Development of a Diagnostic Platform to Detect Protein Biomarkers of Infectious Diseases
Date
2020-04-28
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Abstract
Production limiting diseases are a major cause of economic losses to the dairy industry and often affect animal welfare. The implementation of robust diagnostic tests that can detect diseases at an early stage is essential to decrease the high costs associated with the treatment of chronic cases and lost productivity. In this study, we focus on the development of diagnostic tests for two important production limiting diseases, mastitis and enzootic bovine leukosis (EBL). Somatic cell count is the gold standard test for the diagnosis of subclinical mastitis, but an increase in somatic cell count may not always correlate to infection of the udder as it is influenced by multiple factors. On the other hand, the most common methods to diagnose EBL are agar gel immunodiffusion or ELISA. A major limitation of these tests is the requirement of manual handling of samples and the long processing time, which makes them unsuitable as on-farm diagnostic tests. Therefore, the development of reliable point-of-care technology to detect these diseases in a convenient and cost-effective manner is required. In this regard, biomarkers of infection and disease can be detected with a novel biosensor, based on a novel split trehalase (TreA) enzyme, developed in our lab. Glucose is the output signal for this biosensor platform which can be detected by a colorimetric enzymatic assay or a handheld glucometer. In this thesis, we have applied the existing biosensor platform to detect BLV antibodies in clinical serum by using bacterial surface complementation of fusion proteins, containing split TreA fragments fused to antigens expressed on the outer membrane of E. coli. Our results indicate that using this assay, it is possible to detect anti-BLV antibodies in clinical serum and distinguish between healthy and BLV positive samples. We also applied the split TreA biosensor platform to detect lactoferrin, which is the biomarker of subclinical mastitis in cattle. A phage display library approach was used to identify the peptides with affinity for LF. In conclusion, we expanded the existing split TreA biosensor platform with tests for the detection of production diseases like EBL and mastitis that are compatible with on-farm use.
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AT Autotransporter, IMI Intramammary infections, PCA Protein complementation assay, TEV Tobacco etch virus, BLV Bovine leukemia virus, EBL Enzootic bovine leukosis, TreA E. coli periplasmic trehalase, LF Lactoferrin
Citation
Mukherjee, S. (2020). Development of a Diagnostic Platform to Detect Protein Biomarkers of Infectious Diseases (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.