Development of a PCR for Identification of Bordetella hinzii Author(s): Karen B. Register Source: Avian Diseases, 57(2):307-310. 2013. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10433-102212-ResNote.1 URL: http://www.bioone.org/doi/full/10.1637/10433-102212-ResNote.1 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. AVIAN DISEASES 57:307–310, 2013 Research Note— Development of a PCR for Identification of Bordetella hinzii Karen B. RegisterA Ruminant Diseases and Immunology Research Unit, Agricultural Research Service, United States Department of Agriculture, National Animal Disease Center, 1920 Dayton Avenue, Ames, IA 50010 Received 31 October 2012; Accepted 7 February 2013; Published ahead of print 11 February 2013 SUMMARY. Bordetella hinzii infects primarily poultry and immunocompromised humans. It is closely related to the etiologic agent of turkey coryza, Bordetella avium. Distinguishing between B. avium and B. hinzii is difficult, and there is no method for identification of B. hinzii suitable for use by diagnostic laboratories. This report details the development of a B. hinzii–specific PCR targeting the ompA gene. Assay sensitivity is 100% based on analysis of 48 B. hinzii isolates from diverse geographic locations representing all known ribotypes. Evaluation of 71 isolates of B. avium and 20 other bacterial isolates from poultry, comprising gram-negative and gram-positive commensals and pathogens of nine genera, demonstrated an assay specificity of 100%. The ompA PCR is a rapid, reliable, and accurate method for identification of B. hinzii and provides a valuable new tool for veterinary diagnostic laboratories investigating poultry respiratory disease outbreaks. RESUMEN. Nota de Investigacio´n—Desarrollo de un me´todo de PCR para la identificacio´ndeBordetella hinzii. La Bordetella hinzii infecta principalmente a las aves de corral y a seres humanos inmunodeprimidos. Esta bacteria esta´ estrechamente relacionada con el agente etiolo´gico de la coriza de los pavos, Bordetella avium. La diferenciacio´n entre B. avium y B. hinzii es difı´cil, y no hay un me´todo para la identificacio´ndeB. hinzii que sea adecuado para su uso en los laboratorios de diagno´stico. Este informe detalla el desarrollo de un me´todo de PCR especı´fico para B. hinzii dirigida al gene ompA. La sensibilidad del ensayo fue del 100%, con base en el ana´lisis de 48 aislamientos de B. hinzii aislados de diferentes sitios geogra´ficos que representan a todos ribotipos conocidos. La evaluacio´n de los 71 aislamientos de B. avium y de otros 20 aislamientos bacterianos de aves comerciales, que incluye bacterias comensales y pato´genas, gram-negativas y positivas de nueve ge´neros, demostro´ que el me´todo tenı´a una especificidad del 100%. El me´todo de PCR basado en el gene ompA es un me´todo ra´pido, confiable y preciso para la identificacio´ndeB. hinzii y proporciona un nuevo y valioso instrumento para los laboratorios de diagno´stico veterinario que investigan brotes de enfermedades respiratorias en la avicultura. Key words: Bordetella hinzii, ompA, PCR Abbreviations: ATCC 5 American Type Culture Collection; NADC 5 National Animal Disease Center Bordetella hinzii infects primarily poultry and immunocompro- reference strains, and 37 strains whose characterization has been reported mised humans but it also has been isolated from rabbits (11) and previously (11,13,14). The 71 B. avium isolates tested include the type mice (5). Although initially thought to be nonpathogenic in poultry, strain, four reference strains, and 49 strains whose characterization has it was recently shown that some strains cause disease in turkey poults been reported previously (11,13,14). In addition to the ATCC type that is indistinguishable from the clinical presentation of turkey strain and one reference strain, 17 avian isolates of Bordetella bronchseptica were analyzed, including 11 described in prior publications coryza caused by Bordetella avium (9). Bordetella hinzii and B. avium (8,10,11,14,15). Bordetella isolates not included in previous reports are are closely related and share many genetic and phenotypic traits. well-characterized strains from a collection maintained at the National Only a few phenotypic tests can delineate these species, and results Animal Disease Center (NADC). All PvuII ribotypes known to occur may vary depending on inoculum size, culture conditions, and the in avian isolates (11) are represented within the group tested for each specific procedure used (1,2,6,7,16). Ribotyping and restriction Bordetella species. Bordetella hinzii and B. avium were cultivated for enzyme analysis reliably distinguish between B. avium and B. hinzii 18–36 hr at 37 C on 5% sheep’s blood agar. Although sheep’s blood (11,14), but these tests are not readily carried out in most diagnostic agar also supports the growth of B. bronchiseptica, cultures for this laboratories. There is currently no rapid method for identification of study were grown for 24–36 hr at 37 C on Bordet-Gengou agar B. hinzii suitable for use in a diagnostic setting. To address this need, supplemented with 10% sheep’s blood to be consistent with laboratory a highly sensitive and specific PCR targeting the ompA gene of B. research protocol. Other bacteria represent avian pathogens or normal hinzii was developed. flora of the turkey respiratory tract, the latter obtained by swabbing the upper third of the trachea of clinically healthy birds with BBL CultureSwabs (Becton Dickinson and Company, Sparks, MD). Swabs MATERIALS AND METHODS were streaked on the day of collection onto duplicate 5% sheep’s blood agar plates that were incubated at 37 C for 24–48 hr with or without Bacterial isolates and growth conditions. The host and geographic 5% CO2. A representative of every distinguishable colony type was origin of bacterial isolates included in this study are indicated in selected for identification. Isolates from tracheal swabs were evaluated Tables 1 and 2. Forty-nine isolates of B. hinzii were evaluated, including by Gram’s stain and identified using the MicroLog System (BioLog, the American Type Culture Collection (ATCC) type strain, four Inc., Hayward, CA). PCR. Boiled lysates prepared from a single colony as described previously (12) were used as template. Ten microliters of each PCR was ACorresponding author. E-mail: [email protected] analyzed by agarose gel electrophoresis in 3:1 NuSieve (Lonza Rockland, 307 308 K. B. Register Table 1. Bordetella spp. isolates included in this study. Table 2. Additional avian isolates included in this study. Organism Host Geographic origin n Organism Host Geographic origin n B. hinzii Human United States 3 Alcaligenes faecalis Turkey North Carolina 2 Human Switzerland 2 Escherichia coli Turkey Iowa 1 Human Spain 1 Chicken unknown 1 Turkey United States 1 Enterococcus gallinarum Turkey Iowa 1 Turkey Minnesota 7 Enterococcus columbae Turkey Iowa 1 Turkey Ohio 5 Enterococcus avium/faecalis Turkey Iowa 1 Turkey Iowa 4 Ornithobacterium rhinotracheale Turkey Minnesota 2 Turkey California 3 Pasteurella multocida Turkey West Virginia 1 Turkey New York 1 Turkey California 1 Chicken Australia 1 Pseudomonas aeruginosa Turkey Iowa 1 Chicken Belgium 1 Staphylococcus aureus Turkey Iowa 1 Turkey or chicken Ohio 12 Staphylococcus hyicus Turkey Iowa 1 Rabbit Hungary 1 Staphylococcus delphini Turkey Iowa 1 Mouse Missouri 1 Staphylococcus xylosis Turkey Iowa 1 Unknown Minnesota 5 Staphylococcus intermedius Turkey Iowa 1 B. avium Turkey North Carolina 1 Turkey Iowa 12 Turkey California 6 Turkey Minnesota 5 Turkey Ohio 1 AM748263–AM748265), representing ,540 bp of the 39 portion Wild turkey New Jersey 1 of the open reading frame, was used to design PCR primers to Turkey South Africa 3 amplify and sequence ,520 bp of the gene. An alignment of the Turkey Germany 1 Mallard New Jersey 7 sequences from PCR products obtained with a subset of the B. hinzii Canada goose New Jersey 1 isolates included in Table 1, representing different hosts and PvuII Saw-whet owl Virginia 1 ribotypes, revealed three sequence variants. A representative of each Unknown Ohio 15 ompA sequence variant was aligned with ompA gene sequences in Unknown Minnesota 2 GenBank from several additional Bordetella species to identify regions Unknown Iowa 7 Unknown North Carolina 3 Unknown Germany 5 B. bronchiseptica Turkey Ohio 4 Turkey Iowa 4 Turkey California 3 Turkey Minnesota 2 Turkey Wisconsin 1 Turkey United States 1 Turkey Germany 1 Turkey Unknown 1 Dog United States 1 Rabbit United States 1 Inc., Rockland, ME) containing a 1:10,000 dilution of GelRed (Phenix Research Products, Candler, NC). Chromosomal DNA purified using a commercially available kit (Promega, Madison, WI) was used to assess the limit of detection. Purified DNA was quantified with PicoGreen (Invitrogen, Carlsbad, CA). DNA sequencing. PCR products were purified with spin columns (QIAGEN, Valencia, CA) and sequenced directly at the NADC’s Genomics Unit using BigDyeH Terminator version 3.1 on a 3130 XL Genetic Analyzer sequencer (Applied Biosystems, Foster City, CA). Sequence data were analyzed using Vector NTI Suite software (Invitrogen). Final consensus sequences were derived from a minimum of three sequence reads with at least one from each strand.