Team:Uppsala

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• Overview
• Team
• Project
• Modelling
• Human Practice

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

Cyathostominae

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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]. In addition, due to studies which has shown that treatments with deworming drugs can trigger the encysted larva in the intestines to re-emerge, small strongyle has gotten increased attention within the field [1].

Statistics Box:

In 461 horses in Sweden, an overall of 53,7 % of all horses was discovered to be infected [1]. In the period July to September the prevalence was estimated to 78,4% in the horse population, indicating seasons affect the infection rate [1]. Similar trends are seen in Europe and North America showing reported cases with 100% of horses showing small strongyle infections [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

Strongylus Vulgaris

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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 hence preventing infection

Methods for Detection

To be able to count either small or large strongyles, differentiation by studying them at a certain stage in the development is necessary. Eggs in feces are incubated for 10 days to let the parasite reach the right state. Afterwards the large or small strongyles can be successfully counted. Unfortunately this method only works for adult strongyles that have laid eggs. What is worth noting is that this technique only makes it possible to detect strongyles after the infection which obviously is a disadvantage, especially in the case for the large strongyle.

An alternative to this is to use ELISA, which could be used in the bloodstream, and PCR based methods, although the ELISA method need to be carried out in specialised laboratory settings and are not commercially available. Regarding PCR and ELISA they tend to become very expensive [12, 13, 17].

What is currently needed is a simple detection method, which can be used on spot by the farmer without the need to send samples to specialised labs as is the case with the above mentioned methods. Therefore we aim to develop a detection method that can be applied to pastures which would give horse owners the tools to prevent infection by large strongyles.

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.

Creating the Worm Buster

A step by step guide

So, how will we achieve this?

How can we use synthetic biology and a genetically designed bacteria to solve both these issues?

The first thing is to define the desired characteristics of each worm buster. To face this problem the team need to develop a bacteria which can:

Bacteria 1

Live in the intestinal tracts of horses and report a quantitative signal of small strongyle in faeces. The small strongyle buster can thereby 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

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. We choose two distinct approaches. The first of them, transcriptomic analysis, 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 at the presence of the nematode. The promoters of these genes can then be used to develop a biosensor.

The second approach utilizes libraries of phages expressing a set of peptides on their surface. By 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 and a camera.

Complementing Research of Cyathostominae

Proteomics

A second part will attempt to explore the known concerted action behaviour of the parasites.Seasonal encystation and emergence of larvae occurs in a coordinated fashion. 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 test, 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. If we can find it we could further develop diagnostic tools for guidance of treatment of deworming as treatment could be dissuaded at cases where amount of grown worms is low and amount of cysts is high. Deworming drugs have no effect on encysted worms and killing of the relatively few grownups would induce cyst bursting. Detection and describing of the eventual molecule demands different approaches depending on what structure we assume the molecule to have.

Modelling

Grass Sampling

Methods for detecting a strongyle infected field do exist, but is a long and arduous process. Our aim is to build predictable 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.

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

Global Worming problem and society

Integrated Human practice

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. Will society accept our solution?

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. Understanding what consequences resistance amongst strongyles has on these aspects and how the worm buster project would change the conditions within the field is what the human practice and modelling groups are all about.

Our approach to these aspects of the project is to perform market analyses, economic modelling and a report about the spreading resistance. Furthermore, our goal is to reach out to the people that are affected the most, such as horse owners, with information and advice regarding resistance since the issue lacks awareness.

Raising Awareness (light bulb)

Even if new diagnostic methods are introduced, solving the resistance issue requires a long term commitment which demands that safety precautions are routinely met. As some of the current deworming drugs have damaging effect on wildlife it is of utter importance to follow guidelines regarding how animals undergoing treatment should be kept.

While performing the market analysis we are also informing people affected by this issue. This is planned do be done by sending a questionnaire together with a compilation of a background consisting of information about the strongyles, resistance and guidelines for dosages and timing of deworming treatments. By spreading this information we hope to build better understanding on drug usage, which is a key action in order to prevent resistance development.

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.

Lastly, we have also been in direct contact with platforms such as Ridsport, a popular magazine in the community of horse owners, to spread the information of our project. We also hope to feature on “Vetenskapsradion”, a radio station updating about the news in diverse research fields.

Resistance in a larger scope (pill bottle)

The level of antibiotic resistance is increasing in several parts of the world and this is one of the major problems humankind is facing today. Apart from reducing the abuse of antibiotics and inventing new and functional alternatives, there is no solution to this growing problem. In the future, horses can suffer by parasites following the same path of survival mechanism, development of resistance. This would in worst case make the deworming drug treatments useless.

This issue also has the potential to grow even larger by breaking the borders in the long term view and hence reach a larger range of hosts. Shedding light to this area in purpose of increasing the awareness would result in horse owners and hopefully the equine industry in general being more controlling of the deworming treatment.

Financial perspective (dollar sign)

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 survey from 2009 where most of the participants were in the field of training, breeding and food production, showed that the estimated cost for a horse used for sports below elite level in Sweden is around 6-10 000 EUR while the monthly cost is estimated to 250-400 EUR. In Europe the mean cost for a horse in the same genre is 6 625 EUR and the mean monthly cost is 374 EUR [21]. According to “The Horse” (an american magazine about equine health) the minimal monthly cost is 179 - 257 EUR (2500 - 3600 USD per year) [22]. The amount above is not an estimation for horses used at elite level, hence can only be used to get an absolute minimum value of how much money that is spent.

If the resistance issue becomes widespread and effective deworming treatments cease to exist, the death ratios among horses will increase. Since this scenario is highly likely, the market interest in diagnosis and treatment systems is expected to increase. The economic loss expected by horse breeders in case of uncontrolled infections, include the daily expenses since the horse was born, breeding fees and negated profits that could have been achieved thanks to the horse.

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.

Uppsala 2018 - Our planned outreach

For the future of iGEM

In order to promote iGEM and synthetic biology among the public, we are working with several mini-projects such as preparing lectures, writing a booklet and organizing a panel debate. By working with different approaches we hope to raise the general awareness of synthetic biology as a research and occupational field. We try to draw attention to the challenges and possibilities that come with the profession within the field, as our goal is to inspire new generations of synthetic-biology enthusiasts.

Media Outreach

We think this project has great potential not only in the field of veterinary diagnostics, but also in the promotion of synthetic biology overall. Therefore we want to reach out to as many individuals as possible. There is also the potential of inspiring people outside or within the field to give the iGEM competition more consideration. This can be achieved by being active on our Facebook, Instagram, Twitter and Youtube accounts. The content we are uploading will be both educational and informative. Another approach has been to reach out to the local university newspaper “Techna”, that has published an article about our project and also about what iGEM is. We are also striving to get a chance to speak at this year's TedX Uppsala event.

Educational Outreach

Another potential collaboration is to reach out to schools and provide an educational lecture about synthetic biology to elementary school, high school and university students. Our idea is to adapt the content of the lecture, to make it useful and interesting for a wider audience. By reaching out to schools, we hope to inspire interest in students for sciences and specifically in the synthetic biology field, and hopefully inspire them to later join the Uppsala iGEM Team. We are also planning on attending events organized by other parties. For example, we will attend the annual conference at Uppsala University - Student Conference in Science and Technology 2018.

Public Discussion

Together with the Stockholm iGEM team, we are arranging a panel debate in which speakers and experts from the respective topic are invited to inform and debate. The purpose of the public discussion is to have an informative session about common challenges within the biotechnology field as well as creating an environment for an open discussion. To address a larger audience, we plan on inviting TV companies, such as SVT and TV4, to be able to broadcast the discussion on television. We also plan to invite other student driven organisations to the debate.

Booklet

To highlight the everyday challenges people within the synthetic biology field face every day, we are addressing an issue that has grown extensively in society. We have decided to shed light on the issue of burning out. The aim is to create a booklet with simple practical exercises as well as information about the issue. Seen through the lenses of iGEM, it will have some focus on the kind of stress that correlates with working on projects, teamwork and research ethics. Nonetheless, we hope that as many people as possible would find some constructive advice for dealing with exhaustion and how to prevent it. In conclusion, this will become helpful not only for the future iGEM Uppsala teams, but also for other teams worldwide.

Collaborations

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.

Sponsors

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

Acknowledgements

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

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

Maria Wilen, Consult at Biology Education Centre, Uppsala Universitet

References

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[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. Link:

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

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[6] Matthews JB, Hodgkinson JE, Dowdall SMJ, Proudman CJ. 2004. Recent developments in research into the Cyathostominae and Anoplocephala perfoliata. Veterinary Research 35: 371–381. Link:

[7] Jasovský D, Littmann J, Zorzet A, Cars O. 2016. Antimicrobial resistance—a threat to the world’s sustainable development. Upsala Journal of Medical Sciences 121: 159–164. Link:

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[9] C. Sellon D, T. Long M. Equine Infectious Diseases. Elsevier Health Sciences, 2007 Link:

[10] Colen MA, D. C. K van D, F. N. J. K. Anthelmintic resistance in Cyathostominae. Link:

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[12] Ling J. 2017. Strongylus vulgaris och Anoplocephela perfoliata. WWW-dokument 2017-07-14 Link:

[13] Bracken MK, Wøhlk CBM, Petersen SL, Nielsen MK. 2012. Evaluation of conventional PCR for detection of Strongylus vulgaris on horse farms. Veterinary Parasitology 184: 387–391. Link:

[14] 2013. New Method for Detecting Bloodworms. WWW-dokument 2013-07-27 Link:

[15] Cobb R, Boeckh A. 2009. Moxidectin: a review of chemistry, pharmacokinetics and use in horses. Parasites and Vectors 2: S5. Link:

[16] Corning S. 2009. Equine cyathostomins: a review of biology, clinical significance and therapy. Parasites and Vectors 2: S1. Link:

[17] Nielsen MK, Scare J, Gravatte HS, Bellaw JL, Prado JC, Reinemeyer CR. 2015. Changes in Serum Strongylus Vulgaris-Specific Antibody Concentrations in Response to Anthelmintic Treatment of Experimentally Infected Foals. Frontiers in Veterinary Science, doi 10.3389/fvets.2015.00017 Link:

[18] The Swedish horse sector. WWW-dokument. Retrived 2018-06-23. Link:

[19] Häggblom M, Rantamäki-Lahtinen L, Vihinen H. Equine sector comparison between the Netherlands, Sweden and Finland. 36. Link:

[20] Hästar och anläggningar med häst 2016 - JO24SM1701 - In English. WWW-dokument. Retrived 2018-06-24 Link:

[21] Liljenstolpe C. 2009. Horses in Sweden. Swedish University of Agricultural Sciences. Link:

[22] S. Loving N. 2012. How Much Does a Horse Cost? – The Horse. WWW-dokument 2012-. Retrived 2018-06-24. Link:

[23] The Anthelmintic Ingredient Moxidectin Negatively Affects Seed Germination of Three Temperate Grassland Species. WWW-dokument. Retrived 2018-06-25. Link:

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