RHS Parasitic Worms Quiz – the Answers!

By Jenni McIntyre

We hope you enjoyed the Royal Highland Show. Thanks for taking the time to do the quiz – please find the answers below. For anyone who missed the quiz or the show, here is the online version.

Parasitic gastroenteritis is arguably the primary production limiting disease of sheep in the UK. How much do you know about it?

1. Parasitic gastroenteritis is caused by:

a. Fluke (trematodes)

b. Viruses

c. Roundworms (nematodes)

2. What is the main nematode worm causing parasitic gastroenteritis in lambs in the summer?

a. Teladorsagia (Ostertagia)

b. Oesophagostomum

c. Trichostrongylus

3. In the Springtime which nematode worm can cause diarrhoea and sudden death in lambs?

a. Teladorsagia (Ostertagia)

b. Cooperia

c. Nematodirus

4. How can we diagnose parasitic gastroenteritis?

a. History and examination

b. Postmortem

c. Faecal egg count

d. Rule out other causes of diarrhoea

e. A combination of the above

5. How many anthelmintic classes are there available to treat parasitic gastroenteritis in sheep?

a. 1

b. 4

c. 5

d. 7

6. Worms are able to develop or acquire resistance towards anthelmintics, making them less effective. How many of the available classes of anthelmintics have had reports of resistance in Europe?

a. 1

b. 2

c. 3

d. 4

7. How can we assess the efficacy of an anthelmintic on a farm?

a. Do a faecal egg count

b. Do two faecal egg counts (faecal egg count reduction test)

c. See if diarrhoea stops after treatment

d. See if mites die after treatment

8. A faecal examination usually reports a Nematodirus egg count, a Strongyle egg count and notes any Coccidia species present. A strongyle egg count is what is used to help diagnose parasitic gastroenteritis in sheep. Strongyle eggs all look very similar. Roughly how many different species of strongyle worms are there?

a. 1

b. 3

c. 6

d. 9

9. ‘All strongyle species are equally likely to cause disease’

a. True

b. False

10. ‘A lamb with a high egg count may not necessarily need treatment’

a. True

b. False

Strongyle egg counts can be useful in confirming a disease suspicion and examination of faeces is very helpful in deciding, for example, between anaemia due to fluke and anaemia due to Haemonchus.

Every treatment given selects for resistant worms in the sheep. Worms on the pasture are ‘safe’ and are said to be ‘in refugia’ – they are not affected by the anthelmintic. However, over time the number of resistant larvae on pasture increases and the number of susceptible larvae decrease as the sheep are repeatedly treated with anthelmintic on the holding.

A faecal egg count reduction test can give some indication of how well an anthelmintic is performing, but it uses a strongyle egg count with no knowledge of which species are present – highly pathogenic species may not be affected despite a reduction in egg output! It is important to gain a fuller understanding of what is happening on the farm.

Some farmers are now tending towards breeding resistant or resilient lambs which are less affected by the parasites and require fewer treatments. Others are using Targeted Selective Treatment to reduce anthelmintic use and preserve susceptibility amongst the worm population on the farm so the anthelmintic remains better effective for longer. By recording treatments they are able to choose breeding replacements which are hardier and more productive, improving their flock.

It is time to think about controlling parasites this year in such a way that we can still control them next year too.

1. c (Roundworms), 2. a (Teladorsagia), 3. c (Nematodirus), 4. e (A combination of the above), 5. c(5), 6. d (4), 7. b (Do two faecal egg counts), 8. d (9), 9. b (False), 10. a (True)

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The Faecal Egg Count Reduction Test – don’t put all your eggs in one basket!

By Jennifer McIntyre

Parasitic worms cause disease and reduce productivity in grazing livestock. Faecal Egg Counts (FECs) are widely used to estimate infection levels of various worm species, which often occur as mixed infections. In sheep, the commonest and most important species of worm parasites belong to the strongyle family, which includes Teladorsagia circumcincta and Haemonchus contortus. When a FEC is performed on a farm, it is a ‘strongyle egg count’ that is usually returned, with other species of interest noted and counted if required. Whilst it is recognised that interpretation of a FEC is complex, guidelines will often still include comments about the mean ‘egg per gram’ and how it relates to infection levels within the sheep such as ‘low infection levels’ being less than 250 eggs per gram.

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Faecal Egg Count Reduction Tests
FECs can be used to investigate the efficacy of anthelmintic treatment on a particular farm using the Faecal Egg Count Reduction Test (FECRT). This is increasingly important with the rapid rise in anthelmintic resistance. For the FECRT, FECs are performed before and after anthelmintic treatment (the post-treatment sample time is dependent on which anthelmintic is being used) and the difference between the two is calculated. Ideally the egg count drops to zero, or close to it, so that the anthelmintic can be said to have an efficacy of over 95%. Anything less than this (too high an egg count after treatment) can be interpreted as resistance. Obviously it is important that the FECRT is carried out correctly as otherwise additional factors may confound the outcome – such as under-dosing or the use of an expired product.

