Masters Theses

Date of Award

12-2000

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Robert N. Moore

Committee Members

Habib zaghouam, Phil Bochsler

Abstract

Of the many illnesses that plague the stockyards and feedlots of the world, none is as refractory or costly as bovine pneumonic pasteurellosis, more commonly known as "shipping fever" A vast amount of evidence has implicated Pasteurella haemolytica serotype A1 as a major component m this respiratory disease complex Included in the repertoire of virulence factors employed by this organism is a potent ruminant specific leukotoxin (LktA) that is suspected to contribute to the profound pulmonary lesions and fibrinous pleuritis that are characteristic of acute pneumonic pasteurellosis The unfortunate result of this disease is manifested in staggering economic losses that include treatment costs, market delay, and animal mortality

The importance of this toxin in the disease process has been punctuated by evidence suggesting that neutralizing antibodies against LktA are responsible for attenuating the seventy of the disease The fact that a potent humoral immune response against this critical bacterial epitope was noted in subjects recovering from the disease gave promise of developing an effective vaccine However, despite some very creative efforts, a viable approach to vaccination has eluded scientific and veterinary personnel for years. These strategies have included live vaccines, bacterns, recombinant subunit vaccines, and DNA based approaches While some of these approaches have shown promise in the laboratory, field trials have been disappointing, and at times inconclusive

The focus of this work is to present a foundation for the concept of generating a transiently sustained form of passive immunity that will attenuate, but not completely abrogate the disease state This mechanism of prophylaxis will allow the individual to mount an active response, in the event that it becomes necessary, without suffering the extensive pathology associated with acute pneumonic pasteurellosis.

In sum, I propose a novel approach to passive immunity, whereby a recombinant single chain variable-fragment (scFv) derived from a defined LtxA neutralizing antibody is fused with an IgGl signal peptide on the ammo terminus and an Igκ constant domain on the carboxy terminus The novel DNA construct is placed under the influence of a viral promoter/enhancer element in a eukaryotic expression vector and transfected into SP2/0 and COS-7 cell lines to demonstrate the feasibility of heterologous gene expression in lymphoid and non-lymphoid cell lines

The results of this study indicate that detectable expression levels of the fusion construct were appreciated m the COS-7 cell line but not the SP2/0 line Furthermore, detectable levels of protein were not appreciated in the extracellular milieu, suggesting a possible defect in post-translational processing or folding of the molecule Expression alone demonstrates the feasibility of this strategy, however the issue of secretion and functionality are central to the idea of DNA based passive immunity and remain to be resolved

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