Screening anthelmintic resistance to triclabendazole in Fasciola hepatica isolated from sheep by means of an egg hatch assay

Robles-Pérez et al. BMC Veterinary Research (2015) 11:226 DOI 10.1186/s12917-015-0543-1 RESEARCH ARTICLE Open Access Screening anthelmintic resistance to triclabendazole in Fasciola hepatica isolated from sheep by means of an egg hatch assay David Robles-Pérez1, José Manuel Martínez-Pérez1, Francisco Antonio Rojo-Vázquez1,2 and María Martínez-Valladares2* Abstract Background: In the present study, the detection of anthelmintic resistance to triclabendazole (TCBZ) in sheep infected by Fasciola hepatica was studied using an egg hatch assay (EHA). F. hepatica eggs were recovered from bile and faeces of infected animals by isolates with different grade of anthelmintic resistance to TCBZ: i) a resistant isolate (RT); ii) a susceptible isolate (ST); iii) naturally infected sheep by a susceptible field strain (FST). In the EHA the percentage of hatched eggs were calculated according to the following concentrations of TCBZ diluted in dimethyl-sulfoxide (DMSO): 0.05, 0.2, 1, 5, and 25 μg/ml. Results: In relation to the EHAs carried out with the eggs from bile of sheep infected by ST, differences were found in the percentage of hatched eggs between the control well, only with DMSO, and the two highest concentrations of TCBZ (5 and 25 μg/m) (p < 0.05). However, when we tested the drug with the eggs from the bile of sheep infected by RT, the percentage of hatched eggs was similar among all concentrations. Since the range of hatching varied between isolates, we calculated the ratio of the results of each concentration to its control value confirming the higher hatching in RT than in ST. We developed an EHA with eggs recovered from faeces in order to avoid the slaughter of sheep. The results of the EHAs with the isolate ST showed differences in the percentage of hatching between the highest concentration (25 μg/ml) and the control well (p < 0.05); however, these differences were not confirmed under field conditions with the strain FST. Conclusions: The ovicidal effect of TCBZ in F. hepatica eggs from bile was shown using a commercial formulation diluted in DMSO with a minimum concentration of 5 μg/ml. However, in eggs recovered from faeces the results are not conclusive. The cleaning of eggs recovered from faeces is an important issue that should be reviewed and standardized before comparing results between susceptible and resistant isolates in this kind of EHA. Keywords: Fasciola hepatica, Anthelmintic resistance, Egg hatch assay, Triclabendazole Background Fasciola spp. infects mammals worldwide, mainly ruminants, but also humans can become infected. In ruminants, and especially in sheep, the infection reduces feed conversion, growth, and meat and milk production. Moreover, it is one of the major causes of liver condemnations at abattoirs and interferes with fertility and fecundity [1]. * Correspondence: 2 Instituto de Ganadería de Montaña (CSIC-ULE), Finca de Marzanas, 24346 Grulleros, León, Spain Full list of author information is available at the end of the article The infection is usually caused, in temperate areas of the world, by the common liver fluke F. hepatica. Its prevalence is rising nowadays due to different factors such as climate change, man-made environmental modifications or the presence of anthelmintic resistance (AR) [1, 2]. AR is the result of repeated administration of the same anthelmintic; moreover, its development has been favoured by ineffective treatment due to the underdosing. AR is the result of repeated treatments of the same anthelmintic although its development is also favoured by the administration of underdosing or overdosing [3]. © 2015 Robles-Pérez et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Robles-Pérez et al. BMC Veterinary Research (2015) 11:226 Currently, the most commonly used drug to control fasciolosis belongs to benzimidazole (BZ) family and is the triclabendazole (TCBZ). TCBZ has been the drug of choice for treating liver fluke infections in livestock for over 20 years [4] since it is the only anthelmintic effective against both F. hepatica stages, immature and mature flukes [5]. However, there are several reports describing resistant strains of F. hepatica to TCBZ all around the world, in Australia [6], Argentina [7] and also in different European countries [8–11]. Therefore, early detection of resistance is essential, since reversion to susceptibility does not seem to occur [12]. Some in vivo and in vitro tests have been developed to detect the AR in ruminants. Among the in vivo tests, the faecal egg count reduction test (FECRT) is based on the reduction of the number of eggs in faeces after the anthelmintic treatment [13]. Regarding in vitro tests, an egg hatch assay (EHA) has been described to detect BZ resistance in Trichostrongylidae [14, 15]. The EHA is based on the ovicidal properties of some BZs, and on the capacity of eggs from resistant isolates to embrionate and hatch at higher concentrations than those ones from a susceptible isolate [16]. Although the EHA was originally designed to detect AR in gastrointestinal nematodes (GIN), some studies have been carried out with F. hepatica eggs from gall bladder and/or faeces using TCBZ, albendazole (ABZ) and their sulphoxide metabolites [17–19]. The aim of this study has been to characterize the susceptibility and resistance of F. hepatica isolates to TCBZ by means of an EHA using eggs from gall bladder and faeces. Page 2 of 5 Egg hatch assays A commercial formulation of TCBZ (Fasinex®) diluted in dimethyl sulfoxide (DMSO) was used to carry out the EHAs. The concentration of TCBZ in this commercial formulation was 50 mg/ml. Dilutions of 10, 40, 200, 1000 and 5000 μg/ml were prepared to obtain a final concentration in the wells of 0.05, 0.2, 1, 5, and 25 μg/ ml after adding 10 μl of each dilution to a total volume of 2 ml. In all EHAs, control wells with 10 μl of DMSO were included. Eggs from faeces were obtained by sedimentation [22] from animals infected by ST and from a pool of faeces of sheep naturally infected by FST. Four sheep, two infected with ST and two with RT, were killed by injection of sodium pentobarbital (Dolethal®) into the jugular vein in order to recover eggs from the bile. F. hepatica eggs were directly recovered from the gall bladder and washed several times with tap water by sedimentation. The slaughter of animals complies with national regulations (R.D. 53/ 2013) and with all animal welfare standards, taking (...truncated)