Development and Evaluation of Live-Attenuated Paratuberculosis Vaccine Candidates
Creators: name: Eshraghisamani, Razieh
Publisher: name: Graduate Studies
Year: 2023
Subjects: subject: mycobacterium avium subsp. paratuberculosis
subject: Johne’s disease
subject: live-attenuated vaccines
subject: modified live vaccine
subject: oral vaccine
subject: enteric/mucosal vaccine
subject: dairy calves
subject: immune responses
subject: Education--Agricultural
subject: Education--Health
subject: Education--Sciences

description: Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis transmitted among ruminants through the fecal-oral route. It economically challenges the dairy industry, raises animal welfare concerns, and presents a potential public health risk. JD control faces obstacles due to insufficient sensitivity of current JD diagnostics, ineffective inactivated vaccines, and costly management practices. This thesis's primary objective was to develop an oral live-attenuated vaccine by targeting essential MAP genes, ensuring that the vaccine is attenuated for calf tissue colonization while retaining its ability to trigger immune responses. The first objective of this study was to identify MAP essential genes for persistence in calf tissue. Genome-wide transposon mutagenesis and high-throughput sequencing were combined to evaluate the essentiality of each coding region in the bacterial genome to survive in dairy calves. A total of 430 in vivo essential genes (ES) and 260 in vivo growth-defect (GD) genes were identified in MAP genome. Subsequently, two genes were selected for deletion: MAP 1643 (encoding isocitrate lyase) from the ES category and MAP 1531c (encoding the ABC transporter BacA) from the GD category. This resulted in the development of two live attenuated vaccine candidates, IcL and BacA, which were then assessed using a calf oral infection model. Both vaccine candidates were attenuated for colonization in calf tissues, while the deletion of these genes did not diminish their immunogenicity. Instead, inoculation with BacA vaccine led to a greater upregulation of proinflammatory cytokines compared to IcL and the wild-type strains, along with a more significant expansion of cytotoxic and memory T-cells compared to uninfected control. Finally, the effectiveness of the BacA oral vaccine in controlling MAP intestinal infection was assessed in a vaccination-challenge trial, where calves were first vaccinated and then exposed to wildtype MAP. Immunization with BacA reduced MAP colonization in the ileum and ileocecal valve, but no protection was observed in the jejunum. This partial protection was associated with increased CD4+ and CD8+ T cells with a pro-inflammatory phenotype (IFN-γ+/TNF-α+) in the bloodstream and Peyer's patches of vaccinated animals compared to the infection control group. Further investigation indicated that the variation in protection between the ileum and jejunum is primarily influenced by their tissue-specific transcriptome profiles, rather than differences in immune cell composition in response to MAP infection. In summary, the oral live-attenuated vaccine BacA was generated by removing an essential MAP gene. This vaccine candidate is attenuated for tissue colonization and can elicit MAP-specific immune responses, offering partial protection against MAP infection, especially in the ileum and ileocecal valve.