Project Description

Johne’s disease affects livestock via bacterial infection in the intestinal lymph nodes, and is a contributing cause of economic loss within the dairy industry globally as well as causing suffering to our livestock. The pathogen, Mycobacterium avium spp. paratuberculosis(MAP), is transmitted between cattle within farms and is absorbed by macrophages which then reside in the lymphatic nodes of the intestinal wall. The bacterium can reside within the animal for as long as 8 years before suddenly falling ill and rapidly deteriorating in condition. The aim for our project was to find an alternative diagnostic test for Johne’s diseases which will produce a result within a significantly shorter period of time than those commercialized tests being used currently. Due to Johne’s disease having no practical cure, the objective for these tests is to rapidly diagnose to allow farmers to remove infected individuals and prevent further spread to other, healthy, livestock. The mechanism for this detection involves a fusion protein which contains a split luciferase fused with monomers of a MAP-specific encapsulin protein, where a characteristic presence or absence of bioluminescence produced by the luciferase can be used to develop the diagnosis. This is contained within a small, enclosed device, which contains a photodiode capable of both detecting the presence of bioluminescence and converting this into a signal which will allow the device to then show whether or not the sample contains the presence of MAP, providing the necessary diagnosis.

Johne’s disease is one which acts on a global scale, with 10 year average annual losses across the European Union totalling $364.3 million USD, annual loss in the United States around $198.4 million USD and $25 to $56 million USD each year here in New Zealand.(1,2) The wellbeing of the cow also requires attention, as the livestock which go undiagnosed will continue to suffer until they succumb to the fatal symptoms of infection. Testimonials from local farmers at Canterbury Grasslands Limited, ANZCO foods and Craigmore Sustainables showed us that there was a clear problem with the current testing, where samples of cattle bodily fluids are sent to be tested in off-site laboratories with a result being returned around a week later.(3,4) These tests were used to confirm what had already been identified in the visual examination, and, with a week between suspicion and diagnosis, further infection could take place before the cow was removed. They made it very clear to us that our idea for a rapid, on-site test would provide a vast improvement on the current methods, and could allow them to limit the spread of infection within their livestock. MAP is transmitted between cattle via fecal contamination and pre- and post-natal interactions between mother and calf, at which point the infection begins. From here, MAP embeds itself in the intestinal wall and begins to proliferate and establish a parasitic relationship with the host. After a certain period of time, which can range from 2 to 8 years, serious symptoms of enteritis become apparent and the cow begins wasting. At this point, there is a high presence of MAP within the intestines so high levels of shedding (removal into the fecal matter) of the MAP occurs, which then allows the infection process to restart. The original host has lost a significant amount of its body weight, can no longer be used for any agricultural output and will eventually die.(5,6,7,8,9) The progression of MAP infections is displayed in Figure 1.

The UCNZ iGEM team is developing LuMOO, the luminescent MAP on-site observation kit, which will come in the form of a small, enclosed device which the farmer can simply add their serum to, with the device registering a positive or negative result within a few minutes. So how does it work? We have selected a target protein which is highly specific to the MAP bacteria, called MAP-specific encapsulin 2A (also named MAP2121c and major membrane protein). This protein is a part of the encapsulin 2A family of proteins, which means it forms 60-mer nanocages via the association of many monomers and holds cysteine desulfurase, an enzyme pertinent to cell starvation.10 It is due to this distinct feature that we developed the assay mechanism, which detects the formation of these cages as a means to detect the presence of the MAP pathogen within the Bovis serum.(11) The team engineered a fusion protein which combines the encapsulin monomers with a split luciferase system developed by Promega, NanoLuc Binary Technology (herein NanoBiT), which consists of Small BiT or SmBiT, and Large BiT or LgBiT that when brought in close proximity, associate to form the NanoBiT luciferase and present a bioluminescent glow.(12) With encapsulin monomers attached to both SmBiT and LgBiT, when the cages form the SmBiT and LgBiT will be brought in close proximity to each other and bind to form the NanoLuciferase enzyme. Addition of the substrate NanoLuc, a glow can be detected. However, in the presence of MAP, the wild type encapsulin monomers are present, which will separate the fusion-encapsulin monomers within the cage, which will reduce or eliminate the glow as the SmBiT and LgBiT are distantly spaced and unable to associate and glow. The association and dissociation of the MAP encapsulin cage can be seen in Figure 2. The presence or absence of bioluminescence will be detected by the engineered device’s TSL237 photodiode, which will transfer the signal to the attached Arduino, where it will be translated to a positive or negative result for the farmer to see. This system is held within a compact, light sealed box which will allow the farmers to add a sample and receive a result in a fast and efficient manner.This rapid-action test will both save the farmers from a loss of output which would come about due to further infected cattle, as well as allowing those infected individuals to be humanely culled rather than prolonging the suffering caused by the disease.