Restriction Endonuclease Analysis Discriminates Bordetella bronchiseptica Isolates

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2000, p. 4387 4393 Vol. 38, No. 12 0095-1137/00/$04.00 0 Restriction Endonuclease Analysis Discriminates Bordetella bronchiseptica Isolates RANDY E. SACCO,* KAREN B. REGISTER, AND GWEN E. NORDHOLM USDA/Agricultural Research Service, National Animal Disease Center, Ames, Iowa 50010 Received 10 April 2000/Returned for modification 16 August 2000/Accepted 21 September 2000 One hundred ninety-five Bordetella bronchiseptica isolates from 12 different host species worldwide were characterized by restriction enzyme analysis (REA). These isolates had previously been categorized into 19 PvuII ribotypes. Twenty restriction endonucleases were evaluated for use in REA. Digestion of chromosomal DNA with HinfI, followed by submarine electrophoresis in agarose gels and staining with ethidium bromide, produced DNA fragments in the 4.0- to 10-kb range, which readily discriminated B. bronchiseptica isolates, resulting in 48 fingerprint patterns. Moreover, AluI digestion of chromosomal DNA produced 39 distinct fingerprint profiles with DNA fragments ranging from 6.0 to 20.0 kb. While REA frequently provided more discriminatory power than ribotyping, there were examples where the use of ribotyping was more discriminatory than REA. Passage of selected isolates up to passage 25 did not change the REA profile. Moreover, the Bvg phase did not alter the fingerprint profile of chromosomal DNA from B. bronchiseptica strains digested with HinfI or AluI. Based on the results presented herein, the combination of REA and ribotyping should provide valuable information in understanding the molecular epidemiology of B. bronchiseptica infections. Bordetella bronchiseptica is a common respiratory pathogen in a number of animal species. It is an etiologic agent of swine atrophic rhinitis and bronchopneumonia, canine tracheobronchitis, and bronchopneumonia in laboratory and companion animals. In rare instances, B. bronchiseptica has been reported to infect humans; a majority of these infections have occurred in patients with underlying conditions such as cystic fibrosis, AIDS, Hodgkin s disease, or leukemia (2, 3, 8, 23). The types of infections seen in these patients have included pneumonia, tracheobronchitis, sinusitis, peritonitis, meningitis, and septicemia (2, 3, 8, 23). Until recently, there has been a lack of a simple and reliable method for typing of B. bronchiseptica isolates for classification. Ribotyping has been utilized to characterize B. bronchiseptica isolates from several animal species and was shown to provide a basis for grouping these organisms into distinct types (13 15). Keil and Fenwick (5) utilized random amplified polymorphic DNA fingerprinting and ribotyping to evaluate the genetic diversity among 26 canine B. bronchiseptica isolates. Methods such as restriction enzyme analysis (REA) of chromosomal DNA may also have power in discriminating among B. bronchiseptica strains. In fact, REA and ribotyping have been utilized in molecular epidemiologic studies of other bacterial species (1, 4). We have previously reported that REA and ribotyping could be utilized to discriminate Bordetella avium and Bordetella hinzii isolates (17). In the present experiments, REA was utilized as a method for characterizing B. bronchiseptica isolates previously grouped on the basis of ribotyping. This study represents the first examination of the potential usefulness of REA as a method of classifying B. bronchiseptica isolates from several host species. MATERIALS AND METHODS Bacterial strains. A total of 195 B. bronchiseptica isolates were examined (113 laboratory strains and 82 field isolates). Strains B58, B65, and 5203 (7) were obtained from Tibor Magyar, Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest, Hungary. Strain St. Louis was obtained from Tom Milligan, St. Louis University Hospital, St. Louis, Mo. Strains with the descriptor MBORD were generously provided by David Dyer, University of Oklahoma, Oklahoma City (11). The 113 laboratory strains utilized in the present study were obtained from 11 different host species from diverse geographic locations (Table 1). MBA-4, a bvg isogenic mutant of MBORD846 (produced in the laboratory of Jeff Miller, University of California Los Angeles), was kindly provided by David Dyer. Eighty-two field isolates were included from the following sources. B. bronchiseptica isolates obtained from seals during a phocine morbillivirus outbreak were supplied by Geoff Foster, Scottish Agricultural Colleges Veterinary Science Division, Drummonhill, United Kingdom (15). Thirty turkey isolates were kindly provided by Y. M. Saif, The Ohio State University, Wooster. Swine isolates from a field case of atrophic rhinitis were obtained from the Diagnostic Laboratory, Iowa State University College of Veterinary Medicine, Ames. REA. (i) Chromosomal DNA isolation. Bacterial strains were grown on blood agar base slants (Difco, Detroit, Mich.) for 48 h at 37 C. Bacterial cells were harvested and adjusted to a similar concentration in 0.85 M NaCl. A 1.5-ml aliquot of the bacterial cells was centrifuged at 16,000 g for 4 min. The supernatant was decanted; pellets were stored at 70 C. DNA was isolated using a commercially available kit (DNAzol; Gibco BRL, Gaithersburg, Md.) according to recommendations of the manufacturer. (ii) Restriction enzyme digestion, electrophoresis, photography, and analysis. The following restriction enzymes (Gibco BRL) were examined: AluI, BglII, ClaI, DraI, DdeI, EcoRI, EcoRV, HaeIII, HhaI, HindIII, HinfI, HpaI, HpaII, MvaI, NciI, PvuII, PstI, RsaI, TaqI, and XbaI. Digestion of chromosomal DNA with each restriction enzyme was carried out via the recommendations of the manufacturer. The reactions were stopped by the addition of 5 l of stop solution (0.25% bromophenol blue, 0.25% xylene cyanole, 25% Ficoll 400) to 21 l of reaction mixture. The digested DNA fragments were electrophoresed in 0.7% agarose gels using TBE buffer (0.089 M Tris, 0.089 M boric acid, 2 mm EDTA, ph 8.0). A HindIII digest of lambda phage DNA was used as a molecular size marker. Gels were stained and photographed as previously described (17). Photographs were scanned for computer analysis using a Scanjet IIcx with DeskScan software (Hewlett-Packard, Boise, Idaho). GelCompar software (Applied Maths, Kortrijk, Belgium) was used for comparison of fingerprint profiles. Similarity between all possible pairs of fingerprint profiles using the coefficient of Dice (18) was calculated by the cluster analysis module of the software. Dendrograms were derived from a matrix of similarity values by the unweighted pair-group method using arithmetic averages. RESULTS * Corresponding author. Mailing address: USDA/ARS, National Animal Disease Center, P.O. Box 70, 2300 Dayton Rd., Ames, Iowa. Phone: (515) 663-7354. Fax: (515) 663-7458. E-mail: rsacco@nadc.ars.usda.gov. REA. Twenty restriction endonucleases were evaluated for use in REA of B. bronchiseptica isolates. Of the endonucleases examined, digestion of B. bronchiseptica chromosomal DNA with HinfI or AluI resulted in well-separated and distinct bands 4387

