Abstract
Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. In this study, we identified a mutant strain RA2640 by Tn4351 transposon mutagenesis, in which the AS87_04050 gene was inactivated by insertion of the transposon. Southern blot analysis indicated that only one insertion was found in the genome of the mutant strain RA2640. SDS-PAGE followed by silver staining showed that the lipopolysaccharide (LPS) pattern of mutant strain RA2640 was different from its wild-type strain Yb2, suggesting the LPS was defected. In addition, the phenotype of the mutant strain RA2640 was changed to rough-type, evident by altered colony morphology, autoaggregation ability and crystal violet staining characteristics. Bacterial LPS is a key factor in virulence as well as in both innate and acquired host responses to infection. The rough-type mutant strain RA2640 showed higher sensitivity to antibiotics, disinfectants and normal duck serum, and higher capability of adherence and invasion to Vero cells, compared to its wild-type strain Yb2. Moreover, the mutant strain RA2640 lost the agglutination ability of its wild-type strain Yb2 to R. anatipestifer serotype 2 positive sera, suggesting that the O-antigen is defected. Animal experiments indicated that the virulence of the mutant strain RA2640 was attenuated by more than 100,000-fold, compared to its wild-type strain Yb2. These results suggested that the AS87_04050 gene in R. anatipestifer is associated with the LPS biosynthesis and bacterial pathogenicity.
Highlights
Riemerella anatipestifer is a Gram-negative, non-motile, nonspore forming, rod-shaped bacterium that causes disease such as fibrinous serositis, sometimes with caseous salpingitis and vegetative disorder [1]
In the initial screening of R. anatipestifer Yb2 mutants carrying transposon Tn4351, we identified one mutant strain RA2640 by a slide agglutination test, which lacked serum agglutination ability to serotype 2 positive sera (Fig. 1A)
Our previous study has identified 33 genes which were involved in R. anatipestifer biofilm formation by random transponson mutagenesis [17]
Summary
Riemerella anatipestifer is a Gram-negative, non-motile, nonspore forming, rod-shaped bacterium that causes disease such as fibrinous serositis, sometimes with caseous salpingitis and vegetative disorder [1]. R. anatipestifer infection is probably the most economically important disease of farmed ducks worldwide, but only a few virulence factors have been established so far, including VapD, CAMP cohemolysin and OmpA [2,3,4,5]. Lipopolysaccharide (LPS) is a major virulence factor of most Gram-negative bacteria [6,7]. The LPS molecule is typically composed of three parts: lipid-A, core-polysaccharide (core-PS) and O-antigen repeats. The O-antigen repeats is known to be responsible for antigenicity and sero-specificity which is displayed on the surface of the bacterial cells [8]. The genes for the synthesis of LPS have been characterized in several species of bacteria [11,12]. The genes responsible for R. anatipestifer LPS synthesis have not been characterized yet
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