Liver fluke isolates: a question of provenance

Veterinary Parasitology 176 (2011) 1–8

Contents lists available at ScienceDirect

Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Review

Liver fluke isolates: a question of provenance I. Fairweather ∗ Parasite Therapeutics Research Group, School of Biological Sciences, Medical Biology Centre, The Queen’s University of Belfast, 97 Lisburn Road, Belfast, BT9 7BL, United Kingdom

a r t i c l e

i n f o

Article history: Received 3 June 2010 Received in revised form 3 December 2010 Accepted 9 December 2010 Keywords: Liver fluke Fasciola hepatica Published articles Fluke provenance

a b s t r a c t A survey of literature on experimental infections with the liver fluke, Fasciola hepatica published between 2005 and 2009 has revealed a general lack of information on where fluke material (i.e. metacercariae) was sourced from. Even less information was given on the drug status of the fluke isolate used, which is a particular concern for those studies that involved anthelmintics. In these two respects, information on the liver fluke lags far behind that for nematodes, where such information is given almost as a matter of course. Of additional concern is that, at times, information about the source and drug history of fluke isolates was incorrect. The overall aim of the review is to demonstrate why it is important to provide as much information as possible on what fluke material is being used. It also attempts to correct some of the errors in the literature and gather together what information is available about the provenance of those isolates that have been used in recent experimental studies. © 2010 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Descriptions of drug sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Variation between isolates with respect to their sensitivity to the same drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Variation in drug sensitivity at different stages of development within a single isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Verification of reports of drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological differences between isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Details of defined isolates used in recent studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. TCBZ-susceptible isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Cullompton isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2. Fairhurst isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3. Sunny Corner isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. TCBZ-resistant isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1. Sligo isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2. Oberon isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.3. Dutch isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.4. Leon isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Tel.: +44 28 90972298; fax: +44 28 90975877. E-mail address: [email protected] 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.12.011

2 2 2 2 2 3 3 4 4 4 4 5 5 5 5 5 6

2

I. Fairweather / Veterinary Parasitology 176 (2011) 1–8

7.

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction There has been a growing perception in recent years that little information on the provenance of fluke isolates is given in publications on Fasciola hepatica. Also, that the situation for fluke lags far behind that in studies on nematode parasites, where mention of the isolate(s) used is almost de rigueur and there is a chain of information providing detail on drug sensitivity. Publications dealing with research on F. hepatica should be aiming towards this model. To test whether the lack of information is a correct perception or not, survey was undertaken of publications on F. hepatica over the 5-year period between 2005 and 2009. Initially, the ISI Web of Science database was searched, using “Fasciola hepatica” as search term. Publications were selected that dealt with experimental infections involving the use of fluke metacercariae. The publications included studies on ruminant (sheep and cattle) and rodent (mice, rat and rabbit) hosts. One hundred and one papers were identified and classified according to whether they gave information on the source of the metacercariae or not and whether they indicated a particular isolate was used and what its drug sensitivity was. In this time period, papers published by our group at Queens have included such information on the different isolates we have used. Removing the Queens papers (27) from the survey, the source of material was given in 55 of the remaining 74 papers; no information on source was given in 13 of the papers and in only 6 papers were details provided on the isolate used and its source. While drug sensitivity may not be a major concern for studies on basic fluke biology or vaccine trials, for example, 13 of the studies (representing 17.5% of the total) involved anthelmintics, where drug susceptibility would be an issue. Of these 13 papers, only 5 gave details of both isolate and its source. The purpose of this communication is an attempt to show why it is important to know the background of the fluke isolate that is being studied. 2. Sources of material A number (4) of the sources were commercial suppliers (see Table 1). Any material provided by the VLA since 1998 is likely to belong to the Cullompton isolate. In 6 other papers, the source of material was said to be Compton Paddock Laboratories, Newbury, Berkshire, UK. That is incorrect. The laboratory ceased to produce metacercariae in ∼2002 and the equipment was passed on to Dr. Coles in Bristol. So, any material purported to come from Compton Paddock most likely was sub-contracted to either Bristol or VLA. Moreover, the VLA has now stopped producing metacercariae as well. In other studies, the metacercariae used were produced in the researchers’ own laboratory and no detail was given on the treatment history or drug sensitivity of the isolate. There appear to be a number of laboratories throughout

6 6 6

Table 1 Sources of metacercarial material quoted in publications in the 2005–2009 publication survey. 1. Commercial suppliers Baldwin Aquatics Inc., Monmouth, Oregon, USA (6 papers). Veterinary Laboratories Agency (VLA), Weybridge, Surrey, UK (5 papers). Dr. Gerald Coles, Department of Clinical Veterinary Science, University of Bristol, UK (6 papers). Elizabeth Macarthur Agricultural Institute, New South Wales Agriculture, Merangle, New South Wales, Australia (5 papers). 2. Research labs with the capacity to produce metacercariae Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, Sofia, Bulgaria. South Valley University, Qena, Egypt. University of Limoges, Limoges, France. INRA, Nouzilly, France. Universidad Nacional Autónoma de Mexico, Mexico City, Mexico. ´ ´ University of Gdansk, Gdansk, Poland. Pomeranian Medical University, Szczecin, Poland. W. Stefanski Institute of Parasitology, Warsaw, Poland. Instituto Nacional de Saúde Dr. Ricardo Jorge, Porto, Portugal. Universidad de Valencia, Valencia, Spain. Faculty of Veterinary Medicine, Lugo, Spain. Universidad de la República, Montevideo, Uruguay. 3. More recent commercial suppliers Ridgeway Research Ltd., St. Briavels, Gloucestershire, UK.

