Masters Theses

Date of Award

12-2001

Degree Type

Thesis

Degree Name

Master of Science

Major

Comparative and Experimental Medicine

Major Professor

David A. Bemis

Committee Members

Roger C. Carroll, Stephen A. Kania, Melissa A. Kennedy

Abstract

Atrophic rhinitis (AR) is an economically important upper respiratory tract disease of pigs, and is of multifactorial etiology. Bacterial agents of importance are toxigenic strains of Pasteurella multocida and Bordetella bronchiseptica alone or combined. Physical and chemical irritants like dust or ammonia in an animal's.environment may also contribute to the severity of disease. Many capsular type D and occasional type A strains of P. multocida produce a potent, intracellular, heat labile, 146 kD mitogenic toxin called Pasteurella multocida toxin (PMT). PMT is the major virulence factor that correlates significantly with progressively severe AR. A single component P. multocida toxoid vaccine was sufficient for inducing immunity against experimentally induced AR. In this study subunit fragments of PMT were genetically constructed and evaluated for immunogenicity. Three C-terminal constructs (685, 302 or 155 amino acids from the C-terminus) and two N-terminal constructs (1130 or 482 amino acids from the N-terminus) of PMT were cloned into expression vectors pET 29b+ and pPROEXHTc. A full-length recombinant PMT was also cloned and expressed in pET 29b+ vector. The recombinant proteins were purified by affinity chromatography. Six to eight week old BALB/c mice were immunized with these recombinant proteins and their immunogenicity was evaluated using ELISA and toxin neutralizing assay on Vero cells. Protective efficacy of immunization by these fragments was determined using lethal challenge with recombinant PMT. Immunization by the 685 amino acid C-terminal fragment and the 1130 amino acid N-terminal fragment protected mice from lethal challenge with rPMT. Recent studies that attempted to identify a biological activity domain of PMT reported contradictory results. While one study reported the requirement of N-terminus 500 ammo acid of the toxin for biological activity, two other studies reported the importance of C-terminus on the biological activity of toxin. However, in this study the protective N - terminal fragment lacked the putative C-terminal activity region and the protective C terminal fragment lacked the putative N-terminal activity region. The results of this study indicate that PMT may be a multifunctional toxin and either one of these regions is sufficient to induce a protective immune response in mice. Further studies are required to clarify the regions involved in binding and activity of the toxin. Correlation of the mouse protection assay with prevention of AR in pigs merits further study. The PMT fragments produced in this study will provide a basis for development of more refined subunit vaccine candidates.

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