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Proteomic and immunological investigation of Mycobacterium avium subspecies paratuberculosis secreted proteins

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Title: Proteomic and immunological investigation of Mycobacterium avium subspecies paratuberculosis secreted proteins
Author: Facciuolo, Antonio
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Mutharia, Lucy
Abstract: Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of Johne’s disease, an enteric infection and production-limiting disease of ruminants globally. Fecal-oral transmission is the predominant route of infection whereby asymptomatic cattle intermittently shed Map in feces and milk sustaining pathogen transmission. Current diagnostic tests display poor sensitivity in identifying asymptomatic animals due to the use of undefined antigens and knowledge gaps in Map components eliciting pathogen-specific immunity. In this thesis a proteomic investigation of the Map secretome identified 162 proteins; among which 66 had not been previously reported in Map culture filtrates. Serum antibodies from Map-infected cows identified 11 antigenic targets in the secretome that were subsequently cloned and expressed revealing a subset that could differentiate (P < 0.05) uninfected- (n > 30) from Map-infected cows (n > 30) in ELISAs and immunoblotting. Antisera for six of the antigens showed reactivity to the recombinant and native Map protein, and minimal reactivity to a closely related Mycobacterium avium protein extract suggesting the recognition of Map-specific epitopes. The immunogenicity of these proteins during early Map infection was evaluated at Peyer’s patches (PP) in surgically isolated intestinal compartments during experimental infection of calves. Map-infection of jejunal, but not ileal PPs, induced mucosal IgA B cell responses to 9 proteins in the majority of calves (n=7) assayed at 1–2 months post-infection. Secretory IgA specific to these 9 proteins was detected in the jejunal and ileal contents consistent with the homing of B-lymphocytes to the gut mucosa. These data report for the first time: that Map uptake occurs in jejunal PP, in addition to ileal PP; secreted Map proteins induce both systemic IgG and mucosal IgA antibody responses during the early stages of Map-infection; and, there is a functional dichotomy in mucosal immunity at jejunal and ileal PPs in calves. These secreted proteins provide novel diagnostic reagents for the advancement of both serological and fecal IgA-based early detection of Johne’s disease in cattle. In addition, these immunogenic proteins provide pathogen-specific tools to further interrogate both host systemic and mucosal immune responses occurring in cattle during Map-infection.
Date: 2015-08
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