the world that have the capacity to produce metacercariae on occasion or perhaps to obtain metacercariae from field-collected snails (see Table 1). There may be other laboratories as well as these twelve, but this selection was gleaned from the 2005 to 2009 survey. 3. Descriptions of drug sensitivity 3.1. Variation between isolates with respect to their sensitivity to the same drug For example, in relation to nitroxynil action (and in terms of the morphological responses observed in vitro) the following isolates have been ranked in order of their sensitivity: Cullompton > Sligo > Oberon > Fairhurst (McKinstry et al., 2007, 2009). This hierarchy is different from their response to triclabendazole (TCBZ) treatment: Cullompton > Fairhurst > Oberon > Sligo (Walker et al., 2004; also, Robinson et al., 2002; Halferty et al., 2009a; Toner et al., 2009, 2010a,b,c, in press). So, it seems that the Sligo isolate is particularly susceptible to nitroxynil, while the Fairhurst isolate is more refractory. However, as indicated below, this susceptibility applies only to adult flukes. 3.2. Variation in drug sensitivity at different stages of development within a single isolate Information relating to the susceptibility of different fluke isolates to TCBZ in sheep is presented in Table 2. For some isolates, the sensitivity to a particular drug remains unchanged throughout its development: this is true of

I. Fairweather / Veterinary Parasitology 176 (2011) 1–8

3

Table 2 Efficacy of TCBZ against different isolates of Fasciola hepatica in sheep. Fluke isolate

TCBZ efficacy at specified times post-infectiona

Reference

Cullompton

100% at 4 wpi 98.12–100% at 12 wpi

Fairhurst

78.4% at 2 wpi 86.1% at 4 wpi 98.5% at 6 wpi 100% at 12 wpi 78.3% at 2 wpi 77.0% at 4 wpi 98.4% at 6 wpi 100% .v. adult 0% at 3 dpi 0% at 4 wpi 0% at 12 wpi 0% at 2 wpi 5% at 4 wpi 0% at 12 wpi

McCoy et al. (2005) McConville et al. (2009a) Flanagan et al. (in press) Walker et al. (2004) Flanagan et al. (in press)

Sunny Corner

Sligo

Oberon

Boray (personal communication)

McConville et al. (2009a) Coles and Stafford (2001) Walker et al. (2004) Flanagan et al. (in press)

wpi: weeks post-infection. a dpi: days post-infection.

the TCBZ-resistant Oberon and Sligo isolates and of the TCBZ-susceptible Cullompton isolate in response to TCBZ treatment. In contrast, the Sligo isolate has been shown to be susceptible to nitroxynil at the adult stage (Coles and Stafford, 2001), but this does not extend to the juvenile (4-week) stage (efficacy of 18.9%: McCoy et al., 2005). Different trends have been established for the Fairhurst and Sunny Corner isolates. For the Fairhurst isolate, the efficacy of TCBZ increases with the age of the fluke infection (Table 2). In cattle, the efficacy of TCBZ is 89% at 2 wpi and 100% at 12 wpi (Walker et al., 2004). In one study (Hutchinson et al., 2009), the Sunny Corner isolate of F. hepatica was described as being “fully susceptible” to TCBZ, with reference to the abstract of Boray et al. (1997) to support this assertion. However, no mention of the isolate was made in that abstract. In another paper, the isolate is described as “a known triclabendazole reference strain”, without any explanation of what this means (Sargent et al., 2009). The isolate is fully susceptible at the adult stage, but not at the immature (2- and 4-week) stages (see Table 2 and Section 6.1.3). The results of the studies illustrate the point that field isolates display different drug sensitivities – and at different stages in their development – and that this could be important in interpreting the results of drug studies and when designing drug control strategies. 4. Verification of reports of drug resistance Reports of resistance to fasciolicides (particularly TCBZ) appear in Journals from time-to-time: e.g. Anon (2006, 2008a,b,c, 2009) and Mooney et al. (2009). However, they do not necessarily represent genuine cases of resistance at all. Other factors can account for reduced efficacy: incorrect (under-) dosing, product failure, reduced metabolism as a result of liver damage, inadequate and incorrect diagnostic tests, even variable quality of drug formulations. Therefore, the reports should be considered to be cases of treatment failure until they can be verified by strict, controlled experimental efficacy studies. This has been done for the isolates

described below. Often, the cases of potential resistance turn out not to be so at all and this can give a false impression of the extent of drug resistance and the efficacy of a drug such as TCBZ. In turn, this could lead farmers to use alternative, less active and even possibly inappropriate drugs. This would result in inadequate control of fasciolosis, leading to its spread and the greater risk of development of drug resistance. This is an important point, because once mis-information has been published it tends to be repeated and become established: this should be avoided if at all possible. 5. Biological differences between isolates Two studies have shown interesting differences between isolates. In the first (McConville et al., 2009a), carried out in sheep, the Cullompton and Sligo isolates were compared. The Sligo flukes reached the bile ducts 1 week earlier than the Cullompton flukes (7 wpi as against 8 wpi) and produced eggs 2 weeks earlier (60 days post-infection (dpi) as against 75 dpi). This would be an advantage in the field, but is offset by the fact that the Sligo flukes are smaller than Cullompton flukes, produce relatively fewer eggs and their metacercariae are less infective to sheep, with only 24% of the dose reaching maturity; by comparison, 57% of Cullompton metacercariae reached maturity (McConville et al., 2009a). The Cullompton isolate is unusual in that it is aspermic and triploid (Fletcher et al., 2004; Hanna et al., 2008), yet produces eggs that can develop and hatch normally to release miracidia and carry on the life cycle. The observation raises the possibility that it would be possible for clonal populations to evolve rapidly in the field once resistance had been selected for. The second study (Walker et al., 2006) monitored the development of the Fairhurst and Oberon isolates through one complete life cycle. The Oberon flukes were faster to egg hatch (12 days as against 14 days); faster to produce cercariae (49 days as against 53 days); and produced more than 4 times as many cercariae as the Fairhurst isolate. Moreover, the metacercariae were more infectious to

