Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.     

Innate and adaptive resistance of Indonesian Thin Tail sheep to liver fluke: a comparative analysis of Fasciola gigantica and Fasciola hepatica infection

In the current study, three independent trials directly compared Fasciola gigantica and Fasciola hepatica infection of ITT sheep. In all trials, F. hepatica infection resulted in higher worm burden recoveries and greater physiological damage to ITT sheep. Developmental differences of the two Fasciola species were also observed during the first twelve weeks of a primary infection, where the migration and growth of F. hepatica was more rapid than F. gigantica. Various immunological blood parameters were measured and indicated similar kinetics in the humoral and cellular responses during the time course of infection with each Fasciola species. In contrast to F. hepatica infection, we demonstrate an innate and adaptive comparative ability of ITT sheep to resist the early stages of infection with F. gigantica infection. Unraveling the mechanisms leading to this differential resistance may potentially lead to new methods for the control of fasciolosis and other human liver flukes.     

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.     

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.     

Evaluation of cercarial antigen for the serodiagnosis of fasciolosis in experimentally and naturally infected sheep

The value of cercarial antigen for diagnosis of experimental and natural sheep fasciolosis was studied by enzyme linked immunosorbent assay (ELISA) and enzyme linked immunotransfer blot (EITB). In ELISA, the antibody levels of experimentally infected sheep with Fasciola gigantica appeared at 2 weeks post infection (PI), gradually increased till 7 weeks PI and nearly remained at the same level from 7 to 13 weeks PI (the end of experiment). Also, the sensitivity and specificity of cercarial antigen for diagnosis of naturally sheep fasciolosis were 100 and 90%, respectively.In EITB, in the sheep experimentally infected with F. gigantica, the band of 32.5kDa molecular weight polypeptide appeared at 2 weeks PI and continued till the end of experiment. Also, the cercarial antigen recognized 32.5kDa molecular weight band with all sera from naturally infected sheep with fasciolosis (n = 25). This band did not cross-react when tested with sera from infected sheep with Cysticercus tenuicollus (n = 20). This study suggests that, the 32.5kDa molecular weight polypeptide could be used as sensitive and specific epitope for the serodiagnosis of sheep fasciolosis.     

Humoral response (IgG) of goats experimentally infected with Fasciola hepatica against cysteine proteinases of adult fluke

The use of cysteine proteinases from Fasciola hepatica adult flukes for the serodiagnosis of caprine fasciolosis by means of an indirect ELISA test was studied. Two proteolytic fractions from adult fluke homogenates, with apparent molecular weights of 28 and 34 kDa (P28 and P34 respectively), were characterised as cysteine proteinases using azocasein assays and gelatin gel analysis. Both P28 and P34 fractions were electroluted and used as antigens in two different indirect ELISA tests. Serum IgG levels against P28 and P34 in goats given an experimental primary infection with 200 metacercariae or in goats given two experimental infections with 200 metacercariae were determined and compared with those observed in an uninfected control group. ELISA tests using both cysteine proteases showed a rapid and consistent detection of specific IgG in all experimentally infected goats. The IgG response to P28 was the first to be detected as early as 2-3 weeks post-infection and remained elevated throughout the experiment. The response to P34 was detected later (4-6 wpi) and disappeared in some animals at 18 wpi, while flukes were still present in the bile ducts. No significant differences were observed between the anti-P28 and anti-P34 IgG responses between animals receiving a primary or a challenge infection. The results of our study, although preliminary, are promising since the P28 ELISA described here may be a reliable method for the immunodiagnosis of F. hepatica infection in goats.     

两种片形吸虫感染家兔的试验比较

用大片形吸虫和肝片形吸虫感染家兔以便选择大片形吸虫对动物的最佳感染量,及明确肝片形吸虫和大片形吸虫的生物学和对动物宿主的致病力的差别。结果显示肝片形吸虫虫体在兔体内发育成熟的时间早于大片形吸虫,感染成活率更高,对动物的病理损害明显比感染大片形吸虫兔的病变要轻。本试验证实这两种片形吸虫除了形态学的差异外,在对动物致病力、病理损害等方面确实存在差别。     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fag) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fag and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

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.     

Comparison of two assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance to triclabendazole in Fasciola hepatica in sheep

A sheep trial was performed to evaluate two diagnostic assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance of Fasciola hepatica to triclabendazole (TCBZ). The FECRT defines successful TCBZ treatment as a 95% or greater reduction in fluke faecal egg counts (FECs) at 14 days post-treatment (dpt). The CRT defines effective TCBZ treatment as faeces negative for Fasciola coproantigens at 14dpt, as measured by the commercial BIO K201 coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium). Forty-nine indoor-reared sheep were split into four trial groups and each sheep was infected with 200 metacercariae of 1 of 4 F. hepatica isolates, previously described as susceptible (Cullompton and Fairhurst) and resistant (Leon and Oberon) to TCBZ action, respectively. TCBZ treatment was administered at 12 weeks post-infection (wpi) to one sub-group in each infected sheep group, and these sheep were culled at 4 weeks post-treatment (wpt). Untreated sheep sub-groups, were culled at a parallel time-point, that is, at 16wpi. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period, sub-sampled and examined by a standardised egg sedimentation protocol and by the BIO K201 ELISA. Results supported the use of both the FECRT and the CRT for the diagnosis of resistance of F. hepatica to TCBZ. In addition, the study confirmed the TCBZ susceptibility of the Cullompton and Fairhurst F. hepatica isolates and the TCBZ resistance of the Oberon F. hepatica isolate. However, the Leon F. hepatica isolate was found to be susceptible, rather than resistant, to TCBZ action.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

Identification of the Egyptian species of Fasciola.

