Team:Uppsala/99

Uppsala iGEM 2018

Nematode parasites cost the agricultural industry lots of money and grief each year due to the many consequences they cause. Among many we can find severe health issues and resistance development in the most commonly occurring family of strongyles. There are currently no easy methods for the diagnosis of these parasites. By reprogramming a smart bacteria to detect and report the presence of the parasites, we aim to develop a simple diagnostic method. This will provide the tools necessary to help farmers both to make decisions on whether to treat their animals and prevent infection.

Small Strongyles

Small Strongyles or Cyathostominae are among the most common equine parasites, with more than 52 species in their family [2]. The infectious stage of small strongyles is when they’ve developed into larvae while still lurking in the grass. While horses graze, they consume the worms and the small strongyles continue to develop in the horses’ intestines, forming cysts in the intestinal wall [1]. When further evolved, these small strongyles can burst out from their cysts during late winter or early spring, moving up towards the intestinal lumen where they become adult worms [1, 9].

Statistics Box:

In a study of 461 horses in Sweden, 53,7 % of all horses were discovered to be infected [1]. From July to September the prevalence was estimated to 78,4% in the horse population, indicating a seasonal trend for infection rate [1]. Similar trends are seen in Europe and North America showing reported cases with 100% of horses showing small strongyle infection [2].

Symptoms

The release of larvae from cysts can lead to lesions, diarrhea, and potential weight loss. This condition is called cyathostominosis [1]. When untreated, the death tolI can reach number up to 50%. During the seasonal rupture of cysts, millions of larvae can be released at the same time, which can result in severe and life-threatening consequences [6]. The infection of small strongyles is not one of presence, but one of quantity. They are not dangerous in small amounts and therefore it is difficult to tell whether a horse needs to be treated or not [10].

By providing a better diagnostic tool, farmers will be able to know when to treat the horse in the purpose of avoiding mass rupture and severe consequences.

Methods of Detection

Currently the only method for detection and counting of how many worms there are in an animal is counting nematode eggs in fecal samples. This method is not reliable and also requires farmers and ranchers to send in fecal samples to a lab with trained personnel [4]. This technique is expensive, inconsistent, and requires timely shipping of samples. Currently many horse owners are reluctant to conduct the testing and treat their horses regardless of need, contributing to the problem of resistance [10].

Resistance development

Unfortunately, the extensive overuse of deworming drugs has now lead to the detection of worms that are resistant to the most commonly used drugs [2,5]. Since no new deworming drugs have been approved for use in horses the whole equine industry relies on macrocyclic lactones, currently the most common type of deworming drugs used. Unfortunately, cases of resistance among nematode adults have been spotted for macrocyclic lactones as well. So far, four studies with similar results spanning from Europe to North America have been published with concrete data, linking certain small strongyle species to reduced time until detection of eggs after deworming treatment, showing an increase in resistance[2].

Moxidectin is a very common drug used. It is both hazardous for the environment and is also losing its effectiveness towards worm populations [15,16].

Due to all of these facts, we aim to develop a diagnostic tool to give horse owners a better estimation of the amount of worms in the horse. This would allow them to adapt the deworming treatment based on the level of infection.

Large Strongyles

The small strongyles larger cousin, Strongylus Vulgaris, is the most pathogenic parasite in horses, posing a significant threat[1]. They, like small strongyles, live in the grass and and infect the horse after being ingested[4]. During the different larval stages inside the horse, the parasite enters the intestinal blood vessels as a part of their life cycle[4,11]. Because of this, they generally can’t be targeted by deworming drugs or detection methods and can cause major problems for domestic animals[8]. The large strongyle therefore needs to be detected in pasture in order to prevent infection.

Symptoms

When the worms migrate in the arteries they cause inflammation in the arterial wall and induce the formation of blood clots. When a small blood clot forms, it may form an embolism, which can detach and travel in the bloodstream until it reaches and blocks smaller blood vessels. This prevents oxygen and nutrient supply to surrounding tissues and may result in colic[14]. This can cause problems in the animals, with the worst case scenario being death[4].

Having these parasites is obviously a huge problem and there is great need for methods that can detect the parasites before ingestion, preventing infection.

Methods for Detection

To be able to count either small or large strongyles, the current methods require growing their eggs from samples and counting them. The disadvantage is that this method only works for adult strongyles that have already laid eggs! This technique only makes it possible to detect strongyles after the infection which is a huge disadvantage! Other methods for detection, such as using an ELISA test or a PCR based method are expensive and require a high level of expertise [12, 13, 17].

What is currently needed is a simple detection method which could be used on site without specialised equipment. To fulfil this need we aim to develop a detection method that can be applied to pastures in order to prevent infection to begin with!

