Detection of Gallibacterium spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization

Abstract

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 crossreactivity 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.

Identification and Characterization of Potential Performance-Related Gut Microbiotas in Broiler Chickens across Various Feeding Trials

“…Gallibacterium spp. have been found to be present in the upper respiratory tract as well as the lower genital tract of chickens (8). The 16S rRNA gene sequences related to G. anatis were isolated from the guts of healthy male and female birds across trials.…”

“…This was somewhat surprising, as both of these bacteria have the potential to be pathogenic. G. anatis is capable of causing serious systemic infections affecting multiple organ systems, although the mechanism of pathogenesis remains obscure (8). Gallibacterium spp.…”

Identification of Gallibacterium species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry evaluated by multilocus sequence analysis

“…These strains have previously been characterized in detail by phenotypic methods, and a number of these strains have also been characterized by genotypic methods as shown in Table 1 Bojesen et al, 2003aBojesen et al, ,b, 2007Bisgaard et al, 2009). Pasteurella multocida 08/14290 field strain was identified by phenotypic methods and Biotyper reference database library version 3.…”

Assessing pathogenicity ofGallibacterium anatisin a natural infection model: the respiratory and reproductive tracts of chickens are targets for bacterial colonization

“…The last mentioned criterion was scored as "normal", "moderate changes" and "severe changes". Bacterial colonies noticed under microscopic examination were confirmed as G. anatis by specific in situ hybridization based on the protocol of Bojesen et al (2003a).…”

“…Furthermore, Gallibacterium anatis has two biovars: G. anatis haemolytica and G. anatis anatis. Bacterial identification can be performed on the basis of phenotypic properties, but in addition G. anatis-specific polymerase chain reaction (PCR), quantitative PCR, fluorescent in situ hybridization and matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) techniques have been shown to be promising as a means of accurate diagnosis (Bojesen et al, 2003a(Bojesen et al, , 2007Alispahic et al, 2011;Huangfu et al, 2012). Also, MALDI-TOF MS is well established for the characterization of Gallibacterium isolates into existing species, in agreement with multi-locus sequence typing (Alispahic et al, 2011).…”