Skip to main content

Comparison of four commercially available ELISA kits for diagnosis of Fasciola hepatica in Irish cattle



Fasciola hepatica is a liver parasite of mammals and it results in poor welfare outcomes and economic losses in ruminants. While faecal egg count is the test most commonly used for diagnosis, it does not indicate presence of migrating immature stages. Serological techniques increase sensitivity at all stages of the liver fluke infection. The aim of this study was to compare four commercially available ELISA tests for the diagnosis of F. hepatica. For this purpose, we tested three sample types; (i) known F. hepatica status sera from an experimental infection for the comparison of sensitivities and specificities, (ii) sera from pre- and post-flukicide-treated (albendazole, closantel, nitroxynil and triclabendazole) beef cattle to contrast the differences of seropositivity before and after treatment, and (iii) bulk tank milk samples from dairy herds sampled during high and low F. hepatica exposure periods for assessing seasonal variations with the four tests available. Samples were tested using ELISA kits supplied by four manufacturers (Ildana Biotech, IDEXX, Svanova, and Bio-X). Samples were analysed simultaneously and in duplicate.


In the control population Ildana, IDEXX and Bio-X presented 100% sensitivity (Se) and specificity (Sp), Svanovir presented a Se of 59% and a Sp of 96%. In flukicide-treated beef cattle, kits highlighted decreasing antibody levels 90 days post-treatment in variable degrees. Finally, bulk milk showed a significant decrease in ELISA value between high and low fluke exposure periods with all tests studied.


Se and Sp found in the present study, confirm that Ildana, IDEXX and Bio-X are accurate for the detection of F. hepatica exposure in Irish cattle. Svanovir Se and Sp in this population, indicate that a larger study is necessary to confirm this test characteristic in Irish herds. In post-treatment use, Bio-X showed a consistent and significant decrease of ELISA value in all groups treated, denoting to be a reliable tool for assessing treatment effect at 90 days post-treatment. Finally, all tests showed to be a reliable tool for the F. hepatica monitoring of high and low exposure seasons, using bulk tank milk samples.


Fasciola hepatica, commonly known as the common liver fluke, is a trematode parasite [1, 2] of mammals [3]. Its clinical manifestation is fasciolosis and it has a worldwide distribution [4, 5] reflecting a marked capacity for adaptation of both the causal agent and its intermediate mollusc host [5]. This adaptability, combined with the effects of global warming, increases the potential for F. hepatica related losses in livestock [6, 7] and increased prevalence in humans [5].

Fasciolosis is an important disease of domestic livestock [8] and both immature and mature stages of the parasite in the final host result in a 15% decrease in milk yield [9], an average reduction of 1.5 kg [10] or 0·7 kg milk/cow per day [11]. Annual losses have been estimated to be around €2.5 billion to livestock and food industry worldwide [12]. The presence of F. hepatica may also impact the shedding of Escherichia coli O157 in cattle destined for the human food chain [13]. Although livestock fasciolosis does not correlate with human fasciolosis [14], veterinary public health measures and food safety practices are recommended to reduce the risk of infection [5].

Fasciola hepatica has a preference for temperate climatic zones as its 18 to 30 week life cycle [4] requires mild temperatures and high humidity for the development of the intermediate host and free-living stages [15,16,17,18]. It can, however, also be found in areas of the tropics in conjunction with Fasciola gigantica [14]. The requirement for specific weather patterns for completion of its lifecycle leads to seasonal variations in livestock infection [19]. In temperate climatic zones without large seasonal climatic variations such as Ireland, management factors strongly influence the exposure and spatial distribution of the parasite [20, 21].

The definitive diagnostic test for F. hepatica is liver necropsy, which provides a highly accurate diagnosis of fasciolosis when bile ducts are dissected [22]. This is not practical as a herd or flock management tool as it can only be carried out post-mortem [23]. The most frequently used ante-mortem diagnostic test is the detection of eggs in faeces by sedimentation or flotation techniques, which are expressed as faecal egg count (FEC) [4] and had shown to have high specificity, detecting current infection [24], however, the accuracy of detection of small numbers of eggs in faecal samples is determined by the volume of sample available [22, 25] which constitutes a difficulty for diagnosis. The test can also be a poor indicator of infection when the parasite burden is low or when non-reproducing immature stages are migrating [26, 27], although increasing sample size and repeated sampling can increase both specificity (Sp) and sensitivity (Se). These diagnostic tools are laborious, time consuming, require skills for the identification of eggs and immature flukes, and are also unsuitable for a large scale or herd level testing [27, 28].

To prove effective as part of a control programme, diagnostic methods for herd screening must be reliable, easy to perform [28], and the cost of testing must relate to the benefit obtained by the diagnosis. Ideally, the diagnostic test should allow for early diagnosis of infection, and have the capability to detect seasonal differences in infection thus informing treatment decisions [29]. To meet these requirements, liver fluke specific enzyme-linked immunosorbent assays (ELISA) have been developed and are being routinely used in cattle [19, 30, 31]. F. hepatica ELISAs are adaptable tests which detect specific antibodies or antigens in faeces and pooled or individual milk and sera. Failure to diagnose immature migrating stages of liver fluke in the final host is a disadvantage of faecal egg counts, therefore the use of ELISA tests with the capacity of early diagnosis is a major advantage. The most damaging stage of this infection in the final host occurs during the migration of immature stages. The use of ELISA techniques for F. hepatica diagnosis has demonstrated improved sensitivity of diagnosis over coprological techniques, and has the improved advantage of detection of pre-patent infections [11, 31, 32]. Additionally, detection of F. hepatica antigens in faeces is also feasible; using available copro-antigen ELISA kits, which have shown to have high sensitivity and specificity [31, 33].

