The diagnosis of fasciolosis in feces of sheep by means of a PCR and its application in the detection of anthelmintic resistance in sheep flocks naturally infected

Abstract

The aim of this study was to develop a PCR for the diagnosis of Fasciola hepatica infection in feces of sheep based on the ribosomal internal transcribed spacer. Detection of infection was possible from the second week post-infection in experimentally infected sheep by amplification of a 292bp fragment. This PCR was employed for the detection of anthelmintic resistance (AR) in naturally infected sheep flocks, and results were compared with techniques such as the fecal egg count reduction test (FECRT) and the copro-antigen reduction test (CRT). The FECRT was carried out in two flocks, Santillan de la Vega (SV) and Corullón (CR), with sheep treated with albendazole (ABZ), clorsulon (CL), or triclabendazole (TCBZ). Feces were collected from individuals on days 0, 7, 15, and 30 post-treatment (pt). The FECRT showed adult F. hepatica to be resistant to ABZ and CL in both flocks. All parasite stages in the SV flock were susceptible to TCBZ, while in the CR flock, adult flukes showed resistance and immature forms were susceptible to the treatment. To compare FECRT and the PCR results, we calculated the percent of positive sheep on day 1 pt. In both flocks, the percent positive sheep was consistently higher by PCR than by sedimentation, confirming that the PCR is a more sensitive method of diagnosing infection and therefore to detect the resistance in infected animals. The CRT was carried out in the SV flock using a sandwich ELISA kit. The percent of sheep found positive by PCR was higher than with ELISA. Comparison of FECRT, CRT, and PCR for the detection of AR showed PCR to be the most sensitive.

Keywords: Anthelmintic resistance; CRT; FECRT; Fasciola hepatica; PCR.