Research paperAnalysis of daily variation in the release of faecal eggs and coproantigen of Fasciola hepatica in naturally infected dairy cattle and the impact on diagnostic test sensitivity
Introduction
Fasciola hepatica (F. hepatica), more commonly known as liver fluke, has serious production-limiting impacts in dairy cattle, affecting milk production, milk quality, weight gain and fertility (Schweizer et al., 2005). In the UK the economic cost of reduced production due to fluke infections is estimated at £300 million per year (Williams et al., 2014). In Australia in 2020, reduced milk production due to F. hepatica infection was estimated to cost the Victorian dairy industry USD 101 million per year (Kelley et al., 2020). A robust quantitative diagnostic test that can identify F. hepatica-infected individuals and herds would allow for prompt intervention and treatment of cattle with appropriate flukicides. Europe has moved towards screening dairy herds with an ELISA detecting antibodies in bulk tank milk (BTM) samples which are readily available and easy to collect (Pritchard et al., 2005; Salimi-Bejestani et al., 2005; Charlier et al., 2007; Bennema et al., 2009; McCann et al., 2010; Kuerpick et al., 2013; Selemetas et al., 2014; Bloemhoff et al., 2015; Howell et al., 2015; Novobilský et al., 2015). The bulk tank milk sELISA (BTM sELISA) was first described by Salimi-Bejestani et al. (2005): this assay has a high sensitivity (96 %), and moderate specificity (80 %) and can accurately identify herds that are incurring production losses when herd prevalence is in excess of 25 % (Charlier et al., 2007). However, the BTM ELISA has its drawbacks as anti-Fasciola antibodies in milk can persist for up to six months even after successful treatment with a flukicide (Salimi-Bejestani et al., 2005). In Australia, most dairy herds use a split calving system, which means that flukicide treatments occur at different times of the year for individual herds. As a result, antibodies found in milk could arise from treated and untreated cattle which complicates the interpretation of a positive BTM ELISA test.
As a consequence, a different approach has been used to screen dairy cattle in Australia. Brockwell et al. (2014), Elliott et al. (2015) and Kelley et al. (2020) each used the commercial coproantigen ELISA (BIO K 201 kit, Bio X Diagnostics) test to screen multiple herds for F. hepatica as coproantigen release ceases 7 days after effective treatment with a flukicide (Brockwell et al., 2013). The coproantigen ELISA (cELISA) detects infection in cattle from >6 weeks post-infection (PI) and has a high sensitivity 77 %–100 % and specificity >99 % (Mezo et al., 2004; Brockwell et al., 2013; Mazeri et al., 2016). In addition, correlations between F. hepatica burden and OD were observed in cattle by Charlier et al. (2008) (R 0.60) and Brockwell et al. (2013) (R2 0.8718) although recent work by Martínez-Sernández et al. (2016) found a somewhat weaker correlation (R2 0.2998). However, Brockwell et al. (2013) observed a 2–6 fold variation in coproantigen release from cattle over a 5 day period. To address the variable release of coproantigens the cELISA kit was modified by Martínez-Sernández et al. (2016) increasing the sensitivity from 0.60 ng/mL to 0.15 ng/mL; however, the variability in the cELISA in daily samples increased by 6–12 fold (Mezo et al., 2004). There is a consensus in the literature that the cELISA kit-cut off recommended by the commercial manufacturer is too high to accurately distinguish between positive and negative cattle. As a result, studies have used various ELISA OD cut-offs for detecting F. hepatica infections in cattle: 0.114 OD (Mezo et al., 2004) 0.030 OD (Charlier et al., 2008), 0.014 OD (Brockwell et al., 2013), kit cut off × 0.67 (Palmer et al., 2014), 0.084 OD (Martínez-Sernández et al., 2016) as well as 1.3 % (Brockwell et al., 2014) or 1.6 % (Elliott et al., 2015) of the OD value of the positive control. The lack of consistency between reports makes it difficult to determine the sensitivity of the cELISA, but several studies in cattle have reported that the assay can detect as few as 1, 2, and 15 flukes in the liver (Mezo et al., 2004; Brockwell et al., 2013; Martínez-Sernández et al., 2016).