At present, several F. hepatica ELISA kits for sera and milk are commercially available, each comprising of different antigens, methods, sample dilutions, S/P% calculations and thresholds. Comparison of these tests under identical conditions has not been previously assessed and the wide range of commercial ELISA tests generates indecision in the related community as to which test to use and the significance of results. It is important to F. hepatica management in cattle herds that commercially available kits can not only detect infection but can do so in a timely manner with the ability to detect seasonal variations and post-treatment effects. The present study, therefore, aimed to evaluate and compare four commercially available ELISA kits for milk and sera, in their ability to detect exposure to F. hepatica in Irish cattle of known status and in naturally infected herds; pre-and post-flukicide treatment (sera), and in bulk tank milk (BTM) samples taken over a 12 months period.


Assay sensitivity and specificity

In all, 24 pre-colostral samples and 44 experimentally infected samples (22 at four wpi and 22 at 10 wpi) were tested across both groups. No liver fluke eggs were evident in experimentally infected calves at 4 weeks post- infection. However, all infected calves recorded positive FECs by 10 wpi and infected livers post-mortem (data not shown).

Results from pre-colostral and experimentally infected calves (4 and 10 wpi) across the four tests can be seen in Table 2 and Fig. 1. Of the kits examined Ildana, IDEXX and Bio-X correctly identified all 24 pre-colostral samples as negative and all 22 experimentally infected samples at both four and ten wpi as positive (Table 2). This yielded Se and Sp for Ildana, IDEXX, and Bio-X kits of 100 and 100%, respectively, from at least four wpi (Table 2). The Svanovir kit classified 23 of the 24 pre-colostral samples as negative and 13 of 22 experimentally infected samples at 4 and 10 wpi as infected with F. hepatica with likely production losses (Table 2). This yielded a Se of 59.1% and Sp of 95.8% for the Svanovir test (Table 2).

Fig. 1
figure 1

Scatter plots outlining ELISA results from pre-colostral and experimentally infected calves (4 and 10 weeks post-infection (wpi)) across (a) ILDANA, (b) IDEXX, (c) SVANOVIR and (d) BIO-X F. hepatica test kits. Positive cut-off values for each kit are represented by the dashed line (−---)

For all commercial kits examined, the number of positive and negative cases at four and ten wpi remained consistent. However, variations in S/P% and ODRs were observed principally in Ildana (P ≤ 0.0001), IDEXX (P ≤ 0.0001) and Svanovir (P ≥ 0.05) (Fig. 1), although all samples remained clearly positive. These variations in Ildana and IDEXX were characterised by a decrease in the range of positive S/P% (i.e. Ildana- 4wpi: 30 to 400%, 10wpi: 30 to 130%; IDEXX- 4wpi: 200 to 590%, 10wpi: 80 to 170%) (Fig. 1). In contrast, the Svanovir test showed an increase in this range (4wpi: 0.4ODR to 0.8ODR, 10wpi: 0.4ODR to 1.5ODR), but this change was not significant (Fig. 1). No evident or significant changes were observed with the Bio-X kit.

Pre- and post-treatment kit variations in naturally infected beef cattle

Ten animals were allocated to each treatment group and in total 50 individual sera samples were tested on two occasions (before and after the administration of a flukicide treatment). A boxplot outlining pre- and post-treatment results for each test kit across the five groups investigated are included in Fig. 2.

Fig. 2
figure 2

Boxplot of pre- and post-treatment results from naturally infected beef cattle across test kit and five flukicide treatments. Ildana, IDEXX and Bio-X kits recorded results as S/P% (left Y axis) and the Svanovir kit recorded results as ODR (right Y axis). IL = Ildana Biotech Fasciola ELISA test kit. ID = IDEXX Fasciola hepatica antibody test kit. SV = SVANOVIR Fasciola hepatica antibody test. BI = Bio-X Diagnostics Fasciola hepatica ELISA kit. Pre = value prior to treatment i.e. Day 0. Post = value 90 days post-treatment i.e. Day 90

Wilcoxon matched-pairs signed rank test for determining the significance of post- treatment variations determined significance in nine of the 20 comparisons (Table 3). The Bio-X test showed a decrease of S/P% in albendazole (z = 2.85, P < 0.01), closantel (z = 2.67, P = 0.01), nitroxynil (z = 2.76, P = 0.01) and triclabendazole (z = 2.76, P = 0.01) groups and for these groups, the decrease was observed in nine of the ten animals evaluated (Table 3). In contrast, the Svanovir test only showed a significant decrease of ODRs 90 days after albendazole treatment (z = 2.40, P = 0.02) (Table 3).

Ildana and IDEXX tests detected an increase of S/P% 90 days after first sample in the no treatment group (z = − 1.96, P = 0.05 and z = − 2.40, P = 0.02, respectively), this increase resulted from six and nine animals, respectively. Additionally, the Ildana test detected a significant decrease of S/P% in eight of the ten animals treated with nitroxynil.

