Team:Uppsala/testing2
Uppsala iGEM 2018
Nematode parasites 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 to solve this problem. While working towards the goal of creating a reprogramed smart bacteria (nicknamed the worm buster) to detect and report the presence of the small strongyles, we discovered new useful applications of existing techniques along the way. This has made our vision about the worm buster more realistic, which in the future will provide the tools necessary to help farmers make a decisions 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 third generation sequencing provide groundwork for further expansions in the field of veterinary diagnostics.
The Targets
Figure 1: 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, 8].
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 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 [5]. 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 [9].
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 avoid unnecessary use of anthelmintics by raising the awareness in farmers regarding when they actually need to treat their horses.
Current Methods of 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 [4]. 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 [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 [2,4]. 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[2].
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.
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 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 [5]. 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 [9].
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 avoid unnecessary use of anthelmintics by raising the awareness in farmers regarding when they actually need to treat their horses.
Current Methods of 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 [4]. 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 [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 [2,4]. 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[2].
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.