Design

Prototype

Design 1 - the Eppendorf

We first assumed that our testing could take place in a 1.5ml eppendorf tube. Our cell free system currently takes up a volume of 50μl, leaving ample space for a few drops of blood to be added. If RNA in this blood sample is associated with bovine TB (bTB), it would trigger our toehold switch, causing a reporter to be expressed (eg, a colour change). An eppendorf tube is lightweight, durable, and can be totally sealed to prevent outside contamination. The environment in which the test is performed can be unclean and full of possible contaminants, so this seal is an important design feature.

Design 2 - the grip tube

Following a meeting with Phil Leighton, a vet who conducts bTB tests in the south west, we realised our prototype needed some major changes. We learned that blood tests in cattle take place behind the tail, as opposed to the current skin test which takes place in the neck. According to Phil, the blood test is easier and safer to perform, although there is still some risk of kicking from the cows hind legs. This risk is significantly reduced as the cow is normally held in a crush, preventing the rear legs from kicking backwards and letting the test stand to the side of the animal. However, he also told us that an eppendorf tube would be too small and fiddly for the purpose of a blood test. The current blood test tube is around 110mm long with a 12mm diameter, bulky enough for the vet performing the test to get a good grip on the tube whilst drawing blood from behind the cow. The environment in which the test takes place is unclean and frantic, so our next design took elements from the current blood test tube. We designed a larger test tube with a vacuum sealed lid, shown here. Inside the main tube is a smaller eppendorf tube containing the cell-free test system.

Design 3 - the filter

Our own research into bovine blood testing and some more advice from Phil brought up a new issue: bovine blood tests can draw about 7ml of blood. Our test only requires a few drops, but the vet performing the test would not have time to measure delicate amounts of blood whilst behind an agitated cow. This lead us to move the eppendorf containing our cell free kit to the base of the blood tube. We then needed to design a filter that allows only a small amount of blood to get through to our test. Ideally this membrane would let only a few drops of blood through whilst keeping the remaining blood above the membrane. This method lets the tester collect a blood sample as normal. Another benefit is that in a future design the cell free kit could snap off of the blood tube, this allows for both a TB test to be carried out and a blood test. By associating these two parts together, most likely with a tear off label, the blood could be tested in more detail if required.

The 3D mockup shows the vacutainer with an eppendorf in the base containing our cell free kit, the filter sit above this. This design allows the blood to be kept away from the cell free kit, while the filter lets the correct about of blood into the eppendorf tube. This should hopefully allow the vet to take a blood sample as normal, but also ensure the cell free kit isn’t saturated with blood.

The Current Methods and Their Limitations

The final prototype of our bovine TB test has been honed and revised several times as we considered the practicalities of TB testing for the vet performing the test. We took on advice from vets local to the southwest (namely Phil Leighton and Dick Sibley) and incorporated this into our prototype design. This allowed the design to ‘evolve’ and advance; we now have a final prototype which should maximise the ease of testing and minimise any error in the testing.

Our first consideration in testing was where we would take the sample from. The current skin test is performed on the neck, which must be shaven and then injected with avian and bovine TB. The vet then returns 72 hours later to diagnose the cattle based on the skin’s reaction to the injected strains. Phil told us that neck testing is awkward and time-consuming for the vet, and that blood tests are easier to perform.

As the RNA associated with a TB infection (but not present in the BCG vaccine) is found in the blood of infected cows, we further discussed this option with Phil. He told us that bovine blood tests are usually drawn from a prominent vein behind the tail of the cow, and that this kind of test has many benefits over the cumbersome skin test. These are outlined:

• The blood test is much easier and quicker to perform. There is no need to shave the area, it is a simple matter of inserting a needle and drawing blood.
• The blood test is a safer option, as the tester can stand to the side of the cow (avoiding kicks from the hind legs) to draw blood quickly, and the thick skin in this area means that the cow is unlikely to feel much pain from the test.
• The current skin test can agitate the cow as the neck is a sensitive area, which must be shaved and injected.
• The vet must only make one visit to the farm to draw blood samples, whereas with the current test the vet visit twice returning 72 hours after injection to diagnose.

