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The field evaluation of albendazole and triclabendazole efficacy against Fasciola hepatica by coproantigen ELISA in naturally infected sheep
Veterinary Parasitology 190 (2012) 272–276
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
The field evaluation of albendazole and triclabendazole efficacy against Fasciola hepatica by coproantigen ELISA in naturally infected sheep Adam Novobilsky´ a,∗ , Helen Björk Averpil b , Johan Höglund a a Swedish University of Agricultural Sciences (SLU), Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, Box 7028, 75007 Uppsala, Sweden b Lindholmen Veterinärpraktik, Sandövägen 470, 43494 Vallda, Sweden
a r t i c l e
i n f o
Article history: Received 19 March 2012 Received in revised form 18 June 2012 Accepted 19 June 2012
Keywords: Coproantigen ELISA Fasciola hepatica Sheep Albendazole Triclabendazole Anthelmintic resistance Treatment efficacy
a b s t r a c t Albendazole (ABZ) has been used for control of ovine fasciolosis in Sweden for several decades. However, increasing prevalence of Fasciola hepatica in Sweden requires attention to diagnostic tools and current control strategies. The coproantigen reduction test (CRT) using commercial Bio-X K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) was recently suggested as a novel method for diagnosis of patent F. hepatica infection. The aim of this study was to compare the efficacy of albendazole and triclabendazole (TCBZ) treatment against F. hepatica in naturally infected sheep in south-western Sweden by CRT, and also to evaluate the usefulness of this test as a diagnostic marker for anthelmintic efficacy following treatment with ABZ. Three weeks after housing, 24 serologically positive ewes on a commercial farm in Sweden were randomly allocated into three groups, which were either dewormed with 5 mg/kg ABZ, 10 mg/ml TCBZ or left untreated. Twenty-six days after initial administration of these anthelmintics, all of the sheep in the ABZ group and untreated control groups were treated with 10 mg/kg TCBZ. While TCBZ caused elimination of coproantigen and eggs in faeces 7 days after application, ABZ treatment failed completely. Neither anthelmintic resistance, underdosing, nor lack of efficacy due to the presence of immature flukes can be out ruled as possible causes. Despite some deviations in conformity between coproantigen levels and presence of fluke eggs, the CRT was a useful tool for measuring treatment efficacies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The faecal egg count reduction test (FECRT) is the gold standard for the detection of clinical levels of anthelmintic resistance in nematode parasites of ruminants (Coles et al., 1992). Although FECRT has also been widely used to assess drug efficacy against Fasciola hepatica, it has several limitations when used with this trematode. First, the continual release of eggs from the gall bladder even after removal of
∗ Corresponding author. Tel.: +46 18671208; fax: +46 18673334. E-mail addresses: [email protected], ´ [email protected] (A. Novobilsky). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.06.022
adult flukes from the liver causes problems (Fairweather, 2011). Also, intermittent egg shedding and an inability to measure effects during the pre-patent period make it problematic to interpret the results (Mitchell et al., 1998). Recently, a coproantigen enzyme-linked immunosorbent assay (ELISA) was described, measuring levels of specific F. hepatica antigen in ruminant faeces (Mezo et al., 2004, 2008; Valero et al., 2009). Besides being a novel diagnostic tool for patent fluke infection, this test was claimed to be useful assessing anthelmintic efficacy, because unlike the sedimentation technique it allows detection of adult flukes even 4–7 weeks before onset of patency (Valero et al., 2009). Until now, the coproantigen reduction test (CRT) using the commercial Bio-X K201 Fasciola coproantigen
A. Novobilsk´ y et al. / Veterinary Parasitology 190 (2012) 272–276
ELISA assay (Bio-X Diagnostics, Jemelle, Belgium) has, according to our knowledge, been validated only in experimentally infected sheep to assess anthelmintic efficacy and resistance against triclabendazole (TCBZ) (Flanagan et al., 2011a,b). Control of fasciolosis in sheep in Sweden relies mainly on an annual use of the only registered drug, albendazole (ABZ) (Valbazen, Pfizer), which is commonly used during the winter period. On some farms, TCBZ is increasingly applied under special license1 , especially in situations where sheep are suffering from acute fasciolosis caused by immature migrating flukes in the liver. Owing to the increasing prevalence of F. hepatica at Swedish abattoirs in recent years (Dimander, 2009), there is a strong need to evaluate efficacy of treatment and to improve current control strategies. Therefore, this study compares the efficacy of ABZ and TCBZ on a sheep farm in the field using CRT. The aims of the study were both to compare the efficacy of ABZ and TCBZ treatment in a sheep flock that has been suffering from fasciolosis for the last two years, and also to evaluate the usefulness of the CRT as diagnostic marker. 