Liver fluke control on sheep farms in Northern Ireland: A survey of changing management practices in relation to disease prevalence and perceived triclabendazole resistance
C. McMahon a , H.W.J. Edgar b , R.E.B. Hanna b , S.E. Ellison a , A.M. Flanagan a , M. McCoy c , P.-E. Kajugu b , A.W. Gordon d , D. Irwin e , J.E. Barley b , F.E. Malone b , G.P. Brennan a ,
I. Fairweather a,∗
aParasite Therapeutics Research Group, School of Biological Sciences, Medical Biology Centre, The Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom
bVeterinary Sciences Division, Agri-Food and Biosciences Institute (AFBI) Stormont, Belfast BT4 3SD, United Kingdom
cDepartment of Agriculture and Rural Development, Dundonald House, Upper Newtownards Road, Belfast BT4 3SB, United Kingdom
dBiometrics Division, Agri-Food and Biosciences Institute (AFBI), Newforge Lane, Belfast BT9 5PX, United Kingdom
eAgriculture Branch, Agri-Food and Environmental Science Division, Agri-Food and Biosciences Institute (AFBI), Hillsborough BT26 6DR, United Kingdom

a r t i c l e i n f o

Article history:
Received 19 October 2015 Received in revised form 25 November 2015
Accepted 30 November 2015

Keywords: Fasciola hepatica Liver fluke Northern Ireland Questionnaire Survey
Faecal egg count reduction test Coproantigen ELISA reduction test
a b s t r a c t

Reports of resistance to triclabendazole (TCBZ) among fluke populations have increased in recent years. Allied to this, there has been a rise in the prevalence of the disease, which has been linked to climate change. Results from questionnaire surveys conducted in Northern Ireland (NI) in 2005 (covering the years 1999–2004) and 2011 (covering the years 2008–2011) have provided an opportunity to examine the extent to which fluke control practices have changed over a prolonged time-frame, in light of these changes.
A number of differences were highlighted. There was a significant shift away from the use of TCBZ over time, with it being replaced largely by closantel. The timing of treatments had moved earlier in the year, perhaps in response to climate change (and an altered pattern of disease). In relation to the frequency of drug treatments, there were no major changes in the overall pattern of drug treatments between the two survey points, although on both occasions approximately one-third of flock owners gave more than 3 treatments per year to ewes. In lowland areas in 2011, flock owners were rotating drug classes more often (each year and at each treatment) than in 2005, whereas in upland areas, flock owners were rotating less often and more were not rotating at all. Between 2005 and 2011, the percentage of flock owners giving quarantine treatments to bought-in stock had halved, to a very low level (approximately 10%).
Using data from a complementary TCBZ resistance survey (Hanna et al., 2015), it has been shown that the way in which data are selected and which efficacy formula is applied can influence the calculation of drug efficiency and impact on diagnosis of resistance.
© 2015 Elsevier B.V. All rights reserved.


Successful control of fasciolosis is confronted by two major chal- lenges: an increased incidence of the disease and the spread of resistance to triclabendazole (TCBZ), the drug most active against acute and chronic fluke infections. The upsurge in incidence has been attributed to the effect of climate change, which has favoured the snail intermediate host, and long-range forecasts have sug-

∗ Corresponding author. Fax: +44 28 90975877.
E-mail address: [email protected] (I. Fairweather). 0304-4017/© 2015 Elsevier B.V. All rights reserved.

gested that this trend will become more pronounced (Fox et al., 2011). TCBZ resistance has emerged in several areas of the World and, again, the situation is likely to become more serious in the future. Allied to this is the problem of accurate diagnosis of resis- tance, so that genuine cases can be separated from reports that are simply cases of “treatment failure”, for whatever reason. For a more detailed discussion of these topics, the reader is referred to the reviews by Fairweather (2011a,b).
Of crucial significance to fluke control are the management strategies put in place by farmers; also, how well the farmers per- ceive the issues described above and how they are responding to them. Farmers are able to source and obtain advice from veter-

inarians, the agricultural press, farm advisors, suitably qualified personnel (SQP), other farmers, government agencies and drug company representatives, for example. However, few large-scale surveys have been carried out to determine farmers’ attitudes and control practices. Of those that have, more surveys have been con- cerned with dairy cattle (eg Mezo et al., 2008; Bloemhoff et al., 2014; Selemetas et al., 2015) than sheep (e.g. Morgan et al., 2012; Rojo-Vazquez and Hosking, 2013) and not all were restricted to fluke. This communication presents information derived from two Questionnaire surveys in Northern Ireland (NI), conducted in 2005 and 2011. They were designed to gather data on control and sheep management practices in relation to fasciolosis and how the latter may have contributed to the levels of resistance described in a sep- arate study (Hanna et al., 2015). Particular attention was given to the pattern of anthelmintic use, the timing and frequency of treat- ments, drug rotation and quarantine strategy. The Questionnaires covered a time-span of more than a decade and so provide a good view of changing practices over a relatively prolonged period of time.
This paper also extends the analysis of data stemming from a field survey of TCBZ resistance in NI carried out in 2011 and pub- lished by Hanna et al. (2015). Data taken from that survey has been used to test how the calculation of reductions in faecal egg count (FEC) and coproantigen levels can be affected by the selection of data and efficacy formulae applied. The results of the 2011 survey will be compared with those of a similar field survey carried out in 2008. The combined field efficacy and Questionnaire data should give a good overall view of fluke control in the Province. The data on fluke will complement that of nematode control in NI published previously (McMahon et al., 2013a,b,c).

2.Materials and methods

2.1.TCBZ field resistance surveys

2.1.1.Field survey
The survey was carried out on 13 sheep farms, designated A–M. For details of experimental design and FEC and coproantigen ELISA methodology, see Hanna et al. (2015).

2.1.2.Field survey
The protocol as described for sampling in 2011 was based on a similar survey conducted in 2008 on 12 of the aforementioned hold- ings (Flanagan, 2010; Chapter 5). Raw data were obtained from the author and analysed using the approach described in the next sec- tion (Section 2.2). The inclusion of these data is intended to provide a comparison of efficacy results between different time points.

