At each site, one group of cattle or sheep was treated with a combined formulation (Fasimec Cattle or Fasimec Sheep), another received ivermectin and triclabendazole separately. In trials on lice infestation, an additional group remained untreated. Samples for faecal egg counts were collected on days -7, 0 (treatment day), +7, +14 and +21 after treatment. Lice assessments were carried out on days -7, 0, +7, +14, +28, +42 and +56. RESULTS: Both treatments were highly efficacious (> 98% efficacy) against liver fluke in cattle and sheep, against three sucking lice species of cattle and against gastro-intestinal nematodes in sheep. There was also no significant difference between treatments in efficacy. Against gastro-intestinal nematodes, Fasimec Cattle was significantly (P < 0.01) more effective than the separately applied ivermectin and triclabendazole treatment. Mean efficacy for the Fasimec Cattle and Ivomec/Fasinex 120 groups respectively, was 97.6% and 94.2% on Day +7, 98.9% and 91% on Day +14 and 98.5% and 92.6% on Day +21. CONCLUSION: The efficacy of Fasimec' Cattle and Fasimec Sheep was at least equal to that of currently registered products (with the same active ingredients) used to control these parasites.     

Efficacy of two formulations of albendazole against liver flukes in cattle.

Albendazole (10 mg/kg of body weight) was administered as a drench suspension or as a feed additive to 24 cattle with naturally acquired infections of Fasciola hepatica and Fascioloides magna. Cattle were euthanatized 16 to 30 days after treatment, and the number of viable flukes was counted. Viable F hepatica and F magna were decreased by 91.4% and 70.6% for drench administration and by 82.9% and 71.9% for the feed additive treatment, respectively. There was no significant difference between the efficacy of the 2 formulations in decreasing viable fluke numbers, compared with untreated controls.     

The specificity of antibody responses in cattle naturally exposed to Fasciola hepatica

Fasciola hepatica causes significant morbidity and mortality in dairy cattle in the Andean region of Cajamarca, Peru, where prevalence of infection of up to 78% has been reported. ELISA and Western blot analyses were used to characterise antibody responses in dairy cattle to adult F. hepatica to excretory-secretory (E/S), somatic (SO) and surface (SU) antigens. Three groups of dairy cattle - calves, heifers and adult cows - naturally exposed to F. hepatica in this region, were monitored every 2 months over a 2-year period. Calves, heifers and adult cows all had antibodies which recognised a 28kDa protein in the SO preparation, whereas only adult cows had antibodies that recognised a 28kDa protein in E/S products. All three groups of cattle responded to a 60-66kDa group of proteins in E/S and SU preparations and a 17kDa antigen in SO products was recognised by antibodies from cows and heifers but not calves. The total antibody response to E/S antigens measured by ELISA, increased over time in calves and remained constantly high over the 2-year period in all three groups of cattle. Slight fluctuations in the antibody response occurred in the group of heifers and cows coinciding with seasonal changes in the level of challenge.     

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.     

Field trial on comparative efficacy of four fasciolicides against natural liver fluke infection in cattle

A controlled trial was conducted to evaluate the current efficacy of albendazole (ABZ), rafoxanide (RFX), triclabendazole (TRC) or clorsulon (CLS) against Fasciola hepatica in naturally infected cattle. This trial was conducted in Turkey during the spring, the time of year when liver fluke infection is endemic. Fifty crossbred cattle were selected for inclusion in the trial based on finding eggs of F. hepatica in the feces. The cattle were weighed and randomly allotted into five groups of 10 cattle and treatments were as follows: Group 1 served as non-treated control (CONT), Group 2 was treated orally with ABZ at 12 mg/kg, Group 3 was treated orally with RFX at 10 mg/kg, Group 4 received TRC orally at 12 mg/kg and Group 5 received CLS administered subcutaneously (s.c.) at 2 mg/kg. On day 0 (inclusion day), individual fecal samples were collected on days 0 (inclusion day), 7, 14, 28 and 56, after treatment. The drug efficacy was assessed as a percentage of the egg or fluke reduction and body weight gain relative to the untreated control. The results in the study showed a mean reduction of egg counts by 66.7%, 68.2%, 78% and 84.2% in Groups 2-5, respectively. In conclusion, our results indicate that CLS is a highly effective compound for the treatment of F. hepatica in cattle under these field conditions.     

The effectiveness of the drugs Dovenix and Bilevon(R)-Injection against liver fascioliasis (Fasciola hepatica, Linné 1758) in cattle]

Dovenix and Bilevon-injection (manufactured by SPECIA, France and BAYER, West Germany, respectively) were tested for their anthelmintic efficacy against Fasciola hepatica in cattle. The drugs proved highly effective against both adult and immature flukes. The faeces of the treated animals were negative for F. hepatica eggs when examined 91 days after the treatment. In experiments with rats Dovenix and Bilevon-injection were tolerated up to eight times and ten times higher doses than the normal therapeutic dose, respectively.     

Bulk milk-estimated seroprevalence of Fasciola hepatica in dairy herds and collecting of risk factor data in East Frisia, northern Germany

The liver fluke Fasciola (F) hepatica is one of the most important trematodes in cattle farming worldwide. Fasciolosis in dairy cows is leading to production losses due to decreased milk yield, liver condemnation and impaired reproduction. The treatment of dairy cows is unsatisfactory, because available drugs are either effective against adult flukes only or have long withdrawal times or in some countries may not be used at all. In the present study the prevalence of F. hepatica in dairy farms located in East Frisia, which is part of the federal state Lower Saxony, was investigated. East Frisia is considered a high risk area for Fasciola hepatica infections, because of its coastal location, high precipitation and moist pastures. About 750 bulk milk samples were collected in January and November 2006 and analysed for F. hepatica antibodies using the Pourquier ELISA. In addition, questionnaires, which were answered by 260 of the participating farmers, were evaluated to analyse management-related factors associated with fasciolosis. In January and November, 52.1% and 53.6% of the bulk milk samples, respectively, showed positive results. Thereby, 88.1% of the examined farms showed an unchanged infection status, whereas 6.2% of the farms became seropositive during the grazing season and 5.8% of the dairy herds turned seronegative. Statistical analysis revealed a significant negative association between average annual milk production and the frequency of infections with F. hepatica.     