4

I. Fairweather / Veterinary Parasitology 176 (2011) 1–8

rats and reached patency more quickly (59 dpi as against 70 dpi). Adding up the time differentials across the life cycle, the Oberon isolate would gain ∼2.5 weeks over its Fairhurst counterpart, a considerable advantage (Walker et al., 2006). Moreover, the study showed that the development of resistance by the Oberon isolate has not led to a reduction in fitness: indeed, it maintained a higher level of fitness than the Fairhurst isolate throughout the life cycle (Walker et al., 2006). Fitness was measured in terms of reproductive success, with the Oberon isolate being faster to egg hatch and faster to produce cercariae than the Fairhurst isolate. Moreover, it produced more cercariae and the metacercariae were more infectious to the rat host and the flukes reached patency more quickly (Walker et al., 2006). The combined data, albeit limited, indicates that there are variations between different fluke populations and that these differences need to be taken into account when attempting to understand the epidemiology of fascioliosis. 6. Details of defined isolates used in recent studies In this section, as much information as possible has been gathered together to demonstrate the drug susceptibility of individual isolates and to provide an overview of what studies they have been used for. 6.1. TCBZ-susceptible isolates 6.1.1. Cullompton isolate The Cullompton isolate was first obtained (in 1998) as a field isolate from sheep slaughtered at an abattoir in Cullompton, Devon, UK. It was originally established at VLA, in order to provide researchers with fluke material. The isolate has been maintained in Queens since 1999. It is fully susceptible to TCBZ at adult and juvenile stages (Table 2). In the rat host, the efficacy of TCBZ was shown to be 95.3% against 12-week-old adult flukes (Keiser et al., 2007). The Cullompton isolate is more sensitive to TCBZ action than the Fairhurst, Oberon and Sligo isolates (Walker et al., 2004). In addition to these efficacy studies, the susceptibility of the Cullompton isolate to TCBZ has been demonstrated in a number of in vivo studies: Meaney et al. (2006, 2007), Halferty et al. (2008), McConville et al. (2009a), Hanna et al. (2010), Flanagan et al. (2011, in press) and Toner et al. (2010b,c, in press). The studies by Toner et al. (2010a,b,c, in press) and Hanna et al. (2010) have established the time-course of drug action in the sheep host. The isolate has also been used for a large number of in vitro studies involving TCBZ and its metabolites, as indicated below. The Cullompton isolate has also been used in studies on the impact of TCBZ and TCBZ.SO on egg hatching (Alvarez et al., 2009). The Cullompton isolate is also sensitive to other fasciolicides: albendazole, clorsulon and nitroxynil (Buchanan et al., 2003; McKinstry et al., 2003, 2007, 2009; Meaney et al., 2003, 2004, 2005a,b, 2006, 2007; McConville et al., 2006). In relation to nitroxynil, it is more sensitive than the Sligo, Oberon and Fairhurst isolates (McKinstry et al., 2009). It has been used in studies to assess the activity of a number of experimental compounds: artemisinin derivatives,

the plant product, genistein (Toner et al., 2008), and the triclabendazole derivative, compound alpha. Compound alpha has an efficacy of 99.87% against the adult fluke (12 weeks old), but only 62.5% against 4-week-old juvenile flukes (McConville et al., 2009a). Morphological changes to the Cullompton fluke following compound alpha treatment, both in vivo and in vitro, have been described by McConville et al. (2006, 2008, 2009a,b,c, 2010) and Hanna et al. (2010). Following treatment with the artemisinin derivative, OZ78 100% worm burden reductions have been achieved against juvenile (3 wpi) and adult (8–9 wpi) infections of the Cullompton isolate in rats (Keiser et al., 2006b). OZ78 is relatively more effective than the related derivatives, artemether and artesunate and the latter two drugs show greater efficacy against the adult fluke than the juvenile parasite (for details, see Keiser et al., 2006a,b). A number of morphological studies have been carried out to examine surface and internal changes to the fluke induced by these compounds (Keiser et al., 2006a,b; Keiser and Morson, 2008a,b; Halferty et al., 2009b). The testis morphology of the Cullompton isolate has been described by Hanna et al. (2008). The isolate is aspermic and triploid, spermatogenesis not proceeding beyond the primary spermatocyte stage, as described in Section 5. The following in vitro studies on TCBZ and its metabolites have involved the Cullompton isolate: Robinson et al. (2002, 2004), Mottier et al. (2006), Alvarez et al. (2005, 2009), McConville et al. (2006), Meaney et al. (2006, 2007), Devine et al. (2009, 2010a,b,c, 2011), Halferty et al. (2009a) and Toner et al. (2009, 2010a). 6.1.2. Fairhurst isolate The Fairhurst isolate was originally obtained from the Compton Paddock Laboratory, Newbury, Buckinghamshire, UK by Dr. J.C. Boray in 1985. It had no history of exposure to anthelmintic drugs and has been shown to be susceptible to a number of fasciolicides (closantel, rafoxanide, clorsulon and luxabendazole) (Boray, 1990). In a number of publications, it has been referred to as the Compton isolate (Boray and De Bono, 1989; Boray, 1990; Miller et al., 1994). The isolate was maintained in Australia by Dr. J.C. Boray and its name changed to Fairhurst. It has been maintained in Queens since 2001. Recent claims of the use of the Compton isolate probably represent use of the Cullompton isolate. A recent controlled trial in sheep has confirmed its sensitivity to TCBZ at the adult (12-weekold) stage of infection: efficacy was 100% (Flanagan et al., in press). Efficacy data from cattle and sheep trials carried out by Dr. Boray has been presented in Tables 1 and 2 of Walker et al. (2004). In cattle, the efficacy of TCBZ was 89% at 2 wpi and 100% at 12 wpi; the sheep data is given in Table 2 of this review. In relation to TCBZ action, the Fairhurst isolate is less sensitive than the Cullompton isolate, but more sensitive than the Oberon and Sligo isolates (Walker et al., 2004). The reproductive fitness of the Fairhurst isolate relative to the Oberon isolate during the course of the life cycle has been described by Walker et al. (2006) (see Section 5). It has been shown to be less sensitive to nitroxynil treatment