High and low exposure season detection in naturally infected bulk tank milk samples

A total of 103 BTM samples from 29 herds were analysed using the four ELISA kits. In all, 14 herds supplied samples for all 4 time points. The mean number of samples received per month was 26 samples (range 20 to 29). Herd sizes ranged from 60 to 310 milking cows, the mean herd size being 157 cows. All study herds were specialist dairy enterprises with no additional livestock species such as fattening of cattle or sheep on the farm. All cows were grazing from February until November and were housed in December and January.

Test results from the four ELISA kits revealed the highest S/P% and ODR medians in December 2010 and the lowest in July 2011 (Ildana (3.31 S/P%), IDEXX (47.81 S/P%) and Svanovir (0.41 ODR)), the Bio-X test showed the lowest S/P% in April 2011 (10.3 S/P%) (Fig. 3).

Fig. 3
figure 3

Box plots of bulk tank milk ELISA results across test kit (a) Ildana, (b) IDEXX, (c) SVANOVIR, (d) BIO-X and time (December 2010, April 2011, July 2011, October 2011)

Generalized estimating equation (Table 4) confirmed the higher risk and lower exposure seasons observed in the previous descriptive analyses. All tests showed significant decreasing antibody levels in April, July and October against December. A decrease in July alongside April was significant with the Ildana, IDEXX and Svanovir test (P = 0.006, < 0.001 and 0.021, respectively). A significant decrease in S/P% was observed in October in comparison to April using the IDEXX test (Coefficient = − 27.04; P = 0.003), on the contrary the Svanovir test showed a small significant increase (Coefficient = 0.09; P = 0.044). Finally, higher S/P% were detected in farms which treated with a flukicide in contrast with farms which did not use any flukicide treatment.


The aim of this study was to compare and evaluate four commercially available ELISA kits (Ildana, IDEXX, Svanovir, Bio-X) for diagnosis of F. hepatica in Irish cattle, as a comparative study of the four tests available has not been previously reported. For this purpose, samples from three different populations were evaluated, including sera from a known-status population (known positive and negative sera), sera from naturally infected beef cattle (before and after treatment with different flukicides) and naturally infected BTM samples from 31 dairy herds (collected in four different months within a year). To evaluate these tests a paired design was applied, offering advantages like the minimisation of between-subject variability and elimination of confounding [29]. Also, the evaluation of these tests in naturally infected populations gives a real view in day-to-day parasite control measures practiced.

The Ildana assay is based on a recombinant antigen [34] (Table 1) and has previously been used in multiple Fasciola hepatica studies in Ireland [19, 21, 37]. Sensitivity and specificity ratios reported previously were 98% in bovine sera [37]. Previous studies, which also used recombinant antigens, have reported similar sensitivities and specificities at variable times post- infection [38,39,40], and no cross-reaction with other present parasites was observed [39]. The IDEXX kit (f2 antigen) (Table 1) (originally the Pourquier ELISA) has shown to be very reliable, as previous experimental infections reported sensitivity and specificity ratios of 100% [38, 41] and close to a 100% in natural infections [42, 43]. However, a study by Simões et al. in 2017 reported a specificity ratio of 56% in Brazil [44]. The E/S antigen, in the Svanovir kit (Table 1), has previously shown a strong correlation between F. hepatica antibody levels, intra-hepatic fluke frequency and production parameters [11, 45] and a sensitivity and specificity of 92 and 88%, respectively, in BTM samples compared to sera [30]. With regards to the Bio-X kit, containing the CL1 antigen (Table 1), an earlier study found a strong correlation between an in-house ELISA, which used the same antigen as Svanovir (E/S), and Bio-X in sera from non-infected and naturally infected cattle [46].

Table 1 Summary of ELISA kit characteristics
Table 2 Classification of samples from pre-colostral and experimental infected animals at 4 and 10 weeks post-infection (wpi) by four commercial F. hepatica test kits
Table 3 Wilcoxon matched-pairs rank-signed analysis of individual cow pre- and post-treatment serum ELISA S/P values across four test kits and five flukicide treatments
Table 4 Generalized estimating equation analysis of bulk tank milk continuous ELISA results

In the known status population all tests detected F. hepatica antibodies 6 weeks prior to the detection of eggs by FEC. This early diagnosis has been widely described in the literature [38, 41] and the present study confirms this characteristic for the four different commercial tests assessed. Ildana, IDEXX and Bio-X tests presented a 100% Se and Sp in an experimental population and is in line with sensitivity and specificity ratios previously reported. Although the Svanovir test did not reach the 100% Se and Sp it showed to be suitable for the use with BTM samples for determination of in-herd prevalence and seasonal changes. In the present study, the Svanovir test detected one naïve animal as positive, suggesting a possible attachment of unspecific antibodies to the antigen. Cross-reaction could only be possible in the presence of other worm infections [38] and sera used as known status- negative to F. hepatica were collected from naïve animals, with no previous exposure to pasture helminths (pre- colostral). This finding suggests that further research needs to be conducted with this test in Irish cattle as no previous reports of this finding are available in the literature.

