Parasitol Res (2004) 94: 427–438 DOI 10.1007/s00436-004-1222-5
O R I GI N A L P A P E R
S. M. Walker Æ B. McKinstry Æ J. C. Boray G. P. Brennan Æ A. Trudgett Æ E. M. Hoey H. Fletcher Æ I. Fairweather
Response of two isolates of Fasciola hepatica to treatment with triclabendazole in vivo and in vitro Received: 6 August 2004 / Accepted: 28 August 2004 / Published online: 28 October 2004 Springer-Verlag 2004
Abstract In this study, the susceptibility of two isolates of Fasciola hepatica—the Fairhurst and Oberon isolates—to treatment with triclabendazole was investigated, both in vivo and in vitro. The Fairhurst isolate originated in England, but has since been maintained in Australia; the Oberon isolate originated in Australia. Triclabendazole had a very high efficacy against the Fairhurst isolate. In sheep (dose: 10 mg/kg), the efficacy ranged from 78.4% at 2 weeks post-infection to 98.5% at 6 weeks post-infection. In cattle (dose: 12 mg/kg) efficacy was 89% at 2 weeks post-infection and 100% at 12 weeks. In contrast, against the Oberon isolate, triclabendazole had 0% efficacy against 2-week-old flukes in sheep (dose: 10 mg/kg) and 5% against 4-weekold flukes. Surface changes to flukes of the two isolates were assessed by scanning electron microscopy following treatment in vitro for 24 h in triclabendazole sulphoxide (15 and 50 lg/ml). Disruption took the form of blebbing, swelling and furrowing of the tegument and was greater in the Fairhurst than the Oberon isolate. Surface changes generally were more severe in the anterior than posterior region of the fluke and the dorsal surface was also consistently more severely affected than the ventral surface. Disruption was more severe at the higher drug concentration for both isolates. The morphological data is consistent with the efficacy data, which indicates that S. M. Walker Æ B. McKinstry Æ G. P. Brennan Æ A. Trudgett E. M. Hoey Æ H. Fletcher Æ I. Fairweather (&) Parasite Proteomics and Therapeutics Research Group, School of Biology and Biochemistry, Medical Biology Centre, The Queen’s University of Belfast, 97 Lisburn Road, BT9 7BL Belfast, Northern Ireland E-mail:
[email protected] Tel.: +44-28-90972298 Fax: +44-28-90335877 J. C. Boray Elizabeth Macarthur Agricultural Institute, NSW Agriculture, Camden, NSW, Australia J. C. Boray Consultant for Veterinary Parasitology Research and Development, 1 Pedaman Place, NSW 2259 Jilliby, Australia
the Fairhurst isolate of F. hepatica is susceptible to triclabendazole treatment, whilst the Oberon isolate is refractory.
Introduction There has been a dramatic resurgence of fasciolosis, due to Fasciola hepatica, in recent years as a result of climate change and the advent of milder, wetter weather (Mitchell 2002). In parts of the United Kingdom, for example, liver condemnation rates are as high as 50% for cattle and more than 30% for sheep; these figures probably underscore the true extent of the disease. The effect of wet and mild conditions has been similar in Ireland, France, the endemic areas of Spain and several central and eastern European countries, including the Baltic states, the Ukraine and Russia. The problems of Australia and New Zealand can also be included, as well as tropical fasciolosis due to Fasciola gigantica. Calculated from the number of ruminants grazing endemic areas, the annual economic loss due to the disease has been estimated to be 2–3.2 billion US dollars (Boray 1985; Spithill et al. 1999). There are effective anthelmintics available, but the emergence of resistance to salicylanilide compounds and particularly to triclabendazole may result in problems for the control of the disease. The most commonly used drug world-wide is triclabendazole, due to its high activity against the migrating, tissue-invading and most damaging immature stages. The potential for development of resistance to triclabendazole was first reported in Australia in laboratory studies (Boray 1990). The occurrence of a high level of resistance in an irrigation area of Victoria, Australia was described by Overend and Bowen (1995) and Boray (1997). Since then, resistance has been described in the Netherlands, in various parts of the United Kingdom, Ireland and Australia (Mitchell et al. 1998; Fairweather
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and Boray 1999; Moll et al. 2000; Thomas et al. 2000; Gaasenbeek et al. 2001). In order to study mechanisms of drug action and resistance, it is important to identify fluke isolates of known susceptibility or resistance to triclabendazole and establish experimental infections of them. Initial studies on the action of triclabendazole were carried out using a field (presumed susceptible) isolate of F. hepatica supplied by the Central Veterinary Laboratory, Weybridge, Surrey, UK (Stitt and Fairweather 1992, 1993, 1994, 1996; Stitt et al. 1995). More recent studies on the mechanism of resistance have involved the triclabendazole-susceptible Cullompton and the triclabendazoleresistant Sligo isolates (Robinson et al.2002, 2004). For the Sligo isolate, the experimental work has been supported by efficacy data (Coles et al. 2000; Coles and Stafford 2001). In this paper, efficacy data on the activity of triclabendazole against two further fluke isolates—the Fairhurst and Oberon isolates—is presented and supported with morphological data to confirm their susceptibility and resistance, respectively, to triclabendazole.