2. Material and methods 2.1. Animals and location of the trial The study was performed in a commercial sheep flock from Halland County in south-western Sweden with 80 ewes of the Gotland breed. Based on recent data from meat inspection, F. hepatica has been found at low prevalence in the flock since the autumn 2010. During the last 2 years the whole flock has been dewormed every year during the housing period with ABZ at the recommended dose rate of 5 mg per kg (Fass vet) and the last treatment was carried out in April 2011 before spring turn out. Before that, ABZ has been used less frequently since a decade. The whole flock was housed in late October 2011 (21 days before start of the study) and all animals were then kept indoors throughout the study period, with water and silage provided ad libitum. 2.2. Study design Eleven days before the start of the study (day − 11), serum samples were collected from all ewes in the herd and examined by indirect ELISA (see below). A total of 24 highly seropositive (sample/positive control ratio > 50) ewes were selected and based on the results of serology, randomly allocated in blocks of three treatment groups, each containing 8 sheep. Ewes were in the range of 2–6 years old and all of them were in the first period of pregnancy. The ewes in group A were treated with 10 mg/kg TCBZ (Fasinex, Novartis Animal Health), those in group B were treated with 5 mg/kg ABZ (Valbazen, Pfizer), and sheep in group C remained as untreated controls. Then 24 days after the ewes in both groups B and C were treated with 10 mg/kg
1 Fasinex is not registered in Sweden and therefore, not available at the market. Veterinarians and farmers may apply to the Swedish Board of Agriculture for exception of using Fasinex and import the drug from abroad.
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TCBZ. The efficacies of both anthelmintics were evaluated by coproantigen reduction test (Flanagan et al., 2011a), whereas patency of infection was monitored by a qualitative sedimentation test (see below). Faecal samples were collected from individual sheep at days 0, 7, 24, 46 and 74. Day 0 was the day of the first application of anthelmintics to groups A and B. Individual faecal samples were collected into plastic gloves which were knotted, coded and stored at 4 ◦ C for up to two days until parasitological analyses. Blood samples for indirect ELISA were collected again at day 24 and were stored at −20 ◦ C until use. 2.3. Parasitological analyses Determination of F. hepatica coproantigen levels in individual faecal samples were performed by Bio-X K201 Fasciola coproantigen ELISA (Bio-X cAg ELISA) (Bio-X Diagnostics, Belgium) according to the manufacturer’s instructions. All individual samples were run in duplicate. Results were expressed as the percentage positivity according to formula: % = OD of sample × 100/OD of positive control. The cut-off value determined by the producer was 6.6% positivity. Qualitative examination of faeces for the presence of F. hepatica eggs was performed blind by a sedimentation technique in an independent veterinary diagnostic laboratory specializing in the monitoring of sheep parasites (Vidilab, Enköping, Sweden), and as described in Thienpont et al. (1979). Antibodies in sera were determined by indirect ELISA using a native excretory/secretory F. hepatica antigen according to Novobilsky´ et al. (2007). Serological results are given as the mean of the optical density (OD) derived from duplicate samples, expressed as the sample/positive control ratio (S/P ratio): S/P ratio = mean OD of sample × 100/mean OD of positive control. Negative and positive controls were included and tested in duplicate on each plate. Reference negative and positive sera were used to establish the cut-off value of 10%. Negative sera were obtained from 50 sheep on 4 Swedish farms without a previous history of fasciolosis, whereas positive sera were collected from various sources of F. hepatica positive sheep flocks. 2.4. Statistics Statistical analysis and graphs were performed using Graph Pad Prism 5.02 (GraphPad Software, USA). Tables were produced using Microsoft Excel (Microsoft Office 2007). The nonparametric Kruskal–Wallis test was used to compare differences of coproantigen levels between the treatment groups. The cut-off value in indirect ELISA was obtained by a receiver operating characteristic (ROC) curve analysis. One way ANOVA was used to compare the differences in S/P ratio obtained by the indirect serum antibody ELISA. 3. Results The seroprevalence in the herd was 67% (n = 80) immediately before the trial was started (day − 11). The course of coproantigen levels and the number of egg-positive sheep in all three groups during the study period are shown in
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Table 1 Numbers of coproantigen positive and egg positive animals during the trial. At day 0, group A and B were treated with triclabendazole (Fasinex) in dose 12 mg/kg and albendazole (Valbazen) in dose 5 mg/kg. At day 24, group B and C were treated with triclabendazole (Fasinex) in dose 12 mg/kg.
