Type of Document	Dissertation
Author	Jablonski, Lynn McGonagle
URN	etd-05042006-164516
Title	Development of a cloning system for gene expression in Pasteurella multocida
Degree	PhD
Department	Veterinary Medical Sciences
Advisory Committee	Advisor Name Title Sriranganathan, Nammalwar Committee Chair Boyle, Stephen M. Committee Member Carter, G. R. Committee Member Larson, Timothy J. Committee Member Schurig, Gerhardt G. Committee Member
Keywords	Pasteurella multocida Genetics Chicken cholera Immunological aspects Chicken cholera Genetic aspects
Date of Defense	1993-01-14
Availability	restricted
Abstract To identify antigens unique to live Pasteurella multocida P1059, 10 week old specific pathogen-free (SPF) chickens were vaccinated three times with one of the following: viable cells from P. multocida P1059, 3865, 3866, or cells from formalin-killed strain PI059 or formalin-killed strain P1059 that were opsonized with antiserum directed against killed strain PI059 prior to immunization. Vaccinated birds were challenged with 1.5 x 107 CPU of live strain P1059. Eight, 71, 86, and 50% of the birds that received live strains P1059, 3865, 3866 and killed strain P1059 (respectively), exhibited clinical signs of fowl cholera. Antisera directed against live strain PI059 recognized 23 proteins ranging from 14- to 92-kilodaltons (kDa); 20 of which were adsorbed by strain 3865. The molecular masses of the three remaining proteins were 25-, 30- and 43-kDa. A genomic library of strain P1059 was constructed using the plasmid vector pUC-19 and screened with antisera against live strain P1059; 12 out of 4,100 clones were recognized. The inserts of the plasmids from these clones ranged from 0.48- to 6.S-kilobases (kb) in length. Five of the 12 clones expressed proteins with molecular masses of 34-, 37-, 42-, 46- and 55-kDa. Escherichia coli CSR603(pOP43- 2G) and CSR603(pOP33-SF) expressed proteins recognized by antisera directed against live strain P1059. E. coli CSR603(pOP43-2G) expressed an epitope(s) which was recognized by antisera directed against strains 3865 and 3866. Conditions for transformation were optimized and attempts were made to create a shuttle vector in order to establish a cloning system for gene expression in P. multocida. The highest efficiency of transformation (1.25 x 10 7 CFU/p.g DNA) was obtained when 7.6 x 1010 cells of P. multocida R473 were electroporated at 12.5 kV cm-1 for 10 ms with 5 ng of the plasmid, p VM109. Of the six strains tested, representing serogroups A, B, D and E, all were transformed successfully. Vectors including pBR322, pUC19, pJFF224-NX and pSP329 were unable to transform P. multocida. To create a shuttle vector for gene expression in P. multocida, a Pasteurella plasmid (pLAR-1) was cloned in both orientations into the BamH I site of pBR322. These plasm ids, pLRBR .. 21 and pLRBR-67, had a transformation efficiency of 4.5 to 8 x 104 CFU/μg of DNA in strain R473. Chromosomal DNA containing the Brucella abortus copper-zinc superoxide dismutase gene was cloned into the Cia I site of pLRBR-21. The 1.8-kb fragment encoding a 42-kDa Pasteurella protein was cloned into an additional unique site (Nru 1) of pLRBR-21 to determine if this plasmid was a viable shuttle vector for gene expression in P. multocida.
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