Materials and methods
Table 1 Efficacy of triclabendazole against the Fairhurst isolate of Fasciola hepatica in sheep Treatment
Number of flukes at necropsy
Group 1, untreated controls
146 160 184 143 195 191 158 Mean 44 9 22 40 133 7 0 Mean 90 66 5 0 0 0 3 Mean 4 0 12 0 2 0 0 Mean
Recovery: 56% Group 2, TCBZ 10 mg/kg, week 2
Efficacy: 78.4% Group 3, TCBZ 10 mg/kg, week 4
Efficacy: 86.1% Group 4, TCBZ 10 mg/kg, week 6
F. hepatica isolates 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 has been maintained in Australia since 1985 by Dr J.C. Boray and its name has been changed to Fairhurst. The Oberon isolate was first identified in 1999 on a farm property in Oberon, New South Wales, Australia where resistance to triclabendazole was suspected. It has been maintained by Dr J.C. Boray since that time. 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 as described below. Protocol for in vivo efficacy studies in sheep Fairhurst isolate in sheep A total of 28 Merino ewes, aged 3–4 years, were established as fluke-free by analysis of faecal samples. The sheep were inoculated with 300 F. hepatica
Efficacy: 98.5%
168.1 (SD 21.5)
36.4 (SD 45.7)
23.4 (SD 38.0)
2.6 (SD 4.4)
metacercariae each and maintained on fluke-free pasture with supplementary feeding. After weighing, each sheep was allocated to one of four groups of seven that were ranked according to body weight, group 1 being the untreated control group. At 2, 4 and 6 weeks postinfection (p.i.), groups 2, 3 and 4, respectively, were treated with triclabendazole at a dose of 10 mg/kg. At week 16 p.i., all sheep were slaughtered, their livers removed and flukes recovered and counted. Fairhurst isolate in cattle Twelve Hereford·Aberdeen Angus heifers aged 18 months were established as fluke-free, ranked according to weight and allocated to one of two groups of six, group 1 being the untreated control group. They were inoculated with 600 metacercariae each and kept on fluke-free pasture with supplementary feeding. At 2 weeks p.i., group 2 was treated with triclabendazole at a dose of 12 mg/kg. All cattle were slaughtered at week 16 p.i., their livers removed and flukes recovered and counted. In a separate trial, 12 Aberdeen Angus calves, aged 6–8 months, were secured as being flukefree by analysis of faecal samples. They were ranked
429 Table 2 Efficacy of triclabendazole against the Fairhurst isolate of F. hepatica in Hereford · Aberdeen Angus 18-month-old heifers and Aberdeen Angus calves Treatment for 18-month-old heifers
Number of flukes at necroscopy
Group 1, untreated controls (Hereford · Aberdeen Angus heifers)
163 99 126 183 89 117 Mean 129.5 (SD 36.6) 6 33 15 7 14 11 Mean 14.3 (SD 9.8)
Recovery: 25.9% Group 2, TCBZ 12 mg/kg, week 2
Efficacy: 89.0% Treatment of Aberdeen Angus calves Group 1, untreated controls (Aberdeen Angus calves)
Recovery: 25.4% Group 2, TCBZ 12 mg/kg, week 12
Efficacy: 100%
84 63 141 141 126 54 Mean 101.5 (SD 39.4) 0 0 0 0 0 0 Mean 0
Table 3 Efficacy of triclabendazole against the Oberon isolate of F. hepatica in sheep Treatment
Number of flukes at necropsy
Group 1, untreated controls
67 66 44 39 62 71 73 61 Mean 60.4 (SD 12.4) 11 69 70 44 104 103 90 144 Mean 79.4 (SD 40.7) 72 21 57 82 63 54 27 83 Mean 57.4 (SD 23.2)
Recovery 20.1% Group 2, TCBZ 10 mg/kg, week 2
Efficacy: 0% Group 3. TCBZ 10 mg/kg, week 4
Efficacy: 5%
In vitro drug treatment according to body weight and allocated to one of two groups of six, group 1 being the untreated control group. All individuals were inoculated with approximately 400 metacercariae each and maintained on fluke-free pasture with supplementary feeding. At 12 weeks p.i., group 2 was treated with triclabendazole at a dose of 12 mg/kg. All calves were slaughtered at week 16 p.i., their livers removed and flukes recovered and counted.