2.2.Statistical analysis of field survey data

Initial analysis of the FEC and coproantigen ELISA data was car- ried out as described by Hanna et al. (2015).
A second analysis was performed to examine the effect of dif- ferent formulae on calculating treatment efficacy and anthelmintic resistance. As per WAAVP guidelines (although developed for gas- trointestinal nematodes and not fluke specifically), anthelmintic resistance was declared when the calculated treatment efficacy was <90% (Coles et al., 2006). Reductions in FECs and coproantigen levels were calculated using Microsoft Excel. Percentage reduc- tion was based either: on the formula of Kohapakdee et al. (1995), where percentage reduction = [(T1 - T2)/T1] × 100, where T1 is the arithmetic mean FEC pre-treatment and T2 is the arithmetic mean FEC post-treatment (pt) for a group of treated animals (referred to subsequently as FECRT1); or on the iFE- CRT3 formula of Cabaret and Berrag (2004), where percentage reduction = (1/n)ti(100 × (1 - [Ti2/Ti1])), where Ti2 is pt and Ti1 is pre-treatment eggs per gram (epg) of faeces in host I from a total of n hosts. Each host serves as its own control. Referred to subsequently as FECRT2, it is the individual animal counterpart of FECRT1. Using the available data, 3 datasets were constructed. They rep- resented different combinations of FEC and coproantigen ELISA data and were used to assess their bearing on the calculation of drug efficacy. The datasets were: Dataset 1. Pre- and post-treatment epg averages, calculated using all 20 (or total number of collected) faeces samples; Dataset 2. The pre-treatment epg average, calculated using only the values generated from sheep with a current infection (i.e. pos- itive by coproantigen ELISA). The pt average was calculated using all 20 (or total number of collected) faeces samples; and Dataset 3. Pre- and post-treatment epg averages, calculated using only the values generated from sheep with a current infection. 2.3.Questionnaire surveys 2.3.1.Questionnaire The questionnaire contained 42 questions, which covered con- trol of gastrointestinal nematodes, liver fluke and ectoparasites. The analysis of the nematode control data has been published by McMahon et al. (2013a,b). In relation to fluke, questions referred to product use over a 5-year period (2000–2004), the timing and frequency of treat- ment, product rotation, quarantine treatments and perception of the prevalence of resistance. The survey was conducted by personal interview, with 81 farmers. For further details of the survey, see McMahon et al. (2013a,b). 2.3.2.Questionnaire The questionnaire comprised 51 questions, organised into three main sections, namely, parasite control practices (Section 1, McMahon et al., 2013a), farm management (sheep) (Section 2, McMahon et al., 2013a,b) and farm management (cattle) (Section 3, nematode control, McMahon, 2015 Thesis). Questions included the same topics as listed for the 2005 edition, but covering the period 2008–2011. Between May and September, 2011, the questionnaire was sent to 1,000 farmers, amongst whom were sheep-only farmers, cattle- only farmers and mixed sheep and cattle farmers. There were 305 returns, of which 252 were relevant to sheep. For further details of the survey, see McMahon et al. (2013a,b). 2.4.Recorded cases of acute fluke 2.4.1.Diagnoses of acute fasciolosis, post-mortem findings (Great Britain) Since 1975, the Veterinary Investigation Diagnosis Analysis Database (VIDA) has documented the findings from every sub- mission made to regional laboratories of the Animal Health and Veterinary Laboratories Agency and Scottish Agricultural College Disease Surveillance Centre ( veterinary-investigation-diagnostic-analysis-vida-reports). The aggregate data of these 23 centres provide a useful indication of the level of disease reported across England, Scotland and Wales. 2.4.2.Diagnoses of acute fasciolosis, post-mortem findings (Northern Ireland) Similar data are recorded for NI in the 2 diagnostic centres: AFBI Omagh (west of the Province) and AFBI Stormont (east of the Province). Information was retrieved from the AFBI Pathology Database and was limited to diagnoses of acute fluke between 2000 and 2014. The limitation to acute fluke enabled direct comparison of recorded cases in NI and GB. The data were filtered to remove carcasses or viscera received from independent researchers, other research institutes, pharmaceutical companies and “in-house” ani- mals on VSD premises. Results were limited to flocks held in NI. The recorded numbers of cases per year in the VIDA database were compared graphically with the recorded numbers of cases per year in the AFBI Laboratory Information Management System (LIMS) database Table 1 Faecal egg count reduction test (FECRT) values of TCBZ treatment on Farms A–E using the constructed datasets and described formulae (2011 field resistance survey data). Farm A Farm B Farm C Farm D Farm E 2011 survey n-Pre 19 20 20 20 20 n-Post 18 18 16 13 18 2.5.Literature search for reports of “triclabendazole resistance” Dataset 1 FECRT1 76.3 FECRT2 0.0 0.0 4.1 8.6 0.0 0.0 0.0 37.7 33.7 Using “triclabendazole resistance” as the initial search term, the ISI Web of Science database was searched: the database returned publications detailing studies in ruminants (sheep and cattle) and in humans. Publications were selected that dealt with both experimental infections and/or natural infection. One hun- dred and sixty-two papers were identified; removing publications which related to human fasciolosis (6 papers), or other verte- brate infection with different trematode species [Fasciola gigantica (10), Schistosoma mansoni (2), Clonorchis sinensis (1) and Parago- nimus westermani (4)] left 138 database entries. These remaining Dataset 2 FECRT1 80.9 FECRT2 8.8 Dataset 3 FECRT1 80.3 FECRT2 14.4 2008 survey n-Pre 15 n-Post 14 Dataset 1 FECRT1 84.2 FECRT2 80.5 Dataset 2 FECRT1 82.4 FECRT2 86.2 Dataset 3 FECRT1 94.1 FECRT2 86.2 0.0 0.0 0.0 0.0 15 12 100 100 100 100 100 100 0.0 0.0 0.0 0.0 – – – – – – – – 0.0 0.0 0.0 0.0 12 9 34.4 20.5 34.4 20.5 37.2 53.4 37.7 33.7 31.9 43.4 – – – – – – – – entries were classified as belonging to one of the following cate- gories: diagnosis of infection (3), review article (27), histological or morphological investigation (85) or diagnosis of resistance (23). Considering only the diagnosis of resistance publications, abstracts were mined for experimental methodology behind the diagnosis of resistance and the number of holdings investigated. The results of this search are discussed in Section 4.3. 3.Results 3.1.General Results are presented in 4 Sections: the TCBZ field resistance surveys; the impact of calculation formula on drug efficacy eval- uation; analysis of the questionnaire surveys; and a comparison between the number of cases of fasciolosis recorded in NI and the rest of the UK. 3.2.TCBZ field resistance surveys 3.2.1.Field survey The results of the survey have been published elsewhere (Hanna et al., 2015). Briefly, the FECRT data revealed evidence for substan- tial TCBZ resistance on 5 of the 13 farms studied (farms A–E). Each of these 5 farms had a high level of fluke infection (determined by pre-treatment FECs) in comparison to the remaining 8 farms. This result was supported by those of the coproantigen reduction test (CRT) and fluke histology. Nitroxynil (NIT) and closantel (CLOS) were fully effective against the fluke populations in these flocks (Hanna et al., 2015). 3.2.2.Field survey TCBZ resistance was diagnosed by both FECRT and CRT on farm D only, amongst the 12 farms studied (Flanagan, 2010). 3.2.3.Analysis of the effect of calculation formula on the level of resistance declared On 5 of the 13 farms studied in 2011 (farms A–E), all of which managed animals in lowland areas, the pre-dose F. hepatica FEC values indicated that a significant level of chronic fluke infection was present in the flocks. On the remaining farms in the study, the pre-dose F. hepatica FECs were too low to allow meaningful comparison of pre-treatment and pt faeces samples and, in these cases, post-dose coproantigen ELISA testing was not carried out. The remaining results in this section will be limited to flocks A–E for the 2011 data. Note: FECRT1 = method as described by Kohapakdee et al. (1995); FECRT2 = method as described by Cabaret and Berrag (2004); n-Pre = number of animals included in pre-treatment mean calculations; n-Post = number of animals included in post- treatment mean calculations. On 3 of the 12 farms visited in 2008, all of which were in low- land areas, there was sufficient evidence of liver fluke infection (eggs in faeces) in pre-treatment samples to warrant the collec- tion of pt samples. In the remaining 9 flocks, no further analyses were conducted. Coproantigen ELISA testing was conducted on the 3 holdings where fluke infection was evident, but no optical density (OD) data remain available, and the status of infection was recorded as either positive or negative. As (mostly) the same holdings were visited in both surveys, the designation of each holding is conserved, i.e. farm A from 2008 is the same premises as farm A from 2011. egg count reduction testing (FECRT). Considerable variation was observed between holdings and between animals on each holding, which reflects the over-dispersed nature of nat- ural fluke infections. TCBZ resistance, or TCBZ treatment failure, is defined as less than a 90% reduction in FEC (Brockwell et al., 2014). Substantial levels of resistance were found in 4/5 (farms B–E) properties in 2011 (Table 1), an increase over the (1/3, Farm D) lev- els seen in 2008, with another (farm A) holding falling below the 90% threshold in 2011 (Table 1). Both efficacy calculation meth- ods detected resistance using the group data, but the percentage reductions varied: FECRT2 generally indicated higher levels of TCBZ resistance than FECRT1. Calculation of treatment efficacy in the individual animal (Table 2), rather than the efficacy per group of animals, revealed considerable variation. Typically, when an efficacy of 100% was observed, pre-treatment FEC values were low and, in many instances, egg counts rose between sample points, resulting in “negative” efficacy values (recorded as 0.0 in Table 2). The fact that a greater number of animals could be included in the latter (2011) survey than the initial (2008) survey (Table 3) may be indicative of a greater incidence of fluke at pasture, although that cannot be explicitly determined from the available data. Where calculated, treatment efficacy in the individual animal was higher in 2008 (Table 3) than in 2011 (Table 2). reduction testing. As stated previously, OD values were not available for the survey data of 2008 and so the following is limited to the 2011 data. TCBZ resistance, or TCBZ treatment failure, is defined as less than a 90% reduction in CRT Table 2 Efficacy of TCBZ treatment in the individual sheep calculated using the formula of Kohapakdee et al. (1995). Values were calculated for each animal sampled on both day 0 and day 21 for field resistance survey data in 2011. Survey. Over the period 1999–2004, TCBZ was consistently the most frequently selected anthelmintic (>60%) (Fig. 1A). The relative contribution of CLOS to treat fasciolosis, both as a sin- gle active and in combination, increased over the 5-year period.

