Date

2014

Author

Çırçır, Ayça

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Mannheimia haemolytica A1 and Pasteurella multocida serotype A:3 are gramnegative bacterial pathogens which are considered the causative agents of bovine respiratory disease (BRD) or shipping fever in cattle. The foremost of several virulence factors of M. haemolytica A1 which allows lung colonization and establishment of infection is leukotoxin (Lkt). The others include adhesin, capsule, outer membrane proteins, and various proteases used for attachment, spreading and evading innate and adaptive host immune responses. Similarly, the major virulence factors of P. multocida are capsule, lipopolysaccharide (LPS), toxin (PMT), outer membrane proteins (OMPs), especially PlpE and OmpH, adhesins and type IV fimbriae (pili). BRD has considerable economic importance to the global cattle and feedlot industry. Effective control of BRD is challenging due to inconsistencies in diagnosis and treatment regimes. Extensive antibiotic usage in cattle industry, both prophylactically and therapeutically, leads to development of antibiotic resistance. Therefore, development of vaccine strategies against BRD to demonstrate protection becomes an important issue for cattle industry. P. multocida A:3 lipoprotein E (PlpE) and outer membrane protein H (OmpH) and M. haemolytica A1 leukotoxin (LtkA) have been selected as the antigens for vaccine development studies in our laboratory. PlpEC-OmpH and OmpH-LktA recombinant fusion proteins were previously constructed, expressed and purified. When PlpEC-OmpH was used as an universal vaccine candidate in combination with a mineral oil-based CpG adjuvant, it conferred 100% protection against P. multocida A:3 in BALB/c mice. In the present study, OmpH-Lkt recombinant protein was formulated as an oil-based vaccine candidate and BALB/c mice were immunized with this vaccine formulation. In two independent sets of mice challenge-protective potency experiments for each pathogen, %100 and %50 protection was demonstrated against M. haemolytica A1 and P. multocida A:3, respectively. Additionally, lktA fragment was cloned from the genomic DNA of M. haemolytica A1 at the upstream of plpEC-ompH fusion and the resulting lktA-plpEC-ompH triple fusion was constructed. In vitro expression of this fusion was shown in E.coli BL21 (D3) cells and the purified protein was used for vaccine preparation. The vaccine candidate comprising LktA-PlpECOmpH was formulated with a mineral oil-based adjuvant and the sera collected from the immunized mice were used for the determination of specific antibody and serum IFN-γ titers. Although the antigen-specific IgG1 and IgG2a levels were higher in immunized group, there was no significant difference between control and immunized mice groups in terms of IFN-γ levels. Protective capacity (potency) of this vaccine candidate was also evaluated via mice challenge experiments with lethal doses of P. multocida A:3 and M. haemolytica A1 separately, which did not give consistent results in two independent sets of experiments for each pathogen.

Subject Keywords

Pasteurella multocida., Respiratory organs, Bovidae., Vaccines.

URI

http://etd.lib.metu.edu.tr/upload/12618291/index.pdf
https://hdl.handle.net/11511/24283

Collections

Graduate School of Natural and Applied Sciences, Thesis