苏皖水牛肝片吸虫和前后盘吸虫感染情况的调查

苏皖两省１０县（市）共调查３０２农户的３０７头水牛,粪便学检查肝片吸虫阳性４３头,阳性率１４．０％,其中安徽的阳性率为１１．６１％（１８／１５５）,江苏阳性率为１６．４８％（２５／１５２）;母牛阳性率为１１．８％,公牛为１１．４％;前后盘吸虫阳性１７０头,阳性率为５５．４％（１７０／３０７）,其中安徽的阳性率为５８．１％（９０／１５５）;江苏的阳性为５２．６％（８０／１５２）,母牛的阳性率为６０．１％（１３７／２２８）,公牛的阳必国４１．８％（３３／７９）,两种吸虫的阳性率均呈现随年龄增加而提高的趋势,经统计学分析,两种吸虫的感染与地形无显著差异（Ｐ〉０．０５）,肝片吸虫的感染与性别、年龄亦无显著差异（Ｐ〉０．０５）,但对前后盘吸虫来说,感染与性别、年龄的差异显著（Ｐ〈０．０５）。而各县间两种吸虫感染则有显著关     

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).     

Immune responses in rats and cattle to primary infections with Fasciola hepatica

Antibody and lymphocyte proliferative responses to fluke antigens were compared in rats and cattle following infection with Fasciola hepatica. Antibody responses differed between rats and cattle in that rats responded more quickly and with a greater rate of synthesis over the first three weeks of infection than did cattle. Lymphocyte proliferative responses developed and disappeared at similar times in both cattle and rats, being detectable early in infection, but returning to background levels by week 6 after infection. The presence of antibody but not of lymphocyte proliferative responses correlated with the timing of the development of resistance in cattle and rats as described by others. Sensitisation by intraperitoneal injection of fluke antigens in Freund's incomplete adjuvant (FIA) induced antibody production but not lymphocyte proliferative responses in both rats and cattle. Serum antibodies continued to rise after oral infection of sensitised animals although this was much more marked in the cattle than in the rats. On the other hand lymphocyte proliferative responses were absent when sensitised cattle were infected, while the findings with rats were much more variable. Cattle sensitised by intraperitoneal injection of fluke antigens in FIA were not protected against subsequent infection with F hepatica.     

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.     

应用酶标SPA Dot-ELISA同时检测牛羊血吸虫病和肝片吸虫病

采用给雄性家兔注射黄牛、水牛、山羊ＩｇＧ混合液（１∶１∶１）制备的兔抗黄牛、水牛和山羊ＩｇＧ连接酶标ＳＰＡ，解决了酶标ＳＰＡ与牛羊血吸虫和肝片吸虫免疫复合物相结合的技术关键，并制成了可同时用于牛羊血吸虫病和肝片吸虫病诊断的酶试剂。建立了Ｄｏｔ－ＥＬＩＳＡ同时检测牛羊血吸虫病和肝片吸虫病技术，能测出人工感染１～１５０条虫体７～４２ｄ病畜抗体，阳性检出率达１００％。     

The disposition kinetics of albendazole following the administration of single and divided doses to cattle and buffalo

Concentrations of albendazole sulphoxide and its sulphone metabolite in plasma in cattle and buffalo were measured by high-performance liquid chromatography after single and divided intraruminal administration of albendazole at the recommended nematocidal and fasciolicidal dose rates of 7.5 and 15.0 mg/kg body weight, respectively. No significant differences in the plasma concentrations of the metabolites or their pharmacokinetic parameters were observed between cattle or buffalo at either dose rate. Pharmacokinetic analysis and the disposition curve of the metabolites indicated increased uptake of the drug in both cattle and buffalo when the same total amount of the drug was given in divided doses compared to a single dose (p<0.05). The divided dose schedules of administration could possibly be exploited to extend the life of the available benzimidazole anthelmintics.     

A survey of fascioliasis in beef cattle killed at abattoirs in southern Queensland

The overall prevalence of fascioliasis in beef cattle found in an abattoir survey completed in May 1983 in southern Queensland, was 1.1%. Prevalence was 36% in the Stanthorpe Shire, varied from greater than 1 to 10% in 12 shires and was 1% or less in 24 shires. Fascioliasis was not recorded from the other 46 shires in the area of the survey. Prevalence was less than 1% in female cattle of all ages. In males, prevalence increased with age from 0.6% in 17-month-old cattle to 4.7% in 4-year-old cattle. The differences in sex-specific prevalences may be due to differences in susceptibility to the parasite between sexes, differences in susceptibility to the parasite between sexes, differences in management between sexes, or artefacts associated with sampling for a disease of very low prevalence. The presence of the exotic snail species, Lymnaea columella and L. viridis was confirmed in southern Queensland in 1977. These snails are suitable intermediate hosts for Fasciola hepatica and may widen the endemic area for liver fluke because of their more aquatic character and greater tolerance to high temperatures than L. tomentosa, the traditional snail host in Queensland. We suggest that introduced snails have not yet had a substantial impact on the importance of fascioliasis to the beef industry of southern Queensland. The survey provides baseline data for the future monitoring of this potentially important parasitic disease.