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What is well known is that the ‘strongyle egg count’ is not providing information about just one species, but rather a wide range of nematode species. Some of these are serious pathogens, like T. circumcincta, whilst others are mild pathogens causing negligible damage. Some of these nematodes have eggs which are shaped differently from the others. For example, Cooperia curticei, a mild pathogen, has eggs which are ‘straighter’ than the other species. For the most part, however, it is very difficult to distinguish one species of worm from another and thus understand the significance of the egg count by looking at the eggs alone.

How to overcome these limitations of the FECRT?
Eggs can be cultured and species identified after a week to ten days by examining larvae. The two larvae below can be differentiated by their tail length, along with other aspects of their anatomy.

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Alternatively, DNA-based methods (such as PCR), which are highly specific, can be employed to determine species identity within only a few days following sample collection.
If we rely on the FECRT alone to determine the efficacy of an anthelmintic on farm, we can fall into a trap. Below are two examples of real–life farms where FECRTs have been carried out. The differences are significant!

First we counted the eggs…
Farm 1 carried out a FECRT using a benzimidazole anthelmintic (class 1-BZ). The FEC fell by 80%. This would suggest resistance is present (remember, resistance is present if the efficacy is less than 95%), but that the drug still has a good effect; after all, the count has still dropped to only 20% of the original FEC before treatment.
Farm 2 also carried out a FECRT. They used ivermectin, an anthelmintic in the 3-ML class. Their egg count dropped by 75%. So a similar result to Farm 1. Resistance is present but the ivermectin is still usable.

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Next we speciated the strongyles…
We decided to hatch the strongyle eggs from both farms before anthelmintic treatment and culture them over ten days to become larvae. We then picked almost 100 larvae at random and speciated them by PCR to find out what was present. 86% of the population on Farm 1 was identified as T. circumcincta, the nematode we’re most concerned about. But this worm only made up 25% of the sample population collected from Farm 2.

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How can we interpret this? Why does it matter?
This means that an 80% reduction in the ‘strongyle egg count’ on Farm 1 will definitely include T. circumcincta, our worm of interest (present at a proportion of 86% in the original population).
But on Farm 2 a reduction of 75% could mean that T. circumcincta, the serious pathogen, has been unaffected by the ivermectin as it only made up 25% of the original sample population.

Can we confirm this?
We decided to look at what was left post treatment. The faeces collected from the lambs after benzimidazole treatment on Farm 1 had mainly mild pathogens, with less than 5% of the sample identified as T. circumcincta. A good outcome.
In contrast Farm 2 had only T. circumcincta in the samples collected after treatment of the lambs with ivermectin. When we looked at the proportion of eggs per gram represented by each of the nematode species we had found after culture, the egg count for T. circumcincta hadn’t changed at all. Ivermectin did not appear to have any effect on this serious pathogen on this farm. So despite each farm having similar results based on the FEC alone, the species identification showed something very different. These results will allow the farmers to make more informed decisions as to their flock management than if only egg counts alone had been used to assess anthelmintic efficacy on the farm.

Genome assembly is a bit of a puzzle

By Alan Tracey and Stephen Doyle, Parasite Genomics, Wellcome Trust Sanger Institute

One of the core aims of the BUG project is to complete our work on the Haemonchus contortus genome and to generate a high quality reference genome assembly for Teladorsagia circumcinta. However, the process by which a high quality genome assembly is generated is a non-trivial matter. In an ideal world, we would simply be able to “read” each base from one end of a chromosome to the other, telomere to telomere. Although recent advancements in DNA sequencing technologies have transformed our ability to analyse genomes, unfortunately it is still extremely difficult with current technologies to assemble medium to large scale genomes. This is because we are limited to breaking the genome into a very large number of very small fragments that are sequenced individually in high-throughput DNA sequencers (an approach called “shotgun sequencing”), leaving us with the challenge of trying to assemble these short sequences back together. Ultimately the aim is to assemble the short sequences together so that they perfectly reflect the true genome sequence.  This process is very much like doing a very difficult jigsaw puzzle that may consist of as little as thousands, but typically millions or billions of pieces, many of which are very similar to each other.

The analogy of a jigsaw puzzle can be used to highlight some of the difficulties faced during the assembly of eukaryotic genomes of organisms such as H. contortus and T. circumcincta. These challenges include: (i) the presence of long stretches of repeated nucleotides (imagine a puzzle of a vast expanse of cloudless blue sky), (ii) biases in sample preparation and sequencing mean that some regions of the genome are poorly represented or missing (lost puzzle pieces result in permanent holes. However, we often don’t know they are missing, or where the holes are, until the rest of the puzzle is close to completion), (iii) the presence of other organisms or tissues in the sample that leads to sequence contamination (somehow pieces from another jigsaw puzzle have found their way into our jigsaw puzzle box), and (iv) genetic differences, whereby the same positions of the genome can be represented in subtly different ways (multiple variations [for example, two in a diploid organism] of a puzzle piece that can fit in the same location). Given that these genomic “jigsaws” consist of so many pieces, we rely heavily on high performance computing and assembly algorithms designed to help put these pieces together; however, both are not perfect and often the pieces are incorrectly assembled together, for example some pieces joined in the wrong order.

Fortunately, we have access to, and are using, some of the latest sequencing technologies available that can make genome assembly easier. “Third generation”, single molecule sequencing such as that produced by the Pacific Biosciences RSII sequencing machine produce much longer reads (thousands of nucleotides long) than the dominant previous generation sequencing technology (from the company Illumina; typically between 100-250-bp long). These longer “Pacbio” reads often enable us to assemble difficult regions such as repeats, which we may not have previously been be able to do in a short-read only assembly. Having bigger, more unique jigsaw puzzle pieces allow us to make more confident joins, resulting in an easier assembly process. To compliment these new long read sequences, we have also been using optical mapping approaches, which give us an independent long-range view of our assembly. Optical mapping employs restriction endonucleases that cut the template DNA in specific places in the genome; by comparing the pattern of these cut sites in hundreds-to-thousands of kilobase-long DNA molecules to our DNA assembly, we can generate very long, contiguous DNA sequences (Figure 1). Quality optical maps can be very difficult to produce due to the challenges associated with extracting and purifying high molecular weight DNA that is essential for this application. However, the challenges with making an optical map are worth overcoming; having an optical map is very much like being able to see the picture on the jigsaw box as we try to assemble all of the pieces, and this gives us invaluable large-scale context to our assembly and ensures that long-range problems, such as incorrectly joined chromosomes, do not happen.

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Figure 1. Example of a H. contortus optical contig (top) aligned against a sequence-derived contig (bottom). The vertical black lines in both sequences represent the actual (top) and in silico (bottom) predicted restriction endonuclease cut sites that are used to orientate the two sequences.

At the Wellcome Trust Sanger Institute, we synthesise several types of sequence and mapping evidence (currently Illumina short read, PacBio and optical mapping) and use many years of genome assembly experience to improve genome assemblies beyond what is currently possible by solely automated means. This manual curation process employs a tool called “GAP5”, which allows us to view, edit and reassemble regions of the genome in the final stages of assembly. This approach has been used to assemble the H. contortus genome into chromosomal-scale pieces, which are essentially complete from telomere to telomere (Figure 2). Small improvements are now being made to account for some of the variation seen in this highly polymorphic genome prior to release as a publically available resource for researchers working on Haemonchus and related species.

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Figure 2. A “Circos” plot showing syntenic (highly similar) regions shared between H. contortus (purple bars) and Caenorhabditis elegans (numbered, coloured bars). Each coloured line linking a C. elegans chromosome to H. contortus sequence represents a highly similar, translated region shared between the two genomes. This plot demonstrates that much of the genome is shared, and that it exists in a similar orientation between the two organisms.

As we near completion of the H. contortus genome assembly, our next challenge is to assemble the T. circumcinta genome. We are currently working on optimising the Pacbio and optical mapping conditions to maximise sequence lengths of the raw data, which we believe will be crucial in making sense of this genome, which appears to be considerably larger and potentially more complex even than that of H. contortus.

Meeting report – Bangalore and Lucknow

Following on from the Antimicrobial Resistance (AMR) meeting in Barcelona, I took part in a meeting in February in Bangalore, India on AMR in Veterinary Infectious Diseases. The meeting was held under the auspices of the UK Department of Business, Innovation and Skills (BIS), with the aim of supporting delivery of the global strategy on AMR. It was organized by Professor Utpal Tatu from the Indian Institute of Science, Bangalore, aided by Swati Saxena, the senior science and innovation adviser of the British High Commission in New Delhi. There were five delegates from the UK and several participants from India, covering a wide range of areas. Helminths and anthelmintic resistance were included in the programme and alongside myself, Professor Raman from Chennai gave an excellent overview of the problems associated with anthelmintic resistance in Indian livestock. Prof Raman is a close collaborator of fellow BUG Consortium member Professor Neil Sargison, who was also in India at the same time, but at the Indian Parasitology meeting in Chennai. The Bangalore meeting covered many interesting areas related to AMR, including food hygiene and AMR, antimicrobial stewardship, vaccine production as an alternative to drug therapy, indigenous plant-based therapies, and epidemiological factors determining the spread of resistant microbes.

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We were very warmly welcomed by the Indian scientists, by Prof Tatu and his wife, and by the Deputy High Commissioner, Dominic McAllister at his home in Bangalore

On the Saturday, the highlight of the meeting was a field trip to a ‘Camp’ in the countryside a couple of hours outside Bangalore, which Prof Tatu has been involved in establishing. Local farmers bring their livestock for diagnosis and treatment by veterinarians.

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Livestock at the camp (photo courtesy of Dr Ron Dixon)

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Veterinary work at the camp (photo courtesy of Dr Ron Dixon)

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Although most of the animals seen at the camp were cattle, we also saw many flocks of sheep and goats in the countryside around Bangalore

Given that a recent review in the Lancet Infectious Diseases suggested that the worldwide cost of dealing with the issue of AMR would fall somewhere between the £6 billion of the Large Hadron Collider and the £96 billion of the International Space Station, it is abundantly clear that no one country can tackle the problem in isolation. This was an excellent scoping meeting, which hopefully will lead to further collaborations between UK and India on this important topic.

Following Bangalore, which is in Karnataka, southern India, it was off to Lucknow in Uttar Pradesh in the north, to the Central Drug Research Institute (CDRI) for the 2016 meeting “Current Trends in Drug Discovery and Research”.

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Visitors to the Institute were greeted with a beautiful artwork in the hallway, constructed from coloured sand, to celebrate the 2016 meeting

This was a wide-ranging meeting covering many aspects of drug discovery and research, from novel approaches to chemical synthesis to the application of new drugs in many different human diseases. The neglected tropical diseases (NTDs) were well represented with interesting talks on novel drugs/approaches for malaria, filariasis and leishmaniasis.

The meeting had a strong cultural theme; it was hosted at the CDRI and all meals, consisting of delicious traditional Indian food, were taken in a large marquee in the grounds. Another large tent in the gardens hosted the poster sessions, where graduate students and young post-docs presented their work. These sessions were of an extremely high standard and speak highly of the training provided at CDRI and other Indian Institutes. We were treated to an impressive cultural evening of music and dance in the local Kathak style. Invited speakers were also presented with a memento of the meeting, while session Chairs received a commemorative medal.

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Memento for invited speakers

Science Update

It is now 9 months since the BUG project started and we are making good progress in several areas. Our major goal is to develop better diagnostics for detecting anthelmintic resistance in the field, but in order to identify the important genetic markers, we first need to generate the genomic resources. Since starting the project, considerable progress has been made by James Cotton and the team at the Wellcome Trust Sanger Institute (WTSI) in finishing the genome of Haemonchus contortus. This has involved intensive manual curation of the Illumina draft sequences and the use of PacBio long sequence reads to fill in gaps and join scaffolds. Optical mapping technology, which allows the construction of genome-wide restriction maps from strands of fluorescently labelled DNA, has also been used to guide long range assembly. Progress has been excellent and the aim of completing all six chromosomes and providing a reference quality genome is well in sight. Work is also underway on the sequencing and assembly of the draft Teladorsagia circumcincta genome. DNA samples for Illumina sequencing, PacBio and optical mapping have been prepared in Glasgow and sent to WTSI, where assembly of the genome has begun, guided by the most successful approaches for H. contortus.

Meanwhile, two genetic crosses between drug sensitive and drug resistant H. contortus isolates have been generated at the University of Edinburgh and Moredun Research Institute by Neil Sargison and Dave Bartley. The aim of this part of the project is to aid genome assembly and to identify regions of the genome involved in drug resistance. Preliminary analysis of the first (F1) generation is already yielding novel findings with respect to benzimidazole resistance and plans to undertake drug selection on the F3 generation of the crosses are in place. In Glasgow, we have been producing DNA libraries from individual F1 larvae for whole genome sequencing and the generation of a genetic linkage map. We are also optimizing a technique to sample large numbers of markers throughout the genome (Rad-Seq) for individual L3 larvae, which will be used to compare parasite populations harvested pre- and post- anthelmintic treatment on UK farms.

In Bristol, Eric Morgan and the team have been busy surveying farms for H. contortus infections with the aim of identifying appropriate premises for sampling this coming season. In Edinburgh, the sampling of local flocks for T. circumcinta has identified a number of farms for continuing surveillance and has provided field populations of L3 larvae to begin analysis. Meanwhile Cath Milne from SRUC held a well-attended focus group in the Scottish Borders to ascertain the opinion of farmers on worm control, a very important aspect of our project.