Adaptation and evaluation of three different in vitro tests for the detection of resistance to anthelmintics in gastro intestinal nematodes of cattle

Abstract

Three different in vitro methods, the Larval Development Test (LDT), the Larval Migration Inhibition Test (LMIT) and the Micromotility Meter Test (MMT) have been adapted to detect anthelmintic resistance in cattle nematodes. Nematode eggs and third stage larvae of different Ostertagia ostertagi and Cooperia oncophora isolates were obtained from faecal cultures of experimentally infected calves. Additionally, adult C. oncophora were evaluated in the MMT for the detection of resistance to ivermectin (IVM). For all three in vitro tests standard operating procedures (SOPs) were established and successfully used for the detection of responses of non-parasitic and parasitic stages to different anthelmintic substances and the description of dose–response curves. In the LDT ivermectin (IVM) and thiabendazole (TBZ) were tested, in the LMIT IVM and levamisole (LEV) and in the MMT only IVM was evaluated. Susceptible isolates of C. oncophora and O. ostertagi, an IVM-resistant isolate of C. oncophora and a TBZ-selected isolate of O. ostertagi were used in all ( C. oncophora) or only some of these tests ( O. ostertagi). For all isolates sigmoidal dose–response curves and EC 50 values for the tested substances were obtained using a four-parameter logistic model. For the LDT, the previously reported problem in development of larvae was successfully overcome with mean development rates between 80% and 87% in negative controls. Following optimization of incubation times, temperatures, mesh sizes (LMIT only), nutritive medium (LDT only) and group size (MMT only) all three test systems reliably detected significant differences in the response to IVM between the susceptible and IVM-resistant isolate of C. oncophora ( p < 0.0001), resulting in an resistance ratio (RR) value of approximately 5 for IVM and 2.8 for LEV in C. oncophora. The LDT also detected differences in the response to TBZ between the susceptible and BZ-selected O. ostertagi isolates ( p < 0.001) with an RR of 2 for TBZ. With the standardization of the described tests we report reproducible and reliable in vitro methods for the detection of resistance to IVM (LDT, LMIT and MMT) and TBZ (LDT) for cattle parasitic nematodes.