Bo-Find
The Alberta cattle industry accounts for over $470 million towards Alberta’s GDP (Stats Can, 2017). Since it is a huge economic driver for the province, maintaining healthy agricultural practices are extremely important. BRD is currently treated by giving the cattle antibiotics with the hope that the infection will pass (Taylor et al., 2010). This affects farmers as it can cause cattle and economic losses.
Currently, no vaccines against these BRD infections are used by producers. Treatment of sick cattle is usually through mass antibiotic administration through feed or water. Calves that are high risk for BRD are metaphylactically treated with antibiotics (Taylor et al., 2010). However, mass antimicrobial administration represents major antimicrobial consumption and does not always result in decreases in cattle mortality (Baptiste & Kyvsgaard, 2017). Unnecessary blanket treatment of animals can also lead to the increased spread of antimicrobial resistance genes and the decrease of antibiotic efficacy (Whiteley et al., 1992).
We envision Bo-Find as a hand-held device that vets or farmers can use to detect Bovine Respiratory Disease (BRD) in cattle with minimal sample preparation (Figure 1). Current detection methods require extensive culturing and DNA preparation prior to pathogen identification or detection. Bo-Find will use a simple colour-change to give users a yes/no readout for infection. All of our tests will be able to be carried out within one hour, speeding up the detection process.
Figure 1. Bo-Find will incorporate on-site nasal swabs with recombinase-polymerase amplification technology to get rapid diagnostic results.
Bo-Find would require vets or farmers to take a deep nasopharyngeal swab of the cattle. These swabs have been validated as a collection method for BRD testing (Crosby et al., 2022). DNA from the collected samples is typically isolated using a commercial kit, such as DNeasy Blood & Tissue Kit or QIAamp PowerFecal DNA Kit, both from Qiagen (Conrad et al., 2020; Reuter et al., 2020). However, we propose that may be unnecessary and a simple heat treatment step has been shown to be sufficient for sample preparation (Sumit Jangra et al., 2021). In our design, nasopharyngeal swabs would be added to a tube with a prepared extraction buffer solution. This tube would then be heated to 100°C for 10 minutes (Figure 2). The sample would then be transferred to the detection tubes. Each detection tube contains a different set of primers for pathogen detection. These primer sets were validated previously and have been shown to work with RPA detection (Conrad et al., 2020). As a proof-of-concept, we will begin our detection testing using purified plasmids from our E. coli library. The RPA reaction will be allowed to start upon addition of the prepared sample DNA mixture, then incubated at 37°C for 30 minutes.
Figure 2. Hand-held temperature unit for sample incubation and detection (A). Nasal swab samples are first heat treated (B). Heat-treated nasal swab samples are loaded into individual detection tubes (C). Each tube contains a different primer pair for strain detection. A colour change indicates a positive result (D).
Bo-Find is advantageous in various ways in comparison to currently available solutions, largely owing to its ease of use. The system is fully contained in a user-friendly handheld device with color readout. This means the device can be used directly by the researchers or producers working with cattle, rather than relying on labs which consume more resources, incur higher costs, and can take multiple days for results to come back. Bo-Find takes less than an hour after initial testing onsite to give results. In addition to the convenience, a shorter detection time means the disease can be caught earlier before it proliferates, and action can be more effectively taken to treat affected cattle and stop the spread of disease. More effective detection can also lead to more targeted and efficient use of antibiotics given to cattle.
Bo-Find aligns with the United Nations’ Sustainable Development Goals. The 12th Sustainable Development Goal calls for responsible consumption and production. Bo-Find addresses this by reducing the need for use of prophylactic antibiotics, which are overused on healthy cattle to prevent BRD. By detecting pathogens earlier, it allows for targeted treatment of only infected cattle rather than preventative treatment of the entire herd. Bo-Find also addresses the 13th Sustainable Development Goal, climate action. The testing kit will be primarily made from recycled and/or biodegradable materials, preventing high volumes of testing waste from entering landfills. If possible, we would also like to work with users to hold a recycling treatment initiative, which would allow us to collect used kits to be recycled for future use.
While superior in theory, the kit’s design will need to be tested and improved for practical use. The device’s current design allows for testing of only one cow at a time. This can make Bo-Find unfeasibly slow if large-scale detection is required, so we plan to find a way to improve the design to allow for faster testing of multiple animals at once. TwistDx currently holds a commercial license for use of RPA, which Bo-Find requires for its lack of temperature fluctuation needed during DNA amplification. To safeguard against license infringement, consultation with an intellectual property lawyer will be necessary before the product is released for commercial use. We will largely be testing the detection system using E.coli. While a safer and easier option than working with Bovine Respiratory Disease pathogens, the system may behave differently with the two bacteria. Due to this uncertainty, we will have to ensure before commercialization that the system effectively detects its intended pathogen.
To overcome these potential problems we can consider developing a larger testing device that will be able to hold more rows on test, thus allowing for less waste while still testing the same amount of cattle. In feedlots cattle are usually divided into multiple pens. In these pens they are in close proximity to each other. Because of this, they develop very similar microbiomes within around 14 days from initial arrival at the feedlot. Consequently, only one to three cows would need to be tested per pen, depending on the size of the pen. This would allow for use of less tests, creating less product waste and less time for farmers or researchers to test each cow.