A decrease in S/P% was observed with the Ildana and IDEXX kits (Fig. 1) at 10 weeks post-infection suggesting final stages of the primary immune response [47], however, further research is needed to confirm this. The decrease in S/P% observed in the present study has previously been reported with IDEXX, with positive cases still remaining positive after treatment [41] as in the present study. But IDEXX has also shown to maintain constant antibody detection after shorter periods of time (21 to 42 days post- infection) depending on infection dose [38]. In the present study, Svanovir and Bio-X showed more stable S/P% and ratios at 10 weeks post- infection (Fig. 1), the differences observed in ODR at 10 weeks post infection could be explained by variations in the stability of the binding reaction between antigen and antibody [48], however, further study would be needed to confirm this observation.

As natural infections are usually constant during high risk grazing periods [19], adult animals present higher levels of detectable antibodies as they have been exposed to more high risk seasons. Having said that, the experimental infection method used in the present study included a single infective dose, containing 115 METs and was administered to young animals. Experimental infection does not necessarily equate the response measured by these tests in adult cow populations, as adult cows have been exposed repeatedly through their productive lives, with the possible build-up of specific antibodies. This is of special importance in pasture-based systems, like Ireland.

In general, the naturally infected-beef population presented decreases of ELISA values 90 days after the application of treatment. The Svanovir kit has previously shown a significant decrease of ODRs at 3–6 months [49] and 1 year [50] post treatment. These previous reports and the present results propose the use of the Svanovir kit more than 90 days after treatment. In comparison, results obtained from the Bio-X test confirmed that a 90 days period after treatment is adequate to measure its effects.

In the naturally infected populations studied, the four kits showed a general agreement in the detection of F. hepatica antibodies in the different groups, this effect was especially evident in the BTM population. The Svanovir test discrepancies seen in the known status population were not evident in the BTM sample group, this could be attributed to a larger sample size, the dilution of antibodies in bulk tank milk samples [51] or higher concentration of antibodies in adult animals.

Changes in BTMs antibody detection were dependent on the seasonal exposure variations (Fig. 3 and Table 4), as previously described in Europe [15], Germany [52] and Ireland [19], which defined winter as high exposure season and summer as low. Conventionally, ELISA testing has been used with individual sera or even with pooled sera for herd diagnosis. However, ELISAs are being widely used on BTM samples [19, 51] because of the practicality for the determination of herd-level status, making the BTM antibody ELISA an attractive alternative [30, 31]. The inconvenience of the use of ELISA tests for the detection of F. hepatica antibodies is that results do not necessarily indicate the presence of active infection, as antibodies will still circulate after treatment [51] and the levels of exposure would also be related with age and stage of the milking period, it is important to consider the treatment measures applied, age and milking period of the herd before interpreting BTM ELISA results. Nevertheless, the four tests studied detected the classical seasonal dependent variations on antibody levels in Irish dairy herds (Fig. 3).


It is clear that fasciolosis represents a major risk for the health and production of cattle worldwide, moreover, a potential increase of F. hepatica burden has been predicted as a result of climate change [7]. For the appropriate treatment and control of liver fluke, diagnosis is key. As previously mentioned, the diagnostic test should allow the early diagnosis of the disease, be able to detect seasonal differences in infection and thus informing treatment decisions [29]. The present study highlights the early and reliable diagnostic capacity of the four commercially available tests assessed for fasciolosis, although, the Svanovir kit presented lower sensitivity and specificity under experimental conditions. For a better understanding of the Svanovir results in relation with sensitivity and specificity, further studies need to be performed using the Svanovir test in Irish cattle. All tests detected changes in antibody levels 90 days post-treatment and Bio-X showed greater accuracy in this detection as all changes after treatment were significant. However, a larger sample population and/or longer sampling time would be necessary to confirm the findings using the Svanovir kit as observed by Köstenberger et al. in 2017 [50] and Charlier et al. in 2012 [49]. The use of all four tests with BTM samples showed to be a reliable tool for the determination of high and low exposure seasons and in-herd prevalence throughout the year, however, results must be interpreted considering herd health management, Fasciola hepatica life cycle and herd milking pattern.


Sample populations

Calves of known F. hepatica status – control population

To source negative samples of known F. hepatica status, blood from 50 neonatal pre-colostral, housed calves born in January and February 2016, were collected into plain vacutainers. Calves were either Holstein-Friesian or Jersey-cross breeds. All blood samples were collected within the first hour post-calving. These calves were born and housed at Teagasc (Irish Agriculture and Food Development Authority), Moorepark, County Cork, Ireland. Samples were collected by a parallel study under license from the Health Products Regulatory Authority (HPRA) (AE19132/P044) and approved by the Teagasc Animal Ethics Committee (TAEC).

For the purposes of obtaining positive samples of known F. hepatica status, blood samples were collected from Holstein-Friesian calves (n = 25) experimentally infected with F. hepatica metacercariae (MET). Experimental infection was achieved by orally dosing each calf with 115 METs, after 10 weeks grazing period, this pre-infection grazing period was carried out for acclimatisation and ensuring infection. Calves were housed immediately post-infection. Grazing and housing took place at Teagasc farm. These animals were infected as part of a 75-calf vaccination trial funded by the Irish Department of Agriculture, Food and the Marine and licensed by the HPRA (AE18982/P088) and TAEC. Only non-vaccinated calves (control infected group) were included in the current study. Blood and faecal samples were collected on the fourth week post infection (wpi) for assessing the detection of immatures stages and on the tenth wpi for assessing mature parasites. For the purpose of post-mortem evaluation, infected animals were transported and slaughtered according to the Irish Slaughter of Animals Act [53] 3 months post-infection.

