Bacterial determinants of importance in the virulence of Gallibacterium anatis in poultry

Abstract

Gallibacterium anatis, a member of the Pasteurellaceae family, constitute a part of the normal micro-flora of the upper respiratory tract and the lower genital tract in chickens. However, increasing evidence indicate that G. anatis is also associated with a wide range of pathological changes, particularly in the reproductive organs, which leads to decreased egg production, lowered animal welfare and increased mortality. As a recently defined opportunistic pathogen limited focus has been placed on the pathogenesis and putative virulence factors permitting G. anatis to cause disease. One of the most studied virulence determinants is a large RTX-like toxin (GtxA), which has been demonstrated to induce a strong leukotoxic effect on avian macrophages. A number of fimbria of different sizes and shapes has been described. Particularly fimbriae belonging to the F17-like family appears to be common in a diverse selection of G. anatis strains. Mutants lacking the FlfA fimbria were severely attenuated in experimentally infected chickens. Additional characteristics including the ability to express capsular material possibly involved in serum resistance; secretion of metalloproteases capable of degrading immunoglobulins, and hemagglutinins, which may promote biofilm formation are all factors likely linked to the virulence of G. anatis. A major advantage for the study of how G. anatis interact with its host is the ability to perform biologically relevant experimental infections where natural routes of exposure allows reproduction of lesions observed during spontaneous infections. This review summarizes the current understanding of the G. anatis pathogenesis and discusses the contribution of the established and putative virulence factors described for this bacterium to date.

Figure 1

Biovars of Gallibacterium anatis show difference in hemolytic properties. Colonies of G. anatis biovar (bv.) haemolytica strain 12656–12 and G. anatis bv. anatis strain F149. Hemolysis is seen as a clearing zone around the colonies of G. anatis b haemolytica. Colonies are 1–2 mm. greyish, smooth, semitransparent, slightly raised and circular with an entire margin when incubated for 24 h at 37 °C on nutrient-rich plates containing blood. Strains of G. anatis biovar haemolytica and genomospecies 1 and 2 shows β-hemolytic zones (1–2 mm) around the colonies on agar plates with blood from calf, horse, swine, sheep, rabbit or chicken [3,10,12,13,14].

Figure 2

GtxA is a novel RTX-like protein with an unusual domain organization. Schematic presentation of the domain organization of GtxA from G. anatis biovar haemolytica strain 12656–12 [RefSeq:WP_013746567] compared to the typical domain organization of RTX toxins represented by HlyA from E. coli [PRF:225074]. HlyA has 34% coverage and 29% identity with GtxA. Regions of homology are shown with dotted lines. The GtxA toxin is comprised of 2038 amino acids (aa). In the N-terminal part of GtxA (approximately aa 1 – 950), two overlapping regions with weak homology to a membrane protein with unknown function (COG1511) is present. The remaining C-terminal part of GtxA has homology to HlyA by having a RTX N-terminal domain (pfam02382), the Ca²⁺-binding repeats (COG2931) and a domain classified as peptidase M10 serralysin C terminal (pfam08548). GtxA also contains the lysine residues, Lys1484 and Lys1607, required for activation of the toxin by acetylation by GtxC. The homologue lysine residues in HlyA are found at Lys564 and Lys690. In addition to the described domains, HlyA also contains a RTX C-terminal domain (pfam08339). Domains were identified using NCBI conserved domain search with default settings.

Figure 3

The F17-like fimbria, FlfA, is exposed on the surface of G. anatis . FlfA on the surface of G. anatis strain 12656–12 is shown by immunogold electron microscopy using anti-FlfA serum, followed by a secondary antibody conjugated to 10-nm gold particles. Picture modified from Copyright © American Society for Microbiology, [63].

Figure 4

Outer membrane vesicles (OMVs) are naturally secreted from G. anatis . OMVs are seen as spherical structures in sizes ranging from 20 nm and up to 160 nm. The OMVs were isolated from G. anatis strain 12656–12 and viewed by transmission electron microscopy with 2% (w/v) phosphotungstic acid staining [69].

Figure 5

Capsular material is present on the surface of G. anatis . A thin capsular structure can be seen on the surface of G. anatis strain 12656–12 using uranyl acetate staining followed by transmission electron microscopy [80].