An experimental study on triclabendazole resistance of Fasciola hepatica in sheep

Veterinary Parasitology 95 (2001) 37 43 An experimental study on triclabendazole resistance of Fasciola hepatica in sheep C.P.H. Gaasenbeek a,, L. Moll b, J.B.W.J. Cornelissen a, P. Vellema b, F.H.M. Borgsteede a a Institute for Animal Science and Health (ID-Lelystad), PO Box 65, 8200 AB Lelystad, Netherlands b Animal Health Service (Location Drachten), PO Box 361, 9200 AJ Drachten, Netherlands Received 24 May 2000; received in revised form 5 September 2000; accepted 14 September 2000 Abstract The efficacy of triclabendazole in sheep experimentally infected with Fasciola hepatica was studied. Two groups of 12 lambs were infected with a susceptible (S) or a resistant (R) strain of F. hepatica. Eight weeks after infection, six lambs of each group (ST and RT) were treated with triclabendazole (10 mg/kg). The other lambs were used as untreated controls (SC and RC). The parameters studied were: GLDH, -GT, ELISA measuring antibodies against recombinant cathepsin-l 1 and eggs per gram faeces (epg). The lambs were slaughtered 16 weeks after infection and the number of flukes counted. The GLDH, -GT levels and the OD value of the ELISA decreased as a result of the treatment in group ST. Patent infections were observed in all animals of groups SC, RT and RC. In group ST, occasionally a few eggs were found in five lambs. The percentage of flukes was 31.3 in SC and 37.6 in RC. In the treated groups ST and RT, the percentage of flukes was 0.06 and 33.6, respectively. These results corresponded to efficacies of 99.8% in the susceptible and 10.8% in the resistant strain. Since the resistant strain was isolated from a mixed cattle and sheep farm, it confirms the presence of triclabendazole resistance in the Netherlands. 2001 Elsevier Science B.V. All rights reserved. Keywords: Fasciola hepatica; Drug resistance; Triclabendazole; Sheep-Trematoda; The Netherlands 1. Introduction The liver fluke, Fasciola hepatica, is a common parasite of ruminants in many countries in the temperate climates and often causes severe economic losses. Effective strategies for the control of fasciolosis are mainly based on the use of drugs. Triclabendazole (TCBZ) Corresponding author. Tel.: +31-320-238238; fax: +31-320-238050. E-mail address: c.p.h.gaasenbeek@id.wag-ur.nl (C.P.H. Gaasenbeek). 0304-4017/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0304-4017(00)00413-1

38 C.P.H. Gaasenbeek et al. / Veterinary Parasitology 95 (2001) 37 43 (Fasinex, Novartis) is worldwide one of the most used drugs for the control of fasciolosis. The efficacy of TCBZ in experimentally infected sheep, 6 weeks after infection, was 100% at a dosage rate of 10 mg/kg (Boray et al., 1983; Turner et al., 1984). Smeal and Hall (1983) found efficacies of 99% in infections from 1 to 12 weeks and Boray (1997) found efficacies of 99 100% in infections from 4 to 14 weeks old. However, resistance of the liver fluke to TCBZ in naturally infected sheep has been reported in Australia (Overend and Bowen, 1995), in Ireland (Lane, 1998; O Brien, 1998), and in Scotland (Mitchell et al., 1998). Recently, resistance to TCBZ was observed on a farm with cattle and sheep in the Netherlands (Moll et al., 2000). The aim of the present experimental study in sheep was to compare the efficacy of TCBZ against the resistant liver fluke strain described in the study of Moll et al. (2000) with a susceptible laboratory strain. 2. Materials and methods 2.1. Animals The experimental study was performed with 24 four-month old Texel male and female lambs. The lambs had been reared indoors and had never been exposed to any helminth infection. The parasite-free status was checked by faecal examination for nematode and liver fluke eggs and by faecal culture for nematode larvae. The lambs were allocated to four groups on the basis of body weight and sex. 2.2. Experimental design and treatment At week 0, 12 lambs were infected with 500 metacercariae of the susceptible strain (S). This strain has been maintained for 30 years on the Institute for Animal Science and Health (ID-Lelystad) and has never been in contact with anthelmintics. The other 12 lambs were infected with 500 metacercariae of the resistant strain (R). This strain was isolated from a sheep which had been treated several times with TCBZ at the recommended dosage rate of 10 mg/kg body weight without a decrease in egg output (Moll et al., 2000). Eight weeks after infection, six lambs of each group were treated orally with the recommended dose of 10 mg/kg body weight TCBZ (ST and RT). The animals in the other groups were used as untreated controls (SC and RC). All lambs were slaughtered 16 weeks after infection. 2.3. Serological and parasitological examinations Blood samples were collected weekly to analyse serum levels of glutamic dehydrogenase (GLDH) and -glutamyl transpeptidase ( -GT). Antibody titres in serum against F. hepatica were tested with an indirect ELISA assay. This assay uses a recombinant F. hepatica cathepsin-l 1 as antigen and was evaluated for its ability to detect antibodies against F. hepatica in sheep and cattle (Cornelissen, personal communication). Blood samples were taken 20 h after the administration of TCBZ to analyse the presence of TCBZ and its metabolites in plasma.

