Resistance in Agriculture: Investigation of Anthelmintic Drug Uptake and Resistance Mechanisms in Gastrointestinal Nematode Parasites of Livestock.

The parasitic nematodes are important pathogens of man and domestic animals that can cause chronic debilitating diseases. The trichostrongylid nematodes represent common and economically important pathogens of grazing livestock and have a worldwide prevalence. The free-living stages are taken up with pasture and pathogenic stages are ultimately found in the gastro-intestinal tract. These parasites are common throughout the United Kingdom. The numbers of reported clinical outbreaks and the geographic range of these parasites are increasing, with a contributing factor being our increasingly warmer and wetter climate prolonging the survival of the free-living stages. Control of these parasites is heavily dependent on a limited number of anthelmintics and is estimated to cost the sheep farming industry in excess of £84 million per annum. The anthelmintic drug ivermectin is a mainstay for control of these common parasitic nematodes, and the importance of this drug to control both animal and important human nematode infections was recently recognised by the award of a Nobel Prize in 2015 to the researchers who discovered it.
As recognised by the BBSRC's Resistance in Agriculture Call, anthelmintic resistance is now becoming commonplace due a number of factors, including over-application. Multiple resistance to different classes of drugs has now also been reported and there is an urgent requirement to understand the precise nature of this resistance in order to preserve the efficacy of the remaining compounds. Resistance to ivermectin and other classes of anthelmintic and indeed multidrug resistance represent a real and deepening crisis afflicting the UK and global livestock rearing industry and this may ultimately become a problem in human medicine where IVM is also a key component of large population-wide worm control programmes in the developing world.
The mode of action of ivermectin is well characterized but very little is known about the actual resistance mechanism to this drug. This proposal aims to understand the nature of both ivermectin resistance, resistance to other anthelmintic classes and multidrug resistance using a multidisciplinary approach. Our hypothesis, based on our preliminary data, states that disruption of IVM uptake is key to generation of widespread resistance. We propose that anatomical changes in neuronal circuitry of the nematode's "nose" is linked to resistance to this drug. We propose that similar mechanisms may be responsible for the structurally distince drugs of the Benzimadazole, Levamisole and Monepantel classes and may indeed be involved in multidrug resistance that we see in the feild. We plan to prove this hypothesis and therefore develop a deeper understanding of how drugs get into nematodes, how resistance occurs, how the spread of resistance can be monitored and controlled, and finally how we can develop new, more effective anthelmintics while extending the "shelf-life" of our currently available drugs.
This proposal will increase our basic knowledge of parasitic nematodes that are a major cause of serious economic losses to livestock farming industries worldwide. A proportion of this work will be carried out in the well-characterised, genetically-amenable model system, Caenorhabditis elegans, thereby reducing the use of animals in our experiments. The timing of this proposal is important as the technology and tools (next generation sequencing of C. elegans mutants and well completed genomes of the key parasitic nematodes) are now available to address the nature of resistance in these important pathogens. Treating nematode infections effectively in livestock will also provide economically and environmentally sustainable agriculture in the UK that will ultimately help maintain the UK's food security. Using a multidisciplinary approach to address the challenge posed by resistance makes this work an ideal fit for the Resistance in Agriculture Call.

Trichostrongylid nematodes impact on the health and welfare of grazing ruminants and represent a significant economic burden to the global livestock farming industry due to production losses. Control measures depend upon the administration of a limited number of anthelmintics; widespread resistance to many of these drugs means an uncertain future for this sector. The macrocyclic lactone, ivermectin (IVM) is the most important drug available to control these endemic parasites and while the mode of action is clearly understood, having been uncovered in the model nematode Caenorhabditis elegans, the precise mode of resistance is currently unknown but is believed to be multi-factorial. Our preliminary results indicate that the anterior amphidial sensory structures play a critical role in IVM uptake. Genetic and structural defects are implicated in IVM resistance in both C. elegans and in the ovine parasite Haemonchus contortus. Using genetics, chemical biology and molecular biology techniques combined with C. elegans and parasite-based approaches we will prove this link and identify the amphid genes associated with resistance to IVM. This approach will be complemented by applying our labelled IVM probes and we will likewise develop probes to the additional anthelmintic classes (Levamisole, Benzimadazole and Monepantel) thereby allowing their uptake and role in resistance to be analysed. We will assess field and laboratory derived nematode parasites with respect to labelled drug uptake, anthelmintic class resistance and multidrug resistance. Finally we will clone and characterize key amphid associated resistance genes from the parasites H. contortus and Teladorsagia circumcincta. Together, this multidisciplinary approach will advance our understanding of anthelmintic resistance in nematodes and will provide tools that in the future will be invaluable in tracking, assessing and controlling the spread of anthelmintic resistance in key livestock infections.

Nematode parasites represent a serious constraint on efficient and sustainable livestock production in the UK and throughout the World, with significant impacts on health and welfare. Control is heavily dependent on the prophylactic and therapeutic use of a limited number of anthelmintic compounds, with a heavy reliance on the macrocyclic lactones such as ivermectin. The macrocyclic lactones were initially developed to control parasites of veterinary importance but are also a mainstay in the control of soil transmitted helminths and filarial nematodes that are responsible for significant physical and mental disorders in billions of humans globally, hence they epitomize the "One Health" concept. Understanding and combating antimicrobial resistance (including anthelmintic resistance) in livestock infections represents a key priority area for the BBSRC and the current call "Understanding Resistance in Agriculture" and will be directly addressed in this proposal. Ivermectin, levamisole, albendazole, monepantel and multidrug resistance is emerging against the key veterinary nematodes and IVM resistance is expected to arise as a result of the large-scale human nematode control programs currently underway. Despite the key role that ivermectin and the other classes of anthelmintics play in the control of parasitic nematodes, there still exists a relatively poor understanding of the mode of action and molecular nature of drug resistance in these common pathogens. A clearer understanding of how ivermectin and the other main classes of anthelminthics enter nematodes and how they subsequently become resistant to these drug will be crucial in the slowing of this inevitable resistance process, prolong the usefulness of these drugs by allowing selective application and will contribute to the future development of more effective anthelmintics.
There is also a real need to develop anthelmintic resistance markers. Field and laboratory measurement of IVM and anthelmintic resistance in parasitic nematodes is a relatively time-consuming process, involving the examination of pre- and post-treatment faecal egg counts. Currently, there are no universally applicable/reliable in vitro tests available to assess IVM resistance (SCOPS.org.uk). As the BBSRC has recognised in the Resistance in Agriculture Call, understanding the development of anthelmintic resistance and how parasites evolve in the face of anthelmintic pressure is essential for the implementation of strategies to preserve treatment efficacy. Identification of markers that detect specific changes that are responsible for, or are associated with, anthelmintic resistance is the first stage in controlling the development and dissemination of anthelmintic resistance. Markers would enable the early detection of resistance, the prevalence of resistance to be charted, and the impact of traditional treatment strategies on resistance development to be assessed. In this context, it is important to note that we will make the fluorescent probe molecules available to the community through links with commercial suppliers. The elucidation of drug uptake and resistance mechanisms may also allow the modification of existing drugs, for example by allowing access via the gut or cuticle, thereby bypassing the amphids. Resolving the mechanism of anthelmintic resistance may also provide structural insights that can be informative for the future development of novel compounds.