Detection of circulating 54 kDa antigen in sera of bovine calves experimentally infected with F. gigantica

The antibody response and circulating antigen levels in bovine calves, infected experimentally with Fasciola gigantica, were monitored using enzyme-linked immunoelectrotransfer blot (EITB) and sandwich ELISA, respectively. By EITB, the infected calves' sera recognized the polypeptides in the range of 54-58 kDa as early as 2 weeks post-infection. By 12th week post-infection, the lower two polypeptides of 12 and 8 kDa had disappeared. In sandwich ELISA, the circulating 54 kDa and whole worm antigen of F. gigantica were detected in the sera samples of infected calves as early as 2 weeks post-infection and persisted until the end of experiment (26th week PI). The 54 kDa antigen of F. gigantica appears to be specific and possesses promising immunodiagnostic potential for early prepatent diagnosis of bovine fasciolosis.     

28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis

Coprological confirmation of ovine fasciolosis in the field, prior to out breaks of the disease and/or strategic antifluke medication, seem to be of little consequence. Efforts are, therefore, being made to evolve a putative antigen specific to serodiagnostic test for early diagnosis during prepatency. In the present investigation, 28 kDa cysteine proteinase was used in ELI SA and Western blot to detect Fasciola gigantica antibodies and further Dipstick-ELISA was developed for field application, using known positive monospecific sera from experimentally infected sheep with 100 F. gigantica metacercariae. Isolation of 28 kDa cysteine proteinase was achieved from bubalian origin flukes. The specific antigen, recognised homologous antifluke antibodies by Western blot as early as 2nd week post-infection (wpi) with 100% sensitivity, in sera samples of sheep harbouring 38 flukes and by 10th wpi in sheep harbouring 3-8 flukes. All sheep were found positive for the infection when ELISA and/or Dipstick-ELISA was applied from 4th wpi. In pooled sera of infected sheep, these were positive during 4th wpi.     

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.     

Immunoglobulin E recognition of Dirofilaria immitis antigens is more specific than immunoglobulin G.

The chronological development of the serum IgE and IgG response to microfilaria, third and fourth stage larvae, and male and female adult Dirofilaria immitis was examined by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blot (EITB). Dirofilaria immitis-specific IgE and IgG levels peaked 16-18 weeks post-infection after increasing in response to the fourth larval molt. Specific IgG levels plateaued after patency, while IgE continued to decline. The use of ammonium sulfate cut sera showed there was no quenching or blocking of IgE binding by IgG in the ELISA and EITB methods used in this study. IgE-specific EITB showed 30-49 bands for the five respective extracts that were identified by M(r) or relative mobility. Eighty-five to 100 bands were visualized by IgG-specific EITB for the same five extracts. The isotype-specific ELISA and EITB were shown to be closely related by significant correlations (P < 0.0001) between S/N ratios and the number of bands found on blots. The isotype-specific EITB bands non-specifically recognized were greater in size than 21 kDa for IgG and 45 kDa for IgE. Recognition of bands changed over time with some bands being recognized only by prepatent sera. Ten antigen bands of seven M(r) were consistently and specifically recognized by IgE in the five-stage extracts by sera from prepatent and patent infections; only one such M(r) at 13.9 kDa, was described for IgG. A potentially diagnostic 31.9 kDa antigen band was identified on the IgE-specific EITB of D. immitis female extract and was shown to be recognized by IgE in sera from all infected dogs at all time points examined from 2 weeks until 1 year post-inoculation. Overall, IgE reactivity was more specific for D. immitis infections than IgG reactivity.     

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.     