C.P.H. Gaasenbeek et al. / Veterinary Parasitology 95 (2001) 37 43 39 From 8 weeks onwards, faecal samples were taken. The number of F. hepatica eggs per gram faeces (epg) was determined with the Dorsman technique (Dorsman, 1965). All lambs were slaughtered 16 weeks after infection. The livers were examined and the number of flukes was counted (Ross and O Hagan, 1966). 2.4. Statistical analysis The efficacy percentage was calculated for the susceptible and the resistant strain using the following formula: Percentage efficacy = mean number of flukes of group SC or RC mean number of flukes of group ST or RT mean number of flukes of group SC or RC 100 A paired t-test was used to perform statistical analysis. Significance of differences in the number of liver flukes between the groups was analysed with the method of least-squares. 3. Results 3.1. Clinical observations In group RC, one sheep died 13 weeks after infection from a cause other than fasciolosis. The flukes of this animal were collected and used as normal data. 3.2. Serological examinations The GLDH serum levels indicated that the infection had well established in all lambs (Fig. 1). The GLDH serum levels of group ST decreased significantly (P <0.05) within 1 week after treatment. In addition, the -GT serum levels started to elevate when the liver flukes arrived in the bile ducts 8 weeks after infection in the groups SC, RC and RT. The -GT level in group ST remained on a low level (P <0.05) and indicated that the treatment had killed the liver flukes (Fig. 2). Antibodies of F. hepatica measured in sera with the indirect ELISA test increased after 3 weeks in all groups (Fig. 3). After treatment, the OD value of the ST group decreased significantly (P <0.05) compared with groups SC, RC and RT, but still showed a positive extinction. From 8 weeks onwards, a titration from 1/100 to 1/12 800 of the sera of all groups was performed. A seroconversion was seen in groups SC, RC and RT at a dilution of 1/6400. Group ST showed a seroconversion at a dilution of 1/800 (data not shown). Plasma levels of sulphoxide and sulphone metabolites of TCBZ taken 20 h after treatment varied between 9.650 14.878 and 5.214 12.052 g/ml, respectively.

40 C.P.H. Gaasenbeek et al. / Veterinary Parasitology 95 (2001) 37 43 Fig. 1. Mean GLDH values of sheep in group SC (closed square), group ST (open square), group RC (closed circle) and group RT (open circle). Arrow indicates treatment with TCBZ. Fig. 2. Mean -GT values of sheep in group SC (closed square), group ST (open square), group RC (closed circle) and group RT (open circle). Arrow indicates treatment with TCBZ. 3.3. Faecal examinations The results of the faecal examinations are illustrated in Fig. 4, eggs of F. hepatica were found in faeces from 10 weeks post-infection. In groups SC, RC and RT, the epg increased until the end of the experiment. A few eggs per gram (0.3 1) were observed in the faeces of five of the lambs in group ST. 3.4. Post-mortem worm counts Table 1 summarizes the mean number of flukes and the percentage efficacy of the anthelmintic treatment. In group SC, 31.3% of the original challenge of F. hepatica was