I. Fairweather / Veterinary Parasitology 176 (2011) 1–8

in vitro than the Cullompton, Sligo and Oberon isolates (McKinstry et al., 2009). 6.1.3. Sunny Corner isolate This isolate was isolated in 1989 in Australia by Dr. Joe Boray and is fully susceptible to TCBZ at the adult stage in sheep. TCBZ has reduced activity against 2-week-old fluke (78.3%) and 4-week-old fluke (77.0%), but greater activity against 6-week-old fluke (98.4%) (Boray, personal communication). The isolate has been used in studies on the efficacy of different formulations of combination products (Hutchinson et al., 2009; Sargent et al., 2009). Synergy between TCBZ and closantel has been demonstrated for this isolate and an ivermectin + TCBZ combination was slightly more effective than TCBZ alone against immature flukes (Boray, personal communication). A low level of synergy between ivermectin and clorsulon against this isolate has also been demonstrated (Boray, personal communication). 6.2. TCBZ-resistant isolates 6.2.1. Sligo isolate The Sligo isolate was originally derived from sheep on a farm in Sligo, Ireland in 1998, following failure of TCBZ (in combination with levamisole) to control liver fluke. A laboratory culture was established from eggs collected from treated ewes, subjected to further selection and TCBZ resistance in adult fluke was confirmed (Coles et al., 2000; Coles and Stafford, 2001). Not only that, the isolate was shown to be susceptible to other fasciolicides at the adult stage: closantel, nitroxynil, oxyclozanide (in combination with levamisole), albendazole and (to a lesser extent) clorsulon (in combination with ivermectin) (Coles et al., 2000; Coles and Stafford, 2001). The susceptibility to nitroxynil does not extend to the juvenile (4-week) stage (McCoy et al., 2005). Subsequent sheep trials have shown that the isolate is resistant to TCBZ at 3 days and 4 weeks post-infection, as well as at the adult stage (McCoy et al., 2005; McConville et al., 2009a). It has been maintained in Queens since 1999. It is less sensitive to TCBZ action in vitro than the Cullompton, Fairhurst and Oberon isolates (Walker et al., 2004). The Sligo isolate has been used in experiments on the effect of TCBZ and TCBZ.SO on egg hatching (Alvarez et al., 2009). The isolate has been used in a number of in vitro studies to determine the mechanism of resistance to TCBZ (Robinson et al., 2002, 2004; Alvarez et al., 2005; Mottier et al., 2006), and to sequence ␣- and ␤-tubulin isotypes (Ryan et al., 2008). Altered drug influx/efflux and drug metabolism appear to be more important in the development of resistance than any changes in tubulin isotypes. With regard to other fasciolicides, the Sligo isolate has been shown to be less sensitive to nitroxynil than the Cullompton isolate, but more sensitive than the Oberon and Fairhurst isolates (McKinstry et al., 2009). Treatment with albendazole sulphoxide did not affect tubulin staining in Sligo flukes (McConville et al., 2006). It is fully resistant to the TCBZ derivative, compound alpha, which had 0% efficacy in sheep at 3 dpi, 4 wpi and 12 wpi (McConville et al., 2009a). Morphological changes in adult and juvenile Sligo

5

flukes in response to compound alpha treatment in vitro have been described by McConville et al. (2006, 2007) and histological changes in the reproductive organs following in vivo treatment described by Hanna et al. (2010). Normal testis morphology has been described by Fletcher et al. (2004) and Hanna et al. (2008). The isolate has been used in a study to examine morphological changes following treatment with the artemisinin derivative, artemether (O’Neill et al., 2009). 6.2.2. Oberon isolate The Oberon isolate was first identified in 1999 on a farm property in Oberon, New South Wales, Australia where resistance to triclabendazole was suspected, and was originally maintained by Dr. J.C. Boray. The fluke isolate was challenged with triclabendazole at the dose rate of 10 mg/kg in sheep 6 weeks after inoculation with 300 metacercariae. The selected population was maintained in the laboratory in Lymnaea tomentosa through several generations and the isolate became fully resistant to triclabendazole. It has been maintained at Queens since 2002. In vivo efficacy data from a sheep trial carried out by Dr. Joe Boray was presented in Table 3 of Walker et al. (2004). The isolate has been shown to be resistant to TCBZ at the 2 wpi, 4 wpi and adult stages (Table 2); it is also fully resistant at the 6 wpi stage (Boray, personal communication). In a rat infection, the efficacy of TCBZ was 4% against adult (12–15 week-old) flukes (Keiser et al., 2007). The Oberon isolate is more sensitive to the action of TCBZ than the Sligo isolate, but less sensitive than the Cullompton and Fairhurst isolates (Walker et al., 2004). The isolate is moderately resistant to closantel and to rafoxanide at 4–6 wpi (closantel efficacy of 69.3% against 6-week-old fluke: Boray, personal communication). The Oberon isolate is more sensitive than Fairhurst in response to nitroxynil treatment in vitro, but less sensitive than the Cullompton and Sligo isolates (McKinstry et al., 2009). In response to other compounds, the Oberon isolate has been shown to be 100% susceptible at the adult (12–15 wpi) stage to the artemisinin derivatives, artemether and OZ78 in a rodent model (Keiser et al., 2007). The morphology of the testis has been described by Hanna et al. (2008) and the reproductive fitness of the isolate during the life cycle assessed by Walker et al. (2006), as discussed above (Section 5). Tubulin isotypes of this isolate have been sequenced by Ryan et al. (2008). The Oberon isolate has also been used in a number of in vitro studies on the role of altered drug metabolism in the development of resistance to TCBZ (Devine et al., 2009, 2010a,b,c, 2011). The results have provided further support for the contribution of increased metabolism to the mechanism of resistance. The concept has been extended to the in vivo situation, in a study involving rats (Devine et al., in press). Changes to the surface morphology of the Oberon isolate following treatment with artemether and artesunate have been described by Keiser and Morson (2008a). 6.2.3. Dutch isolate Resistance to TCBZ was first demonstrated in natural infections in both cattle and sheep on a mixed farm in the province of North Holland, The Netherlands by Moll

