Characterization and cross-protection evaluation of M949_1603 gene deletion Riemerella anatipestifer mutant RA-M1

Abstract

Riemerella anatipestifer infection causes high mortality for ducks which results in major economic losses in the duck industry. In this study, we identified a mutant strain RA-M1 by Tn4351 transposon mutagenesis, in which the M949_1603 gene encoding glycosyl transferase was inactivated. PCR analysis revealed that M949_1603 gene is specifically existed in R. anatipestifer serotype 1 strains. RA-M1 presented no reactivity to the anti-lipopolysaccharide (LPS) MAb in an indirect ELISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting demonstrated that RA-M1 LPS had a deficiency in ladder-like binding pattern to rabbit antiserum against R. anatipestifer serotype 1 strain CH3, indicating that the O-antigen structure of RA-M1 was changed. RA-M1 showed significant attenuated virulence in ducks and higher sensitivity to normal duck serum, compared with its parent strain CH3. Furthermore, cross-protection of RA-M1 for R. anatipestifer serotypes 1, 2, and 10 strains was evaluated. Ducks that received two immunizations with inactivated RA-M1 vaccine were 100% protected from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2, and serotype 10 strain HXb2. No changes were observed in the liver, heart, or spleen samples from the protected ducks during autopsy and histological examination. Furthermore, vaccination generated high antibody titers of 1:12,800 against serotypes 1, 2, and 10 strains and enhanced production of interleukin 2 (IL-2) and IL-4 in ducks. These results suggested that M949_1603 gene is associated with serotype 1 O-antigen biosynthesis, and mutant RA-M1 could be used as a novel cross-protection vaccine candidate to protect ducks against R. anatipestifer infection.

The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

“…An indirect ELISA was used to screen the Tn4351 insertion mutant library for strains with defective reactivity to the anti-CH3 LPS monoclonal antibody (mAb), as described previously [ 18 ]. Briefly, each well of a 96-well ELISA plate was coated with whole mutant cells at 10 9 CFU/well in 50 μL of carbonate-buffered saline (pH 9.6).…”

“…In several species of bacteria, such as Escherichia coli and Salmonella enterica , the genes involved in LPS biogenesis have been well characterized [ 16 , 17 ]. 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 ].…”

Deletion of AS87_03730 gene changed the bacterial virulence and gene expression of Riemerella anatipestifer

“…In view of its veterinary importance, many investigations have been carried out on the virulence factors of R. anatipestifer , including CAMP cohemolysin, OmpA, nucleoside-diphosphate-sugar epimerase, and glycosyl transferase etc. 4 5 6 7 . Recently, 49 virulence associated genes were identified by random transponson mutagenesis 8 .…”

SURVEY OF ARIEMERELLA ANATIPESTIFEROUTBREAK IN SOUTHERN TAIWAN DUCK FARMS

“…13 Inactivated bacterins and live vaccines have successfully conferred protection against homologous strains of RA, but were unable to protect against heterologous serotype exposure. 14,15 An important challenge for veterinary vaccines is to improve cross-serotype protection of RA infection.…”