C.P.H. Gaasenbeek et al. / Veterinary Parasitology 95 (2001) 37 43 41 Fig. 3. Mean OD values in the indirect F. hepatica ELISA test of sheep in group SC (closed square), group ST (open square), group RC (closed circle) and group RT (open circle). Arrow indicates treatment with TCBZ. Fig. 4. Mean number of epg of sheep in group SC (closed square), group ST (open square), group RC (closed circle) and group RT (open circle) following treatment with TCBZ at week 8. Table 1 Mean number of flukes (±S.E.M.) recovered from sheep infected with F. hepatica and slaughtered after 16 weeks Group Number of flukes (±S.E.M.) Range Percentage recovery Percentage efficacy Susceptible not treated (SC) 156.67 (14.17) 108 193 31.33 Susceptible treated a (ST) 0.33 (0.23) 0 1 0.06 99.78 Resistant not treated (RC) 188.33 (24.86) 105 276 37.66 Resistant treated a (RT) 168.17 (27.80) 52 230 33.63 10.78 a Treatment: 10 mg/kg TCBZ.

42 C.P.H. Gaasenbeek et al. / Veterinary Parasitology 95 (2001) 37 43 recovered. In group RC, the comparable recovery was 37.6%. Treated sheep showed a mean reduction of fluke burden of 99.8% (SC vs. ST) and 10.8% (RC vs. RT). The number of flukes in group ST was significantly reduced compared with groups SC, RC and RT (P <0.05). The fluke burdens of groups SC, RC and RT were not significantly different. Post-mortem results showed that not all flukes were in the adult stage. In one sheep of the SC group, the majority of the flukes was immature. 4. Discussion The present experiment confirms the resistance to TCBZ of the F. hepatica strain isolated from a mixed cattle and sheep farm in the Netherlands (Moll et al., 2000). All serological and parasitological parameters, GLDH, -GT, ELISA and epg showed that the infection had established well in all lambs. GLDH and -GT provided indication of liver damage associated with pre-patent F. hepatica infection as described previously (van Tiggele, 1978). The low -GT, GLDH and epg values in group ST following treatment all provided evidence of efficacy of TCBZ against susceptible flukes. The indirect ELISA assay based on recombinant F. hepatica cathepsin-l 1 provides a useful method of detecting antibodies against F. hepatica at an early stage, the assay has a specificity of 95.7% and a sensitivity of 100% (Cornelissen, personal communication). However, the effect of the treatment in group ST was only seen when a titration in the indirect ELISA assay was performed. Following administration of TCBZ at 10 mg/kg, the plasma metabolite concentrations fell within the expected range suggesting that liver damage could be excluded as a possible cause of reduced efficacy of TCBZ. The efficacy of TCBZ against the susceptible strain was 99.8% eight weeks after infection. This is comparable with other results in sheep (Boray et al., 1983; Boray, 1997; Dorchies et al., 1983; Smeal and Hall, 1983; Turner et al., 1984). According to Fairweather and Boray (1999), no new fasciolicides are expected to be marketed in near future, so different strategies have been proposed to slow down the development of anthelmintic resistance. Strategic treatment with anthelmintics based on the knowledge of liver fluke epidemiology and a good farm management can reduce infection to a low level and control disease with a low treatment frequency (Boray et al., 1985; Malone, 1997). Reduction of treatment frequency may also reduce the chance of the development of drug resistance, especially in high risk areas where more treatments appear to be necessary. In these areas, it may be necessary to develop strategic treatment regimes which minimise treatment frequency and to monitor treatment efficacy. Obviously, the faecal egg count reduction test only provides an indication of efficacy during patency and cannot detect developing resistance amongst immature stages. Although studies in Australia have demonstrated that the use of combination of drugs with different modes of action can be effective against resistant strains of F. hepatica (Boray, 1993, 1997), this approach has the risk of building up multiple drug resistance. Other alternatives for the control of fasciolosis has led to the testing of vaccines with potential antigens as glutathione-s-transferase and cathepsin-l 1 (Spithill and Dalton, 1998).

