Ho To

Differentiation of Coxiella burnetii by sequence analysis of the gene (com1) encoding a 27-kDa outer membrane protein

The gene (com1) encoding a 27‐kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene‐specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.

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

ABSTRACT 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.

COXIELLOSIS IN DOMESTIC AND WILD BIRDS FROM JAPAN

Serological evidence of infection with Coxiella burnetii was found in 41 (2%) of 1,951 domestic birds and in 167 (19%) of 863 wild birds from 17 and 5 prefectures in Japan, respectively, by microagglutination (MA) test. The bacteriological evidence of the infection was found in 17 (41%) of 41 domestic birds and 37 (22%) of 167 wild birds by the nested polymerase chain reaction (PCR). In addition, C. burnetii was isolated from five each of serum, spleen and fecal specimens from five jungle crows (Corvus macrorhynchos) (whose sera were positive by both the MA test and PCR) by inoculating laboratory mice. Domestic quail (Coturnix coturnix japonica) (3%), domestic muscovy ducks (Cairina moschata) (3%), domestic chickens (2%), domestic mallards (Anas platyrhynchos domesticus) (2%), carrion crows (Corvus corone) (37%), jungle crows (35%), and wild rock doves (Columba livia) (6%) showed serologic evidence of experience with C. burnetii. There was a tendency for a high prevalence among birds living and/or feeding in close proximity to infected livestock. This suggests that these birds are one of the less important links in maintaining the whole cycle of C. burnetii infection.

Phase Variation Analysis of Coxiella burnetii during Serial Passage in Cell Culture by Use of Monoclonal Antibodies

ABSTRACT Antigenic changes in Coxiella burnetii Nine Mile strain phase I during serial passages in cell culture were analyzed with three groups of monoclonal antibodies (MAbs) against lipopolysaccharide. The MAbs of group 1 did not react with organisms that were passaged over five times, and the MAbs of group 2 did not react with organisms that were passaged over eight times. The MAbs of group 3 reacted with organisms passaged up to 15 times but did not react with phase II cells. These results suggest that C. burnetii could be differentiated into four phase states during phase variation.

Antigenic characteristics of polypeptides of Coxiella burnetii isolates.

Eighteen Coxiella burnetii strains from a variety of clinical and geographical sources were screened for antigenic variation of polypeptides by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) coupled with Coomassie brilliant blue (CBB) staining or immunoblotting. These polypeptide profiles showed the greatest variability in the region from 33 to 8.1 kDa. Such differences in the antigenicity of the polypeptides were also recognized by immunoblotting with 15 various mouse anti‐C. burnetii antisera. In addition, we detected a polypeptide at about 28 kDa which was immunodominant in strains from human cases of acute Q fever, milk and ticks but not immunogenic in strains from human cases of chronic Q fever. These findings suggest that this polypeptide is a marker to distinguish between acute and chronic strains.

Characterization of the Coxiella burnetii sucB Gene Encoding an Immunogenic Dihydrolipoamide Succinyltransferase

The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a λ EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced. The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N‐terminal region was only 42%. The gene was expressed in E. coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS‐PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever. The study results suggest that the C. burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever.

Use of Monoclonal Antibodies to Lipopolysaccharide for Antigenic Analysis of Coxiella burnetii

ABSTRACT Antigenic differences among Coxiella burnetii strains were analyzed. The monoclonal antibodies against the lipopolysaccharide outer core did not react with the strains containing a QpRS plasmid or with plasmidless strains, whereas they reacted with strains containing a QpH1 or QpDV plasmid. C. burnetii isolates could be divided into two groups immunologically.