27 kDa Fasciola gigantica Glycoprotein for the Diagnosis of Prepatent Fasciolosis in Cattle

An indirect enzyme-linked immunosorbent assay (ELISA) using 27 kDa glycoprotein of Fasciola gigantica has been evaluated for its potential use in the diagnosis of bovine fasciolosis. Following experimental infection of rabbits, F. gigantica infection-induced antibodies were isolated and later used as ligands in affinity chromatography for isolation of infection-induced antibody-specific proteins. Among the five infection-specific proteins isolated, a glycoprotein of 27 kDa was later isolated by second-step purification using concanavalin A matrix. In crossbred cattle receiving different doses of infection (100, 200 and 400 metacercariae), the anti-27 kDa antibodies were detected as early as the 2nd week post infection. No direct correlation between initial dose, antibody response and fluke establishment was recorded. No cross-reaction was noted with the sera of goats experimentally infected with Paramphistomum epiclitum. ELISA with the 27 kDa glycoprotein could be a feasible diagnostic tool for the early detection of bovine fasciolosis.     

Studies on the prevalence and laboratory transmission of fascioliasis in animals in the Kashmir Valley

In Kashmir, 85.1% of cattle, 51.3% of sheep and 14.8% of goats were found infected with Fasciola spp. The prevalence rate varied from 66.6 to 100.0%, 25.0 to 100% and nil to 66.0% in cattle, sheep and goats respectively in different months of the year. Fasciola gigantica was the predominant species in all animal species but sheep harboured both F. gigantica and F. hepatica. The prevalence of F. hepatica infection in sheep happens to be the first report from India. Lymnaea auricularia sensu stricto supported the development of F. gigantica under laboratory conditions. The incubation temperature affected the shedding of the cercariae. Snails maintained at 25-27 degrees C started cercarial shedding as early as day 20 post-infection (PI), whereas those maintained at 10-12 degrees C commenced it from day 64 PI. One out of three experimentally infected guinea pigs aged 1 month revealed adult flukes in the liver at necropsy on day 52 PI.     

The serum glucose and beta-hydroxybutyrate levels in sheep with experimental Fasciola hepatica and Fasciola gigantica infection

The influence of Fasciola hepatica and Fasciola gigantica infection on serum glucose and beta-hydroxybutyrate (beta-HOB) in sheep was evaluated. This was done by setting up two groups of sheep. The first group (n=13) was split in two sub-groups, one experimentally infected with F. hepatica (n=9) and the other (n=4) as uninfected control. A second group consisting of a sub-group experimentally infected with F. gigantica (n=9) the other sub-group (n=6) left as uninfected control was also set up. The results of weight gain, parasitological and serum liver enzymes activity (glutamate dehydrogenase [GLDH] and gamma glutamyltransferase [gamma-GT]) used in monitoring the infection showed that all infected animals developed fasciolosis. It was observed that a reduction in serum glucose levels was significantly lower (p<0.05) in F. hepatica infected sheep than in uninfected control sheep starting from 5 weeks post-infection (wpi) to the end of the experiment. Similar reduction was recorded in F. gigantica infected sheep between 8 and 19 wpi. In contrast, serum beta-HOB levels were elevated in F. hepatica infected sheep between 6 and 16 wpi and in F. gigantica infected sheep between 7 and 15 wpi. It would appear from these serum glucose and beta-HOB levels that fasciolosis does lead to energy deficiency (low glucose) and ketosis (increased beta-HOB). The decrease in serum glucose and increase in serum beta-HOB levels in infected sheep may help in understanding the interaction between fasciolosis and nutritional status of infected ruminants especially in young growing animals.     

Fasciola gigantica excretory-secretory products for immunodiagnosis and prevention of sheep fasciolosis

