Fasciola hepatica, the liver fluke, causes fasciolosis in cattle and sheep in Australia (Dalton, 1999). In sheep, F. hepatica is considered a significant risk during the summer and winter in high rainfall regions (Cole, 1986) or on irrigated or swampy pastures, and was recently estimated to cost the Australian sheep industry approximately 25 million dollars per year (Lane et al., 2015).
Testing to detect F. hepatica in live ruminants has traditionally involved a faecal egg count after sedimentation (Rojo-Vázquez et al., 2012). However, there are limitations to this approach and several other techniques are now available. So, in this paper we briefly review testing options and report some of the results to date in evaluating a faecal antigen ELISA test as an alternative for checking live sheep for F. hepatica.
It is important to be able to detect F. hepatica when present in live livestock to allow producers to plan appropriate control. Good diagnostics help assess treatment efficacy and also support quarantine programs to prevent the spread of F. hepatica to new regions within Australia.
Faecal egg sedimentation (SEDI) is the recommended approach to detect the golden brown eggs of F. hepatica in the faeces of live animals with patent infections (Rojo-Vazquez et al., 2012). The sensitivity of this technique can be affected by the amount of debris present, and may be as low as 7 per cent, but is typically between 30 and 70 per cent (Anderson et al., 1999; Charlier et al., 2008; Happich and Boray, 1969a, b; Kleiman et al., 2005; Mitchell and Palmer, 2013; Rapsch et al., 2006). The specificity of SEDI is often described as close to 100 per cent (Anderson et al., 1999; Charlier et al., 2008; Rapsch et al., 2006), but it is important to consider the influence on results of morphologically similar eggs which are not from F. hepatica (Gordon et al., 2013). Fluctuations in egg shedding (Flanagan et al., 2011a) and false positives after effective treatment, due to protracted shedding of stored eggs from the host’s gall bladder (Mitchell et al., 1998), are also possible.
Therefore, immunological-based tests have been suggested as alternatives to detect F. hepatica in live animals (Demeler et al., 2012). An antibody ELISA, tested on serum, is currently used in Australia (Hutchinson, 2003). Sensitivity and specificity are high (e.g. 99% and > 95% respectively in cattle) and the test has the advantage of detection from about 4 weeks post infection. However, a blood sample is required from tested animals and antibodies can remain detectable for 12 weeks or more after successful treatment of fluke (Hutchinson, 2003; Brockwell et al., 2013).
The commercial Bio-X K201 ELISA (CAELISA; BIO-X Diagnostics, Belgium) is a sandwich ELISA based on a monoclonal MM3 antibody (Charlier et al., 2014; Mezo et al., 2004). This test detects excretory/secretory products from F. hepatica that pass out of the host animal in the faeces. The CAELISA offers the advantages of faecal sample collection, rather than blood, and possible detection of fluke infestation earlier post infection than occurs with SEDI (Flanagan et al., 2011a, b). Faecal samples can also be stored frozen prior to testing. This test has been evaluated in cattle and sheep overseas (e.g. Gordon et al., 2012; Kajugu et al., 2015) and cattle in Australia with favourable results (Brockwell et al., 2013 and 2014). Little is known about performance using faeces collected from Australian sheep. Palmer et al. (2014) calculated sensitivity between 75 and 91 per cent and specificity between 70 and 100 per cent (Anderson et al., 1999; Palmer et al., 2014). So, CAELISA appeared worthy of further investigation. CAELISA testing is relatively more expensive than SEDI, so the possibility of reducing costs by testing composite faecal samples could be worthwhile for commercial diagnostic testing.
SEDI VERSUS CAELISA FOR SHEEP
To evaluate the CAELISA, a total of 246 sheep faecal samples, collected from five different properties in southern New South Wales, were each tested individually by SEDI and CAELISA. The same operator (TC) conducted all of the testing. Results are summarised:
|SEDI only||SEDI and CAELISA||CAELISA only|
|91 samples were negative by both tests|
Both tests agreed on the results from 138 of the 246 samples tested. The 48 faecal samples that were positive according to CAELISA but negative by SEDI were not unexpected, given the seemingly higher sensitivity of the CAELISA test. A high proportion of false positive CAELISA results was also considered unlikely.
Surprisingly, however, 60 faecal samples were positive using SEDI but negative by CAELISA. This raised concerns of false positive SEDI results due to eggs morphologically similar to but different from F. hepatica eggs and/or the excessive influence of the faecal sub-sampling technique on results. The CAELISA test in sheep only requires a 0.5g sub-sample from each faecal sample, and so may be influenced more by only testing such a small volume.
To investigate this further, an additional 30 sheep faecal samples were collected from an additional commercial sheep property in southern NSW. This time faecal samples were each thoroughly mixed before being sub-sampled for CAELISA testing. Results are summarised:
|SEDI only||SEDI and CAELISA||CAELISA only|
|16 samples were negative by both tests|
Only one sample was positive by SEDI alone. Sixteen samples were negative by both tests, 3 were positive by both tests and 10 faecal samples tested positive by CAELISA but negative by SEDI. This suggested the merit of mixing faecal samples prior to sub-sampling for CAELISA testing, and this approach is being further tested using more faecal samples.
COMPOSITE CAELISA FOR SHEEP
To investigate the potential for CAELISA testing of composite sheep faecal samples, initial work combined subsamples from one of 25 faecal samples that tested positive by both SEDI and CAELISA with subsamples from each of four other faecal samples that tested negative on both SEDI and CAELISA (ie one positive sample mixed with four negative samples). Various methods to compose composites were evaluated, and the best methods (that included mixing of individual faecal samples prior to sub-sampling) showed a sensitivity in excess of 90 per cent, when tested in triplicate. Further testing of this approach is ongoing.
This work suggests that CAELISA could be a useful alternative to SEDI for the detection of Fasciola hepatica infestation in live sheep in Australia. Mixing of individual faecal samples prior to sub-sampling for CAELISA seemed an important additional step, and this will be investigated further.
Completion of the evaluation of testing composite sheep faecal samples with CAELISA is an important step in reducing the overall cost of testing for producers. Still progressing is further work to determine the validity of testing of composite samples using an upgraded version of the commercial test kit (available in March 2016). This will include details for the best way to form composites, including possibly mixing of submitted faecal samples prior to sub-sampling.
The technical contributions to this work from several staff within the School of Animal and Veterinary Sciences at Charles Sturt University, Wagga Wagga, New South Wales is very gratefully acknowledged. Bio-X Diagnostics Limited and R-Biopharm (Lab Diagnostics) Australia also generously supplied some Bio-X K201 ELISA kits free of charge.