Detection of Gallibacterium spp. in chickens by fluorescent 16S rRNA in situ hybridization.
Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, "Actinobacillus salpingitidis," and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens.
Bojesen
Anders Miki
AM
Department of Veterinary Microbiology, Laboratory of Veterinary Pathology, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Copenhagen, Denmark. miki@kvl.dk
Christensen
Henrik
H
Nielsen
Ole Lerberg
OL
Olsen
John Elmerdahl
JE
Bisgaard
Magne
M
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D017403:In Situ Hybridization / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D015661:Pasteurellaceae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016871:Pasteurellaceae Infections / Q000175:diagnosis / Q000473:pathology / Q000662:veterinary*; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification*; D013154:Spleen / Q000382:microbiology / Q000473:pathology
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S