| Riemerella anatipestifer used to be a member of Pasteurella, named Pasteurella anatipestifer. And then, it has been divided into Flavobacterium based on the sequence of16S rRNA in1997. In addition to the specificity in evolution, another important reason for arousing people’s interest is that R.anatipestifer is responsible for infectious serositis of ducklings, which is one of the most important diseases of ducks around the world. The main symptoms, including septicemia, meningitis, pericarditis and peritonitis, always result in huge economic losses. Twenty-one serotypes of R. anatipestifer have been identified, but there is no cross-protective ability in different serotypes. No vaccine has been proved to be safe and efficient. So, novel vaccine, particularly those that provide cross-protection against different serotypes, may aid the control of this disease, though little progress has been made until now.Several complete genome sequences of R. anatipestifer have been published since2011, which is benefit for the high throughput screen of protective antigens. R. anatipestifer serotype2is one of the main factors for outbreak of RA infection. R. anatipestifer serotype2virulent strain RAfl53has been sequenced and detected by immunoproteomics. Twelve immunoreactive proteins have been selected by this assay.We assume that serum prepared by artificial infection of one strain has antibody of another strain. Selection of protective antigens of RAf153and RAf63was perform by immunoproteomics assay using duck convalescent serum against RAf153, followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and peptide mass fingerprinting. Finally, common immunoreactive proteins from RAf153and RAf63were identified as cross-protective vaccine candidates, and so these were cloned and expressed recombinantly. The cross-protection abilities of purified recombinant protein vaccines were tested against homologous and heterologous virulent strains in a challenge model that followed vaccination. Six proteins were identified as cross-protective vaccine candidates. Three of these proteins showed reactivity with convalescent sera after prokaryotic expression, and CR4(OmpA) showed high protective indices against challenges with RAfl53(60%) and RAf63(50%). Conservatism analysis showed that CR1could response to sera against RA serotype1,2,10and13. CR4could response to sera against RA serotype1,2,6,10,11,13,14and17. CR5could response to sera against RA serotype1,2and10. In summary, we have developed a high-throughout, accurate, rapid and efficient method for the successful selection of cross-protective vaccine candidates.Indirect ELISA of immunoreactive proteins OmpA and EfG have been developed in our study.465sera form ducks without vaccinating R.anatipestifer and30sera from360days old breeding geese were detected by OmpA-ELISA. The average positive rate in ducks is48.8%, and is33.3%in breeding geese. Among the positive sera, sera against serotype1,2and10strains account for79.3%.We have developed a PCR assay based on the sequence of16S rRNA published in GenBank. Five positive results have been found when throat swabs of10apparent healthy ducks were detected by this PCR assay. In another case, two R.anatipestifer serotype2strains have been isolated from ten thin, depressed, diarrhoea and ataxic ducks. One of them was mixed infected with E.coli. All the data suggests that the R.anatipestifer infection is serious in Nanjing.Genome sequence of RAfl53has been sequenced (unpublished). Alignment between this genome and genome of serotype1strain RA-GD has been performed by software Mauve2.3.1and SeqManⅡ. Seven specific sequences range from200bp to3000bp have been found from nitheen differences to design primers. Serotype2strains could be successfully distinguished from serotype1strains by these primers. Interestingly, sequence amplified by Primer7is part of gene encoding immunoreactive protein G0121(GenBank accession No. KC905110), which was identified by immunoproteomics assay. But, objective fragments could also be detected in strains of other serotypes. So, more genome sequences should be published if we want to develop target genes for diagnosis. |