Naturally infected herds - blood

Blood samples were collected from a commercial beef herd, containing animals of varying beef breeds, crossbreeds and ages, located in county Clare, Ireland. This herd recorded a prior history of F. hepatica infection by ELISA diagnosis (data not shown). Animals were housed for the winter months and grazed for the rest of the year, the current experiment was carried out during the housing period. Samples were initially collected and analysed for the purposes of evaluation of dosing strategies in farmed beef in Ireland, and when archived, these samples were made available to the current study. Samples were available from five different treatment groups; albendazole (dose rate 10 mg per kg), closantel (dose rate 10 mg per kg), nitroxynil (dose rate 10 mg per kg), triclabendazole (dose rate 12 mg per kg) and a no treatment control group. Individuals were randomly assigned to each treatment group and each group contained 10 animals. Flukicides were administered based on body weight estimates, on a single occasion, and administered orally except for closantel which was a ‘pour on’ preparation. Blood samples were collected from the 50 individuals prior to dosing (Day 0; February 2016) and again 90 days post-treatment (Day 90; May 2016); to allow comparison of pre- and post-treatment ELISA results under farming conditions in Ireland. The dosing strategies experiment which supplied samples for the current study was approved by the TAEC and licenced by the HPRA (AE19132/P031) and funded by the Irish Department of Agriculture, Food and the Marine.

Naturally infected herds - BTM

Archived bulk tank milk (BTM) samples were available from 29 herds, 22 of which were commercial dairy herds and members of the Dairy Management Information System, a discussion group coordinated by Teagasc. The remaining 7 herds were Teagasc dairy research herds. Each herd was requested to submit four BTM samples; in December 2010, April 2011, July 2011 and October 2011. Herd sizes and F hepatica dosing frequency and active ingredient were available for each herd.

Sampling methods

Blood samples from neonatal calves were obtained by jugular venepuncture. All other blood samples were collected using venepuncture of the coccygeal vein. BTM samples were collected by individual farmers and submitted to Teagasc by post in a standardised sampling kit [19]. On receipt at the laboratory, blood and BTM samples were centrifuged (4000 g for 4 mins, blood; 20,000 g for 1 min, BTM). Serum and skim BTM were subsequently collected into 1.5 mL microtubes and frozen at -80 °C until analysed, ensuring only one freeze/thaw cycle. Samples were obtained on the dates specified in the sampling populations sections of the present study. Faecal samples collected from the ground (faecal catch) from the experimentally infected group were stored in sample pots and analysed at the arrival to the laboratory.

Sample analysis

Five grams of faeces were homogenised with water and passed first through a coarse mesh sieve and then a finer, 250 μm mesh sieve. The filtrate was allowed to stand for 5 min to sediment and the supernatant was removed by aspiration. Sedimentation was repeated 1–2 times as required. The supernatant was removed and the sediment was stained with two drops 1% methylene blue. Eggs were counted on a stereomicroscope as outlined by Taylor, et al., 2007 [4]. Results were expressed as presence or absence of liver fluke eggs and all samples were evaluated by the same person.

Blood and BTM samples were analysed concurrently using four commercially available ELISA kits; Ildana Biotech Fasciola ELISA test (Ildana Biotech, Ireland), IDEXX Fasciola hepatica antibody test kit (IDEXX, France), Svanovir F. hepatica-Ab (Svanova, Sweden) and Bio-X Diagnostics Fasciola hepatica Ab ELISA kit (Bio-X Diagnostics, Belgium). All testing was carried out by the same person. All kits have been validated by the manufacturers for use with individual milk, pooled milk and serum samples. Results for Ildana, IDEXX and Bio-X kits were expressed as sample to positive percentage (S/P%) and as optical density ratio (ODR) for the Svanovir test. The specific characteristics of each test are included in Table 1. All assays were completed following manufacturer’s instructions including calculation of S/P% and ODRs.

Sample classification

In addition to continuous ELISA results, serological results were classified as positive or negative according to kit positive cut-off values for three kits (Ildana, IDEXX, Bio-X). The fourth kit (Svanovir) classified samples based on a cut-off value above which animals were deemed infected and whether the potential for production losses existed. These cut-off values are outlined in Table 1 for each assay examined.

Two further categorisations of dairy herds were completed for bulk tank milk analyses. Firstly, herds were classified as small (50 to 120 milking cows), medium (121 to 190 milking cows) or large (over 190 milking cows). These herd size ranges were defined to best represent the data recorded, generating categories of similar size. Secondly, herds were classified on whether or not a F. hepatica dosing protocol was applied in winter 2010.

Statistical analyses

Microsoft Excel (MS Office, 2010) was used for data collation and initial descriptive analyses including scatter plots. Assay Se, Sp and associated statistics were calculated using the MEDCALC diagnostic test evaluation calculator ( Normality of the data was assessed by Shapiro-Wilk normality test and visually using ladder of powers histograms constructed in Stata version 12 (StataCorp, USA). Boxplots, Wilcoxon matched-pairs signed-rank test and Generalised estimating equations (GEE) were completed using Stata version 12. GraphPad Prism 7 (GraphPad Software Inc., 2017) was used to construct the box plot of natural infection-blood data.