Excretory-secretory products (ESP) products of ex vivo Fasciola gigantica adult worms were used for immunodiagnosis of sheep experimental infection with F. gigantica and natural infection with Fasciola spp. by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Specific IgG antibody binding to native or denatured ESP was detected as early as 2 weeks after experimental sheep infection with 100 or 200 metacercariae. No specific IgG antibody binding was displayed by sera obtained from 192 sheep considered to be Fasciola- and other parasite-free by microscopic examination of bile and feces. Additionally, sera from 200 apparently Fasciola-free sheep, yet infected with other parasites, were all negative. The data, thus, indicated that ESP-based ELISA reached nearly 100% sensitivity and specificity in immunodiagnosis of sheep fasciolosis. As expected, the ESP molecules were immunogenic in sheep eliciting interleukin-12p40 mRNA response and considerable amounts of antibodies, which were able to bind to the surface of newly excysted juvenile worms as judged by membrane indirect immunofluorescence, and mediate their attrition via antibody-dependent cell-mediated cytotoxicity. The ESP-induced cellular and humoral immune responses were associated with a modest reduction in worm count, yet with a highly significant (P<0.0001) decrease in size of recovered worms, thus suggesting that ESP immunization might be a safe and cost-effective strategy for reducing transmission of the infection.     

Identification of the antigenic components of paramyxovirus-3, paramyxovirus-6 and Newcastle disease virus in turkeys

The aim of this study was to use the enzyme-linked immunosorbent assay (ELISA) and the Western immunoblotting as possible tools to differentiate infections in turkeys by different paramyxoviruses. Pooled hyperimmune sera of turkeys infected with either paramyxovirus-3 (PMV-3), paramyxovirus-6 (PMV-6), or Newcastle disease virus (NDV) were assayed for antibodies specific to the three viruses by the ELISA and Western immunoblotting. ELISA results showed cross reactions of turkey antibodies between PMV-3 and PMV-6 antigens, while turkey antibodies to NDV did not cross-react with any of the other paramyxoviruses. The immunoblots of sera from birds infected with PMV-3 (Minnesota turkeys and Iowa chickens) reacted to low molecular weight polypeptides of PMV-3 of 29, 32, and 34 kDa, and to a high molecular weight band of 200 kDa. The same Minnesota turkey sera had a cross reaction to the 200 kDa polypeptide of PMV-6, while the Iowa chicken sera did not. Both sera had no apparent reaction to NDV proteins. Western immunoblotting showed that the turkey PMV-3 sera had a specific reaction to a 220 kDa polypeptide present in PMV-3, but not in PMV-6, while the turkey PMV-6 sera had a specific reaction to a 130 kDa polypeptide present in PMV-6, but not in PMV-3. Immunoblots of pooled sera from turkeys infected with PMV-6 (Minnesota source) reacted to the 200 kDa protein present in both PMV-3 and PMV-6; however, no reaction occurred between this sera and NDV proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary experimental infection of riverine buffaloes with Fasciola gigantica.

The clinical course of the primary experimental Fasciola gigantica infection was investigated in riverine buffalo calves of the Murrah breed. Nine male calves aged 12-15 months were randomly assigned to two groups of five (Group I) and four (Group II) animals. Each animal in Group I, was orally infected with 1000 metacercariae (mc) of F. gigantica, whereas Group II animals did not receive any infection dose and served as uninfected controls. No clinical signs of fasciolosis were observed until the sixth week post-infection (PI). Group I animals, however, developed recognised symptoms of acute fasciolosis, comprising apyrexic inappetance, anemia, poor weight gain, diarrhoea and sub-mandibular and facial oedema, respectively, from 5, 6, 8, 16 and 17 weeks PI. The signs were intermittent in nature and of variable duration. The prepatent period was of 92-97 days (mean 95.2 +/- 3.1). One of the five infected animals died on Day 147 PI. At necropsy, 36.8 +/- 11.0% of the infection dose was recovered as adult fluke population. The gross lesions were primarily biliary in nature. Group II, the uninfected controls, throughout the study period of 165 days PI, did not show any symptom and were negative for F. gigantica. The study demonstrated that the onset of adverse effects of F. gigantica on the growth and health of the infected host was mainly noted during late prepatency much before coprological prediction and diagnosis. The significance of preventive therapy against fasciolosis during prepatency has been stressed in endemic areas.     

Serological assay for swine erysipelas using nitrocellulose particles impregnated with an immunodominant 65 kDa antigen from Erysipelothrix rhusiopathiae.

