Elsevier

Veterinary Parasitology

Volume 298, October 2021, 109504
Veterinary Parasitology

Research paper
Analysis 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

https://doi.org/10.1016/j.vetpar.2021.109504Get rights and content

Highlights

  • The liver fluke cELISA and Faecal Egg Counts (LFEC) were evaluated over 5 consecutive days in cows.

  • There was a 2.6–8.9 fold variation between days in the cELISA Optical Density values.

  • There was a 5–16 fold variation between days in the liver fluke LFEC values.

  • The correlation of both tests with fluke counts was higher in the morning sampling.

  • The sensitivity was 100 % for the cELISA and 88 % for LFEC.

Abstract

The liver fluke, Fasciola hepatica (F. hepatica) is a widespread parasite infection in dairy cattle in Victoria, South-eastern Australia. Robust diagnosis of fluke infection is needed in dairy cattle to identify sub-clinical infections which often go unnoticed, causing significant production losses. We tested the coproantigen ELISA (cELISA) and the FlukeFinder faecal egg count kit® on naturally infected cows in a fluke endemic region of Victoria. The aim of the study was to investigate the variation in the release of coproantigens and eggs into faeces over a 5-day period, at the morning (AM) and afternoon (PM) milkings, and to assess the impact of the timing of faecal sample collection on diagnostic test sensitivity. Ten cows were enrolled into the study based on positive F. hepatica faecal egg counts (LFEC) and faecal samples from the ten cows were collected twice daily, at the 7–9 AM and 4–6 PM milking, for five consecutive days. At the conclusion of the sampling period, the cows were euthanized and F. hepatica burden determined at necropsy. A moderate negative correlation between cow age and cELISA optical density (OD) was observed using data from all samples (R -0.63; 95 % CI -0.68 to -0.57). Over the 5-day sampling period, we observed within-animal variation between days for both the cELISA OD (2.6–8.9 fold) and LFEC (5–16 fold), with more variation in values observed in the PM samples for both tests. The correlation with total fluke burden was higher in the AM sampling using both the cELISA and LFEC (R 0.64 and 0.78, respectively). The sensitivity was 100 % for the cELISA using various cut offs from the literature (0.014 OD, 0.030 OD, and 1.3 % or 1.6 % of the positive control). The sensitivity of the FlukeFinder kit® (based on 588 faecal samples and not accounting for lack of independence in the data) was 88 % (95 % CI 85 %–90 %). Seventy one false negatives were recorded from the 588 LFEC tests all of which were observed in the cows with fluke burdens <14 flukes; 42 of the 71 false negative LFECs occurred in one individual cow which had the lowest burden of nine flukes. In dairy cows, the cut-off for production losses due to fasciolosis is estimated at> 10 fluke. Both the cELISA and the LFEC identified all cows that had burdens equal to or greater than this cut-off. Five of the ten cows also exhibited relatively high paramphistome egg counts.

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

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      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.

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