Group A Group B Group C
Day 0
Day 7
Day 24
Day 46
Day 74
4 7 4 7 4 7
0 0 3 4 5 7
0 0 3 5 5 7
0 0 0 0 0 0
0 0 0 0 0 0
Fig. 1, Table 1 and Supplementary Table 1. In group A, rapid reductions of coproantigen levels and parasite eggs in faeces were observed already at 7 days post-treatment (PT) with TCBZ. In contrast, there was a slight increase in mean antigen level between 7 and 24 days PT both in the ABZ treated sheep group in B and in the control sheep in C, suggesting a complete failure of the ABZ treatment. On the other hand, when TCBZ was applied to the animals in groups B and C at day 24, it was apparent that parasite antigens and eggs disappeared both at days 46, 74, and as previously observed in the TCBZ treated animals in group A. As shown in Table 1, there was a lack of conformity between coproantigen levels and egg positivity in several animals. For instance, two ewes in group C were egg positive at days 0, 7 and 24 but were negative by Bio-X cAg ELISA (Supplementary Table 1). Furthermore, we found no significant differences in specific antibody levels determined by indirect ELISA before the start of the trial and at 24 days PT (Fig. 2). 4. Discussion Evaluation of anthelmintic efficacy by coproantigen ELISA has been presented as a novel approach for detection of anthelmintic resistance (AR) in F. hepatica (Mezo et al., 2004, 2008; Flanagan et al., 2011a,b). In this study, the coproantigen reduction test (CRT) was used to compare the
Fig. 1. The course of coproantigen values measured by Bio-X cAg ELISA during the study. Triclabendazole (Fasinex) in dose 12 mg/kg and albendazole (Valbazen) in dose 5 mg/kg was applied to group A and B, respectively, at day 0 after collection of faecal samples. At day 24, groups B and C were treated with triclabendazole (Fasinex) in dose 12 mg/kg. The horizontal line inside each box represents the arithmetic mean; whiskers are the 95% confidence interval. (a–c) Different letters indicate significant difference (P < 0.05) between groups at each time point.
Antigen positive Egg positive Antigen positive Egg positive Antigen positive Egg positive
efficacy of two flukicides during the housing period in naturally infected sheep in a flock from a farm in south-western Sweden, and with a high seroprevalence of liver fluke infection. While TCBZ treatment was successful, we observed treatment failure following deworming with ABZ, which was confirmed both by CRT and qualitative coprological examination. Triclabendazole (TCBZ) is usually the anthelmintic of choice against F. hepatica in livestock, as this drug has high activity against both adult and down to 1 week old juvenile flukes (Fairweather, 2005). It has been suggested that efficient TCBZ treatment by the CRT should be defined as the absence of coproantigen in faeces after 14 days PT (Flanagan et al., 2011b). In a previous study with sheep, coproantigen was not detected in 5 out of 6 TCBZ treated lambs after the first week PT, and fluke antigens in faeces disappeared in all 6 animals after 3 weeks (Mezo et al., 2004). Also, Flanagan et al. (2011a) reported that 2 out of 7 TCBZ treated lambs infected with TCBZ susceptible F. hepatica isolate, were coproantigen positive 7 days PT but all animals were found to be negative 14 days PT. In the current study, coproantigens disappeared by 7 days PT with TCBZ and all ewes were negative until the end of the study (74 days PT). The more rapid reduction of coproantigen levels that we observed herein may be explained by the fact that animals in Group A originally could have a lower number of flukes compared to those in previous studies although this remains speculative. Albendazole (ABZ) is a frequently used flukicide throughout the world; it acts only against adult flukes (Buchanan et al., 2003). ABZ has for several decades been massively employed in Swedish sheep flocks. It is the only
Fig. 2. Mean levels of antibodies (expressed as S/P ratio) measured by indirect ELISA. The horizontal line inside each box represents the arithmetic mean of S/P ratio in each group; whiskers are the 95% confidence interval.
