Uppsala iGEM 2018
Fighting the Hidden Resistance
The nematode parasites Cyathostominae, also known as small strongyles, cost the agricultural industry lots of money and grief each year due to the many consequences they cause. The economic burden of these parasites is forecasted to increase, since these worms are rapidly gaining resistance to most drugs used to combat them. There are currently no easy methods for the diagnosis of these small strongyles. Our vision has been to apply synthetic biology to the untouched field of veterinary diagnostics by developing a better tool for detection, thus decreasing the growth of resistance among small strongyles. While working toward the goal of creating a reprogrammed smart bacteria (nicknamed the Worm Buster) to detect and report the presence of small strongyles, we discovered new useful applications of existing techniques along the way. This has brought our vision about the Worm Buster closer to being achievable, which in the future will provide the tools necessary to help farmers make a decision whether to treat their animals or not.
Our work has laid the foundation for our idea to someday potentially come to fruition. Our novel applications of cutting edge techniques such as phage display and transcriptomics using Oxford Nanopore MinION third generation sequencing provide groundwork for further expansions in the field of veterinary diagnostics.
Small Strongyles or Cyathostominae are among the most common equine parasites, with more than 52 species in their family . 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 . 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 [2, 8].
Figure 1. Small strongyles.
The release of larvae from cysts can lead to lesions, diarrhea, and potential weight loss. This condition is called cyathostominosis . When untreated, the death toll can reach 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 . The threat 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 .
If farmers had the possibility to know when to treat their horses, prevention of mass rupture and other severe consequences like increased resistance development could be achieved. To reach this goal we have developed a model that (based on multiple parameters) calculates the optimal amount of treatments in a specified period. This will help avoid unnecessary use of anthelmintics by raising the awareness among farmers regarding when they actually need to treat their horses.
Current Methods for Detection
Currently the only method for detecting/counting 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 . This technique is expensive, inconsistent, and requires time sensitive shipping of samples. Currently many horse owners are reluctant to conduct the testing; treating their horses regardless of need which contributes to the resistance problem .
Unfortunately, the extensive overuse of deworming drugs has now lead to the detection of worms that are resistant to the most commonly used drugs [1,4]. 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 .
Moxidectin is a very common drug used. It is both hazardous for the environment and is also losing its effectiveness towards worm populations [14,15].
Due to all of these facts, we have during our iGEM project developed a reporter that is suitable for the in vivo environment of horse intestines. Moreover, we have developed new applications on existing techniques to be able to find the promoter that would be coupled to the reporter which would at last create the Worm Buster.
By implementing the model described under Symptoms together with the Worm Buster, horse owners will not only know how large the treatments should be but also when the treatments should happen. Thus they complement each other to minimize the amount of anthelmintics used, thereby helping to prevent resistance development.
The Worm Buster
The first thing the team did was to define the desired characteristics of the Worm Buster and outline the project strategy
- Our bacteria need to live in the intestinal tracts of horses.
- They also need the ability to 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.
The first step was to obtain live nematodes by the recovering them from the feces. After this process the next step would be to divide the large strongyles from the small strongyles using a 3D printed microfluidic chip with a microscope and stored for successive use.
Figure 2. The first stage of worm culturing.
As not a lot is known about our worms of interest, we needed an approach that would allow us to detect the worm without knowing its specific markers. For this, we developed new applications of two existing approaches.
For the first one, we have been developing a custom transcriptomic analysis protocol. This was necessary because transcriptomics is a new application for Oxford Nanopore technology. The transcriptomics procedure relies on the co-culturing of nematodes with E.coli and subsequent sequencing of the bacterial mRNA. This will ideally reveal which genes are upregulated when the bacteria are in the presence of the nematode. The promoters of these genes can then be used to develop a biosensor by linking them to a reporter!
Figure 3. Flowchart representing the trancsriptomics outline.
The second approach used a technique called “Phage Display”, which utilizes a random peptide library expressed on the surface of phages. By repeated rounds of affinity screening, only phages with high affinity to the molecule of interest will be selected. Sequencing the genetic information of these phages has allowed us to construct multiple candidate peptides that may bind to our nematodes’ surface proteins. This would allow the biosensor to aggregate at the detection sites and create a stronger signal.
In our project, phage display has been used in a whole new way. Performing phage display on a whole organism is an unconventional and unpublished procedure. By creating a working protocol for the purpose of finding a specific binder for strongyles we have applied this Nobel-prize winning method in a new way.
Figure 4. Flowchart representing the phage display outline.
After receiving the results from either the transcriptomics or the phage display, they need to be combined with a reporter to get a functioning diagnostic tool (Worm Buster). We have adapted and troubleshot the expression of a fluorescent chromoprotein, UnaG, to be able to detect our worms in both the intestines and in feces. This would enable a relatively simple and quantitative way for ranchers to detect the worms of interest, using a cheap UV lamp, a dark room, and a camera!
In order to make this a viable reporter system, we wanted to make sure the original biobrick part was functional. We show how we tweaked this part in order to study if it works properly so that it could be potentially used in future studies.
Figure 5. We would be able to detect the worms in feces with this method.
Optimization of the Time Between Treatments
To make sure the unnecessary use of anthelmintics is minimized we have created a model that compares regular and optimized usage of anthelmintics. When referring to the optimized use of anthelmintics, this means that the horse only receives treatment when the amount of parasites exceeds a certain threshold. Our model calculates given the initial amount of worms on the pasture and in the horse. Ideally, the information could be used with the Worm Buster to get a better overall result of our project. Our solution will not only be able to tell how large the treatments should be but also when they should be administered. This would guarantee the lowest amount of anthelmintics usage, and hopefully help combat the issue of growing resistance. Due to this fact, our model constitutes a large improvement on the potential implementation of our project since it directly brings us closer to our ultimate goal, which is the decrease in resistance development and the improved physical health of horses.
When we had chosen to create a Worm Buster, we wanted to see if there was a market for this kind of product. To gain knowledge about the market we conducted a market analysis together with the human practices group. The modeling group worked with creating a analysis program that could use the information from the market analysis and see if correlations could be found between different variables.
The small strongyles’ larger cousin, Strongylus Vulgaris, is the most pathogenic parasite in horses, posing a significant threat. They, like small strongyles, live in the grass and and infect the horse after being ingested . During the different larval stages inside the horse, the parasite enters the intestinal blood vessels as a part of their life cycle [4,10]. Because of this, they generally can’t be targeted by deworming drugs or detection methods and can cause major problems for domestic animals .
Due to this, an efficient diagnostic tool that can be applied to grass samples is necessary to prevent ingestion of large strongyles. This is why we looked into the possibility of a second Worm Buster targeting large strongyles could be possible for the future.
Figure 6. Large strongyles.
When the worms migrate into 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 . This can cause problems in the animals, with the worst case scenario being death .
Having these parasites is obviously a huge problem and there is great need for methods that can detect the parasites before ingestion, preventing infection. This makes the need for the second Worm Buster even larger.
Current 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 [11, 12, 16].
In conclusion, the current need is a simple detection method which could be applied to grass samples to be able to check if a pasture is secure for horses to graze on. Therefore we have analysed the possibility of developing the second Worm Buster by studying fluorescence in grass samples. Our results were promising since the fluorescence was clearly visible in the grass!
As you can see these little worms can cause a lot of harm. While we hope that our practical work will help solve the physical problem, 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.
Figure 7. Team member Ricardo Danza with our unofficial mascot Gromit.
We have attacked the problem at several angles to raise awareness for our project. We have performed a market analysis of the problem and sent out questionnaires to better judge the severity of the problem. We’ve even been published in a local swedish magazine, explaining the problem and raising awareness locally.
The equine industry includes many areas like stud farms, breeding, slaughter, trading, training and sports stables, and riding schools . 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 . There are approximately 350 000 horses is Sweden (2016) which gives Sweden a larger horse/person ratio than many other European countries [18, 19]. 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 have worked on the market analysis and economics modelling. The result from these decides if the eventual solutions actually will be possible to implement in real life.
In iGEM Uppsala we are worried about the future of iGEM members wellbeing and have seen multiple cases of burnouts. This is something that has to be changed to make iGEM sustainable for the future. To combat this problem we have hosted an event with iGEM Stockholm and Lund which shed light on the growing problem of burnouts and how to prevent them. To further spread the knowledge we have published a booklet about it! The booklet is a summary of useful guidelines and techniques to prevent team members from burn outs. It also speaks about the current situation in iGEM which we have summarised after performing an international survey with answers from current team members and previous team members all over the world.
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