Description

Abstract

Bovine Tuberculosis (bTB) is a chronic infectious disease predominantly 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 aim to improve the well-being of farmers and protect their livelihoods 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 culls occurring between April 2023 and March 2024 in England - 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 well-being, 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 globally 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 farmers' livelihoods and wellbeing [2,3].

text: bTB predominantly impacts cattle but can also infect wildlife such as deer and badgers. it can even infect humans in close contact with cattle.

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.

text:Devon is considered a high risk zone for bTB, the same county the University of Exeter is in. Map of bTB prevelence in England and Wales

The Issue

bTB has an incubation period that can last from months to years, meaning cattle can be infected with bTB and be infectious long before they start showing physical symptoms, such as lesions on internal organs[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 Mycobacterium 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 M. 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 has presented some of the most pressing issues with the current testing regime:

text: Diagram of how the SICCT Test works, how positive tests require the M.Bovis reaction to be 4mm larger than the M.avium

  1. Stress on Farmers and Cattle


    2. Currently, bTB testing in this region is compulsory and relies on a veterinary intervention that often requires weeks of preparation 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-day 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 fields 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 farmers 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 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 on 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 bTB on those involved with cattle farming, 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

The aim is to detect specific DNA/RNA sequences that show that a bTB infection is present. The first step in this was to produce our Cas 12a/13a proteins that would detect these. We transformed plasmids that coded for the proteins into E. coliBL21(DE3) and overexpressed them in autoinduction media[9]. We then lysed and purified the cultures on an AKTAPure to achieve samples we could use with gRNA to detect the targets. Head to the protein expression and purification section of our results for more information.


Making Cas Specific

To make the Cas proteins specific, we had to first find the crRNA sequences from literature[10,11] to bind the spacer sequence to the protein. Once these were found we had to select spacer sequences that were complementary to sections of either DNA from the bTB genome or RNA sequences that were upregulated in a cows immune response. Once many potential sequences of DNA /RNA were identified, the folding of the spacer sequences when combined with the crRNA had to be tested, to make sure the spacer would remain free to bind to the target sequences. The top 5 sequences for each were identified and ordered as composite parts (see parts collection), transcribed, then combined with the Cas proteins to make them specific, then tested with the target and probe (fluorophore attached to a quencher which is cleaved when Cas is activated). Head to the sgRNA section of our results for more information.

Attaining a Valid Sample

To attain a valid sample, after going through the process of a design engineer cycle we eventually discovered heating the blood sample to be most successful (using methods from Arizti-Sanz, et al paper[12]). This reduces the opacity of the blood, reducing background noise of fluorescence so that a reading of a fluorescent result from bTB can be recorded.

Measuring a Result

The positive result for our test is a fluorescent signal. This can be measured using fluorescent spectroscopy for accurate results. To allow our results to be interpreted by other laboratories and iGEM groups, we have converted our arbitrary units to fluorescein concentration equivalents.

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)[13]. This device is used for detecting fluorescence, which is important as it indicates a positive result for our diagnostics test. For more details, head over to our hardware page.

simplistic and abstract diagram of our hardware components (LED, cuvette, filter, photodiode, microcontroller) which works on the principles of lock in apmplification

Figure 1: Overview of the hardware system. The cuvette containing the sample mixed probes is excited by an LED, causing the probes to fluoresce. This light then passes through the Band-Pass filter, reducing the LED light that will reach the sensor, meaning the fluorescent signal can then measured with reduced noise. The Sensor is an amplified photodiode that converts the fluorescent signal into an electrical signal.

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[13].

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 [13,14].

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 bTB.
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