Pigs (n = 10) that were experimentally challenged with an arthritogenic isolate of Erysipelothrix rhusiopathiae (strain VRS 229; serotype 1a) developed arthritis in at least one of twelve major limb joints. Immunoblots using sera obtained from these pigs at necropsy revealed a major band of immunoreactivity against a subunit polypeptide of apparent molecular mass 65 kDa. The usefulness of the 65 kDa immunodominant subunit as an assay reagent in an ELISA test was examined by presentation of antigen impregnated onto nitrocellulose particles (AINP). This was prepared by electro-transfer of bacterial polypeptides from SDS-PAGE gels to nitrocellulose. Protein bands were visualized by staining with amido black and a strip of nitrocellulose bearing the 65 kDa band was excised and extracted with formic acid. Nitrocellulose particles impregnated with the 65 kDa antigen (65-AINP) were precipitated from solution by neutralization with ammonium hydroxide. 65-AINP was suspended in water and the optimum dilution for ELISA assay was determined by titration to be 0.1 A650 units. Sera from all pigs challenged with VRS 229 reacted against the 65-AINP antigen in the ELISA assay while sera from control, and experimental pigs prior to challenge, failed to do so. The 65-AINP antigen could also be used efficaciously to quantify serological reactivity of pigs experimentally infected with other strains of E. rhusiopathiae representing the three major serotypes (1a, 1b and 2) that are most commonly associated with swine erysipelas infections. Mouse immunizations with 65-AINP also confirmed that nitrocellulose particles bearing the immunodominant subunit antigen will elicit murine antibodies that are monospecific against this determinant.     

Evaluation of an enzyme-linked immunosorbent assay for serological surveillance of infection with Actinobacillus pleuropneumoniae serotype 5 in pig herds

An indirect enzyme-linked immunoassay for serological surveillance of infection of pigs with Actinobacillus pleuropneumoniae (Ap) serotype 5 was developed. The antigen used was prepared from Ap serotype 5b strain L20. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the antigen contained high molecular weight lipopolysaccharide (LPS) and presumably also capsular polysaccharide (CP). The Ap serotype 5 ELISA was tested using sera from pigs experimentally infected with the 12 different Ap serotypes of biotype 1 and with sera from herds naturally infected with Ap serotypes 5, 6, 7 and 12. Cross-reactions were shown in one pig from a herd naturally infected with Ap serotype 7 and in one pig from a herd naturally infected with Ap serotype 12. The herd sensitivities of the Ap5 ELISA and a complement fixation test (CFT) were both estimated to 1.0, on the basis of serum samples from six herds naturally infected with Ap serotype 5. The herd specificities of both tests were estimated to 0.98, based on serum samples from 123 pig herds (10 samples from each herd) from the Danish specific pathogen-free (SPF) programme for pig production.     

Humoral immune response against Fasciola gigantica fatty acid binding protein

Recombinant fatty acid binding protein of Fasciola gigantica was expressed in Escherichia coli and purified by nickel chelating affinity chromatography. The recombinant protein along with native fatty acid binding protein (FABP) isolated from the parasite were evaluated for their potential in the diagnosis of F. gigantica infection in sheep, cattle and buffaloes, both by ELISA and western blotting. Results of this study indicate that there is no humoral immune response generated against this protein in the experimental infection of these ruminants with F. gigantica, thereby limiting the usefulness of this antigen in the early diagnosis of fasciolosis in these animals. Also, the paper discusses the probable reasons for the failure of this protein in detecting humoral response in these animals by ELISA and immunoblotting.     

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.     

Epidemiological survey of Neospora caninum infection in dogs from Romania

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ～ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ～ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes.     