Three databases were created, one for each sampling population (known status, natural infection – blood and natural infection – BTM). The Se and Sp of each kit was calculated for each test using positive and negative samples of known status. Assay Se was calculated as the probability that an experimentally infected calf would be identified as positive (Ildana, IDEXX, Bio-X) or infected (Svanovir) with F. hepatica, based on manufacturer’s interpretation criteria. Sp was calculated as the probability that a pre-colostral calf would be identified as negative (Ildana, IDEXX, Bio-X) or not likely to have been exposed to F. hepatica (Svanovir).

The naturally infected – blood data from beef cattle were analysed by Wilcoxon matched-pairs signed rank test to examine whether significant differences existed between pre- and post-treatment groups across each assay. Additionally, a boxplot was generated to allow visualisation of results across both treatment and ELISA kit used.

To examine whether trends in seasonality could be detected using the assays under investigation, BTM data (continuous) were analysed by GEE. For all continuous GEE analyses, herd was included as a repeated measure and an exchangeable correlation used. A Gaussian family and identity link function was used. Independent variables included in the analyses were herd size (small vs. medium vs. large), dosing protocol (dosed vs. not dosed in winter 2010), and time (December 2010 vs. April 2011 vs. July 2011 vs. October 2011). These variables were forced into the model regardless of their significance level due to their potential impact on BTM results. Finally, results from herds that provided a complete set of four BTM samples were plotted against assay intermediate positive cut-off values to visualise seasonality across assays.

Availability of data and materials

All data is stored in the Teagasc (national food and development authority) database. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.



Bulk tank milk


Enzyme-linked Immunosorbent assay


Faecal egg count


Generalised estimating equations


Health Products Regulatory Authority




Optical density ratio


Sample to positive percentage






Teagasc Animal Ethics Committee


  1. Urquhart G, Armour J, Duncan J, Dunn A, Jennings F. Fasciola hepatica; 1996. p. 103–13.

    Google Scholar 

  2. Borgsteede F. Diseases of Dairy Animals. Parasites, Internal: Liver Flukes. In: Fuquay JW, editor. Encyclopedia of Dairy Sciences. 2nd ed. San Diego: Academic; 2011. p. 264–9.

    Chapter  Google Scholar 

  3. Mas-Coma S, Valero MA, Bargues MD. Climate change effects on trematodiases, with emphasis on zoonotic fascioliasis and schistosomiasis. Vet Parasitol. 2009;163:264–80.

    Article  PubMed  Google Scholar 

  4. Taylor MA, R lL. W RLC. Fasciola hepatica. In: Ltd BS, editor. Veterinary Parasitology. Third ed: Blackwell Publishing; 2007. p. 600.

  5. World Health Organization (WHO). Integrating neglected tropical diseases into global health and development. fourth ed. Geneva: WHO report on neglected tropical diseases; 2017. Accessed 18 Feb 2019

    Google Scholar 

  6. van Dijk J, Sargison ND, Kenyon F, Skuce PJ. Climate change and infectious disease: helminthological challenges to farmed ruminants in temperate regions. Animal. 2010;4:377–92.

    Article  PubMed  Google Scholar 

  7. Fox NJ, White PC, McClean CJ, Marion G, Evans A, Hutchings MR. Predicting impacts of climate change on Fasciola hepatica risk. PLoS One. 2011;6. ISBN: 978-1-118-68711-6

    Article  CAS  Google Scholar 

  8. Olah S, van Wyk JA, Wall R, Morgan ER. FAMACHA © : a potential tool for targeted selective treatment of chronic fasciolosis in sheep. Vet Parasitol. 2015;212:188–92.

    Article  PubMed  Google Scholar 

  9. Howell A, Baylis M, Smith R, Pinchbeck G, Williams D. Epidemiology and impact of Fasciola hepatica exposure in high-yielding dairy herds. Prev Vet Med. 2015;121:41–8.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Mezo M, González-Warleta M, Castro-Hermida JA, Muiño L, Ubeira FM. Association between anti-F. hepatica antibody levels in milk and production losses in dairy cows. Vet Parasitol. 2011;180:237–42.

    Article  Google Scholar 

  11. Charlier J, Duchateau L, Claerebout E, Williams D, Vercruysse J. Associations between anti-Fasciola hepatica antibody levels in bulk-tank milk samples and production parameters in dairy herds. Prev Vet Med. 2007;78:57–66.

    Article  PubMed  Google Scholar 

  12. Animal Health Ireland. Liver Fluke- The facts. 2011. Accessed 18 Feb 2019.

    Google Scholar 

  13. Howell AK, Tongue SC, Currie C, Evans J, Williams DJL, McNeilly TN. Co-infection with Fasciola hepatica may increase the risk of Escherichia coli O157 shedding in British cattle destined for the food chain. Prev Vet Med. 2018;150:70–6.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Mas-Coma S. Human fascioliasis: epidemiological patterns in Uman endemic areas of South America, Africa and Asia. Southeast Asian J Trop Med Public Health. 2004;35:1–11.

    Google Scholar 

  15. Ollerenshaw CB, Smith LP. Meteorological factors and forecasts of helminthic disease. Adv Parasitol. 1969;7:283–323.

    Article  CAS  PubMed  Google Scholar 

  16. Morley NJ, Lewis JW. The influence of climatic conditions on long-term changes in the helminth fauna of terrestrial molluscs and the implications for parasite transmission in southern England. J Helminthol. 2008;82:325.

