Masters Theses

Date of Award

12-1993

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Robert N. Moore

Committee Members

David A. Bemis, Philip N. Bochsler

Abstract

Pasteurella haemolytica Al has been implicated as a causative agent in shipping fever pneumonia. This organism produces a potent leukotoxin which is thought to promote inflammation and potentiate bacterial proliferation in the lungs of infected animals. Although several functional regions within the leukotoxin molecule have been identified, the antigenic regions and mechanism of action of the protein are still unknown. These studies were undertaken to further characterize the location and activity of protective epitopes within the leukotoxin molecule. Two approaches were used to accomplish these goals. In the first series of experiments, activated and unactivated leukotoxin proteins were employed to determine if LKTC gene modification affected recognition of LKTA by a panel of six leukotoxin mAbs. Results indicated that the epitopes for the mAbs were not involved in activation of LKTA by LKTC. Additionally, chimeric toxin molecules, composed of domains of the leukotoxin and the serotype 5 toxin of Actinobacillus pleuropneumoniae, were used to definitively identify the region of the leukotoxin which contained epitopes recognized by the leukotoxin mAbs. Each mAb recognized an epitope contained within amino acids 768-939 of the leukotoxin protein. The second method of examining protective epitopes in the leukotoxin molecule was with the use of anti-idiotypic antibodies. Rabbit polyclonal anti-idiotypic antisera were generated against a leukotoxin-neutralizing mAb, mAb ltx-2. Preliminary evidence indicates that this mAb functions by preventing the leukotoxin from associating with target cells. Anti-idiotypic antibodies of the β or γ subclasses (Ab2β or Ab2γ) were isolated. Subsequent assays tentatively identified these as Ab2β antibodies. Upon injection of these antibodies into mice, Ab3 antibodies were produced in several animals. The Ab3 antibodies recognized the leukotoxin by ELISA and neutralized the leukotoxin in a neutral red cytotoxicity assay. This indicated that the antibodies isolated from the antisera were indeed Ab2B. The Ab2β antibodies were then used to probe for a leukotoxin receptor on leukotoxin target cells. Unfortunately, no receptor was identified using these antibodies. However, the Ab2β antibodies were able to affinity purify antibodies from whole bovine antiserum samples which recognized the same epitope as mAb ltx-2. The results from this study indicate 1) the epitopes for the leukotoxin mAbs are not involved in LKTC-mediated activation and are located within amino acids 768- 939 of LKTA; 2) Ab2β antibodies against a leukotoxin-neutralizing mAb can induce the production of leukotoxin-neutralizing Ab3 antibodies; and 3) the mAb ltx-2 leukotoxin epitope is immunogenic in bovines and such antibodies have activity similar to that of mAb ltx-2.

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