Preventing an Infection

After infestation, the window of opportunity for treatment is comparatively small, as the large strongyle spends most of its life cycle in the blood stream, where the treatment is ineffective. It is therefore important for the farmer to know what levels of infestation are present in the pastures and whether these are safe for their animals to graze on. We aim to develop a tool which allows quantitative measurement of such infestation of pastures, which would decrease the occurrence of infection.

How will we achieve this?

The first thing is to define the desired characteristics of each worm buster. To face the problem the team need to develop bacteria with two different functions. The two different bacteria we will be described below

First Method

The Small Worm Buster

One method is to engineer a bacteria that lives in the intestinal tracts of horses and report a quantitative signal of small strongyle in feces. The small strongyle buster can therefore work as a diagnostic tool and give horse owners the possibility to use individualized dosage of treatment depending on the level of small strongyle infection.

Bacteria 2

The Large Worm Buster

Another method is to engineer a bacteria that can be applied to pasture samples and report the amount of large strongyles in pastures. By detecting large strongyles in pasture the large strongyle “buster” can help horse owners prevent their horses from getting infected since they can see which fields that are infected or not.

Worm culturing

The first step in obtaining live nematodes is the recovery of the eggs from the feces. This is done through filtration of the feces and different centrifugation steps with saturated salt water followed by a bleach containing solution to obtain sterile eggs. The eggs are successively stored in LB media for a period between 7 and 10 days to allow them to hatch and reach the third larval stage. After this process the large strongyles are divided from the small strongyles under a microscope and stored for successive use.

Transcriptomics and Phage Display

As not a lot is known about our worms of interest, we need to choose approaches that allow us to detect the worm without knowing its specific markers. For this, we chose two distinct approaches. For the first one, we’re doing transcriptomic analysis, which relies on the co-culturing of nematodes with E.coli for 2 or 3 hours and subsequent sequencing of the bacterial mRNA, which will reveal which genes are upregulated when the worm is next to the nematode. The promoters of these genes can then be used to develop a biosensor by linking them to a reporter!

The second approach utilizes libraries of phages expressing a set of peptides on their surface. By various rounds of incubation and washing, only phages specific to the strongyle will be found and sequencing of their genetic information will allow us to construct a peptide that is specific to surface markers of the nematode leading to creation of a biosensor

Reporter system

Once we have the results from either the transcriptomics or the phage display, we’ll have to be able to see what we’ve done! We plan on using fluorescent chromoproteins to be able to detect our worms in both grass and in feces. This would allow a relatively simple and quantitative way for ranchers to detect our worms of interest, using a cheap UV lamp, a dark room, and a camera!

Proteomics

Contributing to the lack of Cyathostominae research

A second part will attempt to explore the known concerted action of the parasites. Seasonal encystation and emergence of larvae occurs in a coordinated fashion, so the hypothesis is that the encysted strongyles react to a signaling compound. To find possible candidates for this compound a strategy is employed in which samples of infested host tissue are run through a battery of tests, and occurence of compounds is compared to a control of healthy tissue.

In our project we hope to perform studies to locate and identify this molecule. Since deworming drugs have no effect on encysted worms and killing of the relatively few grownups would induce cyst bursting, it would be important to figure out what the signaling molecule is for further study.

Modelling

Grass Sampling

Methods for detecting a strongyle infected field do exist, but they’re both tedious and time consuming. Our aim is to build reproducible and reliable models based on real world behaviour to reduce the area needed to sample. These models are based on the location and expected movement of the strongyles once outside of the horse.

Economic Modelling

A good model is a great tool to study the simplified behaviour of a real world problem. Often it can be more practical, time- and cost efficient to study the model of the real world system. We aim to showing the economic impact of strongyles and anthelmintic resistance.

In order to truly understand the expected market, cost and reach, we are opting for a predicting market analysis.

As you can see these little worms can cause a lot of harm. While we hope that our practical work will at least will contribute to the solution, all of the problems that come with these worms cannot be solved in a laboratory.

The strongyles harm the horses but the collateral damage is extensive - sentimental value, resistance against anthelmintics, the effect the drugs has on environment and the noteworthy economics are all involved. These are all aspects that make the issue bigger than it appears at first glance.

Raising Awareness

We’re attacking several angles to raise awareness for our project. We plan to perform a market analysis of the problem, send questionnaires out to better judge the severity of the problem and to participate in local radio shows. We’ve even been published in a local magazine, explaining the problem and raising awareness locally.

As our team is partly international we will try to spread the questionnaire to other countries besides Sweden, in hope of getting results reflecting the extent of the issue of strongyles on a bigger scale.

Financial perspective

The equine industry includes many areas like stud farms, breeding, slaughter, trading, training and sports stables, and riding schools [19]. In Sweden the equine industry has a turnover of 450-500 million EUR and horses graze around in fields that in total correspond to 600,000 football fields. [18]. There are approximately 350 000 horses is Sweden (2016) which gives Sweden a larger horse/person ratio than many other European countries [19, 20]. A large amount of money is spent on horses within and without the borders of the EU, so this is clearly a global problem.

Even though all the available data points at a high demand for our product, more data is needed to predict how an eventual new GMO product would be perceived. That is why we are working on market analysis and economics modelling. The result from these decides if the eventual solutions will be possible to implement.

We plan on making our project useful for future iGEM projects by producing informative media for iGEM teams. We plan to provide a Youtube channel explaining scientific concepts, have lectures at local schools and have a public forum discussion in order to better educate the public on what exactly GMOs are! We also plan to publish a booklet outlining how to avoid burnout, and the effects it has on the biotechnology industry.

We are stronger together so when possible we are striving for collaboration with other iGEM teams. So far we are in contact with the teams of Aalto Helsinki, National University of Singapore (NUS), Lund Technical University (LTU), the team of Kungliga Tekniska Högskolan (KTH) and with all other nordic teams at social events and channels. Besides collaboration with teams we also keep a close collaboration with Vidilab, a veterinary diagnostic company.

Vidilab
Besides collaborating with other teams, iGEM Uppsala 2018 has a very close collaboration with Vidilab, a company that works with the current veterinary diagnostic tools. They are offering us usage of parts of their lab and equipment during the summer and potentially during the fall. With Vidilab’s help we have also had the opportunity to collect tissue samples and feces samples for our research.

iGEM Stockholm
Previous years iGEM Uppsala has had a close collaboration with iGEM Stockholm (from KTH), something that is very advantageous to sustain due to the similarities between the teams, not to mention the geographical distance. This year we have two social events planned with the teams, one in Uppsala and one in Stockholm. In addition we have the panel discussion which has been previously mentioned. This year we hope to get the team of Lund Technical University involved as well.

Aalto Hensinki
New for this year is the WIKI and graphical design collaboration with Aalto Helsinki, where a common SLACK channel has been set up for close communication between the members responsible for the WIKI and graphical design in respective team.

Nordic iGEM Collaboration
iGEM Uppsala 2018, also participates in a common SLACK workspace for the project manager of each Nordic team, 'something that has been proven to be very useful in the sense of advice and support from people in the same position. Besides an easy way for direct communication, this year iGEM Uppsala has participated both in the Biobrick Tutorial Weekend arranged by the Technical University of Denmark and the Nordic iGEM Conference in Lund. Both events have been very useful and fun, socially and practically. In summary, both events proved to be wonderful opportunities to connect with other nordic teams.

Another collaboration that we are planning on with other iGEM contacts is a webinar later this summer. This would give us the chance to train in presenting our projects as well as share our experiences from working on iGEM. The general public would have access to the webinar as well.

We would like to extend our greatest gratitude to our kind sponsors:

Without the help and knowledge of these people our project would simply not be possible, we would like to thank the following for their contribution:

Anthony Forster, Professor at Department of Cell and Molecular Biology, Uppsala University

Alice Anlind, Research Engineer, Vidilab, former iGEM participant

Magdalena Haupt, Communications and Parasite expert at Vidilab

Sara Ljungström, Research and Development, Vidilab

Margareta Krabbe, Course administrator iGEM, senior lecturer at Biology Education Centre, Uppsala University

Maria Wilen, Consult at Biology Education Centre, Uppsala Universitet

Ylva Jansson, helpful former iGEMer

Gromit, unofficial team mascot and friend

[1] Karlsson J. Parasite detection in extensively hold Gotland ponies. 50

[2] Molena RA, Peachey LE, Di Cesare A, Traversa D, Cantacessi C. 2018. Cyathostomine egg reappearance period following ivermectin treatment in a cohort of UK Thoroughbreds. Parasites and Vectors 11: 61.

[4] Andersson E. Hur påverkas prevalensen av selektiv avmaskning? 22.

[5] Traversa D, von Samson-Himmelstjerna G, Demeler J, Milillo P, Schürmann S, Barnes H, Otranto D, Perrucci S, di Regalbono AF, Beraldo P, Boeckh A, Cobb R. 2009. Anthelmintic resistance in cyathostomin populations from horse yards in Italy, United Kingdom and Germany. Parasites and Vectors 2: S2