A. Novobilsk´ y et al. / Veterinary Parasitology 190 (2012) 272–276
registered flukicide for sheep, and it is also widely used as a nematicidal drug. Therefore, the failure of ABZ treatment observed in this study must be taken into serious account, and the cause of failure still remains speculative and needs to be further investigated. First, coexistence of mature and immature flukes in one individual animal cannot be excluded despite these animals being treated three weeks post housing, in the second week of November, and when mainly adult flukes are expected to occur. Still, presence of immature flukes at the time of deworming cannot be totally excluded as the cause of treatment failure. Second, the recommended dose of ABZ given by the producer for sheep in many other countries is 7.5 mg/kg (Pfizer, USA). However, ewes in our study were treated with 5 mg/kg, which is the recommended dose according to the Swedish Veterinary Authorities (Fass vet). Third, anthelmintic resistance against ABZ has been reported from Spain (Alvarez-Sánchez et al., 2006). In Sweden, ABZ has frequently been used over several decades both as a nematicide and as the only flukicide in sheep. Also, taking into account that the dosage of ABZ recommended by Swedish Veterinary Authorities is 1.5× lower than in other countries, this may have resulted in a widespread selection for flukes that are resistant to this anthelmintic. Thus, it cannot be excluded that AR is one explanation for the observed treatment failure. At the same time, it has to be admitted that this is still speculative and clearly needs further investigation. Interestingly, the finding of egg positive animals by sedimentation was higher than the coproantigen positive individuals by Bio-X cAg ELISA in all tested groups. In contrast, all coproantigen positive animals contained fluke eggs in their faeces. This discrepancy between the two methods is difficult to clarify, due to an absence of necropsy and the determination of fluke burden in livers. It is well known that F. hepatica eggs are shed inconsistently in the faeces and with a high level of variability (Fairweather, 2011). In addition, fluke eggs can also be detected in faeces as false positives, due to their presence in the gall bladder even some days after successful treatment (Flanagan et al., 2011b). In our study, two individual ewes in Group C were found repeatedly to be fluke egg positive on three consecutive collections (days 0, 7 and 24) while simultaneously they remained negative for the presence of coproantigen (Supplementary Table 1). This indicates that capture of coproantigen by the Bio-X cAg ELISA may cause false-negative results. Although it has been claimed that the coproantigen ELISA has a very high sensitivity, with an ability to detect as few as 1 adult fluke (Mezo et al., 2004), this was not supported by our observations. Similarly, in recent study with naturally infected sheep in Scotland, 5 out of 27 monitored lambs containing 2–6 flukes in livers at slaughtering were found negative by the cAg ELISA (Gordon et al., 2012). False-negative results by cAg ELISA were also reported in cattle (Charlier et al., 2008). Therefore, we suggest that sensitivity of cAg ELISA needs to be reconsidered. One possible explanation for these discrepancies in the sensitivity of the coproantigen ELISA could be due to several modifications in the ELISA procedure. For example, in the original protocol (Mezo et al., 2004) 1 g of ovine
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faeces was mixed with 4 ml of distilled water for faecal supernatant preparation, faecal supernatants were incubated overnight at 4 ◦ C, and absorbances were measured at 492 nm. In contrast, according to the protocol of the commercial Bio-X cAg ELISA it is stated that despite the same mixing ratio as in the original method only 0.5 g of faeces is used, faecal supernatants are incubated for 2 h at room temperature, and the results are measured at 450 nm (Bio-X, Jemelle, Belgium). To the best of our knowledge, the sensitivity of Bio-X cAg ELISA has not been validated against the original method described by Mezo et al. (2004). Although we believe that the Bio-X cAg ELISA could be a very useful diagnostic tool for monitoring of anthelmintic efficacy or resistance, the sensitivity of this method needs to be further verified. In conclusion, this study shows that ABZ was not working as expected in a F. hepatica infected sheep flock. On the other hand, all animals responded and were negative for fluke eggs following subsequent deworming with TCBZ. The reason for the lack of treatment efficacy with ABZ may be due to anthelmintic resistance although this warrants further investigations. The coproantigen ELISA was in general a useful tool for measuring treatment efficacies. However, at the same time we found more egg positive animals with the traditional sedimentation technique, which was superior in this respect. Finally, there were no significant differences in specific serum antibody levels during the course of the study, which further emphasizes that measurement of antibody levels is not an appropriate tool to monitor the effects of deworming. Acknowledgements This study was supported by the Swedish Foundation for Agricultural Research (contract no. H1050003) and the 7th framework program of the EU (GLOWORM, Project FP7-KBBE-2012-288975). We thank to Vidilab (Enköping, Sweden) for performing of coprological examinations. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.vetpar.2012.06.022. References 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. 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. Charlier, J., De Meulemeester, L., Claerebout, E., Williams, D., Vercruysse, J., 2008. Qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fasciolosis in cattle. Vet. Parasitol. 153, 44–51. Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44.
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Dimander, S.O., 2009. Stora leverflundran ett ökande problem? Svenska Djurhälsovården. www.svdhv.org/nyhemsida/Artiklar/090929 not leverflundran.html (in Swedish). Fairweather, I., 2005. Triclabendazole: new skills to unravel an old(ish) enigma. J. Helminthol. 79, 227–234. Fairweather, I., 2011. Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy? Vet. Parasitol. 180, 133–143. Flanagan, A., Edgar, H.W.J., Gordon, A., Hanna, R.E.B., Brennan, G.P., Fairweather, I., 2011b. Comparison of two assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance to triclabendazole in Fasciola hepatica in sheep. Vet. Parasitol. 176, 170–176. Flanagan, A.M., Edgar, H.W.J., Forster, F., Gordon, A., Hanna, R.E.B., McCoy, M., Brennan, G.P., Fairweather, I., 2011a. Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica. Vet. Parasitol. 176, 34–42. Gordon, D.K., Zadoks, R.N., Stevenson, H., Sargison, N.D., Skuce, P.J., 2012. On farm evaluation of the coproantigen ELISA and coproantigen reduction test in Scottish sheep naturally infected with Fasciola hepatica. Vet. Parasitol. 187, 436–444.