Reports on the species of Fasciola present in the Nile Delta, Egypt, appear controversial. Some authors reported the presence of both Fasciola gigantica and Fasciola hepatica, others reported F. gigantica only and mentioned that F. hepatica was found only in imported animals.This study was an attempt to identify the species of Fasciola flukes collected from locally bred animals. Morphologic, morphoanatomic, morphometric, and chemotaxonomic criteria of the fluke isolates were studied. Speciation based on morphologic and morphometric data was not decisive due to overlap in the values of most measurements. Morphoanatomic data proved the presence of both the species, and isoelectric focusing (IEF) of fluke soluble protein confirmed the presence of both F. gigantica and F. hepatica in Egypt.     

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.     

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.     

A comparison of serum biochemical changes in two breeds of sheep (Red Masai and Dorper) experimentally infected with Fasciola gigantica.

Twelve Red Masai and 12 Dorper sheep aged between 6 and 9 months, were acquired from a fluke-free area and sheep of each breed divided into two equal groups of six. Each animal in one group of each breed was experimentally infected with 400 viable metacercariae of Fasciola gigantica. The other groups acted as uninfected controls. Blood samples were taken at weekly intervals for the determination of serum bilirubin, albumin, and gamma glutamyl transferase levels. Following the establishment of infection, albumin levels declined in both breeds of infected animals without any significant difference between the two breeds. However, serum bilirubin and gamma glutamyl transferase (GGT) in the infected animals were elevated significantly more in the Dorper than in the Red Masai sheep. Based on these findings, it would appear that Dorper sheep are more susceptible to the infection than Red Masai sheep.     

Myocastor coypus as a reservoir host of Fasciola hepatica in France

To clarify the role of the nutria Myocastor coypus in the epidemiology of domestic fasciolosis in Loire-Atlantique (department of western France), 438 nutrias were trapped in 9 humid areas of the department and 304 nutrias were trapped in 3 farms where Fasciola hepatica was present; all animals were necropsied. Liver flukes were found in 160 nutrias: 38 nutrias randomly taken in the department (8.7%) and 122 trapped in fasciolosis areas (40.1%). The average parasitic burden was 5.7 flukes per nutria. Sixty-five percent of the liver flukes measured more than 18 mm (size of sexual maturity). The coproscopic examinations carried out on 144 infected nutrias showed that 90% of the infected nutrias shed fluke eggs. The hatching rate was 39.6%. Two groups of 100 Lymnaea truncatula snails, originating from 2 different populations, were exposed to F. hepatica miracidiae hatched from eggs collected from infected nutrias. The prevalence of the infection was 74% and 58.6% in the 2 groups of snails. The average redial burden was 6.2 rediae per snail. The total number of metacercariae was 72.4 metacercariae per snail producing cercariae. Two groups of 5 sheep were orally infected by 150 metacercariae of nutria or sheep origin, respectively. The installation rates of F. hepatica in sheep were respectively 31.6% and 29.6% for the two groups. Specific antibody kinetics of sheep were similar whether the metacercariae were of nutria or sheep origin. M. coypus allows the complete development of F. hepatica and releases parasitic elements that are infective for domestic ruminants. Because of its eco-ethologic characteristics, the nutria could be a potential wild reservoir of F. hepatica in France.     

Transport of serum IgA into bile of sheep infected with Fasciola hepatica

Sheep infected with Fasciola hepatica were studied for their ability to transport intravenously injected radiolabelled IgA from serum to bile. The results show that there was an apparent reduction in the selectivity of transport of IgA into bile in infected animals compared with uninfected controls. However, in infected animals the biliary flow rate was approximately twice that of uninfected animals, and the total amount of radiolabelled IgA transported was similar irrespective of infection status. The possible relevance of the elevated biliary flow rate is discussed with respect to the pharmacokinetics of drugs used for the chemotherapy of helminth infections in sheep.     

Performance characteristics of an enzyme-linked immunosorbent assay for the detection of liver fluke (Fasciola hepatica) infection in sheep and cattle

AIM: To validate an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against liver fluke (Fasciola hepatica) in sheep and cattle sera. METHODS: Gold-standard sera from sheep and cattle of known infection status, i.e. sera from non-infected animals and from animals known to be infected with F. hepatica were assayed with a commercially available ELISA and results analysed by ROC analysis. RESULTS: The ROC analysis suggested cut-offs that were considerably lower than those suggested by the manufacturer, yet the ELISA performed with high sensitivity and specificity, 98 to 100%, respectively for sheep and cattle sera. For bovine sera, particularly good discrimination between positive and negative sera was observed. Infection in experimentally infested animals could be demonstrated 7-8 weeks earlier than with classical parasitological techniques. CONCLUSIONS: The analysis of the ELISA's performance demonstrated high sensitivity and specificity. ROC analyses optimised the cut-off point suggested by the manufacturer of the commercial diagnostic assay. Diagnosis of infection with F. hepatica was achieved much earlier than is possible with current parasitological techniques. This could help with the control of fasciolosis, enabling treatment before clinical manifestation of the disease.     

Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes.