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Infection and Immunity, July 2000, p. 3916-3922, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Inactivation of Pasteurella (Mannheimia) haemolytica Leukotoxin Causes Partial Attenuation of Virulence in a Calf Challenge Model

Sarah K. Highlander,1,* Natalie D. Fedorova,1,dagger David M. Dusek,2,Dagger Roger Panciera,3 Laura E. Alvarez,1 and Carol Rinehart2

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 770301; Bovine Business Unit, Department of Biological Research and Development, Boehringer-Ingelheim Vetmedica, Inc., St. Joseph, Missouri 645062; and Department of Anatomy, Pharmacology and Pathology, Oklahoma State University, College of Veterinary Medicine, Stillwater, Oklahoma 740783

Received 9 February 2000/Returned for modification 22 March 2000/Accepted 30 March 2000

The leukotoxin of Pasteurella (Mannheimia) haemolytica is believed to play a significant role in pathogenesis, causing cell lysis and apoptosis that lead to the lung pathology characteristic of bovine shipping fever. Using a system for Cre-lox recombination, a nonpolar mutation within the lktC transacylase gene of the leukotoxin operon was created. The lktC locus was insertionally inactivated using a loxP-aph3-loxP cassette, and then the aph3 marker was excised from the chromosome by Cre recombinase expressed from a P. haemolytica plasmid. The resulting lktC strain (SH2099) secretes inactive leukotoxin and carries no known antibiotic resistance genes. Strain SH2099 was tested for virulence in a calf challenge model. We inoculated 3 × 108 or 3 × 109 CFU of wild-type or mutant bacteria into the lungs of healthy, colostrum-deprived calves via transthoracic injection. Animals were observed for clinical signs and for nasal colonization for 4 days, after which they were euthanized and necropsied. The lower inoculum (3 × 108 CFU) caused significantly fewer deaths and allowed lung pathology to be scored and compared, while the 3 × 109 CFU dose of either the wild-type or mutant was lethal to >= 50% of the calves. The estimated 50% lethal dose of SH2099 was four times higher than that of the wild-type strain. Lung lesion scores were reduced twofold in animals inoculated with the mutant, while clinical scores were nearly equivalent for both strains. The wild-type and mutant strains were equally capable of colonizing the upper respiratory tracts of the calves. In this study, the P. haemolytica lktC mutant was shown to be less virulent than the parent strain.

* Corresponding author. Mailing address: One Baylor Plaza, BCM-280, Baylor College of Medicine, Houston, TX 77030. Phone: (713) 798-6311. Fax: (713) 798-7475. E-mail: sarahh{at}bcm.tmc.edu.

dagger Present address: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.

Dagger Present address: USDA/APHIS, CVB-LPD, Ames, IA 50010.

Infection and Immunity, July 2000, p. 3916-3922, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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