| Acinetobacter baumannii is non-fermenting pathogen which often infect whit human,its clinical isolating rate is second.A.baumannii is now recognised as causing a broad range of severe nosocomial infections,including skin and soft tissue infections,wound infections,urinary tract infections,secondary meningitis,ventilator-associated pneumonia and bloodstream infections,which were cause of death.However,the reports of infection or isolation of A.baumannii in animals were far less than that of human.Especially,the infection or isolation of A.baumannii in poultry was rarely reported.Therefore,to study the epidemiology of A.baumannii in chickens and demonstrate whether the bacterial isolates originate from human is of highly significant importance to reality and public health.In this paper,we reported the infection of A.baumannii in poultry for the first time,and analized the phylogenetic relationship of epidemic bacterial isolates.In addition,through the prokaryotic expression of outer membrane protein A(OmpA),we analized its immunogenicity,developed an indirect ELISA method for detection of serum antibody against OmpA in chickens and applied in the preliminary application.This study was conducted to isolate and identify A.baumannii from the liver,spleen,lung,trachea,duodenum of chickens about 450 samples From September2014 to October 2015.The pathogenic bacteria was identified by the morphology,cultural characteristics,biochemical identification,susceptibility testing,animal pathogenicity test,specific gene detection.The results showed that two A.baumannii strains were detected,the prevalence rate was only 0.44%(95%CI,0.1-1.6).They were resistant to many antibiotics.They had strong pathogenicity to mice.The value of LD50 both was 7.09×107CFU/0.5 m L.This study had laid the material foundation for the further study of the pathogenic mechanism of A.baumannii and its epidemiological investigation in chicken flocks.In order to understand the phylogenetic relationship among two A.baumannii strains more rationally and determine their evolution origin.This study based on the housekeeping genes rec A,gyr B,rpo B combined with 16 S r RNA gene for phylogenetic analysis of two strains.The results showed that two isolated strains had nearest genetic distance with human source of A.baumannii,and had farthest genetic distance with E.coli.The homology analysis illustrated that the nucleic acid homology of four genes had highest similarity with human source of A.baumannii,which can reached 92%-99.9%.The results showed that the two isolates originated from human,which revealed significant importance to public health.This confirmed the accuracy and reliability of rec A,gyr B,rpo B gene and 16 S r RNA gene for identification of Acinetobacter species.A new method for the identification of A.baumannii was explored.In this study,we designed specific primers,amplified the OmpA gene,cloned to prokaryotic expression vector p ET28a(+),constructed the recombinant plasmid p ET28a-OmpA,and transformed into BL21 cells for expression of induction.By exploring the conditions to determine the best inducing conditions,agarose Ni column was used to purify the recombinant protein and the Western blot was analyzed.The results of SDS-PAGE showed that the optimal induction concentration of IPTG was0.4 mmol/L,and optimal induction time was 5h,optimal induction temperature was37 ?.Fusion protein was 41 k Da which has specific biological activities by western blotting.These results laid the foundation for the further establishment of the rapid and simple immunoassay detection method and the serum epidemiology investigation.By using purified recombinant OmpA protein as coating antigen,an indirect ELISA was established for detecting anti-Ab antibody.The indirect ELISA method established in this paper had good repeatability and specificity.The results showed that the positive rate of 399 serums from chickens in different cities of Henan was3.75%.The indirect ELISA method established in this study could be used for serological detection of clinical samples,which provided an effective method for the clinical diagnosis and epidemiological investigation of A.baumannii in chickens. |
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