WP 5 - New methods for the detection of anthelmintic resistance

Introduction

A key component of the strategies that will be developed by PARASOL are the measurement of anthelmintic efficacy and of the prevalence of resistance alleles in animals/herds to be treated. Treatment with agents that will obviously be ineffective at killing worms will waste resources and is likely to contribute to unnecessary residues and environmental contamination. Detection of resistance alleles before they reach a level that causes obvious clinical failures is an important method for maximising the sustainability of current drugs.

Objectives

We will produce novel in vitro tests for the detection of resistance to anthelmintics, to ensure that resistance to all commonly used drugs in the major nematode parasites of ruminants can be detected reliably and rapidly.

Justification

At present, analysis of the susceptibility of a nematode population against an anthelmintic can be performed by testing adult or larval worm stages in vivo or in vitro, though in vitro tests are not available for all anthelmintics and all species of parasite. The most often used in vivo test, the faecal egg count reduction test (FECRT), requires two samplings of at least 10 animals and microscopic examination of the samples, that is expensive. Of the in vitro tests, the egg hatch test (EHT) has been shown to be suitable for the detection of benzimidazole (BZ) resistance in a range of nematode species small ruminants, cattle, horses and pigs. Tests based on the inhibition of larval development or movement by imidazothiazoles or MLs, like e.g. the micromotility or larval migration tests, allow differentiation between selected and unselected populations from sheep nematode species like Haemonchus contortus. However, there remains a pressing need for a reliable in vitro ML resistance test for cattle nematodes. The most important gastrointestinal parasite species in cattle is Ostertagia ostertagi, though to date ML resistance in Europe has been observed confined to Cooperia oncophora.

Because of the limitations of in vivo and in vitro tests, molecular tests are required to detect the presence of resistance alleles before they cause clinical problems, so that management can be changed to try to prevent clinical resistance emerging. Until now, molecular techniques have only been described for BZ resistance in gastrointestinal nematodes of ruminants, as our knowledge on the molecular mechanism of resistance is confined to the BZs. Several allele-specific polymerase chain reaction methods for the genotyping of different nematode species have been developed. These molecular systems are more sensitive than the in vitro tests described above. All the current molecular tests for AR study the level of resistance by genotyping a representative number, mostly one hundred, individual larval or adult parasites. This is laborious and expensive. Therefore, the molecular tests have so far only been used in research studies. Recent technological innovations like quantitative real-time PCR and Pyrosequencing now offer new opportunities to overcome these limitations.

Expected Results

We will produce new tests for the efficient detection of resistance to anthelmintics in several ruminant worm species. This will enable farmers to use the most effective drugs on their farms. The development of molecular tests will contribute to this process, but also make the monitoring of the spread of resistance alleles within and between populations much more straightforward.

Organization

The activities in this work package are performed through scientists at the University of Veterinary Medicine in Hannover (WP leader), University of Ghent, University of Uppsala, University of Bath and University of Bristol.