Similar problems occur when using F. hepatica faecal egg counts (LFEC). In cattle with low F. hepatica burdens (<10 flukes) false negatives frequently occur (Martínez-Sernández et al., 2016). There are many variations on the LFEC technique, but sedimentation has been found to be the most accurate in cattle (Happich and Boray, 1969; Kajugu et al., 2015). Two studies reported correlations between LFEC and F. hepatica burden. In cattle, the correlation was R2 0.836 and in sheep R2 0.571 (Brockwell et al., 2013; George et al., 2017). LFECs are highly specific >97.5 % (97.5–100%) and egg shedding does not persist after treatment with an effective flukicide (Ibarra et al., 1998; Anderson et al., 1999; Rapsch et al., 2006; Brockwell et al., 2013; Mazeri et al., 2016). However, weekly, daily and hourly variation in F. hepatica egg shedding has been observed in several studies in cattle (Dorsman, 1956, 1960; Hagens and Over, 1966; Brockwell et al., 2013). Based on hourly faecal sampling in cattle, Dorsman (1956) proposed that faecal collection should occur at 1:30 p.m. when the highest egg release was more likely to represent the burden of F. hepatica within the liver. Hagens and Over (1966) reached the same conclusion observing the peak release of eggs between 12:00 p.m.–8:00 p.m., similarly suggesting that this was the most suitable time for sampling cattle. However, the sensitivity of the LFEC is affected by the volume of faeces sampled, the faecal output by the animal, the burden of F. hepatica within the animal, the experience of the technician and the duration of the F. hepatica infection as the test only detects F. hepatica from > 8 PI weeks in cattle (Boray, 1969; Conceição et al., 2002; Rapsch et al., 2006; Charlier et al., 2008; Brockwell et al., 2013; Martínez-Sernández et al., 2016).
Previous studies have investigated the level of variability in coproantigen shedding in animals between weeks and on consecutive days, but not variations within a day. Monitoring of F. hepatica egg shedding variation has been extensive. However, it has never been determined if peak egg shedding from 12:00 p.m. - 8:00 p.m. actually correlates with F. hepatica burden in the liver and is therefore a better time to collect faecal samples from cattle. In this study, recognising the variable release of both coproantigens and eggs, we investigated the sensitivity of two sample points in the morning (AM) and afternoon (PM) milking, the variation in coproantigen and LFEC shedding over a consecutive five-day period and the correlation of coproantigen levels and LFEC with F. hepatica burden in ten naturally infected dairy cows.
Section snippets
Study design
One pasture-fed, split calving dairy herd in Victoria, Australia identified by Kelley et al. (2020) was purposively selected for this study based on the herd owner’s willingness to participate. Thirty cows were screened using the FlukeFinder® kit to determine if they were infected with F. hepatica. Ten cows were selected based on positive LFEC and purchased from the owner. The age of the cows ranged from 2.9 to 11.1 years and the predominant breed was Holstein. Following purchase, the ten
Summary correlation statistics
All ten cows in this study were F. hepatica positive with the number of flukes in the liver ranging from 9 to 72 (Table 1). No correlation was observed between age and TFC (R −0.32; 95 % CI −0.86 to −0.41) or between age and LFEC (R −0.23; 95 % CI −0.30 to −0.15). However, a moderate negative correlation between age and the cELISA OD was observed using data from both AM and PM samples (R −0.63; 95 % CI −0.68 to −0.57) (Fig. 1). At the AM and PM milkings there was a higher positive correlation
Discussion
In this study, we investigated the sensitivity of two fluke diagnostic tests (cELISA and LFEC) in naturally infected dairy cows using faecal samples collected at the AM and PM milking in order to determine the daily variation in coproantigen and egg shedding over 5 consecutive days, to assess the correlation between coproantigen and LFEC levels with F. hepatica burden and determine the impact of this variation on test sensitivity.
CRediT authorship contribution statement
Jane M. Kelley: Methodology, Formal analysis, Investigation, Methodology, Validation, Visualization, Resources, Writing - original draft, Writing - review & editing. Mark A. Stevenson: Formal analysis, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Vignesh Rathinasamy: Investigation, Validation, Writing - original draft. Grant Rawlin: Conceptualization, Funding acquisition, Project administration, Supervision, Resources, Writing - original draft,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors extend their thanks to Stewart and Nita McRae and the veterinarians and animal health officers employed by the State Government of Victoria. The authors would like to thank Jaclyn Swan for her assistance in processing the samples and Gillian Mitchell and Philip Skuce from the Moredun Research Institute, UK, for sequencing the paramphistome DNA. This work was supported by funds from the Gardiner Dairy Foundation, Dairy Australia, La Trobe University and the Victorian Department of
References (52)
- et al.
The sensitivity and specificity of two methods for detecting Fasciola infections in cattle
Vet. Parasitol.