Oberon isolate A total of 24 Merino ewe lambs, aged 10 months, were established as fluke-free by faecal sample analysis. They were inoculated with 300 metacercariae each and maintained on fluke-free pasture with supplementary feeding. After weighing, each lamb was allocated to one of three groups of eight that were ranked according to body weight, group 1 being the untreated control group. At 2 and 4 weeks p.i., groups 2 and 3, respectively, were treated with triclabendazole at a dose of 10 mg/kg. At week 16 p.i., all lambs were slaughtered their livers removed and flukes recovered and counted. All calves were slaughtered at week 16 p.i., their livers removed and flukes recovered and counted.
Male Sprague-Dawley rats, 14 weeks old, were each infected orally with 20 F. hepatica metacercariae of either the Fairhurst or Oberon isolates, under light ether anaesthesia by means of a stomach tube. At 12 weeks p.i., the flukes were removed from the bile ducts of the rats at autopsy, washed under sterile conditions in a laminar flow cabinet in several changes of warm (37C), sterile NCTC 135 culture medium containing antibiotics (penicillin, 50 IU/ml; streptomycin, 50 mg/ml) (NCTC 135 was obtained from Flow Laboratories, Thames, Oxfordshire, UK; antibiotics were obtained from SigmaAldrich, Poole, Dorset, UK). Whole flukes were transferred to fresh NCTC culture medium containing triclabendazole sulphoxide at a concentration of 15 lg/ ml or 50 lg/ml. The former concentration corresponds to maximum blood levels in vivo (13.3 lg/ml following a dose of 10 mg/kg in sheep: Hennessy et al. 1987); the latter to the highest concentration used in vitro (Coles 1986). The drug was initially prepared as a stock solution in dimethyl sulphoxide and added to the culture medium to give a final solvent concentration of 0.1% (v/v). The flukes were incubated for 24 h at 37C. Controls were prepared by incubating whole flukes for 24 h at 37C in NCTC 135 culture medium containing 0.1% (v/v) dimethyl sulphoxide. Zero hour controls were also prepared. After incubation, flukes were processed for scanning electron microscopy.
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Scanning electron microscopy Flukes were processed intact. Initially, they were lightly flat-fixed for 30 min at room temperature in a 3:1 mixture of 4% (w/v) aqueous glutaraldehyde and 1% aqueous osmium tetroxide. The flukes were subsequently free-fixed in fresh fixative for a further 4 h at room temperature, washed in several changes of deionised water, dehydrated in an ascending series of acetone and critical-point dried in carbon dioxide. The specimens were mounted on aluminium stubs, sputter-coated and viewed in a JEOL 6400 scanning electron microscope operating at 10 keV.
Results In vivo efficacy studies The results of the efficacy studies are presented in Tables 1, 2 and 3. The efficacy of triclabendazole increases with the age of the fluke for the Fairhurst isolate and this is true in both sheep and cattle: it ranges from 78.4% (sheep) and 89.0% (cattle) at 2 weeks to 100% at 12 weeks (Tables 1, 2). In contrast, triclabendazole has very low activity against the Oberon isolate in sheep: 0% at 2 weeks and 5.0% at 4 weeks (Table 3).