4388 SACCO ET AL. J. CLIN. MICROBIOL. TABLE 1. Laboratory strains of B. bronchiseptica used in the present study a Strain Host Country of origin MBORD545 Pig The Netherlands MBORD553 Pig United Kingdom MBORD603 Pig Canada MBORD605 Pig Canada MBORD606 Pig Canada MBORD676 Pig Australia MBORD677 Pig Australia MBORD688 Pig United States MBORD790 Pig The Netherlands MBORD791 Pig The Netherlands MBORD792 Pig The Netherlands MBORD793 Pig The Netherlands MBORD795 Pig The Netherlands MBORD796 Pig The Netherlands MBORD797 Pig The Netherlands MBORD798 Pig The Netherlands MBORD800 Pig The Netherlands MBORD801 Pig The Netherlands MBORD802 Pig The Netherlands MBORD803 Pig The Netherlands MBORD804 Pig The Netherlands MBORD805 Pig The Netherlands MBORD846 Pig Switzerland MBORD847 Pig Switzerland MBORD849 Pig The Netherlands MBORD850 Pig The Netherlands MBORD853 Pig Ireland MBORD976 Pig The Netherlands MBORD979 Pig The Netherlands MBORD980 Pig The Netherlands B58 Pig Hungary B65 Pig Hungary 5203 Pig Hungary MBORD590 Dog United States MBORD591 Dog United States MBORD592 Dog United States MBORD594 Dog United States MBORD595 Dog United States MBORD596 Dog United States MBORD599 Dog United States MBORD600 Dog United States MBORD601 Dog United States MBORD602 Dog United States MBORD685 Dog United States MBORD686 Dog United States MBORD732 Dog Denmark MBORD748 Dog Denmark MBORD749 Dog Denmark MBORD750 Dog Denmark MBORD783 Dog The Netherlands MBORD785 Dog The Netherlands MBORD786 Dog The Netherlands MBORD787 Dog The Netherlands MBORD788 Dog The Netherlands MBORD827 Dog Switzerland MBORD839 Dog Switzerland MBORD843 Dog Switzerland MBORD965 Dog The Netherlands MBORD966 Dog The Netherlands MBORD967 Dog The Netherlands MBORD671 Rabbit United States MBORD704 Rabbit United States MBORD705 Rabbit United States MBORD730 Rabbit Denmark MBORD784 Rabbit The Netherlands MBORD823 Rabbit Switzerland MBORD828 Rabbit Switzerland MBORD831 Rabbit Switzerland MBORD833 Rabbit Switzerland MBORD834 Rabbit Switzerland Continued TABLE 1 Continued Strain Host Country of origin MBORD835 Rabbit Switzerland MBORD836 Rabbit Switzerland MBORD837 Rabbit Switzerland MBORD838 Rabbit Switzerland MBORD971 Rabbit The Netherlands MBORD972 Rabbit The Netherlands MBORD981 Rabbit The Netherlands MBORD675 Human Germany St. Louis Human United States MBORD625 Rat United States MBORD629 Cat United States MBORD630 Cat United States MBORD631 Cat United States MBORD635 Cat United States MBORD723 Cat Denmark MBORD733 Cat Denmark MBORD745 Cat Denmark MBORD782 Cat The Netherlands MBORD968 Cat The Netherlands MBORD970 Cat The Netherlands MBORD707 Turkey United States MBORD901 Turkey Germany MBORD681 Koala Australia MBORD698 Koala Australia MBORD700 Koala Australia MBORD626 Leopard United States MBORD627 Guinea pig United States MBORD665 Guinea pig United States MBORD666 Guinea pig United States MBORD668 Guinea pig United States MBORD669 Guinea pig United States MBORD670 Guinea pig United States MBORD673 Guinea pig Germany MBORD674 Guinea pig Germany MBORD762 Guinea pig Ireland MBORD854 Guinea pig Switzerland MBORD624 Horse United States MBORD628 Horse United States MBORD632 Horse United States MBORD633 Horse United States MBORD731 Horse Denmark MBORD982 Horse The Netherlands MBORD983 Horse The Netherlands a There were 113 laboratory strains of B. bronchiseptica isolated from 11 different host species. in the 4 to 10 kb molecular size range. Use of the other endonucleases resulted in bands which could not be readily distinguished, especially in the 3 to 23.1 kb molecular size range, where optimum resolution occurs under the electrophoresis conditions used in this study. Forty-eight distinct fingerprint profiles were found among the 195 B. bronchiseptica isolates following HinfI digestion. These isolates had previously been characterized into 19 distinct PvuII ribotypes (13 15). An example of the REA profiles of selected ribotype 3 B. bronchiseptica isolates is shown in Fig. 1. Based on HinfI restriction enzyme digestion patterns, dendrograms were constructed and similarity between the fingerprint profiles was calculated using the coefficient of Dice by the cluster analysis module of GelCompar software. Genetic diversity among B. bronchiseptica isolates was considerable, with similarity ranging from 68

VOL. 38, 2000 REA DISCRIMINATES B. BRONCHISEPTICA ISOLATES 4389 FIG. 1. Representative REA profiles of selected B. bronchiseptica isolates following HinfI restriction enzyme digestion of chromosomal DNA. These isolates were previously characterized as ribotype 3. Note that REA further differentiated these isolates which were previously characterized by ribotyping. Lanes M, molecular size marker (lambda phage HindIII digest). to 97% (Fig. 2). Even within a host species, the diversity among isolates was striking. For example, there was less than 70% similarity between some swine isolates. Interestingly, the two human isolates clustered with B. bronchiseptica isolates obtained from birds. Thirty-nine distinct fingerprint profiles were found among the 195 B. bronchiseptica isolates following AluI digestion. As was the case for HinfI digestion, REA using AluI frequently provided further discrimination of isolates than ribotyping (Fig. 3). Moreover, genetic diversity among B. bronchiseptica isolates based on AluI restriction enzyme digestion was considerable, with similarity ranging from 46 to 96% (Fig. 4). As was observed for HinfI digestion, diversity of isolates within a species following AluI digestion of chromosomal DNA was remarkable. For example, similarity shown among the dog isolates was less than 50%. By combining the HinfI and AluI data for the 113 laboratory strains, we observed 55 distinct REA profiles (Table 2). In contrast, these laboratory strains had previously been categorized into 16 different PvuII ribotypes (13). While REA generally provided more discriminatory power than ribotyping, there were examples where the use of ribotyping was more discriminatory than REA. For example, MBORD625, MBORD700, and MBORD800 were grouped together by REA as HinfI 004 and AluI 001, but were separated by ribotyping as ribotypes 2, 3, and 6, respectively. As further evidence of the utility of REA analysis for discriminating B. bronchiseptica isolates, we examined the longterm stability of the fingerprint profiles generated by restriction enzyme digestion of DNA from isolates following several in vitro passages. For this purpose, chromosomal DNA was isolated from specific strains following 1, 5, 10, 15, 20, or 25 in vitro passages. The fingerprint profiles generated using either HinfI or AluI restriction enzyme digestion were stable up to passage 25 (data not shown). Most of the known virulence factors of B. bronchiseptica are positively regulated by the products of the bvgas locus (24). When bvgas is active (Bvg phase), known virulence factors are expressed. When bvgas is inactive (Bvg phase), due to mutations in bvgas or modulating environmental signals, most adhesins and toxins are not expressed. Thus, it was of interest to examine whether differences in Bvg phase would alter REA profiles. This was accomplished by comparing the fingerprint profiles of a bvg strain (MBORD846) and an isogenic mutant (MBA-4) that has a deletion in the bvgs gene, resulting in a phase-locked Bvg phenotype. As shown in Fig. 5, MBORD846 and MBA-4 have the same HinfI REA pattern. In addition, these two strains had identical fingerprint patterns following AluI restriction enzyme digestion. Similarly, a bvg strain (B58) and a bvg spontaneous mutant of B58 (B65) had identical REA patterns following HinfI (Fig. 5) or AluI restriction enzyme digestion. Finally, hemolytic (Bvg ) and nonhemolytic (Bvg ) colonies were selected during in vitro passage of specific strains. We found that the HinfI or AluI fingerprint profiles of hemolytic and nonhemolytic colonies of the same strain did not differ. DISCUSSION REA is a highly discriminatory method for determining phylogenetic relationships and has been utilized by previous investigators in examining the molecular epidemiology of genetically diverse strains. In our experiments, REA was utilized in

4390 SACCO ET AL. J. CLIN. MICROBIOL. Downloaded from http://jcm.asm.org/ FIG. 2. Dendrogram showing percent similarity among B. brochiseptica isolates using HinfI restriction endonuclease digestion of chromosomal DNA. Representatives of each of the 48 profiles observed for the 195 B. bronchiseptica isolates are shown. Similarity between fingerprint profiles based on the coefficient of Dice was calculated by the cluster analysis module of GelCompar software. the examination of 195 B. bronchiseptica isolates from 12 host animal species that had been previously characterized by ribotyping. Digestion of B. bronchiseptica chromosomal DNA with HinfI or AluI resulted in DNA fragments in the 4 to 10 kb molecular size range which were more readily distinguishable than fragments generated by digestion with the other restriction enzymes examined. Furthermore, we found that REA is superior to previously described techniques for distinguishing B. bronchiseptica isolates. In fact, as was shown for canine isolates (5), our results indicate that there is more genetic diversity among B. bronchiseptica isolates than previously appreciated. The fingerprint profiles generated by HinfI or AluI restriction enzyme digestion were stable following 25 in vitro passages and were not affected by differences in bvgas expression. Techniques utilized to distinguish among Bordetella isolates have included biochemical and physiological characteristics, whole-cell protein profiles, and fatty acid analysis (10, 16, 21, 22). Additional methods have relied on examining stable genetic elements for characterization of Bordetella isolates, which should be more reproducible than expression-based methods. Indeed, the utility of ribotyping in discriminating among B. bronchiseptica isolates has been proven (5, 13 15). In addition, Keil and Fenwick (5) examined random amplified polymorphic DNA fingerprinting, but this method has its limitations (20). Moreover, macrorestriction fingerprinting using the rare-cutting enzyme XbaI and pulsed-field gel electrophoresis has been utilized by investigators to characterize isolates of B. bronchiseptica (6, 19). However, pulsed field gel electrophoresis protocols typically involve time-consuming procedures for purification of genomic DNA in agarose, and lengthy restriction enzyme digests and electrophoresis times. In their study, Khattak and Matthews (6) had examined restriction fragment length polymorphism analysis of Bordetella species and found that it failed to discriminate among Bordetella pertussis, Bordetella parapertussis, or B. bronchiseptica isolates when chromosomal DNAs were cut with the frequently cutting enzyme EcoRI. Evidently, these investigators did not examine other restriction enzymes for use in restriction fragment length polymorphism analysis. In agreement with these investigators, we found that restriction enzyme digestion with EcoRI produced numerous bands in the 3 to 23.1 kb molecular size range such that discrimination among Bordetella isolates was not possible. Nonetheless, in our experiments we examined 20 different restriction enzymes and were able to demonstrate that digestion of chromosomal DNA using HinfI or AluI restriction endonucleases is useful in discriminating B. bronchiseptica isolates. on July 17, 2018 by guest

VOL. 38, 2000 REA DISCRIMINATES B. BRONCHISEPTICA ISOLATES 4391 TABLE 2. Comparison of REA fingerprint profile and ribotype for laboratory strains of B. bronchiseptica Strain Hinfl profile Alul profile Ribotype MBORD626 001 002 6 MBORD849 001 009 3 MBORD966 001 009 3 MBORD681 001 009 6 MBORD838 002 005 12 MBORD796 002 010 3 MBORD798 002 010 3 MBORD804 002 010 3 MBORD976 002 017 3 MBORD853 002 020 3 MBORD833 002 025 12 MBORD834 002 025 12 MBORD698 003 009 8 MBORD627 003 026 8 MBORD831 003 026 9 MBORD971 003 026 9 MBORD671 003 026 9 MBORD835 003 026 9 MBORD625 004 001 2 MBORD800 004 001 3 MBORD700 004 001 6 MBORD790 004 004 3 MBORD793 004 004 3 MBORD795 004 004 3 MBORD801 004 004 3 MBORD803 004 004 3 MBORD805 004 004 3 MBORD850 004 004 3 MBORD847 004 006 2 MBORD981 004 006 3 MBORD792 004 011 3 MBORD802 004 011 3 MBORD980 004 019 3 MBORD846 005 001 2 MBORD704 005 001 2 MBORD673 005 001 3 MBORD545 005 001 3 MBORD605 005 001 3 MBORD606 005 001 3 MBORD791 005 001 3 MBORD797 005 001 3 MBORD979 005 001 3 MBORD685 005 001 3 MBORD686 005 001 3 MBORD972 005 001 3 MBORD836 005 001 3 MBORD784 005 001 3 5203 005 001 3 B58 005 001 3 B65 005 001 3 MBORD688 005 021 3 MBORD665 006 023 11 MBORD666 006 026 12 MBORD632 006 037 9 MBORD591 008 035 13 MBORD669 012 023 3 MBORD668 012 023 12 MBORD590 014 002 4 Continued TABLE 2 Continued Strain Hinfl profile Alul profile Ribotype MBORD594 014 002 4 MBORD600 014 002 4 MBORD601 014 002 4 MBORD602 014 002 4 MBORD749 014 002 4 MBORD592 014 002 4 MBORD786 014 013 4 MBORD965 014 013 4 MBORD967 014 013 4 MBORD827 014 013 4 MBORD788 014 013 4 MBORD596 014 022 4 MBORD707 015 008 16 MBORD750 015 014 4 MBORD783 015 014 4 MBORD787 015 014 4 MBORD676 016 012 2 MBORD828 016 013 3 MBORD553 017 007 1 MBORD762 017 007 1 MBORD854 017 007 1 MBORD633 018 002 4 MBORD599 018 002 4 MBORD628 018 013 4 MBORD983 018 014 4 MBORD970 019 003 4 MBORD629 019 003 4 MBORD630 019 003 4 MBORD631 019 003 4 MBORD733 019 003 4 MBORD723 019 003 4 MBORD968 019 003 4 MBORD748 020 002 4 MBORD732 020 002 4 St. Louis 022 029 15 MBORD670 023 008 2 MBORD705 024 015 5 MBORD595 025 005 5 MBORD624 026 005 7 MBORD839 027 032 4 MBORD843 028 002 4 MBORD635 029 033 4 MBORD901 030 034 15 MBORD731 031 013 8 MBORD982 032 013 8 MBORD837 033 036 9 MBORD730 034 001 9 MBORD782 035 038 10 MBORD674 036 035 12 MBORD675 038 024 14 MBORD785 039 016 3 MBORD823 040 011 3 MBORD745 041 022 3 MBORD603 042 006 3 MBORD677 043 012 3

FIG. 3. Representative REA profiles of selected B. bronchiseptica isolates following AluI restriction enzyme digestion of chromosomal DNA. These isolates were previously characterized as ribotype 3. Note that REA further differentiated isolates which were previously characterized by ribotyping. Lanes M, molecular size marker (lambda phage HindIII digest). Downloaded from http://jcm.asm.org/ on July 17, 2018 by guest FIG. 4. Dendrogram showing percent similarity among B. brochiseptica isolates using AluI restriction endonuclease digestion of chromosomal DNA. Representatives of each of the 39 profiles observed for the 195 B. bronchiseptica isolates are shown. Similarity between fingerprint profiles based on the coefficient of Dice was calculated by the cluster analysis module of GelCompar software. 4392

VOL. 38, 2000 REA DISCRIMINATES B. BRONCHISEPTICA ISOLATES 4393 REFERENCES An inherent problem in the classification of Bordetella spp. based on phenotypic characteristics is the extensive alterations in expression that can occur depending upon Bvg phase. Thus, while mutations in bvgas could influence the characterization of Bordetella isolates using expression-based methods, we have shown that alterations in bvgas expression as a result of mutation do not affect the REA fingerprint profiles of B. bronchiseptica isolates following either HinfI or AluI restriction endonuclease digestion. As stated above, previous investigators have shown the utility of ribotyping using PvuII to classify B. bronchiseptica isolates. We have shown in the present study that REA can also be utilized in characterizing B. bronchiseptica isolates. While REA generally provided more discriminatory power than ribotyping, there were examples where the use of ribotyping was more discriminatory than REA. 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