Mezo, M., González-Warleta, M., Carro, C., Ubeira, F.M., 2004. An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3). J. Parasitol. 90, 845–852. Mezo, M., González-Warleta, M., Castro-Hermida, J.A., Ubeira, F.M., 2008. Evaluation of the flukicide treatment policy for dairy cattle in Galicia (NW Spain). Vet. Parasitol. 157, 235–243. Mitchell, G.B.B., Maris, L., Bonniwell, M.A., 1998. Triclabendazole-resistant liver fluke in Scottish sheep. Vet. Rec. 143, 399. ´ A., Kaˇsny, ´ M., Mikeˇs, L., Kovaˇrcˇ ík, K., Koudela, B., 2007. Novobilsky, Humoral immune responses during experimental infection with Fascioloides magna and Fasciola hepatica in goats and comparison of their excretory/secretory products. Parasitol. Res. 101, 357–364. Thienpont, D., Rochete, F., Vanprijs, O.F., 1979. Diagnosing Helminthiasis Through Coprological Examination. Janssen Research Foundation. Beerse, Belgium. ˜ L., Mezo, M., Valero, M.A., Ubeira, F.M., Khoubbane, M., Artigas, P., Muino, Pérez-Crespo, I., Periago, M.V., Mas-Coma, S., 2009. MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica. Vet. Parasitol. 159, 77–81.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
The field evaluation of albendazole and triclabendazole efficacy against Fasciola hepatica by coproantigen ELISA in naturally infected sheep Adam Novobilsky´ a,∗ , Helen Björk Averpil b , Johan Höglund a a Swedish University of Agricultural Sciences (SLU), Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, Box 7028, 75007 Uppsala, Sweden b Lindholmen Veterinärpraktik, Sandövägen 470, 43494 Vallda, Sweden
a r t i c l e
i n f o
Article history: Received 19 March 2012 Received in revised form 18 June 2012 Accepted 19 June 2012
Keywords: Coproantigen ELISA Fasciola hepatica Sheep Albendazole Triclabendazole Anthelmintic resistance Treatment efficacy
a b s t r a c t Albendazole (ABZ) has been used for control of ovine fasciolosis in Sweden for several decades. However, increasing prevalence of Fasciola hepatica in Sweden requires attention to diagnostic tools and current control strategies. The coproantigen reduction test (CRT) using commercial Bio-X K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) was recently suggested as a novel method for diagnosis of patent F. hepatica infection. The aim of this study was to compare the efficacy of albendazole and triclabendazole (TCBZ) treatment against F. hepatica in naturally infected sheep in south-western Sweden by CRT, and also to evaluate the usefulness of this test as a diagnostic marker for anthelmintic efficacy following treatment with ABZ. Three weeks after housing, 24 serologically positive ewes on a commercial farm in Sweden were randomly allocated into three groups, which were either dewormed with 5 mg/kg ABZ, 10 mg/ml TCBZ or left untreated. Twenty-six days after initial administration of these anthelmintics, all of the sheep in the ABZ group and untreated control groups were treated with 10 mg/kg TCBZ. While TCBZ caused elimination of coproantigen and eggs in faeces 7 days after application, ABZ treatment failed completely. Neither anthelmintic resistance, underdosing, nor lack of efficacy due to the presence of immature flukes can be out ruled as possible causes. Despite some deviations in conformity between coproantigen levels and presence of fluke eggs, the CRT was a useful tool for measuring treatment efficacies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The faecal egg count reduction test (FECRT) is the gold standard for the detection of clinical levels of anthelmintic resistance in nematode parasites of ruminants (Coles et al., 1992). Although FECRT has also been widely used to assess drug efficacy against Fasciola hepatica, it has several limitations when used with this trematode. First, the continual release of eggs from the gall bladder even after removal of
∗ Corresponding author. Tel.: +46 18671208; fax: +46 18673334. E-mail addresses: [email protected], ´ [email protected] (A. Novobilsky). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.06.022
adult flukes from the liver causes problems (Fairweather, 2011). Also, intermittent egg shedding and an inability to measure effects during the pre-patent period make it problematic to interpret the results (Mitchell et al., 1998). Recently, a coproantigen enzyme-linked immunosorbent assay (ELISA) was described, measuring levels of specific F. hepatica antigen in ruminant faeces (Mezo et al., 2004, 2008; Valero et al., 2009). Besides being a novel diagnostic tool for patent fluke infection, this test was claimed to be useful assessing anthelmintic efficacy, because unlike the sedimentation technique it allows detection of adult flukes even 4–7 weeks before onset of patency (Valero et al., 2009). Until now, the coproantigen reduction test (CRT) using the commercial Bio-X K201 Fasciola coproantigen
A. Novobilsk´ y et al. / Veterinary Parasitology 190 (2012) 272–276
