Description

Abstract

Bovine Tuberculosis (bTB) is a chronic infectious disease predominately infecting cattle and is endemic in South-West England. Testing regimes are critical to managing this disease, however the most common testing system is low in sensitivity, slow, and stressful for cattle and farmers. We hope to improve the wellbeing and protect the livelihoods of farmers by developing a rapid and more specific bTB test. To do this we are developing two testing methods, one with an engineered Cas13a protein to detect RNA immune response biomarkers and the other using a Cas12a protein to detect specific bTB bacteria DNA sequences. These will both have the effect of cleaving the fluorophore from the quencher of the fluorescent probe to create a fluorescent response in the case of a positive result. Our designs have been informed and altered based on the input of relevant stakeholders throughout the entire process.

What is Bovine Tuberculosis?

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB), a chronic infectious disease that predominantly infects domestic cattle and various species of wildlife (e.g. deer and badgers). In the UK, cattle displaying bTB symptoms are systematically culled, with 21,298 culling occurring between April 2023 and March 2024 - a 5% increase from last year's 20,121 culls [1]. This costs the government £120 million a year, of which £50 million falls on farmers alone [1] .

But the impact of bTB extends beyond finances, having significant impacts on farmers and their family's mental wellbeing, not only from the culling of their herds but also from the mere threat of a positive bTB test result [1]. Additionally, while bTB is primarily expressed in cattle, humans in close contact with them are also susceptible to infection. In regions where subsistence farming is more prevalent, bTB transmission from livestock to their handlers results in approximately 140,000 human TB cases and 12,000 deaths per year [2][3]. It is therefore no exaggeration to say that, across the world, bTB represents a serious, global threat to livestock, rural economies and farmer's livelihoods and wellbeing [2][3].


Why We Chose This Project

Team Exeter is based in South-West England, an area that contains approximately a quarter of all English farms and where bTB is now endemic [1]. Consequently, this issue is very apparent in our local surrounding communities and something we wanted to put our efforts towards resolving. Due to the many areas bTB impacts, there are multiple avenues in which bTB could be addressed, a number of which are being enacted by the government. But our team decided to target the current testing regime.


The Issue

Bovine tuberculosis is a slow growing infection, meaning cattle can be infected with bTB and be infectious long before they start showing physical symptoms, such as lesions on internal organs (incubation can last from months to years)[8]. Because of this, routine testing is crucial in managing the spread of the disease [5].

The primary bTB diagnostic tool used in the UK is the tuberculin skin test, a common name for the Single Intradermal Comparative Cervical Tuberculin (SICCT) test[5]. This test works by looking for the immune response rather than the M. bovis bacterium itself due to the aforementioned incubation period. It is conducted by injecting tuberculin extracted from both lab-grown M. Bovis and Mycobacterium avium into the dermis (middle skin layer) of the cow[5]. The cow is deemed infected if the lump reaction created by the M. Bovis injection is 4mm greater than the Mycobacterium avium reaction[8]. While this test has contributed greatly to the controlling of bTB, talking with stakeholders closely involved in the process of cattle farming and bTB testing have presented some of the most pressing issues with the current testing regime:

  1. Stress on Farmers and Cattle


    2. Currently, bTB testing in this region is compulsory and relies on veterinary intervention that often requires weeks of preperation on the farmer's part.[1][4]. Furthermore, to perform the test cattle must be put in a crush twice within a 2 or 3 days period; first to administer the test injections, then again to analyse the results, increasing the risk of harm for all parties involved. According to the farmers interviewed, cattle are normally only brought in from the feilds around 3 times a year, two of which are for bTB testing. This makes the entire bTB testing process unfamiliar and stressful for the cattle, causing them to lose 2-4 weeks of scheduled weight gain.[7].

    On top of this, cattle can be deemed as an Inconclusive Reactor (IR) if the M.bovis lump only exceeds the avium lump by 1-4mm[8], meaning the cow must be isolated and retested 60 days later. In summary, not only can the test itself be a source of stress for both farmer and livestock, but so can the months before and after it.[5].

    text: current skin tests are often stressful for farmers and cattle, putting them at risk of harm. Gif of cattle walking into crush with farmer and cattle look stressed.

  2. Low Sensitivity


    3. The sensitivity of the skin test is between 52%-100% which means that on average 20-25% of TB-infected cattle are missed per round of testing, though this number can be as high as 47%[6]. This gap could lead to the infection spreading in between mandated testing, putting the rest of the herd at risk of infection while waiting for another veterinary visit. This results in a lot of wasted time, energy and stress on cattle and farmers for a testing process that is unable to account for all infected cattle. "Why go through all this effort if the test can't do what it should?" - David Andrews

    text: the current skin test may miss 1 in 5 infected cattle tested. Gif of 5 cows with magnifying glassed scanning them, bacteria is found on 4 but not 1

  3. Speed of Results


    4. As previously mentioned, it takes 2-3 days to get the results of the bTB test. This may not seem like a lot of time but it leaves a window for infection to spread throughout the herd and not be picked up by the time of the second test due to the bTB's incubation period. This has the capability of rendering the results after the second test out of date, forcing the farmer to get additional testing or run the risk of an infection spreading further.

    text: the current skin test takes 2-3 days to produce results, leaving time for the infection to spread. Gif of an infected cow next to 4 healthy cows. Shows the 4 cows slowly becoming infected as time goes on.