We then focused on making our test as easy as possible for the vet to perform. Phil Leighton was invaluable during this process, telling us exactly how we could help vets like him. The current test has several steps: the cow arrives through a press, the number on the cow's ear tag is taken down, it is shaven and injected with the attenuated strains, and then released. 72 hours later, the vet returns. The cow again arrives through a press (in a random order), it’s ear tag number is taken down, the neck is observed and the cow is diagnosed, then the appropriate action is taken. This method has several areas where error can occur.

• The wrong number could be written down at either stage (testing or diagnosis).
• The list of numbers written down can be mixed up, resulting in the diagnosis being matched to the wrong cow.

We aim to help these problems by both devising a new test and a new method of testing. The testing methods contains both a prototype for the test and a method for the test.

Our new test

Our test has fewer steps and should be fairly quick and easy in comparison with the current test. First, the cow arrives through a press, it’s blood is quickly drawn into the test tube, the tube is then labelled with the cow’s ear tag number, and the cow is released. The risk associated with getting the blood is also reduced as the crush prevents the cow from kicking backwards. It also lets the tester stand to the side of the animal when gathering the sample. After a few hours, a diagnosis can be made from the labelled test tubes. In this way, the only possible error source is from incorrect labelling or contamination.

Once the RNA associated with a bovine TB infection has been detected, a reporter gene is expressed. We wanted this reporter to be immediately clear and visible to any untrained personnel, eg. fluorescence was unsuitable as the equipment required to visualise fluorescent proteins is expensive, requires training to use, and is not portable.

Firstly we decided on a simple colour change using chromoproteins. However, taking into consideration the nature of the sample - blood - we were unsure as to whether a colour change would show through the dark colour of the blood. This could be solved by centrifuging the blood and testing the plasma, which has much less colour and so would interfere much less with the chromoproteins. However, we were adamant that our test should require no technical knowledge or equipment, so we have came up with several other possible solutions which we are unable to test due to time constraints:

• Using a metal strip which the chromoprotein could aggregate around, causing a colour change to the strip which could then be removed and examined.
• Using a paper-based system, where a drop of blood could be placed onto a cell-free piece of paper and a diagnosis could be made.
• Using an enzyme indicator - eg. an agglutination assay.

Our New Method

Not only is the current test for TB outdated, the current methods of testing are as well. Currently the cows are recorded using ear tags, these are either plastic or metal, and attached to the cows ears. There are two of them normally and the numbers on them must be written down once the cow is tested. This is a very analogue way of recording the information.

The method we propose is to change the ear tags so that they contain a passive RFID tag, as well as a number. This allows a lot more information to be contained on the tag. Not only this, it also changes the system to a digital version whilst still keeping it familiar to the farmers that use it day to day.

By placing an active RFID reader in the testing facility, this could be used to power the passive RFID tags located on the ear tags. The reader can work up to 100m, whereas the tags work up to 10s of centimetres. If this was placed at the crush containing the cows then as they went through they would be registered as being tested. This removes the chance of human error and also speeds up the testing procedure. The number on the tag would still have to be written on the test tube to associate this with the animal tested.

We were reluctant to have much human involvement but after much deliberation is was decided that writing on the tubes would be the easiest way to link the cow and the tube together. With the RFID tag speeding up the cataloguing of testing.

After waiting for our test to be finished the positive reactors could then be located and the number found. By inputting these number into the computer it would be possible to then walk the cows back through the crush and have an automatic signal for when the infected animal passed through. It would then be easy to find it a remove it from the herd.

For an existing farm they would have to purchase passive RFID tags for their herd, and one active RFID reader located at the crush. A free piece of software would then downloaded onto a laptop to log the cows and located the infected animals.

The cost for these devices are as follows:

• Passive RFID $0.15 each and are stickers so can use on existing tags ^[1]
• Active RFID beacon can be purchased for a maximum of $100 ^[1]

References:

[1] http://atlasrfid.com/jovix-education/auto-id-basics/active-rfid-vs-passive-rfid/