ELISA assay (Bio-X Diagnostics, Jemelle, Belgium) has, according to our knowledge, been validated only in experimentally infected sheep to assess anthelmintic efficacy and resistance against triclabendazole (TCBZ) (Flanagan et al., 2011a,b). Control of fasciolosis in sheep in Sweden relies mainly on an annual use of the only registered drug, albendazole (ABZ) (Valbazen, Pfizer), which is commonly used during the winter period. On some farms, TCBZ is increasingly applied under special license1 , especially in situations where sheep are suffering from acute fasciolosis caused by immature migrating flukes in the liver. Owing to the increasing prevalence of F. hepatica at Swedish abattoirs in recent years (Dimander, 2009), there is a strong need to evaluate efficacy of treatment and to improve current control strategies. Therefore, this study compares the efficacy of ABZ and TCBZ on a sheep farm in the field using CRT. The aims of the study were both to compare the efficacy of ABZ and TCBZ treatment in a sheep flock that has been suffering from fasciolosis for the last two years, and also to evaluate the usefulness of the CRT as diagnostic marker. 2. Material and methods 2.1. Animals and location of the trial The study was performed in a commercial sheep flock from Halland County in south-western Sweden with 80 ewes of the Gotland breed. Based on recent data from meat inspection, F. hepatica has been found at low prevalence in the flock since the autumn 2010. During the last 2 years the whole flock has been dewormed every year during the housing period with ABZ at the recommended dose rate of 5 mg per kg (Fass vet) and the last treatment was carried out in April 2011 before spring turn out. Before that, ABZ has been used less frequently since a decade. The whole flock was housed in late October 2011 (21 days before start of the study) and all animals were then kept indoors throughout the study period, with water and silage provided ad libitum. 2.2. Study design Eleven days before the start of the study (day − 11), serum samples were collected from all ewes in the herd and examined by indirect ELISA (see below). A total of 24 highly seropositive (sample/positive control ratio > 50) ewes were selected and based on the results of serology, randomly allocated in blocks of three treatment groups, each containing 8 sheep. Ewes were in the range of 2–6 years old and all of them were in the first period of pregnancy. The ewes in group A were treated with 10 mg/kg TCBZ (Fasinex, Novartis Animal Health), those in group B were treated with 5 mg/kg ABZ (Valbazen, Pfizer), and sheep in group C remained as untreated controls. Then 24 days after the ewes in both groups B and C were treated with 10 mg/kg
1 Fasinex is not registered in Sweden and therefore, not available at the market. Veterinarians and farmers may apply to the Swedish Board of Agriculture for exception of using Fasinex and import the drug from abroad.
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TCBZ. The efficacies of both anthelmintics were evaluated by coproantigen reduction test (Flanagan et al., 2011a), whereas patency of infection was monitored by a qualitative sedimentation test (see below). Faecal samples were collected from individual sheep at days 0, 7, 24, 46 and 74. Day 0 was the day of the first application of anthelmintics to groups A and B. Individual faecal samples were collected into plastic gloves which were knotted, coded and stored at 4 ◦ C for up to two days until parasitological analyses. Blood samples for indirect ELISA were collected again at day 24 and were stored at −20 ◦ C until use. 2.3. Parasitological analyses Determination of F. hepatica coproantigen levels in individual faecal samples were performed by Bio-X K201 Fasciola coproantigen ELISA (Bio-X cAg ELISA) (Bio-X Diagnostics, Belgium) according to the manufacturer’s instructions. All individual samples were run in duplicate. Results were expressed as the percentage positivity according to formula: % = OD of sample × 100/OD of positive control. The cut-off value determined by the producer was 6.6% positivity. Qualitative examination of faeces for the presence of F. hepatica eggs was performed blind by a sedimentation technique in an independent veterinary diagnostic laboratory specializing in the monitoring of sheep parasites (Vidilab, Enköping, Sweden), and as described in Thienpont et al. (1979). Antibodies in sera were determined by indirect ELISA using a native excretory/secretory F. hepatica antigen according to Novobilsky´ et al. (2007). Serological results are given as the mean of the optical density (OD) derived from duplicate samples, expressed as the sample/positive control ratio (S/P ratio): S/P ratio = mean OD of sample × 100/mean OD of positive control. Negative and positive controls were included and tested in duplicate on each plate. Reference negative and positive sera were used to establish the cut-off value of 10%. Negative sera were obtained from 50 sheep on 4 Swedish farms without a previous history of fasciolosis, whereas positive sera were collected from various sources of F. hepatica positive sheep flocks. 2.4. Statistics Statistical analysis and graphs were performed using Graph Pad Prism 5.02 (GraphPad Software, USA). Tables were produced using Microsoft Excel (Microsoft Office 2007). The nonparametric Kruskal–Wallis test was used to compare differences of coproantigen levels between the treatment groups. The cut-off value in indirect ELISA was obtained by a receiver operating characteristic (ROC) curve analysis. One way ANOVA was used to compare the differences in S/P ratio obtained by the indirect serum antibody ELISA. 3. Results The seroprevalence in the herd was 67% (n = 80) immediately before the trial was started (day − 11). The course of coproantigen levels and the number of egg-positive sheep in all three groups during the study period are shown in
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Table 1 Numbers of coproantigen positive and egg positive animals during the trial. At day 0, group A and B were treated with triclabendazole (Fasinex) in dose 12 mg/kg and albendazole (Valbazen) in dose 5 mg/kg. At day 24, group B and C were treated with triclabendazole (Fasinex) in dose 12 mg/kg.