Animal 1
Farm A 25.0 P
0.0 P 0.0 P 45.5 P 0.0 P 0.0 P 0.0 P 1.2 P 0.0 P 0.0 P 29.7 P 52.8 P
38.8P 0.0 P 62.5 P 100.0 100.0
Farm B
38.9P 0
88.9 0 P
Farm C 0.0
0.0 P 57.9 P 100 54.1 P 0.0 P 0.0 P 3.1 P 0.0 P 66.7 P 100 0.0 P 0.0 P 17.5 P 20.0 P
Farm D 0.0 P
0.0 0.0 P
72.9 P 19.6 P 0.0 P 0.0 P 0.0 P 0.0 P 0.0 P 0.0 P 0.0 P 0.0 P
Farm E 18.1 P
57.3 P 28.8 P 0.0 P 39.7 P 0.0 19.0 61.8 P 0.0 24.4 P 61.3 P 67.0 P 44.9 P 51.0 P 74.7 P 45.4 P 57.2 P 0.0
This was balanced by a decreased contribution of NIT, which fell from approximately 15% to 8% over the course of the survey. Oxy- clozanide (OXY) and albendazole (ALB) comprised less than 5% of all treatments given. Survey. In contrast to the initial survey period, CLOS was the dominant anthelmintic used in the control of fluke during 2008–2011 (Fig. 1B). Approximately 50% of all treatments given comprised CLOS, with a more pronounced contribution of combi- nation products to this total (12.2–19.0%) than in the 1999–2004 survey. Use of TCBZ decreased each successive year through 2008–2011. As before, products containing NIT were the third most commonly identified anthelmintic used. ALB made a greater con- tribution to total anthelmintic use in this period, but remained below 5% of all treatments given. The increased use of OXY between
2010and 2011 may reflect an increased awareness of rumen fluke,

Note: P = positive by coproantigen ELISA on day 21 post-treatment.

Table 3
Efficacy of TCBZ treatment in the individual sheep calculated using the formula of Kohapakdee et al. (1995). Values were calculated for each animal sampled on both day 0 and day 21 for field resistance survey data in 2008.
Animal Farm A Farm B Farm C Farm D Farm E
although that cannot be explicitly determined from the available data.

3.3.2.Timing of treatment
There exists a generally recognised seasonal pattern of liver fluke infection (Fig. 2A) over the grazing season (Abbott et al., 2012; AFBI, 2012). Similarly, the recognised months of highest

100 92.9 P 0

11.6 P 58.1 P 83.4 P 0
64.7 P 100
100 0 P 0

risk for liver fluke infection are January, February, September and October; the medium risk months are March, April, November and December; and the months of lowest risk of fasciolosis are May–August.
Generally speaking, treatments appeared to be given largely to combat acute and sub-acute phases of infection, with less empha- sis placed on chronic infections (Fig. 2B–E). When the patterns are examined, treatment timing generally appeared to be moving ear- lier in the year and the marked decrease in December treatments

Note: P = positive by coproantigen ELISA on day 21 post-treatment.

Table 4
Coproantigen reduction test (CRT) values of TCBZ treatment on Farms A–E using the constructed datasets and described formulae (2011 field resistance survey data).
Farm A Farm B Farm C Farm D Farm E
has split the “treatment window” of August to February seen in 2005 (Fig. 2B and D) into two smaller windows of July to November and January to February, as seen in 2011 (Fig. 2C and E).

3.3.3.Treatment frequency The percentage of flock owners giving no treatments for fluke infections in ewes grazing exclusively lowland areas