6

I. Fairweather / Veterinary Parasitology 176 (2011) 1–8

et al. (2000). In parallel studies on the same farm, closantel was shown to be effective against adult fluke infections in sheep and clorsulon active against adult fluke infections in cattle (Moll et al., 2000). Eggs collected from a sheep on this farm were used to generate metacercariae for an experimental infection in sheep, the result of which confirmed the presence of resistance to TCBZ (Gaasenbeek et al., 2001). Following the demonstration of resistance, the farmer switched to closantel for the treatment of his sheep flock. On returning to the property after 3 years of this regime, the same group of researchers were able to show that the flukes were still resistant to TCBZ, indicating that no reversion to a TCBZ-susceptible state had taken place (Borgsteede et al., 2005). A TCBZ- resistant fluke isolate from cattle on this farm has been maintained at Queens since 2007.

possible to store large quantities of virtually identical infective larvae in liquid nitrogen for use in several experiments, but this is not possible for the liver fluke at the present time. Finally, researchers should be more careful about the information they give on the flukes they work with, so that it is correct and it becomes almost routine for that information to be included in papers, as it is for nematodes. That is what we should be aiming for. The present communication is a beginning: information can be added to the list [of isolates] for new isolates or to extend knowledge on existing isolates. I hope that it will stimulate discussion. It is of more than academic interest, too, as it is important to know the drug sensitivity of fluke populations so that farmers are not given false information on how best to treat their animals.

6.2.4. Leon isolate The first report of resistance to TCBZ in Spain resulted from a study on a natural fluke infection in sheep on a farm in the province of León in north-west Spain (AlvarezSánchez et al., 2006). The same fluke population was shown to be resistant to albendazole and clorsulon (in combination with ivermectin). This observation is potentially significant, as it represents the first instance of multiple drug resistance in the liver fluke. Another publication on this isolate appeared recently: it described the activity of nitroxynil against the adult fluke (Martínez-Valladares et al., 2010). Eggs from the fluke were used to establish the León isolate at Queens in 2006. A subsequent controlled experimental infection in sheep showed that the isolate was not resistant to TCBZ, as first reported (Flanagan et al., in press). The efficacy of TCBZ was 100% against adult (12-week-old) fluke. The efficacy data was supported by results of the coproantigen reduction test (Flanagan et al., in press). The morphology of the testis of the León isolate has been described by Hanna et al. (2008).

I would like to thank the referees for their critical reading of the manuscript and for their constructive suggestions to improve it.

7. Conclusions One immediate conclusion from the survey is that there are relatively few defined isolates of F. hepatica available for study. Also, that information on the drug sensitivity of individual isolates is not yet complete. A greater number of isolates were available and more known about their provenance some 20 or more years ago, thanks largely to the efforts of Dr. Joe Boray (Boray and De Bono, 1989; Boray, 1990; Miller et al., 1994). This is a regrettable situation. What isolates exist need to be preserved and, as new isolates become available, they should be subjected to careful scrutiny to establish how they respond to different fasciolicides. Perhaps part of the reason for this situation is the greater difficulty in maintaining the life cycle of F. hepatica (compared with nematodes). The life cycle is far more complex and includes asexual multiplication in the snail host. The issue of clonal populations of fluke has been addressed by Hanna et al. (2008) (see Section 5). It is possible that an isolate could change drug sensitivity during laboratory culture, but the idea of reversion from a drug-resistant status is unlikely Borgsteede et al. (2005). With nematodes, it is