(1999) - et al.
The use of bulk-tank milk ELISAs to assess the spatial distribution of Fasciola hepatica, Ostertagia ostertagi and Dictyocaulus viviparus in dairy cattle in Flanders (Belgium)
Vet. Parasitol.
(2009) Experimental fascioliasis in Australia
Adv. Parasitol.
(1969)- et al.
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) - et al.
Confirmation of Fasciola hepatica resistant to triclabendazole in naturally infected Australian beef and dairy cattle
Int. J. Parasitol.: Drugs Drug Res.
(2014) - et al.
Associations between anti-Fasciola hepatica antibody levels in bulk-tank milk samples and production parameters in dairy herds
Prev. Vet. Med.
(2007) - et al.
Qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fasciolosis in cattle
Vet. Parasitol.
(2008) - et al.
Evaluation of a simple sedimentation method (modified McMaster) for diagnosis of bovine fascioliosis
Vet. Parasitol.
(2002) - et al.
High prevalence of fasciolosis and evaluation of drug efficacy against Fasciola hepaticain dairy cattle in the Maffra and Bairnsdale districts of Gippsland, Victoria, Australia
Vet. Parasitol.
(2015) - et al.
Application of a coproantigen ELISA as an indicator of efficacy against multiple life stages of Fasciola hepatica infections in sheep
Vet. Parasitol.
(2017)
Dynamics of elimination of the eggs of Fasciola hepatica (Trematoda, Digenea) in the faeces of cattle in the Porma Basin, Spain
Vet. Parasitol.
Epidemiology and impact of Fasciola hepatica exposure in high-yielding dairy herds
Prev. Vet. Med.
Comparison of three ELISA tests for seroepidemiology of bovine fascioliosis
Vet. Parasitol.
Fasciola hepatica: specificity of a coproantigen ELISA test for diagnosis of fasciolosis in faecal samples from cattle and sheep concurrently infected with gastrointestinal nematodes, coccidians and/or rumen flukes (paramphistomes), under field conditions
Vet. Parasitol.
Current threat of triclabendazole resistance in Fasciola hepatica
Trends Parasitol.
Determination of the prevalence and intensity of Fasciola hepatica infection in dairy cattle from six irrigation regions of Victoria, South-eastern Australia, further identifying significant triclabendazole resistance on three properties
Vet. Parasitol.
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.
Paramphistomosis of ruminants: the role of free-living metacercariae
Trends Parasitol.
The field evaluation of albendazole and triclabendazole efficacy against Fasciola hepatica by coproantigen ELISA in naturally infected sheep
Vet. Parasitol.
Estimating the true prevalence of Fasciola hepatica in cattle slaughtered in Switzerland in the absence of an absolute diagnostic test
Int. J. Parasitol.
Performance characteristics of an enzyme-linked immunosorbent assay for the detection of liver fluke (Fasciola hepatica) infection in sheep and cattle
Vet. Parasitol.
MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected withFasciola hepatica and F. gigantica
Vet. Parasitol.
Treatment vs non-treatment of helminth infections in cattle: defining the threshold
Vet. Parasitol.
lme4: linear mixed-effects models using Eigen and S4
J. Stat. Softw.
Determining the prevalence and seasonality of Fasciola hepatica in pasture-based dairy herds in Ireland using a bulk tank milk ELISA
Irish Vet. J.
An analysis of transformations (with discussion)
J. R. Stat. Soc. Ser. B
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2023, International Journal for Parasitology: Drugs and Drug ResistancePrevalence and gross pathology of liver fluke in macropods cohabiting livestock farms in north eastern NSW, Australia, and diagnosis using cELISA
2021, International Journal for Parasitology: Parasites and WildlifeCitation Excerpt :Red deer have also been identified with similar pathology in the liver and this was also thought to contribute to a lower sensitivity of cELISAs assessments in that species (French et al., 2016). Nevertheless, we cannot rule out that daily fluctuations in the release of coproantigens may play a role in the limited sensitivity as coproantigens were recently reported to fluctuate 2.6–8.9 fold in dairy cattle and correlated higher with fluke burden when cELISAs were conducted on faecal samples collected before midday (a.m.) (Kelley et al., 2021). Lastly, the one Macropod with no liver fluke, but positive for coproantigens and fluke eggs in the faeces and gall bladder, most likely occurred as fluke can easily be missed during visual inspection or liver collection given that culling of Macropods was predominately conducted at night.
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Present address: Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Queensland, Australia.