Study On The Characteristics Of RA Naturally Avirulent Strains

Duck Infectious serositis was caused by Riemerella anatipestifer(RA).It's a contagious diseases of domestic duck,turkeys and occasionally wild species.Riemerella anatipestifer infection almost has been found in all the farm ducks worldwide.Duck Infectious serositis has high incidence and mortality rates,is a major cause of disease and economic loss in duck industry.Our laboratory has studied the pathogen epidemiology of RA in last few years,on the basic of isolation and identification of RA throughout the country,more than 500 RA strains was preserved..In this study two strains of low pathogenic Riemerella anatipestifer were selected based on the information of laboratory,through 16 SrDNA nucleotide sequencing,LD₅₀of the ducklings and virulence stability of two strains,it was confirmed that they are Riemerella anatipestifer naturally avirulent strains.Then their invasion ability,cell toxicity of culture filtrate,immunogenicity,culture and biochemical characteristics were studied,to providing materials for further study the virulence factors of Riemerella anatipestifer and attenuated vaccine.The major outcomes of this study are as follows:1.Common bacteria 16 SrDNA primer(16 S-8F/20)and(16 S-1512R/21)was taken as the primer,Riemerella anatipestifer genome DNA as a template to run a PCR.Sequencing results showed,the nucleotide sequence is 1440 bp of 517 strain and 1436 bp of DY strain.Run BLAST in the NCBI,results showed two strains all have 99%homology with reported RA,homologous comparative analysis between 16 SrDNA nucleotide sequence of two strains is more than 99%.2.Riemerella anatipestifer LD₅₀was carried out with 14-day-old nonimmuned health Sichuan duck.The LD₅₀of DY and 517 were 1.18×10¹⁰CFU,2.3×10¹⁰CFU respectively.After 10 successive passages in susceptible animals,strains were regained and then used to challenge duck again,the LD₅₀of DY and 517 were 1.02×10¹⁰and 2.2×10¹⁰CFU respectively.3.Bacteria was cultured by LB,then the lique culture was centrifugalized to discard the cells,after ultrafiltration enrichment,ammonium sulfate precipitation,dialysis and filtration through a membrane of pore size,culture filtrate was obtained.The culture filtrate of avirulent strains was cultured with fibroblasts of duck embryo,in 4 hours the difference of filtrate was not obvious between experiment guoup and blank group,but the positive group(AFstrain)showed membrane disappeared,cytoplasm lost and muclear enrichment etc.Invasiveness was measured by the number of intracellular bacteria grew on the chocolate plate.517 and DY strain was incubated with duck embryo fibroblast for 5h,invasiveness were 1320(CFU/ml)and 1262(CFU/ml)respectively, showed a significant difference(0.01＜p＜0.05)between virulent AF strain1510(CFU/ml).4.Inactivated 517 and DY strain was joined asphalt as adjuvant(5.5×10⁹CFU/ml),5-day-old nonimmuned health Sichuan duck.were inoculated with prepared antigen,0.5ml/per.The antibody levels wre 1.682 and 1.884(A value)after immunization for 21d,and the protection rate were both 100%.The antibody levels wre 1.549 and 1.724after immunization for 28d,and the protection rate were both 100%.Live antigen is about 2.2×10⁷CFU/ml,ducks didn't show obvious clinical manifestations after immunization.The antibody levels wre 1.417 and 1.379 after immunization for 21d,and the protection rate were both 80%.The antibody levels wre 1.289 and 1.491 after immunization for 28d,and the protection rate were 75%and 100%.5.517 and DY naturally avirulent strains are Gram-negative dialister bacterium,encapsulated, which grow well on chocolate plate,the colony is butter-like,and they can grow in LB nutrient agar plate;517 fermented inulin,Arabinose,while DY fermented inulin,Arabinose,and dextrin,other carbohydrates were negative,Catalase test was positive for both strains,gelatin liquefaction tests were negative;517,DY showed resistance to penicillin,ampicillin,and amoxicillin,Furazolidone, SMZ,Netilmicin;grow well in LB.This study showed that Riemerella anatipestifer 517 and DY strain are capsuled,liquefy gelatin negtive,have typical morphous,can induce a good immune responses in animals with a stable virulence.517 and DY strains grow well in LB,would be elementary strains for comparative genomics to study the virulence factor,and to develoPment new vaccine.