Acknowledgements

References Alvarez, L.I., Solana, H.D., Mottier, M.L., Virkel, G.L., Fairweather, I., Lanusse, C.E., 2005. Altered drug influx/efflux and enhanced metabolic activity in triclabendazole-resistant liver flukes. Parasitology 131, 501–510. Alvarez, L., Moreno, G., Moreno, L., Ceballos, L., Shaw, L., Fairweather, I., Lanusse, C., 2009. Comparative assessment of albendazole and triclabendazole ovicidal activity on Fasciola hepatica eggs. Vet. Parasitol. 164, 211–216. Alvarez-Sánchez, M.A., Mainar-Jaime, R.C., Pérez-García, J., Rojo-Vázquez, F.A., 2006. Resistance of Fasciola hepatica to triclabendazole and albendazole in sheep in Spain. Vet. Rec. 159, 424–425. Anon, 2006. Northern Ireland disease surveillance, January to March. Vet. Rec. 159, 615–618. Anon, 2008a. Increase in chronic fasciolosis in sheep in Scotland. Vet. Rec. 162, 199–202. Anon, 2008b. Increase in chronic fasciolosis in sheep in Scotland. Vet. Rec. 162, 537–540. Anon, 2008c. Northern Ireland disease surveillance, January to March. Vet. Rec. 163, 135–138. Anon, 2009. Incidents of coccidiosis and fasciolosis common in Scottish cattle and sheep. Vet. Rec. 164, 483–486. Boray, J.C., 1990. Drug resistance in Fasciola hepatica. In: Boray, J.C., Martin, P.J., Roush, R.T. (Eds.), Resistance of Parasites to Antiparasitic Drugs. MSD AGVET, Rahway, NJ, pp. 51–60. Boray, J.C., De Bono, D., 1989. Drug resistance in Fasciola hepatica. In: Outteridge, P.M., Richards, R.B. (Eds.), Advances in Veterinary Science. The Australian Veterinary Association, Artarmon, Australia, pp. 166–169. Boray, J.C., Sluyter, V., Campbell, N.J., McKinnon, A., 1997. Resistance of immature and adult Fasciola hepatica to triclabendazole in the field. In: Proceedings of the 16th International Conference on World Association for the Advancement of Veterinary Parasitology (WAAVP) , Sun City, South Africa, August 1997, p. 10. Borgsteede, F.H.M., Moll, L., Vellema, P., Gaasenbeek, C.P.H., 2005. Lack of reversion in triclabendazole-resistant Fasciola hepatica. Vet. Rec. 156, 350–351. Buchanan, J.F., Fairweather, I., Brennan, G.P., Trudgett, A., Hoey, E.M., 2003. Fasciola hepatica: surface and internal tegumental changes induced by treatment in vitro with the sulphoxide metabolite of albendazole (“Valbazen”). Parasitology 126, 141–153. Coles, G.C., Stafford, K.A., 2001. Activity of oxyclozanide, nitroxynil, clorsulon and albendazole against adult triclabendazole-resistant Fasciola hepatica. Vet. Rec. 148, 723–724. Coles, G.C., Rhodes, A.C., Stafford, K.A., 2000. Activity of closantel against adult triclabendazole-resistant Fasciola hepatica. Vet. Rec. 146, 504. Devine, C., Brennan, G.P., Lanusse, C.E., Alvarez, L.I., Trudgett, A., Hoey, E., Fairweather, I., 2009. Effect of the metabolic inhibitor, methimazole on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica. Parasitology 136, 183–192. Devine, C., Brennan, G.P., Lanusse, C.E., Alvarez, L.I., Trudgett, A., Hoey, E., Fairweather, I., 2010a. Inhibition of cytochrome P450-mediated

I. Fairweather / Veterinary Parasitology 176 (2011) 1–8 metabolism enhances ex vivo susceptibility of Fasciola hepatica to triclabendazole. Parasitology 137, 871–880. Devine, C., Brennan, G.P., Lanusse, C.E., Alvarez, L.I., Trudgett, A., Hoey, E., Fairweather, I., 2010b. Potentiation of triclabendazole sulphoxideinduced tegumental disruption by methimazole in a triclabendazoleresistant isolate of Fasciola hepatica. Parasitol. Res. 106, 1351– 1363. Devine, C., Brennan, G.P., Lanusse, C.E., Alvarez, L.I., Trudgett, A., Hoey, E., Fairweather, I., 2010c. Enhancement of the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica using the metabolic inhibitor ketoconazole. Parasitol. Res. 107, 337–353. Devine, C., Brennan, G.P., Lanusse, C.E., Alvarez, L.I., Trudgett, A., Hoey, E., Fairweather, I., 2011. Piperonyl butoxide enhance triclabendazole action against triclabendazole-resistant Fasciola hepatica. Parasitology 138, 224–236. Devine, C., Brennan, G.P., Lanusse, C.E., Alvarez, L.I., Trudgett, A., Hoey, E., Fairweather, I., Enhancement of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica by co-treatment with ketoconazole, Vet. Parasitol., doi:10.1016/j.vetpar.2010.12.002, in press. Flanagan, A.M., Edgar, H.W.J., Forster, F., Gordon, A., Hanna, R.E.B., McConville, M., Brennan, G.P., Fairweather, I., 2011. 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., 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., doi:10.1016/j.vetpar.2010.10.057, in press. Fletcher, H.L., Hoey, E.M., Orr, N., Trudgett, A., Fairweather, I., Robinson, M.W., 2004. The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica. Parasitology 128, 69–72. Gaasenbeek, C.P.H., Moll, L., Cornelissen, J.B.W.J., Vellema, P., Borgsteede, F.H.M., 2001. An experimental study on triclabendazole resistance of Fasciola hepatica in sheep. Vet. Parasitol. 95, 37–43. Halferty, L., Brennan, G.P., Hanna, R.E.B., Edgar, H.W., Meaney, M., McConville, M., Trudgett, A., Hoey, L., Fairweather, I., 2008. Tegumental surface changes in juvenile Fasciola hepatica in response to treatment in vivo with triclabendazole. Vet. Parasitol. 155, 49–58. Halferty, L., Brennan, G.P., Trudgett, A., Hoey, L., Fairweather, I., 2009a. Relative activity of triclabendazole metabolites against the liver fluke, Fasciola hepatica. Vet. Parasitol. 159, 126–138. Halferty, L., O’Neill, J.F., Brennan, G.P., Keiser, J., Fairweather, I., 2009b. Electron microscopical study to assess the in vitro effects of the synthetic trioxolane OZ78 against the liver fluke. Parasitology 136, 1325–1337. Hanna, R.E.B., Edgar, H., Moffett, D., McConnell, S., Fairweather, I., Brennan, G.P., Trudgett, A., Hoey, E.M., Cromie, L., Taylor, S.M., Daniel, R., 2008. Fasciola hepatica: histology of the testis in egg-producing adults of several laboratory-maintained isolates grown to maturity n cattle and sheep and in flukes from naturally infected hosts. Vet. Parasitol. 157, 222–234. Hanna, R.E.B., Edgar, H.W.J., McConnell, S., Toner, E., McConville, M., Brennan, G.P., Devine, C., Flanagan, A., Halferty, L., Meaney, M., Shaw, L., Moffett, D., McCoy, M., Fairweather, I., 2010. Fasciola hepatica: histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes collected from experimentally-infected sheep one to four days after treatment with TCBZ and the related benzimidazole derivative, Compound Alpha. Vet. Parasitol. 168, 240–254. Hutchinson, G.W., Dawson, K., Fitzgibbon, C.C., Martin, P.J., 2009. Efficacy of an injectable combination anthelmintic (nitroxynil + clorsulon + ivermectin) against early immature Fasciola hepatica compared to triclabendazole combination flukicides given orally or topically to cattle. Vet. Parasitol. 162, 278–284. Keiser, J., Morson, G., 2008a. Fasciola hepatica: tegumental alterations in adult flukes following in vitro administration of artesunate and artemether. Exp. Parasitol. 118, 228–237. Keiser, J., Morson, G., 2008b. Fasciola hepatica: surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78. Exp. Parasitol. 119, 87–93. Keiser, J., Utzinger, J., Tanner, M., Dong, Y., Vennerstrom, J.L., 2006a. The synthetic peroxide OZ78 is effective against Echinostoma caproni and Fasciola hepatica. J. Antimicrob. Chemother. 58, 1193–1197. Keiser, J., Shu-Hua, X., Tanner, M., Utzinger, J., 2006b. Artesunate and artemether are effective fasciolicides in the rat model and in vitro. J. Antimicrob. Chemother. 57, 1139–1145. Keiser, J., Utzinger, J., Vennerstrom, J.L., Dong, Y., Brennan, G.P., Fairweather, I., 2007. Activity of artemether and OZ78 against

