Markers of anthelmintic resistance in gastro-intestinal parasites of ruminants (PhD)

Results

 Monitoring parasitic gastroenteritis

• PGE can be monitored over time using FECs, however these may be comprised of many different species. The species composition of a FEC could vary between sheep ages and over the season. Recent anthelmintic treatments should be taken into account.
• Ideally, strongyles should be speciated to determine the species composition before choosing to treat sheep based on a high FEC alone.
• Given the diversity of species present within the hosts, and their varying pathogenicity, it is important to remember to monitor weight gain +/- BCS over time. This can detect weight loss due to large numbers of pre-patent larvae within the sheep (which are missed by FEC), or help to determine whether a high FEC requires treatment.

 Monitoring anthelmintic efficacy

• A FECRT can be beneficial to determine the ability of an anthelmintic to reduce pasture contamination with eggs.
• A FECRT, without speciation of the strongyles present pre- and post-treatment, will be limited in it’s ability to identify anthelmintic resistance on a farm. Highly pathogenic species which are highly resistant can be hidden amongst a more sensitive strongyle community.
• The EHT was confounded by the presence of multiple sensitive species within the strongyle population tested. However, it did reflect the proportion of individuals that were BZ resistant. Speciation by PCR of eggs and L1, obtained from the EHT, would be beneficial.
• The LDT used in this study was not helpful to determine IVM resistance in T. circumcincta, or in the mixed species population.
• Genetic tests are only available for BZ resistance, but are currently not commercially available. However, this may change going forward. No genetic tests are available for IVM, but would be useful.

 Identifying markers of IVM resistance for use as a diagnostic test

• Two good quality sequencing resources have been generated, and protocols validated, using two independent UK field populations of T. circumcincta.
• The genomic footprint of IVM resistance (the length of the worm’s genome selected by IVM) is likely to be quite small in the UK field populations.
• Previous studies have suggested IVM resistance is inherited as a dominant trait in T. circumcincta. The sequencing results from this PhD would agree with this. A dominant trait means that individuals can be more rapidly selected, however the potential to maintain susceptible individuals within the population is higher than for a recessive trait.
• IVM causes a flaccid paralysis, and induces starvation in free-living stages. Many interesting genes were identified which might be under selection by IVM. Genes involved in neuronal processes were identified, including those related to feeding and chemosensation. IVM is a lipophilic drug capable of affecting fat stores in worms. Several genes involved in metabolism generally and lipid metabolism and storage specifically were also identified. Other genes which may have regulatory roles were noted within the results.
• Genetic markers of IVM resistance will be identified using an improved genome assembly. The T. circumcincta reference genomes available during this PhD were both highly fragmented (they are in 8000-81,000 contigs, instead of six chromosomes). Both contain technical artefacts, including significant duplication of genes which should be single copy. These genomes may therefore have affected the analysis. When an updated reference genome becomes available the analysis will be repeated. It is hoped that with newer sequencing methodologies, and using protocols to attempt to reduce diversity within T. circumcincta populations, it may be possible to improve the genome in the near future.
• When diagnostic markers become available they will enable researchers to understand how IVM resistance changes over time on a farm. An understanding of how different management practices affect IVM resistance and the benefits of refugia can then be studied.

The challenge

All grazing livestock are infected with gut worms, which they ingest from the pasture. These parasites need to be treated for the health of the animal and to maintain productivity. The main method of worm control is the use of drugs called anthelmintics, but parasites are rapidly becoming resistant to these drugs (like bacteria which become resistant to antibiotics).

With increasing anthelmintic resistance, farmers may find it harder to control disease caused by these worms. Symptoms include diarrhoea, dehydration and poor weight gain. Very severe infections can be fatal. Resistance can lead to welfare and economic problems with under-weight lambs being sold for less or kept on the farm for longer, costing more in labour, feed and time.

There are only a limited number of anthelmintics available and resistance has been reported in four of the five classes of drug. In the UK, the most widely used anthelmintic is ivermectin, which can also be used to treat sheep scab. Unfortunately frequent use of a drug preferentially selects for resistant worms to survive, even when used to treat a different disease, and resistance to ivermectin is thought to be increasing. However, we lack a sensitive diagnostic test.

The project

The most important parasitic worm in UK sheep flocks is Teladorsagia circumcincta. I will be collecting faecal samples from Scottish flocks, before and after ivermectin treatment, to isolate T. circumcincta larvae and analyse their DNA. Anthelmintic resistance is caused by genetic changes and my aim is to identify mutations in the DNA of resistant worms, which can be used in a rapid test for ivermectin resistance. This may also help us better understand how resistance develops and how to counteract it. I will provide feedback to farmers on the information gathered and on best practice to sustainably control parasites on their farms.

Student

Jennifer McIntryre