The path of most resistance

Lurking in the digestive tracts of grazing livestock, parasitic nematodes exact a brutal toll on agriculture. For a long time they have been treated and controlled with a group of drugs known as anthelmintics. However, many of these drugs no longer work effectively because the parasites have developed resistance to them. Here, post-doctoral researcher Dr Roz Laing tells us about the motivation and history behind a new multi-institute project run from the University of Glasgow: the BUG Consortium, which aims to use parasite genomics to help tackle the spread of anthelmintic resistance.

Parasitic nematodes, also known as roundworms, infect grazing livestock worldwide. Adult roundworms live in the digestive tract of the host animal, adversely affecting animal health and productivity, with huge costs to the global economy. The current treatment and control relies on the use of a small number of anthelmintic drugs, but resistance has developed rapidly, analogous to antibiotic resistance in bacteria.

The ability of parasitic nematodes to survive drug treatment has a genetic basis, but the mutations underlying anthelmintic resistance are not well understood despite many years of research. Parasitic nematodes can be difficult to study —for example, adult worms do not survive outside the host animal so cannot be maintained in the lab. But one of the major factors hampering research into anthelmintic resistance is a lack of information about the genetic makeup of parasite populations.

The BUG Consortium is a new BBSRC-funded project to identify the genetic changes underlying anthelmintic resistance and investigate more sustainable methods of parasite control. BUG stands for Building Upon the Genome, and refers to the genome of the sheep parasite Haemonchus contortus, a blood-feeding worm that has rapidly developed resistance to every drug used in its control. The genome provides an extremely powerful tool to study anthelmintic resistance in this species and in related parasites of veterinary and medical importance, but this has been a hard-earned resource. The genome has been exceptionally challenging to assemble and it’s taken nearly ten years to reach this point. But why?

Superficially, nematodes appear to be little more than a mouth, an intestine and a reproductive tract, wrapped up in a protective cuticle. Caenorhabditis elegans (a free-living nematode that we maintain in the lab) is transparent, so we can see this clearly under the microscope. However, when the C. elegans genome was sequenced in 1998, researchers discovered it encoded ~20,000 genes—roughly the same number as the human genome. Worms lack many of the complex organs that we have in our bodies, yet amazingly, the majority of genes associated with disease in humans are also present in C. elegans. This makes it a valuable model to investigate many complex pathways of human disease.

C. elegans is also a useful model for its close relatives, the parasitic nematodes of livestock, such as H. contortus, and the human hookworms. Scientists have gained valuable insights into anthelmintic drug targets and mechanisms of action by examining their effects on C. elegans and have identified mutations that confer drug resistance in the lab. Candidate resistance genes found in parasitic worms can even be isolated and introduced into C. elegans to examine their effect. However, for the most commonly used anthelmintic, ivermectin, this approach has so far proved unsuccessful, perhaps due to subtle yet important differences between target gene families in C. elegans and in parasites.

In 2003, Professor John Gilleard (then University of Glasgow, now University of Calgary) and Dr Matt Berriman (Wellcome Trust Sanger Institute) recognised the need for better genomic resources to study parasitic nematodes directly. To begin to address this, they chose to sequence the genome of H. contortus, the most pathogenic and economically important parasitic nematode of sheep and goats worldwide, and the focus of a large body of research into drug development and resistance. What hadn’t been anticipated was the extremely large size of its genome (roughly three times larger than C. elegans) and the amount of repetitive sequence it contained. This made the project a huge undertaking from the word go.

To complicate matters further, sequencing technologies available at the time required large amounts of DNA to work with, more than is present in a single worm. This meant DNA from many worms had to be sequenced to generate a single genome. Parasitic nematodes have diploid genomes, like humans, meaning the chromosomes occur in pairs (the exception being the sex chromosome in male worms, which has only one copy). To assemble a genome a single consensus sequence is generated for each chromosome, which relies on both copies of the same gene or region of a chromosome being similar enough to merge. This was not always the case for H. contortus, where sequences from the same region of a chromosome could be too different to be recognised as such, and resolving this issue became increasingly complex with increasing numbers of worms.

This phase of the project could be likened to piecing together a hundred thousand piece jigsaw with lots of missing pieces, lots of identical pieces and lots of extra pieces from slightly different jigsaws. However, lots of hard thinking and hard work led to small incremental improvements in the assembly of the H. contortus genome every year. These, in turn, contributed to great improvements in our understanding of many aspects of parasitic nematode biology, as the new sequences were made publicly available at every stage so researchers could use the best assemblies for their work.

It was the advent of ‘next generation sequencing’ technologies that finally provided sufficient sequencing muscle to fully address the issue of the large genome size. These methods rapidly sequence millions of DNA strands in parallel, which vastly increases the amount of sequence that can be generated from small amounts of DNA. This technology, combined with the painstaking development of a less genetically variable population of H. contortus, eventually gave us the advantage we needed. By 2013, the jigsaw was nearing completion – we had a draft genome.

This is the long-awaited resource that underpins our research in The BUG Consortium. The H. contortus draft genome encodes 21,799 genes and has already revealed important differences in anthelmintic drug targets compared to C. elegans, but work to improve and develop the genome as a tool for researchers is ongoing. For more information please visit our website, where my next blog will discuss the genetic analysis of parasitic worms from UK farms, to find where in the genome anthelmintic resistance evolves. If you would like to know more about the project, please contact me (Roz Laing) or Professor Eileen Devaney.

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