Team:Uppsala/Test99
Uppsala iGEM 2018
Nematode parasites of the strongyle family cause the agricultural industry substantial losses and grief each year due to the detrimental effects they have on livestock. Common issues include severe health damage in the host animal as well as resistance development to anthelmintics in the most commonly occurring strongyles. There are currently no easy methods for diagnosing these parasites. By reprogramming a smart bacterium 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 to prevent infection to begin with.
Our Targets:
Small Strongyles
Small strongyles (Cyathostominae) are among the most common equine parasites with more than 52 species in their family. Horses are exposed when they graze on infested pastures, where they consume the worms in their larval stage. The strongyles move to the horses’ intestines, gathering into cysts in the intestinal wall and usually reach very large numbers. These cysts eventually burst and the developed worms moves up towards the intestinal lumen where they become adult worms. The release of the larvae from the cysts can lead to lesions, diarrhea, and potential weight loss in the animal, a condition called cyathostominosis. When untreated, the mortality rate of a small strongyle infection can reach up to 50%.
Large Strongyles
Strongylus vulgaris is the most pathogenic parasite in horses, posing a significant threat to the health of the animal. They, like small strongyles, live in the grass and infect the horse after being ingested. During the larval stages inside the animal, the parasite enters the intestinal blood vessels as a part of their life cycle. This causes them to commonly avoid deworming and detection measures. When the worms migrate in the arteries they cause inflammation in the arterial wall and induce the formation of blood clots. These blood clots may travel in the blood vessels and block smaller passages, inhibiting oxygen and nutrient supply to the surrounding tissues, and may result in colic or in the worst case, death.
The Worm Busters
One approach for designing a diagnostic system against the strongyles is to engineer a “smart” bacterium which will live in the intestinal tract and is capable of reacting to the presence of the parasite by emitting a quantifiable signal. This biosensor may, for instance, induce the production of a detectable fluorescent protein in the feces of the animal. Large strongyles, however, are more elusive and are less frequently present in the intestine during an infection. Here it would be suitable to instead try and detect the presence of the parasite on the pastures as to avoid infection altogether, by developing bacteria responding to the parasite outside of the animal body.
Our work is dependent on finding one or more genes in E.coli which will, exclusively, be highly expressed when the cell is exposed to the parasitic worms. This is done by co-culturing E.coli in liquid medium along with live strongyles, which are harvested from feces and sterilized. From here, the E.coli cells are separated from the solution and their entire transcriptomic suite is extracted and sequenced to detect genes of interest. Any found genes which display promise will have to be validated by qPCR (which is a similar method) in a second run to confirm that they are only expressed due to the strongyle presence. Another approach to tackle our challenge is to screen for interaction between the surface proteins on the strongyle and short peptides. Through affinity screening of a random peptide library displayed on the surface of phages, we can select a peptide with a high affinity to the nematodes surface.
The Worm Busters
One approach for designing a diagnostic system against the strongyles is to engineer a “smart” bacterium which will live in the intestinal tract and is capable of reacting to the presence of the parasite by emitting a quantifiable signal. This biosensor may, for instance, induce the production of a detectable fluorescent protein in the feces of the animal. Large strongyles, however, are more elusive and are less frequently present in the intestine during an infection. Here it would be suitable to instead try and detect the presence of the parasite on the pastures as to avoid infection altogether, by developing bacteria responding to the parasite outside of the animal body.
Our work is dependent on finding one or more genes in E.coli which will, exclusively, be highly expressed when the cell is exposed to the parasitic worms. This is done by co-culturing E.coli in liquid medium along with live strongyles, which are harvested from feces and sterilized. From here, the E.coli cells are separated from the solution and their entire transcriptomic suite is extracted and sequenced to detect genes of interest. Any found genes which display promise will have to be validated by qPCR (which is a similar method) in a second run to confirm that they are only expressed due to the strongyle presence. Another approach to tackle our challenge is to screen for interaction between the surface proteins on the strongyle and short peptides. Through affinity screening of a random peptide library displayed on the surface of phages, we can select a peptide with a high affinity to the nematodes surface.
The Worm Busters
One approach for designing a diagnostic system against the strongyles is to engineer a “smart” bacterium which will live in the intestinal tract and is capable of reacting to the presence of the parasite by emitting a quantifiable signal. This biosensor may, for instance, induce the production of a detectable fluorescent protein in the feces of the animal. Large strongyles, however, are more elusive and are less frequently present in the intestine during an infection. Here it would be suitable to instead try and detect the presence of the parasite on the pastures as to avoid infection altogether, by developing bacteria responding to the parasite outside of the animal body.
Our work is dependent on finding one or more genes in E.coli which will, exclusively, be highly expressed when the cell is exposed to the parasitic worms. This is done by co-culturing E.coli in liquid medium along with live strongyles, which are harvested from feces and sterilized. From here, the E.coli cells are separated from the solution and their entire transcriptomic suite is extracted and sequenced to detect genes of interest. Any found genes which display promise will have to be validated by qPCR (which is a similar method) in a second run to confirm that they are only expressed due to the strongyle presence. Another approach to tackle our challenge is to screen for interaction between the surface proteins on the strongyle and short peptides. Through affinity screening of a random peptide library displayed on the surface of phages, we can select a peptide with a high affinity to the nematodes surface.