Degree Type

Dissertation

Date of Award

2004

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Louisa B. Tabatabai

Abstract

The outer membrane protein P5 from Haemophilus influenzae has been shown to aid colonization in the human nasopharynx by binding to CD66 (CEA) on the surface of mucosal epithelial cells. The intent here was to determine if Haemophilus parasuis contained homologs of the P5 and P2 proteins and to characterize these proteins by SDS-PAGE, IEF, and CEA binding. Using outer membrane protein P5 primers designed from the H. influenzae database, we were able to detect a single PCR product in a H. parasuis field isolate. The PCR product was transformed into a competent E. coli strain and sequenced. DNA sequence analysis revealed homology to the H. influenzae P5 protein. N-terminal sequencing was used to confirm the presence of a 32 kDa P5 protein in both virulent and avirulent reference strains. Immunoblots were performed with H. parasuis reference strains representing all serovars using a P5 monoclonal antibody. A 48 kDa protein was identified in all but one virulent reference strain, whereas a 55 kDa protein was identified in all but one non-virulent reference strain. The 48 and 55 kDa proteins were subsequently identified as P2 by N-terminal sequencing. The outer membrane protein P5 from a Haemophilus parasuis field isolate (serovar 5) was fractionated by anion exchange and size-exclusion chromatography. The isolation of the pure P5 protein from the fraction was performed by elution from a polyacrylamide gel. Identification of the protein as P5 was determined by N-terminal sequencing. The P5 protein was further characterized by isoelectric focusing (IEF). Using a CEA immunoblotting method, it was determined that the purified P5 protein did not bind CEA. However, CEA did bind to a P2 protein in all type strain serovars. The molecular weight of the P2 protein varied between the virulent and avirulent serovars suggesting that it could potentially be used in discriminating between virulent and avirulent strains of H. parasuis.

DOI

https://doi.org/10.31274/rtd-180813-11856

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Jerry Kim McVicker

Language

en

Proquest ID

AAI3136336

File Format

application/pdf

File Size

110 pages

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