Group A Group B Group C
Day 0
Day 7
Day 24
Day 46
Day 74
4 7 4 7 4 7
0 0 3 4 5 7
0 0 3 5 5 7
0 0 0 0 0 0
0 0 0 0 0 0
Fig. 1, Table 1 and Supplementary Table 1. In group A, rapid reductions of coproantigen levels and parasite eggs in faeces were observed already at 7 days post-treatment (PT) with TCBZ. In contrast, there was a slight increase in mean antigen level between 7 and 24 days PT both in the ABZ treated sheep group in B and in the control sheep in C, suggesting a complete failure of the ABZ treatment. On the other hand, when TCBZ was applied to the animals in groups B and C at day 24, it was apparent that parasite antigens and eggs disappeared both at days 46, 74, and as previously observed in the TCBZ treated animals in group A. As shown in Table 1, there was a lack of conformity between coproantigen levels and egg positivity in several animals. For instance, two ewes in group C were egg positive at days 0, 7 and 24 but were negative by Bio-X cAg ELISA (Supplementary Table 1). Furthermore, we found no significant differences in specific antibody levels determined by indirect ELISA before the start of the trial and at 24 days PT (Fig. 2). 4. Discussion Evaluation of anthelmintic efficacy by coproantigen ELISA has been presented as a novel approach for detection of anthelmintic resistance (AR) in F. hepatica (Mezo et al., 2004, 2008; Flanagan et al., 2011a,b). In this study, the coproantigen reduction test (CRT) was used to compare the
Fig. 1. The course of coproantigen values measured by Bio-X cAg ELISA during the study. Triclabendazole (Fasinex) in dose 12 mg/kg and albendazole (Valbazen) in dose 5 mg/kg was applied to group A and B, respectively, at day 0 after collection of faecal samples. At day 24, groups B and C were treated with triclabendazole (Fasinex) in dose 12 mg/kg. The horizontal line inside each box represents the arithmetic mean; whiskers are the 95% confidence interval. (a–c) Different letters indicate significant difference (P < 0.05) between groups at each time point.
Antigen positive Egg positive Antigen positive Egg positive Antigen positive Egg positive
efficacy of two flukicides during the housing period in naturally infected sheep in a flock from a farm in south-western Sweden, and with a high seroprevalence of liver fluke infection. While TCBZ treatment was successful, we observed treatment failure following deworming with ABZ, which was confirmed both by CRT and qualitative coprological examination. Triclabendazole (TCBZ) is usually the anthelmintic of choice against F. hepatica in livestock, as this drug has high activity against both adult and down to 1 week old juvenile flukes (Fairweather, 2005). It has been suggested that efficient TCBZ treatment by the CRT should be defined as the absence of coproantigen in faeces after 14 days PT (Flanagan et al., 2011b). In a previous study with sheep, coproantigen was not detected in 5 out of 6 TCBZ treated lambs after the first week PT, and fluke antigens in faeces disappeared in all 6 animals after 3 weeks (Mezo et al., 2004). Also, Flanagan et al. (2011a) reported that 2 out of 7 TCBZ treated lambs infected with TCBZ susceptible F. hepatica isolate, were coproantigen positive 7 days PT but all animals were found to be negative 14 days PT. In the current study, coproantigens disappeared by 7 days PT with TCBZ and all ewes were negative until the end of the study (74 days PT). The more rapid reduction of coproantigen levels that we observed herein may be explained by the fact that animals in Group A originally could have a lower number of flukes compared to those in previous studies although this remains speculative. Albendazole (ABZ) is a frequently used flukicide throughout the world; it acts only against adult flukes (Buchanan et al., 2003). ABZ has for several decades been massively employed in Swedish sheep flocks. It is the only
Fig. 2. Mean levels of antibodies (expressed as S/P ratio) measured by indirect ELISA. The horizontal line inside each box represents the arithmetic mean of S/P ratio in each group; whiskers are the 95% confidence interval.