Scanning electron microscopy Fairhurst isolate After incubation for 24 h in triclabendazole sulphoxide (TCBZ.SO) at a concentration of 15 lg/ml, all of the flukes appeared motionless and none had visible gut contents. On the ventral surface of the oral cone, the majority of specimens exhibited swelling of the tegument (Fig. 1). The tegument covering the spines was also swollen and the spines appeared slightly sunken due to the swelling of the tegument surrounding them (inset, Fig. 1). Disruption to the dorsal oral cone surface (Fig. 2) was similar to that described for the ventral surface; however, blebbing was also evident, particularly on the tegument between the spines (Fig. 2, inset). Swelling of the tegument was a feature of the anterior midbody, especially along the lateral margins of the flukes (Figs. 3, 4). The swelling was particularly severe on the dorsal surface, which was thrown into a number of deep furrows (Fig. 4). The tegumental surface was covered in tiny blebs, along with a number of larger blebs (Figs. 3, 4). The tegument covering the spines was swollen and the spines appeared to be partially submerged by the swollen tegument surrounding them; this was more evident on the ventral surface (Figs. 3, 4). Although the tegumental changes described were characteristic of the lateral margins of the flukes, they were
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Fig. 1 SEM of the ventral surface of the oral cone region showing swelling of the tegument. Oral sucker (OS), gonopore (G), ventral sucker (VS). Bar 250 lm. Inset shows a higher power image of a group of spines (S) which appear partially sunken by the swollen tegument surrounding them. Bar 17 lm Fig. 2 SEM of the dorsal oral cone, showing swelling of the tegument. Bar 170 lm. Inset shows a higher power image of a group of spines (S). Blebs (B) are present on the tegumental surface. The tegument lining and between the spines is swollen. Bar 17 lm Fig. 3 Lateral margin of the ventral anterior midbody region. The tegument (arrow) between the spines is swollen (arrow) and blebs (B) cover the tegumental surface. The tegument covering the spines (S) is also swollen and the spines appear sunken. Bar 17 lm Fig. 4 SEM of the lateral margin of the dorsal anterior midbody region, showing blebbing (B) of the surface and swelling and furrowing (F) of the tegument between the spines (S). The tegument covering the spines is also swollen. Bar 17lm Fig. 5 Lateral margin of the ventral posterior midbody region. The tegument between the spines (S) is slightly swollen and furrowed (F). The tegument lining the spines is not swollen. Bar 10 lm Fig. 6 Lateral margin of the dorsal posterior midbody region. The tegument is densely covered with blebs (B) of various sizes and is swollen and furrowed (F). Bar 17 lm Fig. 7 SEM of the ventral surface of the tail region showing swelling of the tegument. Bar 170 lm. Inset shows a higher power image of two spines (S) surrounded by swollen tegument. Bar 17 lm Fig. 8 Dorsal tail region. The tegument appears swollen and furrowed (F). Bar 170 lm. Inset shows a higher power image of a spine (S) which appears sunken due to the swollen tegument surrounding it. Bar 17 lm
also evident (to a lesser extent) in the central midbody region. In the posterior midbody region on the ventral surface, there was some swelling of the tegument, but the spines still projected clearly from the tegumental surface and their covering showed only limited signs of swelling (Fig. 5). The tegument was more severely swollen on the dorsal surface, so much so that the spines were totally obscured. The surface was furrowed and carpeted in blebs of varying sizes (Fig. 6). Some of the larger blebs had collapsed, giving them a shrivelled, ‘‘raisin-like’’ appearance (Fig. 6). Again, the central area on both surfaces was less severely affected than the lateral margins of the fluke, though similar morphological changes were observed. The tegumental surface in the ventral tail region appeared relatively normal, apart from slight swelling in the interspine areas (Fig. 7). Swelling was greater on the dorsal surface and the tegumental surface showed signs of furrowing (Fig. 8). The spines projected from the surface in the ventral tail (inset, Fig. 7), whereas they were partially submerged by the surrounding tegument in the dorsal tail (Fig. 8, inset). Overall, the dorsal surface was more disrupted than the ventral surface and the anterior region on both surfaces of the fluke was more severely affected than the posterior region. After incubation for 24 h in TCBZ.SO at a concentration of 50 lg/ml, all of the flukes appeared motionless and none had visible gut contents.
