| Summary: | 2019 Spring. Includes bibliographical references. Mycobacterium bovis, the etiological agent of bovine tuberculosis (bTB) is reported to cause disease in man and animal alike on every continent aside from Antarctica. Although coordinated efforts have been made for over a century in the US to cease transmission of this pathogen, outbreaks still occur. It has been posited that the failure to eliminate transmission of this pathogen is partially due to the diagnostic in use, which lacks critical sensitivity and specificity. To address this gap, we investigated a potential new method of identifying infected animals that is through the detection of a pathogen-derived biomarker. M. bovis phenolic glycolipid (PGL) is a species-specific, highly abundant, and unique glycolipid that comprises up to 2.5% of the dry cell mass. Coupling an LC-ESI-TOF-MS, method with a solid phase extraction, we successfully detected PGL derived from the urine of naturally-infected cattle. With this knowledge, we aimed to generate a detector of PGL that could be applied in a rapid and field friendly diagnostic platform. Using phage display technology, we selected M13 bacteriophage capable of binding M. bovis PGL with specificity that differentiated between M. bovis PGL and M. Canetti PGL, as well as between total lipid fractions of various species of Mycobacterium and other lipids with similar biochemical properties to PGL. These M. bovis PGL specific phage were able to differentiate between unspiked cattle urine and urine spiked with PGL. Lastly, we assessed the relative stability of PGL, specifically contrasted to another highly abundant mycobacterial glycolipid, phosphatidylinositol mannoside (PIM). We found that PGL was the more stable molecule when testing thermal and chemical stability, as well as when treated with protease K. However, when treating these glycolipids with whole cell lysates derived from fresh bovine organs, accurate detection on an LC-ESI-TOF-MS platform was lost. Further studies will be required to probe the ...
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