    Article  CAS  PubMed  Google Scholar 

  17. Relf V, Good B, Hanrahan JP, McCarthy E, Forbes AB, DeWaal T. Temporal studies on Fasciola hepatica in Galba truncatula in the west of Ireland. Vet Parasitol. 2011;175.

    Article  CAS  Google Scholar 

  18. Ducheyne E, Charlier J, Vercruysse J, Rinaldi L, Biggeri A, Demeler J, et al. Modelling the spatial distribution of Fasciola hepatica in dairy cattle in Europe. Geospat Health. 2015;9:261.

    Article  PubMed  Google Scholar 

  19. Bloemhoff Y, Forbes A, Danaher M, Good B, Morgan E, Mulcahy G, et al. Determining the prevalence and seasonality of Fasciola hepatica in pasture-based dairy herds in Ireland using a bulk tank Milk ELISA. Ir Vet J. 2015;68:16.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Bennema SC, Ducheyne E, Vercruysse J, Claerebout E, Hendrickx G, Charlier J. Relative importance of management, meteorological and environmental factors in the spatial distribution of Fasciola hepatica in dairy cattle in a temperate climate zone. Int J Parasitol. 2011;41:225–33.

    Article  CAS  PubMed  Google Scholar 

  21. Munita MP, Rea R, Bloemhoff Y, Byrne N, Martinez-Ibeas AM, Sayers RG. Six-year longitudinal study of Fasciola hepatica bulk milk antibody ELISA in the dairy dense region of the republic Ireland. Prev Vet Med. 2016;134:16–25.

    Article  CAS  PubMed  Google Scholar 

  22. Rapsch C, Schweizer G, Grimm F, Kohler L, Bauer C, Deplazes P, et al. Estimating the true prevalence of Fasciola hepatica in cattle slaughtered in Switzerland in the absence of an absolute diagnostic test. Int J Parasitol. 2006;36:1153–8.

    Article  CAS  PubMed  Google Scholar 

  23. Mazeri S, Sargison N, Kelly RF, deC BBM, Handel I. Evaluation of the Performance of Five Diagnostic Tests for Fasciola hepatica Infection in Naturally Infected Cattle Using a Bayesian No Gold Standard Approach. PLoS One. 2016;11:e0161621.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Charlier J, Vercruysse J, Morgan E, Dijk J, Williams DJL. Recent advances in the diagnosis, impact on production and prediction of Fasciola hepatica in cattle. Parasitology. 2014;141.

    Article  Google Scholar 

  25. Anderson N, Luong TT, Vo NG, Bui KL, Smooker PM, Spithill TW. The sensitivity and specificity of two methods for detecting Fasciola infections in cattle. Vet Parasitol. 1999;83:15–24.

    Article  CAS  PubMed  Google Scholar 

  26. Braun U, Wolfensberger R, Hertzberg H. Diagnosis of liver flukes in cows--a comparison of the findings in the liver, in the feces, and in the bile. Schweiz Arch Tierheilkd. 1995;137:438–44

    CAS  PubMed  Google Scholar 

  27. Brockwell Y, Spithill T, Anderson G, Grillo V, Sangster N. Comparative kinetics of serological and coproantigen ELISA and faecal egg count in cattle experimentally infected with Fasciola hepatica and following treatment with triclabendazole. Vet Parasitol. 2013;196.

    Article  CAS  Google Scholar 

  28. Duscher R, Duscher G, Hofer J, Tichy A, Prosl H, Joachim A. Fasciola hepatica – monitoring the milky way? The use of tank milk for liver fluke monitoring in dairy herds as base for treatment strategies. Vet Parasitol. 2011;178:273–8.

    Article  PubMed  Google Scholar 

  29. Pepe MS. The statistical evaluation of medical tests for classification and prediction. Oxford: Oxford University Press; 2003.

    Google Scholar 

  30. Salimi-Bejestani MR, Daniel R, Cripps P, Felstead S, Williams DJL. Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Fasciola hepatica in milk. Vet Parasitol. 2007;149:290–3.

    Article  CAS  PubMed  Google Scholar 

  31. Charlier J, Meulemeester L, Claerebout E, Williams D, Vercruysse J. Qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fasciolosis in cattle. Vet Parasitol. 2008;153.

    Article  Google Scholar 

  32. Gottstein B, Schneeberger M, Boubaker G, Merkle B, Huber C, Spiliotis M, et al. Comparative assessment of ELISAs using recombinant Saposin-like protein 2 and recombinant Cathepsin L-1 from Fasciola hepatica for the Serodiagnosis of human Fasciolosis. PLoS Negl Trop Dis. 2014;8:e2860.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Takeuchi-Storm N, Denwood M, Hansen TVA, Halasa T, Rattenborg E, Boes J. Farm-level risk factors for Fasciola hepatica infection in Danish dairy cattle as evaluated by two diagnostic methods. Parasit Vectors. 2017;10.

  34. Collins PR, Stack CM, O’Neill SM, Doyle S, Ryan T, Brennan GP, et al. Cathepsin L1, the major protease involved in liver fluke ( Fasciola hepatica ) virulence. J Biol Chem. 2004;279:17038–46.

    Article  CAS  Google Scholar 

  35. Harmsen MM, Cornelissen JB, Buijs HE, Boersma WJ, Jeurissen SH, van Milligen FJ. Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide. Int J Parasitol. 2004;34:675–82.