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The tegument on both ventral and dorsal surfaces of the oral cone region was swollen and covered in blebs, especially in the interspinal areas (Figs. 9, 10). On the ventral surface, the tegument covering the spines was also swollen (inset, Fig. 9). On the dorsal surface, part of the tegument lining the spine tips had been abraded, exposing the crystalline spines beneath (inset, Fig. 10). In the anterior midbody region and particularly along the lateral margins, the body surface was covered in blebs (Figs. 11, 12) and the tegument was very swollen so that the spines were barely visible and did not project above the body surface (Figs. 11, 12). The tegument lining the spines themselves was not swollen. These changes were more evident on the ventral surface (inset, Fig. 11). In the central region of the fluke, on both surfaces, less severe swelling was present than along the lateral margins. In the posterior midbody on the ventral surface, and along the lateral margins, the tegument was swollen and covered in blebs, obscuring the spines (Fig. 13). The swelling was quite severe, throwing the surface into a number of furrows (Fig. 13). On the dorsal surface, swelling of the tegument was less severe so that the spines were more visible; the tegument covering the spines was swollen (Fig. 14). Blebs were present on the surface: some of the larger ones had collapsed, giving them a raisin-like appearance (Fig. 14). Similar morphological changes were observed in the central area on both surfaces of the fluke, though to a less severe extent than along the lateral margins. In the tail region on the ventral surface, swelling and blebbing of the tegument were evident (Fig. 15). Again, the larger blebs showed evidence of collapsing, giving them a shrivelled appearance (inset, Fig. 15). The tegument covering the spines was slightly swollen (Fig. 15, inset). The tegument on the dorsal surface appeared relatively normal, though there was some swelling and the surface showed signs of furrowing (Fig. 16). Overall, the ventral surface was more severely affected than the dorsal surface. There were no regional differences in the severity of the tegumental changes on the ventral surface. Disruption of the dorsal surface was variable, not exhibiting any distinct regional trends. Oberon isolate After 24 h incubation in TCBZ.SO at a concentration of 15 lg/ml, all flukes were alive and moving and two of the six flukes had visible gut contents. The tegument on both ventral and dorsal surfaces in the oral cone region was swollen (Figs. 17, 18, 19, 20). This was particularly evident on the dorsal surface: the tegument between the spines was so swollen that the spines appeared sunken (Fig. 20). Surface blebbing was limited. On the ventral surface, the tegument covering the spines was swollen (Fig. 19). The tegument between the spines was swollen, thrown into distinct furrows and bore a number of small blebs (Fig. 19).
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Fig. 9 SEM of the ventral surface of the oral cone region, showing swelling of the tegument. Gonopore (G), ventral sucker (VS). Bar 250 lm. Inset shows a higher power image of a group of spines (S) surrounded by tegument that is covered by blebs (B). The tegument covering the spines is swollen. Bar 17 lm Fig. 10 SEM of the dorsal surface of the oral cone, showing swelling of the tegument. Bar 17 lm. Inset shows a higher power image of a group of spines (S) which have lost the tegument lining their tips (arrow). Blebs (B) are present on the tegument between the spines. Bar 5 lm Fig. 11 Lateral margin (LM) of the ventral anterior midbody region. The tegument is swollen and covered in blebs (B). Bar 100 lm. Inset shows a higher power image of the blebs (B) on the tegumental surface. Spine (S). Bar 17 lm Fig. 12 Lateral margin (LM) of the dorsal anterior midbody region. The tegument is swollen and covered with blebs (B) that are often larger than the nearby spines. Bar 100 lm Fig. 13 High power SEM of the lateral margin of the ventral posterior midbody region. The tegument covering and between the spines (S) is swollen, furrowed (F) and covered with blebs (B). Bar 17 lm Fig. 14 Lateral margin from the dorsal posterior midbody region. Blebs (B) are present on the tegumental surface between the spines (S). Bar 17 lm Fig. 15 SEM of the ventral tail region showing swelling and blebbing of the tegument over the entire region. Bar 140 lm. Inset shows a higher power image of a group of spines (S). Blebs (B) of various sizes occur on the tegument between the spines. Bar 17 lm Fig. 16 SEM of the surface of the dorsal tail region, showing furrowing (F) of the tegument. Bar 200 lm