C.P.H. Gaasenbeek et al. / Veterinary Parasitology 95 (2001) 37 43 43 In the last 10 years, resistance to TCBZ has been found in Australia and Europe. In order to avoid development of resistance, farmers need advice about the use of anthelmintics using well-calibrated dosing equipment and avoiding underdosing by dosing for the heaviest animal within the group being treated. In the Netherlands, the incidence of fasciolosis has been controlled using an improved liver fluke forecast system, the use of TCBZ in a strategic regime and a good farm management. Despite these measures, resistance is present in the country albeit at not a large scale. In order to minimize the rate of development of drug resistance, it is necessary for producers to use the existing drugs in the most effective way. Acknowledgements The authors acknowledge Novartis Agro Benelux, the Netherlands for the financial support and Dr. M. Jung Novartis Animal Health, Switzerland, for the analyses of the triclabendazole metabolites. We also thank Mr. J. Tibben for his technical assistance. References Boray, J.C., 1993. Synergistic activity of anthelmintics for the control of susceptible and resistant strains of Fasciola hepatica for the prevention or management of anthelmintic resistance. In: Proceedings of the 14th International Conference of the WAAVP, Cambridge, p. 370. Boray, J.C., 1997. Chemotherapy of infections with Fasciolidae. In: Boray, J.C. (Ed.), Immunology, Pathobiology and Control of Fasciolosis. MSD AGVET, Rahway, NJ, pp. 83 97. Boray, J.C., Crowfoot, P.D., Strong, M.B., Allison, J.R., Schellembaum, M., von Orelli, M., Sarasin, G., 1983. Treatment of immature and mature Fasciola hepatica infections in sheep with triclabendazole. Vet. Rec. 113, 315 317. Boray, J.C., Jackson, R., Strong, M.B., 1985. Chemoprophylaxis of fascioliasis with triclabendazole. NZ Vet. J. 33, 182 185. Dorchies, P., Franc, M., De Lahitte, J.D., 1983. Etude de l activité du triclabendazole (DCI) sur Fasciola hepatica chez l agneau. Rev. Med. Vet. 134, 231 234. Dorsman, W., 1965. A new technique for counting eggs of Fasciola hepatica in cattle faeces. J. Helminthol. 30, 165. Fairweather, I., Boray, J.C., 1999. Fasciolicides: efficacy, actions, resistance and its management. Vet. J. 158, 81 112. Lane, G., 1998. Anthelmintic resistance. Vet. Rec. 143, 332. Malone, J.B., 1997. The landscape epidemiology of fasciolosis: geographic determinants of disease risk. In: Boray, J.C. (Ed.), Immunology, Pathobiology and Control of Fasciolosis. MSD AGVET, Rahway, NJ, pp. 65 81. Mitchell, G.B., Maris, L., Bonniwell, M.A., 1998. Triclabendazole-resistant liver fluke in scottish sheep. Vet. Rec. 143, 399. 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. Rec. 91, 153 158. O Brien, D.J., 1998. Fasciolosis: a threat to livestock. Irish Vet. J. 51, 539 541. Overend, D.J., Bowen, F.L., 1995. Resistance of Fasciola hepatica to triclabendazole. Aust. Vet. J. 72, 275. Ross, J.G., O Hagan, J., 1966. A biological technique to assess numbers of Fasciola hepatica metacercariae on pasture. J. Helminthol. 40, 173. Smeal, M.G., Hall, C.A., 1983. The activity of triclabendazole against immature and adult Fasciola hepatica infections in sheep. Aust. Vet. J. 60, 329 331. Spithill, T.W., Dalton, J.P., 1998. Progress in development of liver fluke vaccines. Parasitol. Today 14, 224 228. Turner, K., Armour, J., Richards, R.J., 1984. Anthelmintic efficacy of triclabendazole against Fasciola hepatica in sheep. Vet. Rec. 114, 41 42. van Tiggele, L.J., 1978. Host parasite relations in Fasciola hepatica infections. Immunopathology and diagnosis of liver fluke disease in ruminants. Thesis Rijksuniversiteit Leiden, 164 pp.