Development of tools to detect anthelmintic sensitivity in UK cattle nematodes

Grazing cattle can be infected with a variety of gastrointestinal nematode species. In temperate regions, the most common of these species are considered to be the abomasal parasite, Ostertagia ostertagi, and the small intestinal dwelling nematode, Cooperia oncophora. Control of these nematodes is largely reliant upon the use of three anthelmintic classes: the benzimidazoles, imidazothiazoles and the macrocyclic lactones (ML). Worldwide, reports of anthelmintic resistance in cattle nematodes have increased in recent years; however, little information is available regarding this issue in farmed cattle in the UK. Knowledge is also lacking regarding anthelmintic usage practices, and whether or not the current methods used to detect anthelmintic resistance in cattle nematodes are robust and accurate. In this thesis, a number of approaches were taken to investigate these issues. A questionnaire study was conducted to evaluate anthelmintic usage and nematode control practices on a cohort of UK cattle farms. Descriptive analysis of the responses revealed that first season grazing calves were administered with anthelmintics, on average, twice per year (ranging between once and four times per year). It was also found that farmers depended heavily on ML products, with 80% of respondents administering a ML product in the previous 12 months and only 55% of respondents employing a quarantine treatment for cattle brought onto their farm. Faecal egg count reduction tests (FECRT) were then performed on 20 of the respondents’ farms, using injectable ivermectin (IVM). Two types of faecal egg count (FEC) methodology (a double centrifugation salt flotation method sensitive to one egg per gram and a McMaster method sensitive to 50 eggs per gram) were compared to investigate their utility in determining anthelmintic sensitivity of the derived nematode populations. A number of different statistical analyses were also performed to determine the optimum method of analysis for determining anthelmintic efficacy taking into account the effect of using arithmetic or geometric means, sensitivity of the FEC method and the effect of parametric bootstrapping. Of the 20 farms tested, 13 showed indications of inefficacy of IVM. Genus identification analysis of larvae derived from faecal samples obtained from the FECRT cohort indicated a minimum of 95% Cooperia spp. larvae in the post-treatment samples. Logistic regression analysis was also used to determine associations between management strategy on beef and dairy cattle enterprises and risk factors for IVM resistance. Farmers with dairy cattle were significantly less likely to use FEC (P = 0.013) or isolate new animals at quarantine (P = 0.013) compared to beef cattle farmers. Farmers who routinely monitored FEC were significantly less likely to use anthelmintics than those who did not monitor FEC (P = 0.042) and farmers who sought advice from their veterinary surgeons were less likely to administer anthelmintics according to the average weight of the herd (P = 0.02). Nematodes from two of the farms on which IVM resistance was indicated by the field FECRT were then further passaged and both isolates subjected to a controlled efficacy test (CET). Results from the CET confirmed the presence of IVM resistant C. oncophora (using two different application methods: injectable and pour-on administration), as well as the presence of moxidectin-resistant C. oncophora, the first confirmation in the UK. Analysis of phenotypic parameters was conducted on 679 female C. oncophora recovered at necropsy from all treatment groups. For both isolates, nematodes recovered from calves administered with moxidectin (MOX) pour-on anthelmintic were found to be significantly shorter than nematodes surviving IVM administration and nematodes from untreated control calves. Oviposition was also examined; with no eggs in utero found in any of the nematodes surviving MOX administration. Differences in the numbers of eggs in utero surviving IVM application were observed between the two isolates and also between application methods. Subsequently, analysis of a small section of a glutamate-gated chloride channel (glc-6) gene was conducted to investigate the presence of a single nucleotide polymorphism (SNP) in the glc-6 gene that had been previously proposed to be associated with ML resistance. This was achieved by comparing sequences derived from male and female C. oncophora (from both isolates) obtained from untreated calves with sequences from nematodes that had survived IVM administration. The SNP was not identified in any of the parasites analysed. Due to time and labour costs for conducting FECRTs for detecting anthelmintic resistance, a migration inhibition test was examined for its utility in assessing IVM sensitivity of third stage larvae (L3) derived from populations that had been demonstrated to have varying IVM sensitivity in vivo. Following optimisation, dose response curves and effective concentration (EC50) estimates were generated for all populations, including single species laboratory isolates of O. ostertagi and C. oncophora and mixed species isolates derived from the field studies above. The data failed to correlate with the previously obtained in vivo anthelmintic sensitivity classification for each isolate. Overall, O. ostertagi appeared to be less sensitive to IVM in the LMIT compared to C. oncophora, regardless of the ML sensitivity status of the isolate under study. Thus, these experiments indicated that the LMIT may have limited utility for assessing ML sensitivity of mixed species nematode isolates generated from field samples. In summary, the work in this thesis has found that UK cattle farmers heavily rely upon ML anthelmintics and there are clear differences in parasite control practices between farmers in the dairy and beef sectors, which may influence the development of anthelmintic resistance. As it is unclear when new classes of anthelmintics will become available for cattle, it is imperative to prolong the effectiveness of the current effective classes and to detect anthelmintic resistance as it emerges. This thesis has explored some currently available tools for the detection of ML resistance, with a view to improving them with appropriate best practice advice to help protect the health and welfare of cattle.