The morphological changes described for the oral cone region were also observed in the anterior midbody region, with the dorsal surface being the more severely affected of the two surfaces. In the posterior midbody region and along the lateral margins of the flukes, the tegument was swollen, blebbing was quite extensive and microvillus-like projections were present in areas which did not bear blebs (Figs. 21, 22). Swelling was more severe on the dorsal surface, throwing the tegument into numerous furrows (Fig. 22). The spines were not visible due to the swelling of the tegument between them. The central midbody areas exhibited very little evidence of tegumental disruption. The surface of the ventral tail region appeared relatively normal, apart from some slight swelling of the tegument (Fig. 23). Swelling was more extensive on the dorsal surface, so that the surface was furrowed (Fig. 24). At high power, the surface was seen to be covered with microvillus-like projections and a number of blebs (inset, Fig. 24). Overall, the dorsal surface was more disrupted than the ventral surface and the anterior than the posterior region of the fluke: the anterior-directed disruption was especially evident on the ventral surface. After 24 h incubation in TCBZ.SO at a concentration of 50 lg/ml, three of the six flukes treated were moving with visible gut contents whilst the remaining three were motionless and devoid of any gut contents. In the oral cone region on both surfaces, the tegument was swollen and this partially obscured the spines (Figs. 25, 26). On the ventral surface, large blebs were evident and there seemed to be one of these blebs on
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each spine (inset, Fig. 25). Large blebs were also present on the dorsal surface (Fig. 26, inset). The dorsal surface was generally more roughened and more uniformly covered in tiny blebs and microvillus-like projections than the ventral surface (Fig. 26, inset). In the ventral anterior midbody, the tegument was swollen, though the spines still projected from the tegumental surface. Blebs were present along the lateral margins of the flukes (Fig. 27). Less extensive blebbing occurred along the lateral margins on the dorsal surface, although the tegument was more swollen and generally roughened, due to the presence of microvilli (Fig. 28). Swelling was less severe in the central areas of the fluke on both surfaces. On the ventral surface, blebs were present on the spine tips, and on the dorsal surface microvillus-like projections occurred on the tegument between the spines. In the ventral posterior midbody region, the tegument was swollen, though the spines were distinct, projecting from the surface (Fig. 29). Each spine bore one or two blebs at its tip (Fig. 29). Elsewhere, the surface was relatively smooth. On the dorsal surface there was more severe swelling and more extensive blebbing, such that the spines were obscured (Fig. 30). The larger blebs appeared partially collapsed, giving them a shrivelled, raisin-like appearance (Fig. 30). Swelling and blebbing were less severe in the central areas of the fluke than along the lateral margins, and this was true for both surfaces. The tegument on both surfaces in the tail region appeared relatively normal, apart from some swelling and furrowing (Figs. 31, 32). Overall, the anterior region was more severely disrupted than the posterior, and this was particularly evident on the ventral surface. Generally, the dorsal surface was more affected than the ventral surface. In vitro controls The surface morphology of the control specimens appeared normal.
