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Infection and Immunity, March 2005, p. 1561-1567, Vol. 73, No. 3
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.3.1561-1567.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of an Immunodominant 28-Kilodalton Coxiella burnetii Outer Membrane Protein Specific to Isolates Associated with Acute Disease

Guoquan Zhang,1 Ho To,2 Kasi E. Russell,1 Laura R. Hendrix,1 Tsuyoshi Yamaguchi,2 Hideto Fukushi,2 Katsuya Hirai,2 and James E. Samuel1*

Department of Medical Microbiology and Immunology, Texas A & M University System Health Science Center, College Station, Texas,1 Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, Gifu, Gifu, Japan2

Received 6 August 2004/ Returned for modification 30 August 2004/ Accepted 27 September 2004

Coxiella burnetii causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol. 42:81-85, 1998). In order to clone the adaA gene, the N-terminal amino acid sequence of adaA was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an ~28-kDa mature protein with a typical signal sequence. The adaA (acute disease antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in adaA, and specific antibody to adaA reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that adaA is exposed on the outer surface of C. burnetii. adaA was overexpressed in pET23a as a fusion protein in Escherichia coli to develop anti-recombinant adaA (anti-radaA) specific antibody, which recognized a ~28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that radaA reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an adaA gene-targeted PCR assay and an radaA antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, 407 Reynolds Medical Building, Texas A & M University System Health Science Center, College Station, TX 77843-1114. Phone: (979) 862-1684. Fax: (979) 845-3479. E-mail: jsamuel{at}tamu.edu.

Editor: D. L. Burns

Infection and Immunity, March 2005, p. 1561-1567, Vol. 73, No. 3
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.3.1561-1567.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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