Isolation And Identification Of Proteus Mirabilis From Chicken And Study On Biological Characteristics

Proteus mirabilis disease is newly discovered in recent years, Chickens can be infected in any age.and the age under 7 weeks was the most susceptible time. It also can be passed to generation through the eggs, resulting in a large number of dead chicken.which is a potential threat to farming industry.Sun Qiuyan has made epidemiological investigation of it and result showed that the rate of Proteus mirabilis antibodies is very high in Henan, Anhui, Shandong, Guangxi and other provinces.The traditional method of isolation and identification was mostly used at present. With the development of molecular biology techniques, Related research has been done by some foreign scholars about PCR detection of Proteus mirabilis,and the first related report of the domestic was in 2008. But the PCR and 16srRNA detection of chicken Proteus miarabilis has not been reported in domestic so far.In this study, the strain QY was determined as Proteus mirabilis The phylogenetic tree was established by further determination and analysis gene sequence of its 16srRNA. Then extracted the OMP which was immunized to chicken to detect antibody responses and evaluat protection of it.At the same time, rabbit anti-chicken mirabilis OMP high immune serum was prepared, detection method were built for rapid detection of chicken mirabilis antibodies, it laid a foundation of fast diagnosis for P.mirabilis.The research divides three parts:Part One: Isolation,identification of chicken Proteus mirabilis and phylogenetic analysis of 16SrRNA gene sequenceOne strain disease-causing bacteria marked QY was isolated from infected chicken in Tai an Shandong Province, and identified as P. mirabilis by traditional method of isolation and identification.The result was positive of P. mirabilis serological diagnostic reaction, Infected animal experiments proved that QY was the major pathogen that caused the chicken a large number of sick and death. Antibiotic susceptibility test results showed that it was highly sensitive to Cephalosporin and Enrofloxacin while resistent to Penicillin and Sulfa. The 16 S rRNA gene of 1453 bp was amplified by PCR from QY which was deposited in GenBank (No.GU477712). BLAST analysis showed that the gene shared more than 98.9% homology with the sequences of Proteus mirabilis. then built Phylogenetic Tree with 10 strains of P. mirabilis using DNAStar. it showed that the homology were 98.9%-99.9%, and the homology with AB272366 was up to 99.9%, the QY was confirmed as P.mirabilis from the molecular level.it provided reference for the diagnosis and treatment of infected chicken and the identification of P. mirabilis.Part Two: Extraction and Detect on Immune Effect of Outer membrane protein of P.mirabilisOut membrane proteins of P.mirabilis strains QY was prepared by improved Wooldridge method. the OMP were analyzed by SDS- PAGE.The result showed that: the contents of P.mirabilis OMP was 5.741 mg/mL.Then OMP43.0 was gained by purified.Then OMP,OMP43.0 and whole-cell bacterin (WCB)immunizing antigen were immunized to two groups of chicken of different dose respectively. One group was used to detect antibody responses The other group was registered the rate of protective potency and evaluated protection given by them.Results showed that at 42th day, the titer of serum reach the top: Then, the titer of serum started the slow-moving descent. At 70th day, the titer of serum reach the OMP43.0 (210.2)> OMP (29.1). OMP and OMP43.0 group challenged with QY(100 LD50)can be better efectivly protected than the QY group. the control group was died out. It showed that the OMP,OMP43.0 and whole-cell bacterin of P.mirabilis has good immunogenicity and protection, it lay good theoretical foundation for OMP subunit vaccine. Part three: Preparation of High Titer Immuno Serum and Establishment of detection methods by P.mirabilis OMP with labelled antibody techniqueThe prepared OMP of P.mirabilis strains QY was immunized to rabbits for preparation of high titer immuno serum. And then detections of indirect immunofluorescence and indirect ELISA were established by optimization and screening of conditions. The result showed that the best dilution of high titer immuno serum was 1:160 of IFA test and the best reaction time was 45 min ,the best dilution of FITC labeled goat anti-rabbit IgG was 1:40. the best concentration was 10ug/mL of OMP by Indirect ELISA , the best concen tration of HRP was 1:5000,the best condition was 4℃,24h; 37℃,1h,clinical tested that it can be used to study on the epidemiology and serology of Proteus mirabilis. it proved that the two methods are simple, rapid, and strong specificity. It laid a foundation of fast diagnosis for P.mirabilis ,...