An enzyme-linked immunosorbent assay for antibodies to Hepatozoon canis

Canine hepatozoonosis is a tick-borne protozoal disease caused in the Old World and South America by Hepatozoon canis. An enzyme-linked immunosorbent assay (ELISA) using purified H. canis gamont antigen was applied for the detection of antibodies reactive with H. canis. Evaluation of the ELISA with sera from naturally infected parasitemic dogs indicated that it was sensitive (86%), specific (97%), and comparable to the indirect fluorescent antibody test (IFAT) for the detection of H. canis antibodies. A variable degree of serologic cross-reactivity was found between sera from H. americanum-infected dogs and the H. canis antigen. Dogs experimentally infected with H. canis seroconverted 1-4 weeks post-infection (PI). Antibody levels peaked at 7-9 weeks PI and gradually declined thereafter remaining above the cut-off value until the conclusion of the study 7 months PI. The ELISA will be valuable for serological evaluation of dogs suspected of exposure to H. canis and for epidemiological studies.     

Immunodominant antigens of zoospores from ovine isolates of Dermatophilus congolensis.

Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.     

Cloning and Prokaryotic Expression of Protein Kinase A Catalytic Subunit Gene from Taenia multiceps

To study the potential of protein kinase A catalytic subunit of Taenia multiceps (TmPKA-C) gene as a diagnostic antigen, the TmPKA-C gene was amplified by RT-PCR from the total RNA of Taenia multiceps. The TmPKA-C gene fragment was ligated into prokaryotic expression vector pET-30a and transformed into Escherichia coli Transetta (DE3) strain, then the reactonogenicity of recombinant TmPKA-C protein was analyzed by Western blotting and indirect ELISA. The open reading frame of TmPKA-C gene was 1 032 bp, encoding 343 amino acids. The expression product of TmPKA-C gene mainly existed in the form of inclusion body, and the molecular weight of recombinant protein was about 42 ku. Western blotting analysis showed that the recombinant protein could react specifically with the sera from naturally infected sheep with coenurosis, and it could also have specific reaction with the sera of artificial infected sheep with coenurosis. The indirect ELISA analysis showed that the sera from the coenurosis of sheep could react specifically with the recombinant protein. These results proved that the recombinant protein had good reactionogenicity. This study laid a foundation for the search of new diagnostic antigens for coenurosis of sheep.     

A 2.9 kDa Fasciola hepatica-recombinant protein based ELISA test for the detection of current-ovine fasciolosis trickle infected

The suitability of an enzyme linked immunosorbent assay (ELISA) test with a 2.9 kDa Fasciola hepatica-recombinant protein (FhrAPS) for diagnosing early and current-ovine fasciolosis was analyzed, and compared to that obtained by using a direct ELISA for detecting F. hepatica-circulating FhES antigens and to the coprological sedimentation for fluke egg quantitation. Fourteen Gallega autochthonous breed sheep were experimentally infected with metacercariae by a trickle system (small repetitive infections) and divided into two groups: G-I represented a primary infection for 34 weeks; G-R, animals with primary infection and reinfected 18 w.a.p.i. Seven sheep were left uninfected as the control group (G-C). Serum IgG antibody values against the FhrAPS rose rapidly by 1st w.a.p.i. in all infected sheep. Antibody levels in those with primary infection (G-I, G-C) peaked at 10 weeks, diminishing slightly and levelling from 16 to 34 weeks. Those with primary infection reinfected at 18 weeks had a rebound effect with the highest values observed. Circulating F. hepatica-ES antigens were detected by the 1st w.a.p.i. in all infected groups peaking at 6 weeks, decreasing rapidly to uninfected control values by 10 weeks of infection. Faecal egg-output started 11 weeks after primary infection. An increase in the IgG antibody as well as antigen responses to the FhrAPS and to anti-FhES from the 18 w.a.p.i. was recorded in G-T and G-R after the challenge infection. Antibody levels remained high whereas antigenemia values diminished after 6 weeks. A positive significant correlation between the IgG response against the FhrAPS and the F. hepatica circulating antigens (r2 = 0.428, p = 0.001) was obtained. In conclusion, our standardized diagnostic ELISA for fasciolosis based on the detection of IgG responses to the FhrAPS would be a valuable tool to diagnosis early and current F. hepatica-infections in sheep.