  4. Government Issued Testing


    5. Mandatory testing is only carried out every 6 months in high risk regions (with annual testing being the average) and is managed by government regimes. This gives farmers little agency when it comes to keeping track of the bTB in their herd themselves, as they have to rely on the testing provided for them. Additionally, farmers must wait a considerable amount of time for the required paperwork, all delivered in paper, to arrive, allowing them to continue their practices after the test. This process is made even longer with reported IRs and infected cattle. [7]

    text: Farmers are mostly reliant on government mandated bTB testing, causing them to feel a lack of agency. Gif of stressed farmer standing with a cow. Farmer is relaxed when vet arrives.

Our Aim

Our project aims to address as many of the aforementioned issues related to the current skin test by using synthetic biology to design, construct and validate a practical, sensitive and rapid diagnostic test for bTB that farmers can use to monitor the health of their livestock largely independently. In this case we hope to reduce the direct and indirect impacts of Bovine Tuberculosis on those involved with cattle farmering, protecting their wellbeing and livelihoods. Our proposed test will be informed by the experience of end-users to be simple, self-contained, inexpensive and shelf-stable.

Details

Below you can find the details of the seperate components of our test: how they will be produced, tested and put together.

Click on any of the buttons to read more information about what each step involves:


Producing Cas Proteins


*EDIT Initially for our test we have to produce our Cas proteins. These are specific bTB proteins key to identifying parts of the molecule or an immune response to it in a sample. This involves transforming a plasmid that codes for these proteins into BL21(DE3), a strain of E.coli optimised for protein expression. After successful transformation, the presence of the plasmids can be confirmed with an electrophoresis gel. These bacteria are then inoculated into a larger growth and induced to start the expression, achieved through autoinduction media. Once confirmed that our Cas proteins have been expressed we work on the purification of the samples. A variation of methods are available but we landed on Ni-affinity columns and gel filtration columns run on an AKTAPURE machine. At this point a pure sample of either Cas protein would be obtained.

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Fluorescent spectrophotometry

Background

*PLACE HOLDER TEXT - Fluorescent spectrophotometry is a technique used to quantitatively measure the fluorescence of a fluorophore at a specific wavelength (reference?). This is done by exciting the fluorophore at a specific wavelength, causing the light to be emitted back at the wavelength measured. This technique will eventually be used in our final product to be able to tell if there is a positive result for bovine tuberculosis (bTB). In our experiments we used this technique multiple times and therefore needed to create a measurement to allow our results to be more easily interpreted; this was achieved by designing concentration curves using the same settings and volume of samples that were used to measure our other experiments. These concentration curves could then convert arbitrary units (AU), which could only be interpreted using our spectrophotometer on the right settings, to fluorescein concentration equivalents (CFE) which can be used across all laboratories.

Why we did this

Fluorescein was chosen due to being available in the iGEM 'Fluorescence Measurement Calibrants Kit' therefore allowing future iGEM teams to easily understand our results, as well as being a relatively cheap fluorophore allowing many people to have access to it. Fluorescein was the prefect fluorophore to model a concentration curve around, this is due to the fluorophore in the RNase Alert probes also being fluorescein. This means that our model will have the same extinction coefficient (E=) (Reference) and a very similar fluorescence spectrum to what will be used in our tests, meaning the same excitation and emission wavelengths can be used for greater accuracy of the concentration curve.

Test Kit (Hardware)

Hardware Overview:

Our hardware system uses an open-source, low-cost lock-in amplifier developed by the Norwegian University of Science and Technology (NTNU)[**]. This device is used for detecting fluorescence, which is important as it indicates a positive result for our diagnostics test.

Mechanism:

The process first begins when the fluorophore and quencher of the probes are separated. This leaves the fluorophores isolated, which means they can then be excited at a specific wavelength, causing an emission of light (the fluorescence) at a higher wavelength. A flashing Light Emitting Diode (LED) is used to excite the fluorophore, the fluorescence then passes through an optical band-pass filter, which only allows light within a certain wavelength band to pass through.

Signal Processing:

An amplified photodiode detects the filtered light, converting it into an electrical signal. The amplified photodiode physically converts the input signal that is in a negative to positive voltage range into a modulated (just positive) output voltage range. This analogue signal is then sent to a microcontroller, where digitally the lock-in amplification process begins[**].

Digital Lock-in Amplification:

The analogue input signal is sampled at discrete time, i.e. the voltage is recorded successively at a fixed time interval. After a sample is taken, two reference signals are updated. To ensure the fluorescence signal and reference signal have a constant phase and identical frequency the LED is switched on and off at regular intervals. The individual reference signals and analogue signal are multiplied together, which are then computationally filtered (further reducing noise) using a model exponential curve. These are then combined together to produce a single output [**] [:-)].

Data Transmission and Interpretation:

The processed output is then sent transmitted to a mobile phone app via Bluetooth. The app interprets the data, providing the user both with a read-out of the voltages with respect to time and whether it is likely if the cow being tested has Bovine Tuberculosis.

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