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registered flukicide for sheep, and it is also widely used as a nematicidal drug. Therefore, the failure of ABZ treatment observed in this study must be taken into serious account, and the cause of failure still remains speculative and needs to be further investigated. First, coexistence of mature and immature flukes in one individual animal cannot be excluded despite these animals being treated three weeks post housing, in the second week of November, and when mainly adult flukes are expected to occur. Still, presence of immature flukes at the time of deworming cannot be totally excluded as the cause of treatment failure. Second, the recommended dose of ABZ given by the producer for sheep in many other countries is 7.5 mg/kg (Pfizer, USA). However, ewes in our study were treated with 5 mg/kg, which is the recommended dose according to the Swedish Veterinary Authorities (Fass vet). Third, anthelmintic resistance against ABZ has been reported from Spain (Alvarez-Sánchez et al., 2006). In Sweden, ABZ has frequently been used over several decades both as a nematicide and as the only flukicide in sheep. Also, taking into account that the dosage of ABZ recommended by Swedish Veterinary Authorities is 1.5× lower than in other countries, this may have resulted in a widespread selection for flukes that are resistant to this anthelmintic. Thus, it cannot be excluded that AR is one explanation for the observed treatment failure. At the same time, it has to be admitted that this is still speculative and clearly needs further investigation. Interestingly, the finding of egg positive animals by sedimentation was higher than the coproantigen positive individuals by Bio-X cAg ELISA in all tested groups. In contrast, all coproantigen positive animals contained fluke eggs in their faeces. This discrepancy between the two methods is difficult to clarify, due to an absence of necropsy and the determination of fluke burden in livers. It is well known that F. hepatica eggs are shed inconsistently in the faeces and with a high level of variability (Fairweather, 2011). In addition, fluke eggs can also be detected in faeces as false positives, due to their presence in the gall bladder even some days after successful treatment (Flanagan et al., 2011b). In our study, two individual ewes in Group C were found repeatedly to be fluke egg positive on three consecutive collections (days 0, 7 and 24) while simultaneously they remained negative for the presence of coproantigen (Supplementary Table 1). This indicates that capture of coproantigen by the Bio-X cAg ELISA may cause false-negative results. Although it has been claimed that the coproantigen ELISA has a very high sensitivity, with an ability to detect as few as 1 adult fluke (Mezo et al., 2004), this was not supported by our observations. Similarly, in recent study with naturally infected sheep in Scotland, 5 out of 27 monitored lambs containing 2–6 flukes in livers at slaughtering were found negative by the cAg ELISA (Gordon et al., 2012). False-negative results by cAg ELISA were also reported in cattle (Charlier et al., 2008). Therefore, we suggest that sensitivity of cAg ELISA needs to be reconsidered. One possible explanation for these discrepancies in the sensitivity of the coproantigen ELISA could be due to several modifications in the ELISA procedure. For example, in the original protocol (Mezo et al., 2004) 1 g of ovine
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faeces was mixed with 4 ml of distilled water for faecal supernatant preparation, faecal supernatants were incubated overnight at 4 ◦ C, and absorbances were measured at 492 nm. In contrast, according to the protocol of the commercial Bio-X cAg ELISA it is stated that despite the same mixing ratio as in the original method only 0.5 g of faeces is used, faecal supernatants are incubated for 2 h at room temperature, and the results are measured at 450 nm (Bio-X, Jemelle, Belgium). To the best of our knowledge, the sensitivity of Bio-X cAg ELISA has not been validated against the original method described by Mezo et al. (2004). Although we believe that the Bio-X cAg ELISA could be a very useful diagnostic tool for monitoring of anthelmintic efficacy or resistance, the sensitivity of this method needs to be further verified. In conclusion, this study shows that ABZ was not working as expected in a F. hepatica infected sheep flock. On the other hand, all animals responded and were negative for fluke eggs following subsequent deworming with TCBZ. The reason for the lack of treatment efficacy with ABZ may be due to anthelmintic resistance although this warrants further investigations. The coproantigen ELISA was in general a useful tool for measuring treatment efficacies. However, at the same time we found more egg positive animals with the traditional sedimentation technique, which was superior in this respect. Finally, there were no significant differences in specific serum antibody levels during the course of the study, which further emphasizes that measurement of antibody levels is not an appropriate tool to monitor the effects of deworming. Acknowledgements This study was supported by the Swedish Foundation for Agricultural Research (contract no. H1050003) and the 7th framework program of the EU (GLOWORM, Project FP7-KBBE-2012-288975). We thank to Vidilab (Enköping, Sweden) for performing of coprological examinations. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.vetpar.2012.06.022. References 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. 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. Charlier, J., De Meulemeester, L., Claerebout, E., Williams, D., Vercruysse, J., 2008. Qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fasciolosis in cattle. Vet. Parasitol. 153, 44–51. Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44.
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