Discussion In the present study, two isolates of F. hepatica were examined for their response to triclabendazole treatment in vivo and to treatment with triclabendazole sulphoxide in vitro. The Oberon isolate of F. hepatica has been shown to be more refractory to triclabendazole than the Fairhurst isolate. The results will be discussed in relation to other isolates of F. hepatica used in previous studies on triclabendazole. The efficacy data has shown that triclabendazole has a very high level of activity against the Fairhurst isolate: against juvenile (2- to 6-week-old) and adult fluke and in both sheep and cattle. The trials were carried out in 2000 (cattle) and 2001 (sheep). The recent sheep trial confirms the susceptible status of the Fairhurst isolate previously
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Fig. 17 SEM of the ventral surface of the oral cone region, showing swelling of the tegument. Oral sucker (OS), gonopore (G), ventral sucker (VS). Bar 500 lm Fig. 18 SEM of the dorsal oral cone, showing swelling of the tegument. Bar 250 lm Fig. 19 High power image of the tegument of the ventral anterior cone. The tegument between the spines (S) is swollen and furrowed (F). The tegument covering the spines is swollen and bears a number of blebs (B). Bar 17 lm Fig. 20 High power image of the dorsal anterior cone, showing that the tegument between the spines (S) is grossly swollen, so that the spines appear sunken. The tegumental surface bears a number of blebs (B). Bar 17 lm Fig. 21 High power image of the lateral margin of the ventral posterior midbody region, showing extensive blebbing (B) of the tegumental surface. The tegument is swollen and furrowed (F) and microvillus-like projections (MV) are present in patches on the surface. Bar 17 lm Fig. 22 High power image of the tegument of the lateral margin of the dorsal posterior midbody region, showing severe swelling of the tegument, which is furrowed (F) and covered in blebs (B) of various sizes. Bar 17 lm Fig. 23 SEM of the surface of the ventral tail region. The tegument is slightly swollen and furrowed. Bar 100 lm Fig. 24 SEM of the dorsal tail region, which is extensively furrowed (F). Bar 170 lm. Inset shows a higher power image of the tegumental surface, which is covered in microvillus-like projections (MV) and single blebs (B). Bar 10 lm
published (and referred to as the Compton isolate) in 1990 as 100% against 6-week-old fluke (Boray 1990). The isolate is also susceptible to other fasciolicides. In contrast, triclabendazole has very limited efficacy against juvenile (2- and 4-week-old) stages of the Oberon isolate, indicating its resistant status. This compares favourably with another triclabendazole-resistant isolate, the Sligo isolate. When first obtained from the field, the efficacy of triclabendazole against the adult fluke was 33% (Coles et al. 2000). After laboratory selection for one generation, the efficacy fell to 0% (Coles and Stafford 2001). In this study, the anterior region of the fluke was more severely disrupted than the posterior region, although this was not true of the Fairhurst isolate at the higher drug concentration. This is in contrast to previous in vitro studies on F. hepatica and F. gigantica involving TCBZ.SO, in which the posterior region of the fluke was more severely affected (Stitt and Fairweather 1993; Meaney et al. 2002). The midbody region of the Cullompton isolate showed the greatest response to TCBZ.SO, in the form of sloughing, although the general surface disruption (in the form of swelling and blebbing) was also more posteriorly-directed. The latter was true for the Sligo isolate, in which the tegumental disruption was very much restricted to the spines (Robinson et al. 2002). In a separate study on the juvenile fluke, no anterior/posterior variation was observed (Stitt and Fairweather 1993). Smeal and Hall (1983) observed an ascending necrosis in flukes recovered from sheep following treatment with triclabendazole, indicating more posteriorly-induced drug damage. The results of the present study revealed that the dorsal
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surface was generally more severely affected than the ventral surface; again, the only exception to this was the Fairhurst isolate at the higher TCBZ.SO concentration used. This is in contrast to a previous study in which no difference was noted between the two surfaces with respect to their response to treatment with TCBZ.SO (Stitt and Fairweather 1993). The result also differs from those obtained with 3-week-old juvenile flukes and with adult F. gigantica, in which the ventral surface was more severely disrupted than the dorsal surface (Stitt and Fairweather 1993; Meaney et al. 2002). With another benzimidazole compound, albendazole sulphoxide, the posterior region of the fluke was more severely affected than the anterior and the dorsal surface more than the ventral surface (Buchanan et al. 2003). Regional differences have been observed in previous anthelmintic studies on F. hepatica and the results linked to oral uptake of drug, which would account for more anteriorly-directed disruption, or to accumulation of drug in the posterior region of the gut, which would account for more posteriorly-directed disruption (see recent papers by McKinstry et al. 2003 and Meaney et al. 2003 for a discussion of this point). Given the