Abstract
In this study, the Riemerella anatipestifer mutant strain RA1062 was obtained by screening a random Tn4351 transposon mutant library. The mutant strain was unreactive with the anti-CH3 lipopolysaccharide monoclonal antibody, as demonstrated with an enzyme-linked immunosorbent assay, and its M949_RS01035 gene was inactivated. When cultured in trypticase soy broth, the late stage growth of the mutant RA1062 was significantly decreased. The mutant RA1062 was stained with crystal violet and presented a rough lipopolysaccharide phenotype, which differed from that of the wild-type strain CH3, suggesting that deletion of the M949_RS01035 gene resulted in defective lipopolysaccharide. Silver staining and Western blot analyses further confirmed that the RA1062 lipopolysaccharide had a deficiency in ladder-like binding pattern, as compared to lipopolysaccharide of the wild-type CH3 strain. In addition, the mutant RA1062 showed a higher susceptibility to complement-dependent killing, increased bacterial adhesion and invasion capacities to Vero cells, decreased blood bacterial loads, and attenuated virulence in infected ducks, when compared to the wild-type strain CH3. Moreover, RNA-Seq and real-time polymerase chain reaction analyses indicated that two genes were up-regulated and two were down-regulated in the mutant RA1062 genome. Furthermore, an animal protection experiment showed that immunization of ducks with inactivated RA1062 bacterin conferred effective cross-protection against challenge with the virulent R. anatipestifer serotypes 1, 2, and 10. This study presents evidence that the M949_RS01035 gene is involved in bacterial phenotype, virulence, and gene regulation in R. anatipestifer. The mutant strain RA1062 could be used as a cross-protective vaccine candidate.
Highlights
Riemerella anatipestifer is a Gram-negative, non-motile, non-spore forming, and atrichous bacterium belonging to the family Flavobacteriaceae in the phylum Bacteroidetes [1]
The transposon was inserted at nucleotide position 318 bp of the M949_RS01035 gene, which is 678 nucleotides in length and encodes intramembrane metalloprotease of the CPBP (CAAX proteases and bacteriocin-processing enzymes) family, which consists of 225 amino acids (Figure 1C)
The results showed that 25% diluted serum was effective in killing the mutant strain RA1062, but not the WT strain CH3, indicating that the mutant strain RA1062 was more sensitive to normal duck sera than the WT strain CH3 (Figure 6)
Summary
Riemerella anatipestifer is a Gram-negative, non-motile, non-spore forming, and atrichous bacterium belonging to the family Flavobacteriaceae in the phylum Bacteroidetes [1]. R. anatipestifer has received considerable attention because infection of this bacterium cause major economic losses to the duck farming industry through high mortality, weight loss, and high treatment costs [2]. R. anatipestifer infection is a highly contagious disease that causes fibrinous pericarditis, airsacculitis, and perihepatitis in ducks [3]. A variety of vaccines to protect farm ducks from R. anatipestifer infection have been investigated, including the chaperonin GroEL and inactivated bacterin [7, 8]. As with other Gram-negative bacteria, lipopolysaccharide (LPS) is probably one of the most important virulence factors of R. anatipestifer. LPS is the major outer membrane component of Gram-negative bacteria and typically comprise three structure domains: lipid A, Dou et al Vet Res (2018) 49:93 a core oligosaccharide, and a polysaccharide O-antigen. In R. anatipestifer, five genes (AS87_04050, M949_1556, M949_1603, M949_1360 and M949_RS01915) associated with LPS synthesis have been characterized in our previous studies [18–22]
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