Browsing by Author "Luo, Yi Yi"

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    Creating a Marked Mycobacterium avium subsp. paratuberculosis Vaccine Strain and Detecting Marker-Specific Immune Responses in Calves
    (2018-04-17) Luo, Yi Yi; De Buck, Jeroen M.; Dong, Tao G.; Abdul-Careem, Mohamed Faizal
    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a chronic, contagious granulomatous enteritis with a high global prevalence in dairy cattle. This disease causes significant economic loss in the dairy industry and has been challenging to control due to its ability to survive in harsh environmental conditions for long periods of time, its easily transmittable nature through the fecal-oral route of transmission, the difficulty to detect the pathogen, and lack of an effective treatment or cure. The difficulty with detecting the pathogen stems from current diagnostic assays lacking specificity, posing issues with mycobacterial cross-reactivity, and low sensitivity, particularly during subclinical stages of disease. Previously developed vaccines do not prevent infection and are not approved in Canada in part due to interference with bovine tuberculosis diagnostics. To remediate this issue, positive and negative immune markers were inserted into a field strain of MAP as part of a vaccine capable of differentiating infected from vaccinated animals (DIVA). Specialized transducing mycobacteriophages were used to replace a gene coding for an immunogenic protein (MAP1693c) in the MAP genome with an HA (human influenza hemagglutinin) epitope-tagged immunogenic gene (PepA) via allelic exchange. Once gene replacement was confirmed, these markers were evaluated in a calf infection trial, where 17 Holstein-Friesian dairy calves were inoculated with either 109 CFUs of the marked strain (n = 6), 109 CFUs of a wild-type (WT) field strain (n = 6), or remained uninfected controls (n = 5). Cellular and humoral immune responses were measured using an interferon gamma release assay (IGRA) and enzyme-linked immunosorbent assay (ELISA), respectively. Marker-specific IFN- release was measured by stimulating whole blood with marker peptides and proteins, while antibody response was assessed by incubating serum with the same marker peptides and proteins. Some calves inoculated with the marker strain showed increased cellular immune responses to the HA epitope positive marker. Unexpectedly, a scrambled version of the HA epitope induced a significant IFN- response in marker-infected calves compared to WT-infected (p = 0.016) and uninfected (p = 0.019) groups at 4.5 months post-inoculation. This positive marker thus holds potential as a valuable diagnostic tool as part of a DIVA vaccine for Johne’s disease. We have shown that immune markers, both immunogenic proteins and epitope tags, can be introduced into the MAP genome using a stable allelic exchange method. The specificity of the scrambled HA epitope as an antigen to test for marker-specific cellular immune responses requires further investigation.