variable response to TCBZ.SO seen in the studies mentioned, it seems that no hard-and-fast rule can be applied to the cause(s) of tegumental disruption by the drug. For both isolates, the surface changes observed were more severe at the higher concentration of 50 lg/ml, although in neither case was any tegumental sloughing seen, as described for the Cullompton isolate (Robinson et al. 2002) and an unknown field isolate (Stitt and Fairweather 1993). However, the changes observed in the current study resembled those occurring in the earlier time periods of TCBZ.SO treatment, prior to complete removal of the tegument (Stitt and Fairweather 1993). With respect to tegumental disruption, the Fairhurst isolate was more severely affected than the Oberon isolate. The Oberon isolate was more severely affected than the Sligo isolate: in the latter case, the changes seen were limited to a swelling of the tegument covering the spines (Robinson et al. 2002). The Fairhurst isolate was less severely disrupted than the Cullompton isolate, in that sloughing of the tegument along the lateral margins of the fluke occurred with the latter (Robinson et al. 2002). However, neither of these isolates was as severely affected as the isolate examined by Stitt and Fairweather (1993). In the latter study, the tegument was completely removed from the fluke following TCBZ.SO treatment. From the various studies on triclabendazole, it is evident that isolates shown to be resistant to triclabendazole action in vivo (i.e. the Sligo and Oberon isolates) also display less surface disruption in vitro following treatment with the active, sulphoxide metabolite of triclabendazole. The converse is also true, namely, that isolates regarded as triclabendazole-susceptible from efficacy studies (i.e. the Cullompton and Fairhurst isolates) show more severe tegumental disruption following drug treatment in vitro. However, within the two groups (triclabendazole-susceptible and triclabendazole-
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Fig. 25 SEM of the ventral surface of the oral cone, showing swelling of the tegument. Oral sucker (OS), gonopore (G), ventral sucker (VS). Bar 250 lm. Inset shows a higher power image of several spines (S) from the ventral cone; each spine has a single large bleb (B) on its surface. Bar 25 lm Fig. 26 SEM of the dorsal cone, showing swelling of the tegument. Bar 200 lm. Inset shows a higher power image of the surface in this region. The tegument is swollen and is covered with microvillus-like projections and blebs (B) of various sizes. Spine (S). Bar 17 lm Fig. 27 Lateral margin of the ventral anterior midbody region. There is some swelling of the tegument between the spines (S). Blebs (B) are concentrated along the lateral margin. The tegument covering the spines is also slightly swollen. Bar 17 lm Fig. 28 SEM of the lateral margin of the dorsal anterior midbody, showing the swelling, blebbing (B) and furrowing (F) of the tegument; it is also covered in microvillus-like projections (MV). Bar 25 lm Fig. 29 High power image of the lateral margin of the ventral posterior midbody, showing the swelling of the tegument between the spines (S). Each spine bears one or two blebs (B) at its tip. Bar 17 lm Fig. 30 High power image of the lateral margin of the dorsal posterior midbody, showing the swollen tegument, especially between the spines (S). The tegument is covered in blebs (B) of various sizes. Bar 25 lm Fig. 31 SEM of the surface of the ventral tail region. The tegument is slightly swollen and furrowed (F). Bar 170 lm Fig. 32 Dorsal tail region. The tegument is swollen and furrowed (F). Bar 170 lm
resistant), there is a gradient of disruption from the in vitro morphological data and it is evident that the morphological data (while it supports the efficacy data) perhaps is not as clear-cut as the latter. In terms of their susceptibility to triclabendazole, the isolates can be ranked in the following order: unknown>Cullompton>Fairhurst>Oberon>Sligo. In conclusion, the triclabendazole sensitivity status of two isolates of F. hepatica has been determined by efficacy data, supplemented by morphological data. The Fairhurst isolate can be regarded as triclabendazolesusceptible, whilst the Oberon isolate is triclabendazoleresistant. The maintenance of these two isolates, in addition to the Cullompton and Sligo isolates, will be of value in studies aimed at elucidating the mechanism of action of triclabendazole against the fluke and the mechanism by which the fluke becomes resistant to triclabendazole action. With the continued widespread use of triclabendazole in the field and the spread of resistance to it, it is important to maintain a library of reference isolates of F. hepatica of established susceptibility/resistance to the drug. There are a number of areas where this would be useful: for evaluating the comparative efficacy of triclabendazole against field populations of fluke and the efficacy of alternative drug therapies; for determining which fasciolicides will kill triclabendazole-resistant fluke, thus enabling veterinary surgeons to advise farmers on the therapy of fluke infections; and for the validation of any test for drug resistance, which would facilitate epidemiological studies to determine the true extent of triclabendazole resistance in the field.
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