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United States Department of Agriculture

Agricultural Research Service

Title: Construction of An Isogenic Leukotoxin Deletion Mutant of Pasteurella Haemolytica Serotype 1: Characterization and Virulence (Final Title)

item Tatum, Fred
item Briggs, Robert
item Ames, Trevor - UNIV.OF MINN.,ST.PAUL,MN.
item Zehr, Emilie
item Sreevatsan, Srinand - UNIV.OF MINN.,ST.PAUL,MN.
item Hsuan, Shih - UNIV.OF MINN.,ST.PAUL,MN.
item Whiteley, Laurence - PROCTOR&GAMBLE,ROSS,OHIO
item Maheswaran, Samuel - UNIV.OF MINN.,ST.PAUL,MN.

Submitted to: Microbial Pathogenesis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 10, 1997
Publication Date: N/A

Interpretive Summary: The bacterium Pasteurella haemolytica causes pneumonia in cattle. This disease results in the greatest economic loss to the cattle industry in the United States. Leukotoxin, a protein product produced by P. haemolytica, is believed to play an important role in the disease process of the organism. In this study we inactivated the leukotoxin gene to generate a P. haemolytica strain devoid of leukotoxin expression. Here we report that in contrast to the parent strain, the P. haemolytica leukotoxin mutant does not kill bovine cells. Further studies with the leukotoxin mutant will provide important information as to the role leukotoxin plays in the disease process.

Technical Abstract: Three classes of Pasteurella haemolytica serotype 1 leukotoxin mutants were constructed by site-directed mutagenesis. As determined by Southern blots, single upstream crossover, single downstream crossover, and double crossover mutants of P. haemolytica. were generated. Western blot analysis indicated single downstream crossover and double crossover mutants produced no reactive leukotoxin product. In contrast, single upstream crossover mutants produced intact leukotoxin. Cytotoxic assay using bovine BL-3 cells revealed single downstream crossover mutants and double crossover mutants were devoid of active leukotoxin and were nonhemolytic. Mutants arising by single upstream crossover possessed approximately one-tenth the cytotoxic activity of the parent and were hemolytic, albeit much less so than the parent.

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