7

triclabendazole-resistant Fasciola hepatica. Trans. Roy. Soc. Trop. Med. Hyg. 101, 1219–1222. Martínez-Valladares, M., del Rosario Famularo, M., Fernández-Pato, N., ˜ ˜ Castanón-Ordó nez, 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. McConville, M., Brennan, G.P., McCoy, M., Castillo, R., Hernández-Campos, A., Fairweather, I., 2006. Adult triclabendazole-resistant Fasciola hepatica: surface and sub-surface tegumental responses to in vitro treatment with the sulphoxide metabolite of the experimental fasciolicide compound alpha. Parasitology 133, 195–208. McConville, M., Brennan, G.P., McCoy, M., Castillo, R., Hernandez-Campos, A., Ibarra, F., Fairweather, I., 2007. Immature triclabendazole-resistant Fasciola hepatica: tegumental responses to in vitro treatment with the sulphoxide metabolite of the experimental fasciolicide compound alpha. Parasitol. Res. 100, 365–377. McConville, M., Brennan, G.P., Flanagan, A., Edgar, H.W.J., McCoy, M., Castillo, R., Hernández-Campos, A., Fairweather, I., 2008. Surface and internal tegumental changes in juvenile Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha. Vet. Parasitol. 153, 52–64. McConville, M., Brennan, G.P., Flanagan, A., Edgar, H.W.J., Hanna, R.E.B., McCoy, M., Gordon, A.W., Castillo, R., Hernández-Campos, A., Fairweather, I., 2009a. An evaluation of the efficacy of compound alpha and triclabendazole against two isolates of Fasciola hepatica. Vet. Parasitol. 162, 75–88. McConville, M., Brennan, G.P., Flanagan, A., Hanna, R.E.B., Edgar, H.W.J., Castillo, R., Hernández-Campos, A., Fairweather, I., 2009b. Surface changes in adult Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha. Parasitol. Res. 105, 757–767. McConville, M., Brennan, G.P., Flanagan, A., Edgar, H.W.J., Castillo, R., Hernández-Campos, A., Fairweather, I., 2009c. Ultrastructural changes to the tegumental system and the gastrodermal cells in adult Fasciola hepatica following in vivo treatment with the experimental fasciolicide, compound alpha. Parasitology 136, 665–680. McConville, M., Hanna, R.E.B., Brennan, G.P., McCoy, M., Edgar, H.W.J., McConnell, S., Castillo, R., Hernández-Campos, A., Fairweather, I., 2010. Fasciola hepatica: disruption of spermatogenesis by the fasciolicide compound alpha. Parasitol. Res. 106, 311–323. McCoy, M.A., Fairweather, I., Brennan, G.P., Kenny, J.M., Ellison, S., Forbes, A.B., 2005. The efficacy of nitroxynil and triclabendazole administered synchronously against juvenile triclabedazole-resistant Fasciola hepatica in sheep. Res. Vet. Sci. 78, 33. McKinstry, B., Fairweather, I., Brennan, G.P., Forbes, A.B., 2003. Fasciola hepatica: tegumental surface alterations following treatment in vivo and in vitro with nitroxynil (“Trodax”). Parasitol. Res. 91, 251–263. McKinstry, B., Brennan, G.P., Halferty, L., Forbes, A.B., Fairweather, I., 2007. Ultrastructural changes induced in the tegument and gut of Fasciola hepatica following in vivo and in vitro drug treatment with nitroxynil (Trodax). Parasitol. Res. 101, 929–941. McKinstry, B., Halferty, L., Brennan, G.P., Fairweather, I., 2009. Morphological response of triclabendazole-susceptible and triclabendazoleresistant isolates of Fasciola hepatica to treatment in vitro with nitroxynil (Trodax). Parasitol. Res. 104, 645–655. Meaney, M., Fairweather, I., Brennan, G.P., McDowell, L.S.L., Forbes, A.B., 2003. Fasciola hepatica: effects of the fasciolicide clorsulon in vitro and in vivo on the tegumental surface, and a comparison of the effects on young- and old-mature flukes. Parasitol. Res. 91, 238–250. Meaney, M., Fairweather, I., Brennan, G.P., Forbes, A.B., 2004. Transmission electron microscope study of the ultrastructural changes induced in the tegument and gut of Fasciola hepatica following in vivo drug treatment with clorsulon. Parasitol. Res. 92, 232–241. Meaney, M., Haughey, S., Brennan, G.P., Fairweather, I., 2005a. A scanning electron microscope study on the route of entry of clorsulon into the liver fluke Fasciola hepatica. Parasitol. Res. 95, 117–128, 96, 189– 198. Meaney, M., Haughey, S., Brennan, G.P., Fairweather, I., 2005b. Ultrastructural observations on oral ingestion and trans-tegumental uptake of clorsulon by the liver fluke, Fasciola hepatica. Parasitol. Res. 95, 201–212. Meaney, M., Allister, J., McKinstry, B., McLaughlin, K., Brennan, G.P., Forbes, A.B., Fairweather, I., 2006. Fasciola hepatica: morphological effects of a combination of triclabendazole and clorsulon against mature fluke. Parasitol. Res. 99, 609–621. Meaney, M., Allister, J., McKinstry, B., McLaughlin, K., Brennan, G.P., Forbes, A.B., Fairweather, I., 2007. Fasciola hepatica: ultrastructural effects of a combination of triclabendazole and clorsulon against mature fluke. Parasitol. Res. 100, 1091–1104.

