Testing albendazole resistance in Fasciola hepatica: validation of an egg hatch test with isolates from South America and the United Kingdom

Abstract

The main goal of the current work was to develop and validate an in vitro fluke egg hatch test, as a method for the detection of albendazole (ABZ) resistance in the liver fluke, Fasciola hepatica. Fluke eggs (200/ml, n= 5) from six different isolates were used in the current experimental work. They were obtained from different geographical locations and named Cullompton (UK), CEDIVE (Chascomus, Argentina), INTA-Bariloche (Bariloche, Argentina), Rubino (Uruguay), Cajamarca (Perú) and Río Chico (Catamarca, Argentina). The fluke eggs were incubated (25 °C) for a 12-h period in the presence of either ABZ or its sulphoxide metabolite (ABZ.SO) (5, 0.5 or 0.05 nmol/ml). Untreated eggs were incubated as a control. Incubated eggs (with or without drug present) were kept in darkness at 25 °C for 15 days. Afterwards, the trematode eggs were exposed to daylight over a 2-h period. Hatched and unhatched eggs were evaluated using an optical microscope, and the ovicidal activity was assessed for each fluke isolate. A very low ovicidal activity ( ≤ 13.4%) was observed in the ABZ-resistant CEDIVE isolate for both ABZ and ABZ.SO. Conversely, in the INTA-Bariloche and Río Chico isolates, which are suspected to be susceptible to ABZ, ovicidal activities ≥ 70.3% were observed after incubation with ABZ at the lowest concentration tested (0.05 nmol/ml). This finding correlates with that previously described for the ABZ-susceptible Cullompton. Finally, the Cajamarca and Rubino isolates behaved as ABZ resistant, since no ovicidal activity was observed after eggs were incubated with ABZ at 0.5 nmol/ml. Considering the specific results obtained for each isolate under assessment, the egg hatch test described here may be a suitable method for detection of ABZ resistance in F. hepatica.

Assessment of flukicide efficacy against Fasciola hepatica in sheep in Sweden in the absence of a standardised test

“…hepatica isolates to ABZ was assessed by in vitro FEHT. The principles of FEHT have been described elsewhere ( Alvarez et al., 2009 , Fairweather et al., 2012 , Canevari et al., 2014 ), although our protocol was somewhat modified as described below. Eggs were obtained from pooled faecal samples before treatment in both herds (farms A and B).…”

“…Hatched and unhatched, including dead eggs and eggs with developed miracidia were counted under an inverted microscope at 40x magnification. Ovicidal activity of ABZ was calculated according to the formula, below: Ovicidal activity (%) = (% of eggs hatched in negative control - % eggs hatched after drug exposure/% of eggs hatched in negative control)*100 ( Alvarez et al., 2009 , Canevari et al., 2014 ).…”

“…No reliable molecular markers for TCBZ resistance have so far been reported ( Elliott and Spithill, 2014 , Hodgkinson et al., 2013 ). However, in parallel with larval in vitro testing in nematodes, the Fasciola egg hatch test (FEHT) has been developed to diagnose susceptibility of F. hepatica eggs to ABZ ( Alvarez et al., 2009 , Canevari et al., 2014 , Robles-Perez et al., 2014 ) and TCBZ ( Fairweather et al., 2012 , Robles-Perez et al., 2015 ).…”

Fasciola and fasciolosis in ruminants in Europe: Identifying research needs

“…In vitro egg hatch assays have been used to detect resistance to albendazole and triclabendazole. Albendazole appears to have an ovicidal effect on F. hepatica, and egg hatch assays appear to discriminate between resistant and susceptible isolates (Canevari et al., ; Novobilský, Amaya Solis, Skarin, & Höglund, ; Robles‐Pérez, Martínez‐Pérez, Rojo‐Vázquez, & Martínez‐Valladares, ). In contrast, only one preliminary study suggested that TCBZ had an ovicidal effect (Fairweather et al., ), others showed that an egg hatch assay was not able to detect resistance to TCBZ in the field (Robles‐Pérez, Martínez‐Pérez, Rojo‐Vázquez, & Martínez‐Valladares, ) and in our experience, the amount of DMSO required to solubilize TCBZ and its metabolites is too toxic, hence if the appropriate controls are included, the assay cannot discriminate between resistant and susceptible field isolates of F. hepatica (Hodgkinson and Williams, unpublished observations).…”

First report of closantel treatment failure against Fasciola hepatica in cattle

“…Although guidelines for diagnosis of anthelmintic resistance in F. hepatica are lacking, there is agreement in the literature that the faecal egg reduction test (FECRT) combined with other methods such coproantigen reduction ( Gordon et al., 2012b; Novobilský et al., 2012; Brockwell et al., 2013; Hanna et al., 2015 ), detection of fluke DNA by polymerase chain reaction (PCR) ( Robles-Pérez et al., 2013 ) and/or in vitro egg hatch test ( Alvarez et al., 2009; Fairweather et al., 2012; Canevari et al., 2014 ) provide reliable information about flukicide efficacy in naturally infected animals. Furthermore, post-treatment fluke histology ( Hanna et al., 2010 ) can show direct morphological damage in flukes, but is not applicable in common veterinary practice.…”