n-Pre 18
n-Post 17
Dataset 1 CRT1 0.0
CRT2 0.0
Dataset 2 CRT1 0.0
CRT2 0.0
Dataset 3 CRT1 0.0
CRT2 0.0
remained roughly constant between survey points (<17.0%, Fig. 3A). The percentages of flock owners giving 1 or 3 treatments per year increased (by 7.9% and 1.8%, respectively), while the percentages of flock owners treating 2 or 4 times per year fell between 2005 and 2011(by 7.9% and 2.1%, respectively). The percentage of flock owners giving no treatments for fluke infections in ewes grazing exclusively upland areas increased by Note: CRT1 = method as described by Kohapakdee et al. (1995); CRT2 = method as described by Cabaret and Berrag (2004); n-Pre = number of animals included in pre-treatment mean calculations; n-Post = number of animals included in post- treatment mean calculations. (Brockwell et al., 2014). There was good agreement between FECRT (Table 1) and CRT data (Table 4), in that resistance was found across all 5 holdings. In general, FECRT2 again indicated a greater level of resistance in the holdings than FECRT1. 3.3.Analysis of the questionnaire survey results The analysis is based on 252 returns from the 2011 survey and 81 returns from the 2005 survey. 3.3.1.Anthelmintic use by active ingredient 18.7% (from 0%) between 2005 and 2011 (Fig. 3B). Small increases in frequency were also observed for 1 treatment and 4 treatments per year (of 2.5% and 1.2%, respectively), while treating 2 or 3 times per year was seen less commonly in the results of the second ques- tionnaire (7.9% and 14.5% less, respectively). In flocks grazed on both lowland and upland pastures, there was a small increase (of 4.3%) in the percentage of flock owners who gave no treatments per year (Fig. 3C). Small increases in frequency were also seen for 2, 3 and 4 treatments per year (of 2.8%, 4.3% and 8.7%, respectively), while a lower percentage (20.1% less) of flock owners treated their ewes once per year between the two time-points. The data can be viewed in a different way, by considering the “intensity” of treatment. This parameter was calculated by multi- plying the percentage by the number of treatments: for example, 41.7% × 2 = 83.4 for treatment of ewes in lowland flocks in 2005. Fig. 1. Anthelmintic use, by active ingredient, over the periods 1999–2004 (A) and 2008–2011 (B). Alb = albendazole, TCBZ = triclabendazole, TCBZ* = triclabendazole plus broad spectrum anthelmintic, CLOS = closantel, CLOS* = closantel plus broad spectrum anthelmintic, NIT = nitroxynil, OXY* = oxyclozanide plus levamisole. Adding the values together showed that the intensity of treatment did not change much between 2005 and 2011 for ewes in low- land flocks (183.4 and 172.5, respectively), although there was a decrease for ewes in upland flocks (from 248.1 to 196.1). For flocks grazed on both lowland and upland areas, there was an increase from 164.0 to 197.2 between 2005 and 2011. Lambs. Generally speaking, lambs grazed exclusively in lowland areas (Fig. 4A), exclusively in upland areas (Fig. 4B), or in both lowland and upland pastures (Fig. 4C) were treated for fluke infection less routinely than their adult counterparts. In lowland areas, there was a lower percentage (6.0% less) of flock owners who gave no treatments per year between survey points (Fig. 4B). Increased percentages of flock owners said that either 1, 3 or 4 treatments were given to lambs per year (15.6%, 1.5% and 1.5%, respectively), while decreased numbers of owners gave 2 treatments per year (12.7% less). In upland areas, the percentage of flock owners that gave no treatments per year was similar at the two survey points (Fig. 4C). A (12.0%) greater percentage of flock owners treated lambs once per year in 2011 than in 2005, while the percentage of owners treating 2, 3 or 4 times per year fell by 3.2%, 2.7% and 4.0%, respectively. For animals that were grazed on both lowland and upland areas, there was an increase (of 5.1%) in the percentage of flock owners who gave no treatments per year. There were smaller increases in those giving 3 or 4 treatments per year (1.4% and 1.6%, respectively). A decrease was seen in the percentage of flock owners giving 1 or 2 treatments per year (5.5% and 2.6%, respectively) (Fig. 4C). For lambs, the intensity of treatment was essentially unchanged between 2005 and 2011 for lowland flocks (66.7 and 67.4) and both lowland and upland flocks (78.0 and 77.9), but there was a decrease for lambs in upland flocks (from 121.4 to 102.9). 3.3.4.Product rotation Flock owners were asked to state the frequency with which products were rotated (considering active ingredients, as distinct from product name). Responses to the 2005 question- naire suggested that no rotation between anthelmintic groups was practised by 54.3%, 7.1% and 11.2% of lowland, upland and both lowland and upland flock owners, respectively; that the same active ingredient would be given for a period of greater than one calen- dar year by 37.1%, 17.9% and 22.2% of lowland, upland and both lowland and upland flock owners, respectively; that only 4.3% of lowland flock owners would rotate anthelmintic class every year; and, lastly, that changing the anthelmintic used with each succes- sive treatment was practised by 4.3%, 75.0% and 66.7%, of lowland, upland and both lowland and upland flock owners, respectively. Fig. 2. Timing of anthelmintic treatments for fasciolosis by calendar year. Data is shown as the percentages (%) of owners who indicate that they treat their animals in each month. (A) Seasonality of liver fluke; (B) treatment of ewes 1999–2004; (C) treatment of ewes 2008–2011; (D) Treatment of lambs 1999–2004; (E) treatment of lambs 2008–2011. Fig. 3. Frequency of treatments given to ewes for F. hepatica infection. A = Lowland; B = Upland; C = Both. Fig. 4. Frequency of treatments given to lambs for F. hepatica infection. A = Lowland; B = Upland; C = Both Analysis of the responses to the 2011 question- naire indicated alterations in the product rotation practices of flock owners, namely, that no rotation between anthelmintic groups was practised by 42.1%, 32.1% and 44.9% of lowland, upland and both lowland and upland flock owners, respectively; that the same active ingredient would be given for a period of greater than one calendar year by 6.8%, 3.8% and 2.0% by lowland, upland and both lowland and upland flock owners, respectively; that rota- tion between anthelmintic groups each calendar year was now a practice employed by 29.5%, 28.3% and 34.7%, of lowland, upland and both lowland and upland flock owners respectively; and, lastly, that changing the anthelmintic used with each successive treat- ment was practised by 20.5%, 35.8% and 18.4%, of lowland, upland and both lowland and upland flock owners, respectively. The data can be evaluated in a different way, by considering the farmers’ “interest in drug rotation”. This parameter was calcu- lated by multiplying the percentage for using the same product for more than 1 year by 1, for rotating each year by 2 and for rotating with each successive treatment by 3. For example, 4.3% × 2 = 8.6 for rotating drugs each year by lowland flock owners in 2005. These values for rotation intensity were then combined to give an esti- mate of the overall interest in rotating drugs. On this basis, there was an increase in interest by lowland flock owners (from 58.6 to 127.3) between 2005 and 2011 and there was more intense rotation on these farms; in contrast, there was a decrease in the percentage for no rotation (from 54.3% to 42.1%). For upland flock owners, the percentage for no rotation increased from 7.1% to 32.1% between 2005 and 2011, but there was a decrease in interest in rotation (from 242.9 to 167.8). For owners of both lowland and upland flocks, the percentage for no rotation rose from 11.2% to 44.9% between 2005 and 2011 and there was a decline in interest for rotation (from 222.3 to 126.6). A summary of the changes in rotation practices between the 2 surveys is presented in Table 5. 3.3.5.Quarantine In the 2005 survey, quarantine treatments (that is, treatment of newly or re-introduced animals) for liver fluke infection were prac- tised by 31.0%, 25.9% and 6.9% of lowland, upland and both lowland and upland flock owners, respectively. By 2011, the percentages had dropped to 15.2% and 13.9% for lowland and upland flock owners, respectively, while the percentage remained the same for both lowland and upland flock owners. 3.3.6.Dosing regime When flock owners were asked which products (in their opinion) no longer worked as well as they once did, respon- dents in the 2005 survey indicated that TCBZ (3 lowland, 6 upland), TCBZ plus levamisole (LV) (1 lowland), CLOS (1 lowland, 1 upland), mebendazole (MEB) plus CLOS (1 lowland), NIT (1 lowland) and LV Table 5 General changes in the product rotation strategies of flock owners between survey time-points. Product rotation Lowland Upland Both No rotation Longer than one year Annual Successive treatments plus OXY (1 lowland) were less effective than they had been pre- viously. It should be emphasised that this opinion represents the farmers’ perception, which may not have been supported by any testing having been carried out. Two respondents who held both upland and lowland flocks indicated that certain products were no longer as effective as they were, but failed to indicate which. Also in this survey, flock owners indicated 8 instances of laboratory-confirmed flukicide resistance, but the identity of the diagnostic laboratory and the method of diagnosis were not queried in the questionnaire, so cannot be corroborated. Such ‘diagnoses’ of flukicide-resistant populations were more common in upland flocks [TCBZ (4), CLOS (1), OXY plus LV (1)], than lowland flocks [TCBZ (1), MEB plus CLOS (1)]. In the 2011 survey, 19 instances of laboratory- confirmed flukicide resistance were noted but, as before, the method and identity of the laboratory were not asked in the ques- tionnaire. In all cases, TCBZ was identified as the active ingredient against which fluke resistance was suspected. Modification to the (then) current parasite control strategies were identified in the 2011 survey and deemed to be necessary by 58%, 40% and 52% of lowland, upland and both lowland and upland flock owners, respectively. 3.4.Comparison between the number of cases of acute fluke recorded across the VIDA diagnostic network (mainland UK) and those recorded at the VSD stormont and Omagh centres (NI) By direct comparison of the total number of cases per annum of acute fluke (as revealed by post-mortem examination of carcases) in the AFBI and VIDA databases, it can be noted that during the very high risk years (2002 and 2012), the two centres in NI (AFBI Stormont and AFBI Omagh) together diagnosed a comparable total number of cases to the combined total number of cases diagnosed by the 23 centres across mainland UK (Fig. 5). This may be indica- tive of a greater prevalence of F. hepatica infections in sheep flocks across NI than in their GB counterparts. 4.Discussion 4.1.General discussion The results of the 2011 TCBZ resistance survey have been dis- cussed in depth by Hanna et al. (2015), so will not be repeated here. Briefly, resistance was identified on 5 farms, all located in lowland areas; this conclusion was based on the application of 3 diagnostic techniques (FECRT, CRT and fluke histology). When the results were compared with those of the 2008 survey, it was revealed that the level of resistance had increased considerably in the 3-year period between surveys. The following discussion will focus on: the relative merits of different tests for the diag- nosis of TCBZ resistance; the possible impact of different formulae on the calculation of reductions in FECs and coproantigen levels; a comparison between reports of TCBZ resistance in NI and else- where in the world (specifically the methods used in the testing for resistance); and a discussion of control and management prac- tices carried out by farmers in the Province and how they changed between the Questionnaire surveys conducted in 2005 and 2011. 4.2.FEC/FECRT and coproantigen ELISA/CRT as diagnostic tools For the detection of infections of F. hepatica, FEC by sedimen- tation is known to have a sensitivity of 30-60% and a specificity of 100% (Daniel et al., 2012). A number of biological drawbacks to this technique are well known: they include no eggs shed for up to 12 weeks post initial infection, followed by intermittent shedding thereafter; difficulty in detecting eggs in dilute faecal samples; and over-dispersion of infection within the flock/herd. Another compli- cation is the periodic release of eggs stored in the gall bladder, even in the absence of an active infection (Flanagan et al., 2011a,b). A disadvantage in the FECRT methodology itself for the diag- nosis of drug resistance in fluke infections is the requirement of a 21-day window between sample points, which presents a long interval to leave non-thriving animals untreated. Similarly, on the receipt of samples, specialist skills (including microscopy and egg identification) are necessary. Lastly, in analysing results, differ- ent methods of calculating FECR (based on different formulae and inclusion/exclusion of certain data points) may lead to a different classification of a farm’s resistance status. By way of example, Farm A in 2011 (Table 1) would no longer be likely to see therapeutic ben- efit of treatment according to the results of FECRT2, although the farmer may still see clinical improvement if the level of resistance was as recorded for FECRT1. Consequently, in the absence of formalised guidelines, the FECRT alone is not suitable for the determination of resistance. The ELISA platform is ubiquitous in diagnostic laboratories and, as an alternative diagnostic test, the CRT offers a number of advan- tages. The advantages are: a good association with parasite burden (Mezo et al., 2004; Brockwell et al., 2013); a reduction in the time interval between sample collection dates, as coproantigen levels reach an optical density of zero within 14 days following success- ful treatment (Brockwell et al., 2014); the stability of coproantigens on freezing, which allows samples to be batched, delivering a more cost-effective analysis (Flanagan et al., 2011a,b); and high speci- ficity (Gordon et al., 2012a,b; Kajugu et al., 2012). Nested PCR has been shown to be more sensitive than both FECRT and CRT in terms of the early detection of fluke infection (Robles-Pérez et al., 2013). However, the necessity for further eval- uation of the PCR techniques has been advised (Brockwell et al., 2014). Until such time as that has been achieved, coproantigen ELISA remains the simplest quantitative technique in the routine diagnosis of fasciolosis in livestock and it provides a robust and accessible test to use in conjunction with the FECRT. 4.3.Reports of resistance from around the world By way of comparison, the methods used to declare the pres- ence of anthelmintic resistance in F. hepatica populations in the last 9 years are presented in Table 6. Most commonly (11/15), reports focussed on a single flock or two flocks, and so widespread preva- lence studies are not the norm. In reports where multiple flocks Fig. 5. The annual prevalence of acute fasciolosis as diagnosed at post-mortem in AFBI and VIDA diagnostic networks. Table 6 The methods used to diagnose the presence of anthelmintic resistance in Fasciola hepatica populations in sheep-producing areas around the world. Country Farms F CRT CET FH GGT/GOT Reference Argentina 1 X Olaechea et al. (2011) – X X Sanabria et al. (2013) Peru 1 X X X Ortiz et al. (2013) 1 X Gomez-Puerta et al. (2012) Australia 15 X X Elliott et al. (2015) 7 X X Brockwell et al. (2014) 18 X Stevenson et al. (2002) Netherlands 1 X X Borgsteede et al. (2008) Spain 1 X Alvarez-Sanchez et al. (2006) 1 X Martínez-Valladares et al. (2010) 1 X Martinez-Valladares et al. (2014) Sweden 1 X Novobilsky et al. (2012) England/Wales 25 X Daniel et al. (2012) Scotland 2 X X Gordon et al. (2012a) 2 X Gordon et al. (2012b) N. Ireland 13 X X X Hanna et al. (2015) 12 X X Flanagan et al. (2011a,b) Ireland 1 X Mooney et al. (2009) Note: F = faecal egg count reduction test; CRT = coproantigen reduction test; CET = controlled efficacy test; FH = fluke histology; GGT/GOT = gamma-glutamyl transpeptidase (GGT) and glutamic-oxaloacetic transaminase (GOT); – = research flock. were examined, multiple methods for resistance determination were not always used: therefore, the reports for NI represent one of the most in-depth analyses of the prevalence of flukicide resis- tance thus far conducted. The FECRT was a linchpin of diagnosis in all but one report, although the analytical methods used varied by country (presence/absence of control groups, group size, sta- tistical calculations), complicating any possibility of international comparisons. 4.4.Fluke control practices and sheep management strategies 4.4.1.Product selection Over the period 1999–2004, TCBZ accounted for over 65% of all products used (Fig. 1A), but in 2011 it accounted for only 25% (Fig. 1B), with the value falling steadily in the intervening years. In the absence of widespread confirmed anthelmintic resistance and industry publications recommending the shift away from TCBZ, this change seems likely to be a response to anecdotal accounts of treat- ment failure amongst the flock owners of NI. This represents the first empirical evidence of a shift away from TCBZ use by the farm- ing community in the absence of widespread resistance testing and confirmed diagnosis. In 1999, CLOS use accounted for 12% of all treatments given to control fasciolosis (4% as single active and 8% in combination with broad spectrum wormer). In 2011, this percentage had increased to 52.5% (35.5% as single active and 19% in combination, respectively). Approximately 12% of all treatments given were NIT-based in the surveyed periods, suggesting that oral drench formulations were preferable to the majority of flock owners over injectable formula- tions. This is similar to previous findings for nematode control in NI (McMahon et al., 2013a,b). NIT is known to be difficult to inject and causes tissue staining. The use of OXY rose sharply between 2010 and 2011, which may be a reflection of increased awareness of rumen fluke infection in the farming community. In the survey carried out by Morgan et al. (2012), which covered Great Britain and Ireland, NIT was the drug most commonly used, followed by TCBZ (mainly in combination with levamisole), CLOS, ALB and OXY. Six per cent of farmers reported the use of inappropriate drugs (mainly macrocyclic lactones); similar low levels of unsuitable drug use have been described in other surveys (3%, Bloemhoff et al., 2014; 2%, Selemetas et al., 2015). Overall, then, the decrease in TCBZ use in NI has been matched largely by the increase in use of CLOS. 4.4.2.Treatment timing As stated previously, May-August represents the lowest period of risk of fasciolosis in sheep flocks in temperate regions; periods of medium risk are considered to be March–April and November–December; and the months of highest risk are January–February and September–October. Between the question- naire surveys of 2005 and 2011, the proportion of flock owners treating at low risk time approximately doubled (9% in 2005 and 19% in 2011, for ewes; 13% in 2005 and 32% in 2011, for lambs). There was a corresponding decrease in those treating at times of medium risk (41% in 2005 and 29% in 2011, for ewes; 40% in 2005 and 27% in 2011, for lambs), while those treating at high risk times remained roughly constant (50% in 2005 and 52% in 2011, for ewes; 47% in 2005 and 41% in 2011, for lambs). Sargison (2011) has described the changes in severity, season- ality and local prevalence of fasciolosis in the UK over the last decade (i.e. 2000–2010). These changes have been linked to chang- ing climate, localised flooding and increased rainfall, ultimately compromising effective management of the disease and leading to production losses. These changes have been forecast to continue well into the future (Fairweather, 2011a,b; Fox et al., 2011). While the effects of climate change on fluke prevalence were beyond the scope of this investigation, their consequences may be behind the observed shift in treating earlier in the year. Alternatively, the change in treatment timing may be linked to the shift in product selection towards CLOS, with the emphasis being placed on adult fluke control earlier in the season, in order to prevent pasture con- tamination, that is, a change in emphasis of control strategy to pasture protection. 4.4.3.Treatment frequency No real changes were seen, the basic pattern of treatment fre- quency remaining the same over time. Two treatments per year for ewes was the most practised option (by ∼1/3 of flock owners). Approximately 30% of flock owners gave 3 or more treatments, while the proportion giving no treatments was small, but rose between 2005 and 2011, particularly in upland areas. For lambs, the basic pattern also remained much the same, the only change involving the use of 1 or 2 treatments. There was a decrease in the number of 2 treatments, particularly in lowland areas, counterbalanced by an increase in the use of a single treat- ment, in both lowland and upland areas. In 2011, ∼ a quarter of flock owners gave one treatment. More than half of flock owners gave no treatments at all and the number of 3 or more treatments was low (<10%). Looking at the intensity of treatment between 2005 and 2011 in different areas for ewes, there was little change in the propor- tion of lowland flock owners treating as opposed to not treating and no real change in the intensity of treatment. On upland farms, there was an increase in the percentage of farmers not treating and a decrease in treatment intensity. On both lowland and upland farms, there was a slight increase in the percentage of flock owners not treating, but this was balanced by a slight increase in intensity of treatment. Decisions to treat ewes and how often may depend on the cost benefits of doing so, bearing in mind the price of sheep meat. Other factors include the farmer’s perception of the fluke bur- den and of the level of TCBZ resistance—in the latter case, whether it is worth treating at all. For lambs, there was a slight reduction in the percentage of low- land farmers not treating between the two time-points, but the intensity of treatment was the same. In upland areas, there was a very slight decline in the proportion of flock owners not treat- ing, although there was a distinct drop in treatment intensity. On both lowland and upland farms, the percentage of flock owners not treating showed a slight increase, although the intensity of treatment remained the same. Overall, there was not much change in treatment frequency, with approximately half of flock owners not treating lambs at all. More treatments were given to lambs in upland areas, presumably because of the longer fattening period, but this would be offset by lower fluke burdens in these areas. Treat- ment of lambs also has to take into account the withdrawal period for drugs (e.g. 56 days for TCBZ). Treatment frequency showed significant regional variation in the survey conducted by Morgan et al. (2012), with lambs in NI receiving more treatments (>6) than the general annual average of 3.6. Overall, approximately two-thirds of farmers treated sheep 3 or 4 times per year 43% and 22%, respectively, with 17% treating two times per year and 18% once (Morgan et al., 2012). Such high figures were not encountered in the present NI survey. The frequency and timing of treatment is important for maintaining effective parasite control. In Ireland, for example, dairy cattle are typically treated just once per year and this is not an optimal strategy (Bloemhoff et al., 2014; Selemetas et al., 2015).

4.4.4.Product rotation
In lowland areas, flock owners rotated more in 2011 than 2005 and the intensity of rotation was greater, which represents a sub- stantial increase in interest in rotation in this group of farmers. Probably, this is because they have a more severe fluke problem in their flocks (and potentially more of a drug resistance problem as well), so they are more aware of the need to rotate drug classes. Faced with the decision to rotate or not, they are more likely to choose to do so. In contrast, in upland areas and both lowland and upland areas, fewer farmers practised drug rotation in 2011 than 2005 and there was a significant decrease in the interest to rotate. Fasciolosis is less of a problem in these areas (as, in all likelihood, is resistance) and there is probably lower drug use overall. Therefore, the farmers are not so appreciative of the need to rotate.
A lack of rotation was highlighted in the survey conducted by Rojo-Vazquez and Hosking (2013), with 42% of farmers not carry- ing this out, although it was not clear whether this figure applied specifically to fluke or to helminths generally. Even amongst those farmers that rotated annually (36%), 25% of the group used the same drug class (the benzimidazoles), simply switching to a different product.

4.4.5.Quarantine treatment
Overall, only ∼ one-fifth of flock owners gave quarantine treat- ments in 2005 and this proportion had dropped by approximately half in 2011. The decreases were seen in lowland and upland areas; in flocks grazed on both lowland and upland areas, the percentage remained unchanged.
The fact that the overwhelming majority of farmers in NI do not carry out routine quarantine treatments of new stock is a con- cern, as it may lead to the introduction of fluke (and possibly drug-resistant parasites) into the flock. The lack of an adequate quarantine strategy has been highlighted in other fluke surveys (Mezo et al., 2008; Morgan et al., 2012; Rojo-Vazquez and Hosking, 2013).


Farmers appear to be responding to the potential impact of cli- mate change on the seasonality/epidemiology of fluke in that the timing of treatment moved earlier in the year. In other respects, a number of shortcomings were identified in fluke control pro- grammes employed by NI flock owners, namely:
A significant proportion of flock owners giving a high number of treatments (three or more) per calendar year (Figs. 3 and 4); reduced use of effective juvenile fluke control (by TCBZ) in the absence of empirical evidence of TCBZ resistance (Fig. 2); and reduced levels of quarantine treatment and quarantine separation between 2005 and 2011, increasing the possibility of importing drug-resistant parasites. These shortcomings have highlighted the need for more robust advice to be given to farmers so that they can implement more effective fluke control programmes. The infor- mation in this paper may help to better inform veterinarians and other advisors on what farmers are actually doing and areas where

improvements are needed, in order for them to (be able to) give appropriate advice on the design, development and implementa- tion of management control programmes.
The use of faecal egg counts (pre-treatment) in local diagnos- tic laboratories provides a simple means of determining parasite burden, and whether there is a need to treat at all. For exam- ple, in the survey conducted by Morgan et al. (2012), two-thirds of farmers said they treated for fluke, even though only one-fifth had previous problems with the parasite. Moreover, in the sur- vey carried out in Spain, farmers reported that, when cattle were treated, all animals in the herd were treated, without any prior diagnosis (Mezo et al., 2008). So, accurate diagnosis of infection may obviate a number of treatments in the calendar year. Like- wise, use of faecal egg counts following TCBZ treatment may restore confidence in this anthelmintic in the absence of formal resis- tance testing and diagnosis. Few farmers test for anthelmintic efficacy and so do not know the resistance status of the farm (Morgan et al., 2012; Rojo-Vazquez and Hosking, 2013). These practices, as well as increased awareness of the importance of quar- antine, present an obvious first step in encouraging effective fluke control.

5.1.Added note

The results of a recent survey have highlighted the lack of knowl- edge among sheep farmers about liver fluke, the stage (in the life cycle) targeted by different drugs and the right timings for their use (Anon, 2015). The responses also indicated the need for vets and suitably qualified persons (SQPs) to be familiar with cur- rent testing options and advice on control, so that they can pass on this information to farmers in clear, practical messages at the appropriate time of year. An example of a potential management strategy for disease control has been put forward by Hanna et al. (2015). It is perhaps appropriate here to re-iterate the recommen- dation made: “With the present circumstances of high penetration of TCBZ resistance throughout flocks in NI, provision of advice regarding chemotherapeutically based management of fasciolosis is difficult. In the current absence of any other licensed prod- uct effective against acute fasciolosis, it is considered advisable to continue to use TCBZ in the autumn, in the hope that at least a proportion of the fluke population remains susceptible, and that the burden can be sufficiently reduced to save some animals in an acute outbreak. Concurrent use of a flukicide active against late immature and adult fluke can ensure removal of infection acquired earlier in the season, and so reduce the overall fluke burden. How- ever, the emphasis in control schemes should be shifted to use of an adulticide such as closantel or nitroxynil in the winter and spring months, to reduce potential pasture contamination for the next season” (Hanna et al., 2015). The necessity for correct diag- nosis is a point that has been raised previously by Fairweather (2011a,b).


We wish to thank John Kenny, Sean Ellison and Velma Beattie (AFBI), who helped to compile the Questionnaire in 2005. We also wish to thank Sophie Tynan, Maria Guelbenzu, Robert Walker and David McCoubrey (AFBI), together with Don Morrow and Steven Johnston (CAFRE) and William Sherrard (Pfizer), who helped to disseminate the Questionnaire in 2011.


Abbott, K.A., Taylor, M.A., Stubbings, L.A., 2012. Sustainable control of parasites in sheep (SCOPS). A technical manual for veterinary surgeons and advisors, 4rd edition.

AFBI, 2012. All-island disease surveillance report.
Alvarez-Sanchez, M.A., Perez-Garcia, J., Cruz-Rojo, M.A., Rojo-Vazquez, F.A., 2006. Anthelmintic resistance in trichostrongylid nematodes of sheep farms in Northwest Spain. Parasitol. Res. 99, 78–83.
Anon, 2015. Raising awareness of sustainable management of liver fluke in sheep. Vet. Rec. 177, 246.
Bloemhoff, Y., Danaher, M., Forbes, A., Morgan, E., Mulcahy, G., Power, C., Sayers, R., 2014. Parasite control practices on pasture-based dairy farms in the Republic
of Ireland. Vet. Parasitol. 204, 352–363.
Borgsteede, F.H.M., Taylor, S.M., Gaasenbeek, C.P.H., Couper, A., Cromie, L., 2008. The efficacy of an ivermectin/closantel injection against experimentally induced infections and field infections with gastrointestinal nematodes and liver fluke in cattle. Vet. Parasitol. 155, 235–241.
Brockwell, Y.M., Spithill, T.W., Anderson, G.R., Grillo, V., Sangster, N.C., 2013. Comparative kinetics of serological and copro-antigen ELISA and faecal egg count in cattle experimentally infected with Fasciola hepatica and following treatment with triclabendazole. Vet. Parasitol. 196, 417–426.
Brockwell, Y.M., Elliott, T.P., Anderson, G.R., Stanton, R., Spithill, T.W., Sangster, N.C., 2014. Confirmation of Fasciola hepatica resistant to triclabendazole in naturally infected Australian beef and dairy cattle. Int. J. Parasitol. Drugs Drug Resist. 4, 48–54.
Cabaret, J., Berrag, B., 2004. Faecal egg count reduction test for assessing anthelmintic efficacy: average versus individually based estimations. Vet. Parasitol. 121, 105–113.
Coles, G.C., Jackson, F., Pomroy, W.E., Prichard, R.K., von Samson-Himmelstjerna, G., Silvestre, A., Taylor, M.A., Vercruysse, J., 2006. The detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 136, 167–185.
Daniel, R., Van Dijk, J., Jenkins, T., Akca, A., Mearns, R., Williams, D.J., 2012. Composite faecal egg count reduction test to detect resistance to triclabendazole in Fasciola hepatica. Vet. Rec. 171, 153.
Elliott, T.P., Kelley, J.M., Rawlin, G., Spithill, T.W., 2015. High prevalence of fasciolosis and evaluation of drug efficacy against Fasciola hepatica in dairy cattle in the Maffra and Bairnsdale districts of Gippsland, Victoria, Australia. Vet. Parasitol. 209, 117–124.
Fairweather, I., 2011a. Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy? Vet. Parasitol. 180, 133–143.
Fairweather, I., 2011b. Raising the bar on reporting ‘triclabendazole resistance’. Vet. Rec. 168, 514–515.
Flanagan, A.M., 2010. Ph.D. Thesis. In: Development of Diagnostic Tests for the Detection of Resistance to Triclabendazole in Fasciola hepatica. The Queen’s University of Belfast, Belfast.
Flanagan, A.M., Edgar, H.W.J., Forster, F., Gordon, A., Hanna, R.E.B., McCoy, M., Brennan, G.P., Fairweather, I., 2011a. Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica. Vet. Parasitol. 176, 34–42.
Flanagan, A., Edgar, H.W.J., Gordon, A., Hanna, R.E.B., Brennan, G.P., Fairweather, I., 2011b. 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. Vet. Parasitol. 176, 170–176.
Fox, N.J., White, P.C.L., McLean, C.J., Marion, G., Evans, A., Hutchings, M.R., 2011. Predicting impacts of climate change on Fasciola hepatica risk. PLoS One 6, e16126.
Gomez-Puerta, L., Gavidia, L., Lopez-Urbina, M., Garcia, H.H., Gonzalez, A.E., 2012. Short report: efficacy of a single oral dose of oxfendazole against Fasciola hepatica in naturally infected sheep. Am. J. Trop. Med. Hyg. 86, 486–488.
Gordon, D., Zadoks, R.N., Stevenson, H., Sargison, N.D., Skuce, P.J., 2012a. On farm evaluation of the coproantigen ELISA and coproantigen reduction test in Scottish sheep naturally infected with Fasciola hepatica. Vet. Parasitol. 187, 436–444.
Gordon, D., Zadoks, R., Skuce, P., Sargison, N., 2012b. Confirmation of triclabendazole resistance in liver fluke in the UK. Vet. Rec. 171, 159.
Hanna, R.E.B., McMahon, C., Ellison, S., Edgar, H.W., Gordon, A., Irwin, D., Barley, J.P., Malone, F.E., Brennan, G.P., Fairweather, I., 2015. Fasciola hepatica: a comparative survey of fluke resistance to triclabendazole, nitroxynil and closantel on selected upland and lowland sheep farms in Northern Ireland using faecal egg counting, coproantigen ELISA testing and fluke histology. Vet. Parasitol. 207, 34–43.
Kajugu, P.E., Hanna, R.E.B., Edgar, H.W.J., Forster, F.I., Malone, F.E., Brennan, G.P., Fairweather, I., 2012. Specificity of a coproantigen ELISA test for fasciolosis: lack of cross-reactivity with Paramphistomum cervi and Taenia hydatigena. Vet. Rec. 171, 502.
Kohapakdee, S., Pandey, V.S., Pralomkarn, W., Choldumrongkul, S., Ngampongsai, W., Lawpetchara, S., 1995. Anthelmintic resistance in goats in Southern Thailand. Vet. Rec. 137, 124–125.
McMahon, C., 2015. PhD Thesis. In: Anthelmintic Resistance in Parasites of Sheep
in Northern Ireland and the Strategic Control of Parasitic Diseases. The Queen’s University of Belfast, Belfast.
McMahon, C., Barley, J.P., Edgar, H.W.J., Ellison, S.E., Hanna, R.E.B., Malone, F.E., Brennan, G.P., Fairweather, I., 2013a. Anthelmintic resistance in Northern Ireland (II): variations in nematode control practices between lowland and upland sheep flocks. Vet. Parasitol. 192, 173–182.
McMahon, C., McCoy, M., Ellison, S.E., Barley, J.P., Edgar, H.W.J., Hanna, R.E.B., Malone, F.E., Brennan, G.P., Fairweather, I., 2013b. Anthelmintic resistance in Northern Ireland (III): uptake of SCOPS (sustainable control of parasites in sheep) recommendations by sheep farmers. Vet. Parasitol. 193, 179–184.

McMahon, C., Bartley, D.J., Edgar, H.W.J., Ellison, S.E., Barley, J.P., Malone, F.E., Hanna, R.E.B.H., Brennan, G.P., Fairweather, I., 2013c. Anthelmintic resistance in Northern Ireland (I): prevalence of resistance in ovine gastrointestinal nematodes, as determined through faecal egg count reduction testing. Vet. Parasitol. 195, 122–130.
Martínez-Valladares, M., Famularo, M.R., Férnandez-Pato, N., Castanón-Ordónez, L., Cordero-Pérez, C., Rojo-Vázquez, F.A., 2010. Efficacy of nitroxynil against Fasciola hepatica resistant to triclabendazole in a naturally infected sheep flock. Parasitol. Res. 107, 1205–1211.
Martinez-Valladares, M., Cordero-Perez, C., Rojo-Vasquez, F.A., 2014. Efficacy of an anthelmintic combination in sheep infected with Fasciola hepatica resistant to albendazole and clorsulon. Exp. Parasitol. 136, 59–62.
Mezo, M., González-Warleta, M., Carro, C., Ubeira, F.M., 2004. An ultra-sensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3). J. Parasitol. 90, 845–852.
Mezo, M., González-Warleta, M., Castro-Helmida, J.A., Ubeira, F.M., 2008. Evaluation of the flukicide treatment policy for dairy cattle in Galicia (NW Spain). Vet. Parasitol. 157, 235–243.
Mooney, L., Good, B., Hanrahan, J.P., Mulcahy, G., De Waal, T., 2009. The comparative efficacy of four anthelmintics against a natural acquired Fasciola hepatica infection in hill sheep flock in the west of Ireland. Vet. Parasitol. 164, 201–205.
Morgan, E.R., Hosking, B.C., Burston, S., Carder, K.M., Hyslop, A.C., Pritchard, L.J., Whitmarsh, A.K., Coles, G.C., 2012. A survey of helminth control practices on sheep farms in Great Britain and Ireland. Vet. J. 192, 390–397.

Novobilsky, A., Averpil, H.B., Hoglund, J., 2012. The field evaluation of albendazole and triclabendazole efficacy against Fasciola hepatica by coproantigen ELISA in naturally infected sheep. Vet. Parasitol. 190, 272–276.
Olaechea, F., Lovera, V., Larroza, M., Raffo, F., Cabrera, R., 2011. Resistance of Fasciola hepatica against triclabendazole in cattle in Patagonia(Argentina). Vet. Parasitol. 178, 364–366.
Ortiz, P., Scarcella, S., Cerna, C., Rosales, C., Cabrera, M., Guzmán, M., Lamenza, P., Solana, H., 2013. Resistance of Fasciola hepatica against triclabendazole in cattle in Cajamarca (Peru): a clinical trial and an in vivo efficacy test in sheep. Vet. Parasitol. 195, 118–121.
Robles-Pérez, D., Martínez-Pérez, J.M., Rojo-Vázquez, F.A., MartínezValladares, M., 2013. The diagnosis of fasciolosis in faeces of sheep by means of a PCR and its application in the detection of anthelmintic resistance in sheep flocks naturally infected. Vet. Parasitol. 197, 277–282.
Rojo-Vazquez, F.A., Hosking, B.C., 2013. A telephone survey of internal parasite control practices on sheep farms in Spain. Vet. Parasitol. 192, 166–172.
Sanabria, R., Ceballos, L., Moreno, L., Romero, J., Lanusse, C., Alvarez, L., 2013. Identification of a field isolate of Fasciola hepatica resistance to albendazole and susceptible to triclabendazole. Vet. Parasitol. 193, 105–110.
Sargison, N.D., 2011. Pharmaceutical control of endoparasite infections in sheep. Vet. Clin. N. Am. Food A 27, 139–156.
Selemetas, N., Phelan, P., O’Kiely, P., de Waal, T., 2015. The effects of farm management practices on liver fluke prevalence and the current internal parasite control measures employed on Irish dairy farms. Vet. Parasitol. 207, 228–240.
Stevenson, C.R., Mahoney, R.H., Fisara, P., Strehlau, G., Reichel, M.P., 2002. The efficacy of formulations of triclabendazole and ivermectin in combination against liver fluke (Fasciola hepatica) and gastro-intestinal nematodes in cattle and sheep and sucking lice species in cattle. Aust. Vet. J. 80, 698–701.