8

I. Fairweather / Veterinary Parasitology 176 (2011) 1–8

Miller, C.M.D., Howell, M.J., Boray, J.C., 1994. Glutathione S-transferases as markers of salicylanilide resistance in isolates of Fasciola hepatica. Int. J. Parasitol. 24, 533–542. Moll, L., Gaasenbeek, C.P.H., Vellema, P., Borgsteede, F.H.M., 2000. Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in The Netherlands. Vet. Parasitol. 91, 153–158. 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. Mottier, L., Alvarez, L., Fairweather, I., Lanusse, C., 2006. Resistanceinduced changes in triclabendazole transport in Fasciola hepatica: ivermectin reversal effect. J. Parasitol. 92, 1355–1360. O’Neill, J.F., Johnston, R.C., Halferty, L., Brennan, G.P., Keiser, j., Fairweather, I., 2009. Adult triclabendazole-resistant Fasciola hepatica: morphological changes in the tegument and gut following in vivo treatment with artemether in the rat model. J. Helminthol. 83, 151–163. Robinson, M.W., Trudgett, A., Hoey, E.M., Fairweather, I., 2002. Triclabendazole-resistant Fasciola hepatica: ␤-tubulin and response to in vitro treatment with triclabendazole. Parasitology 124, 325–338. Robinson, M.W., Lawson, J., Trudgett, A., Hoey, L., Fairweather, I., 2004. The comparative metabolism of triclabendazole sulphoxide by triclabendazole-susceptible and triclabendazole-resistant Fasciola hepatica. Parasitol. Res. 92, 205–210. Ryan, L.A., Hoey, E., Trudgett, A., Fairweather, I., Fuchs, M., Robinson, M.W., Chambers, E., Timson, D., Ryan, E., Fetwell, T., Ivens, A., Bentley, G., Johnston, D., 2008. Fasciola hepatica expresses multiple ␣- and ␤-tubulin isotypes. Mol. Biochem. Parasitol. 159, 73–78. Sargent, R.M., Chambers, M., Elliott, T., 2009. Seasonal differences in the efficacy of pour-on formulations of triclabendazole and ivermectin or abamectin against late immature liver fluke (Fasciola hepatica) in cattle. Vet. Parasitol. 161, 133–137.

Toner, E., Brennan, G.P., Wells, K., McGeown, J.G., Fairweather, I., 2008. Physiological and morphological effects of genistein against the liver fluke, Fasciola hepatica. Parasitology 135, 1189– 1203. Toner, E., McConvery, F., Brennan, G.P., Meaney, M., Fairweather, I., 2009. A scanning electron microscope study on the route of entry of triclabendazole into the liver fluke, Fasciola hepatica. Parasitology 136, 523–535. Toner, E., Brennan, G.P., McConvery, F., Meaney, M., Fairweather, I., 2010a. A transmission electron microscope study on the route of entry of triclabendazole into the liver fluke, Fasciola hepatica. Parasitology 137, 855–870. Toner, E., Brennan, G.P., Hanna, R.E.B., Edgar, H.W.J., Fairweather, I., 2010b. Tegumental surface changes in adult Fasciola hepatica in response to treatment in vivo with triclabendazole in the sheep host. Vet. Parasitol. 172, 238–248. Toner, E., Brennan, G.P., Hanna, R.E.B., Edgar, H.W.J., Fairweather, I., 2010c. Time-dependent changes to the tegumental system and gastrodermis of adult Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host. Vet. Parasitol. 174, 218–227. Toner, E., Brennan, G.P., Hanna, R.E.B., Edgar, H.W.J., Fairweather, I., Disruption of egg formation by Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host, Vet. Parasitol., doi:10.1016/j.vetpar.2010.11.032, in press. Walker, S.M., McKinstry, B., Boray, J.C., Brennan, G.P., Trudgett, A., Hoey, E.M., Fletcher, H., Fairweather, I., 2004. Response of two isolates of Fasciola hepatica to treatment with triclabendazole in vivo and in vitro. Parasitol. Res. 94, 427–438. Walker, S.M., Hoey, E., Fletcher, H., Brennan, G.P., Fairweather, I., Trudgett, A., 2006. Stage-specific differences in fecundity over the life-cycle of two characterized isolates of the live fluke Fasciola hepatica. Parasitology 133, 209–216.