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.

Riemerella anatipestifer is an important pathogen of waterfowl, which causes septicemia anserum exsudativa in ducks. In this study, an AS87_03730 gene deletion R. anatipestifer mutant Yb2ΔAS87_03730 was constructed to investigate the role of AS87_03730 on R. anatipestifer virulence and gene regulation. By deleting a 708-bp fragment from AS87_03730, the mutant Yb2ΔAS87_03730 showed a significant decreased growth rate in TSB and invasion capacity in Vero cells, compared to wild-type strain Yb2. Moreover, the median lethal dose (LD 50 ) of Yb2ΔAS87_03730 was 1.24 × 10 7 colony forming units (CFU), which is about 80-fold attenuated than that of Yb2 (LD 50 = 1.53 × 10 5 CFU). Furthermore, RNA-Seq analysis and Real-time PCR indicated 19 up-regulated and two down-regulated genes in Yb2ΔAS87_03730. Functional analysis revealed that 12 up-regulated genes were related to "Translation, ribosomal structure and biogenesis", two were classified into "Cell envelope biogenesis, outer membrane", one was involved in "Amino acid transport and metabolism", and the other four had unknown functions. Polymerase chain reaction and sequence analysis indicated that the AS87_03730 gene is highly conserved among R. anatipestifer strains, as the percent sequence identity was over 93.5%. This study presents evidence that AS87_03730 gene is involved in bacterial virulence and gene regulation of R. anatipestifer.Riemerella anatipestifer is a significant pathogen of waterfowl, turkey and other birds 1,2 . It causes epizootic infectious polyserositis in ducks characterized by lethargy, diarrhea, respiratory and nervous symptoms, which led to high mortality and consequently to great economic losses 3 . 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 .The luxE gene from a number of bioluminescent bacteria had been well defined by genetic and biochemical analysis, including Vibrio harveyi, Vibrio fischeri, Photobacterium phosphoreum, etc 9-11 . The luxE gene encodes acyl-protein synthetase (LuxE) which activates the fatty acid, results in the formation of a fatty acyl-AMP intermediate and functions as the second step in the bioluminescent fatty acid reduction system 12 . Up to date, most studies focus on the role of luxE in bacterial bioluminescence reaction, and the role of luxE in R. anatipestifer has remained unknown. In this study, a luxE homology gene deletion mutant strain Yb2ΔAS87_03730 was constructed by allelic exchange, and the roles of AS87_03730 gene on bacterial growth, adherence and invasion capability, as well as colonization and development during infection were investigated. Furthermore, the function of AS87_03730 on the gene regulation at genome level of R. anatipestifer was investigated using RNA-Seq, the differentially expressed genes between mu...

Riemerella anatipestifer is one of the most important bacterial pathogen of ducks and causes a contagious septicemia. R. anatipestifer infection causes serositis syndromes similar to other bacterial infections in ducks, including infection by Escherichia coli, Salmonella enterica and Pasteurella multocida. Clinically differentiating R. anatipestifer infections from other bacterial pathogen infections is usually difficult. In this study, MAb 1G2F10, a monoclonal antibody against R. anatipestifer GroEL, was used to develop a colloidal gold immunochromatographic strip. Colloidal gold particles were prepared by chemical synthesis to an average diameter of 20±5.26 nm by transmission electron microscope imaging. MAb 1G2F10 was conjugated to colloidal gold particles and the formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Immunochromatographic strips were assembled in regular sequence through different accessories sticked on PVC plate. Strips specifically detected R. anatipestifer within 10 min, but did not detect E. coli, S. enterica and P. multocida. The detection limit for R. anatipestifer was 1×106 colony forming units, which was 500 times higher than a conventional agglutination test. Accuracy was 100% match to multiplex PCR. Assay stability and reproducibility were excellent after storage at 4°C for 6 months. The immunochromatographic strips prepared in this study offer a specific, sensitive, and rapid detection method for R. anatipestifer, which is of great importance for the prevention and control of R. anatipestifer infections.