    Article  CAS  PubMed  Google Scholar 

  36. Mezo M, González-Warleta M, Carro C, Ubeira FM. An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using new monoclonal antibody (MM3). J Parasitol. 2004:845–52.

    Article  Google Scholar 

  37. Selemetas N, Phelan P, O’Kiely P. Waal T d. weather and soil type affect incidence of fasciolosis in dairy cow herds. Vet Rec. 2014;175:371.

    Article  CAS  PubMed  Google Scholar 

  38. Kuerpick B, Schnieder T, Strube C. Evaluation of a recombinant cathepsin L1 ELISA and comparison with the Pourquier and ES ELISA for the detection of antibodies against Fasciola hepatica. Vet Parasitol. 2013;193:206–13.

    Article  CAS  PubMed  Google Scholar 

  39. Carnevale S, Rodriguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis. 2001;41:43–9.

    Article  CAS  PubMed  Google Scholar 

  40. Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma WJ. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease. Int J Parasitol. 2001;31:728–37

    Article  CAS  Google Scholar 

  41. Reichel MP. Performance characteristics of an enzyme-linked immunosorbent assay for the detection of liver fluke (Fasciola hepatica) infection in sheep and cattle. Vet Parasitol. 2002;107:65–72 .

    Article  CAS  Google Scholar 

  42. Rapsch C, Schweizer G, Grimm F, Kohler L, Bauer C, Deplazes P. Estimating the true prevalence of Fasciola hepatica in cattle slaughtered in Switzerland in the absence of an absolute diagnostic test. Int J Parasitol. 2006;36.

    Article  CAS  Google Scholar 

  43. Molloy JB, Anderson GR, Fletcher TI, Landmann J, Knight BC. Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia. Vet Parasitol. 2005;130:207–12.

    Article  CAS  PubMed  Google Scholar 

  44. Simões AN, de Almeida SLH, da R BÁF, IVF M, Donatele DM, Barioni G. Validity of a commercial kit for detection of antibodies in bovine serum in an endemic area for fasciolosis. Rev Bras Parasitol Vet. 2017;26:372–4.

    Article  CAS  PubMed  Google Scholar 

  45. Charlier J, De Cat A, Forbes A, Vercruysse J. Measurement of antibodies to gastrointestinal nematodes and liver fluke in meat juice of beef cattle and associations with carcass parameters. Vet Parasitol. 2009;166:235–40.

    Article  CAS  PubMed  Google Scholar 

  46. Salimi-Bejestani MR, McGarry JW, Felstead S, Ortiz P, Akca A, Williams DJL. Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test. Res Vet Sci. 2005;78:177–81.

    Article  CAS  PubMed  Google Scholar 

  47. Delves PJ, Martin JM, Burton DR, RIM. Roitt’s essential immunology : Peter J. Delves : 9781118415771. 13th ed. New York: John Wiley & Sons Inc.; 2017.

    Google Scholar 

  48. Gosling JP. Immunoassays : a practical approach: Oxford University Press; 2000. Accessed 22 Mar 2019

  49. Charlier J, Hostens M, Jacobs J, Ranst B, Duchateau L, Vercruysse J. Integrating fasciolosis control in the dry cow management: the effect of closantel treatment on milk production. PLoS One. 2012;7.

    Article  CAS  Google Scholar 

  50. Köstenberger K, Tichy A, Bauer K, Pless P, Wittek T. Associations between fasciolosis and milk production, and the impact of anthelmintic treatment in dairy herds. Parasitol Res. 2017;116:1981–7.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Sekiya M, Zintl A, Doherty ML. Bulk milk ELISA and the diagnosis of parasite infections in dairy herds: a review. Ir Vet J. 2013;66:14.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Kuerpick B, Schnieder T, Strube C. Seasonal pattern of Fasciola hepatica antibodies in dairy herds in northern Germany. Parasitol Res. 2012;111:1085–92.

    Article  PubMed  Google Scholar 

  53. Electronic Irish Statute Book. Slaughter of Animals Act: Office of the Attorney General; 1935. Accessed 27 Apr 2019

Download references


We acknowledge Dr. Yris Bloemhoff and the calf team in Teagasc, Moorepark for the contribution of samples.


This study was funded by Irish Department of Agriculture, Food and the Marine research stimulus funding; project reference 13/ S/405 and Dairy Research Ireland. The funding bodies had no role in the design of the study, sample collection, analysis, interpretation of data and in writing the manuscript or decision to submit results.

Author information

Authors and Affiliations



RS, GM, MS, AMM-I and MPM designed and conceived the study. MPM carried out testing, analysed the data and drafted the manuscript. RR, MS, RS and GM reviewed and revised the manuscript. NB, AK, RS, AMM-I and MPM contributed with sample collection. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Maria Pia Munita.

Ethics declarations

Ethics approval and consent to participate

The entire study was approved by the Teagasc Animal Ethics Committee and licensed by the Health Products Regulatory Authority in Ireland. Also, farmers signed a consent form to participate in the study which satisfies the requirement for experimental studies involving client-owned animals, authors must also document informed consent from the client or owner and adherence to a high standard (best practice) of veterinary care.

Consent for publication

“Not applicable”.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Munita, M.P., Rea, R., Martinez-Ibeas, A.M. et al. Comparison of four commercially available ELISA kits for diagnosis of Fasciola hepatica in Irish cattle. BMC Vet Res 15, 414 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: