title: Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR.
abstract: Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial strains. Twenty-two of the strains represented all of the presently available 13 phenotypic variants of G. anatis originating from different geographical locations. Nine strains represented each of the additional six Gallibacterium species and 21 strains represented other poultry associated bacterial species of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae. Regarding specificity none of non-G. anatis strains tested positive with the proposed assay. To test and compare the qPCR method's ability to detect G. anatis from field samples, the sensitivity was compared to a previously published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10(8) colony forming units (CFU)/ml to as low as 10(0) CFU/ml copies. The proposed qPCR method is thus highly specific, sensitive and reproducible. In conclusion, we have developed a qPCR method that allows species-specific identification of G. anatis.
authors.affiliation: a Department of Veterinary Disease Biology, Faculty of Health and Medical Science , University of Copenhagen , Frederiksberg C , Denmark.
authors.forename: Chong
authors.initials: C
authors.lastname: Wang
authors.affiliation: b Boehringer Ingelheim Vetmedica , Guadalajara , México.
authors.forename: Francisco
authors.initials: F
authors.lastname: Robles
authors.affiliation: c Centro de Investigación y Estudios Avanzados en Salud Animal, Facultad de Medicina Veterinaria y Zootecnia , Universidad Autónoma del Estado de México , Toluca , México.
authors.forename: Saul
authors.initials: S
authors.lastname: Ramirez
authors.affiliation: d Department of Animal Science, Faculty of Science and Technology , Aarhus University , Tjele , Denmark.
authors.forename: Anja Brinch
authors.initials: AB
authors.lastname: Riber
authors.affiliation: a Department of Veterinary Disease Biology, Faculty of Health and Medical Science , University of Copenhagen , Frederiksberg C , Denmark.
authors.forename: Anders Miki
authors.initials: AM
authors.lastname: Bojesen
mesh_terms: D000818:Animals; D002645:Chickens / Q000382:microbiology*; D057230:Limit of Detection; D015661:Pasteurellaceae / Q000235:genetics / Q000302:isolation & purification*; D016871:Pasteurellaceae Infections / Q000382:microbiology / Q000662:veterinary*; D011201:Poultry Diseases / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013045:Species Specificity
keywords: Gallibacterium anatis; culture-independent; qPCR; species specific