Isolation Of Chlamydophila Psittaci From Clinical Samples And Genotyping

Objective:To optimize isolation and culture technique of Chlamydophila psittaci avian strains andanalyze the polymorphism of ompA gene through sequencing and cladograming,whichcan be applied to handle the epidemiologic feature of C.psittaci in Hengyang area.So itcan provide the evidence for prevention,diagnosis and therapeutic measure of C.psittaciinfection.It is helpful to investigating pathogenicity and germ line developmentalhistory of C.psittaci and obtaining a reliable epidemiology data of C.psittaci.Methods:C.psittaci ompA gene was extracted from bird livers which were gathered fromHengyang area by DNA extraction kit. A couple of specificity primers was designedthrough reading literatures,the polymerase chain reactions (PCR) amplification resultwas264bp,and was segregated with1.7%agarose gel electrophoresis. For the PCRpositive clinical samples, the liver tissues were homogenized and used to incubatedwith Hep-2and Vero cell monolayers, and Chlamydia inclusion bodies in two differentcells were detected by immunofluorescence(IF). A other couple of specificity primerswas designed through reading literatures,the PCR amplification result was1000bp, andwas segregated with1.2%agarose gel electrophoresis. For the PCR positive clinicalsamples,the PCR productions were purified and were sent to sequencing.Meanwhile,thereference strains of all types of C.psittaci ompA were gained from Genebank, the bloodrelationships of clinical strains and the reference strains were analyzed by the softwareClustalX2and MEGA3,and the similarity of8clinical strains were analyzed byBLAST. Results:Eight were positive which were from five hundred and ninety one avian samples byC.psittaci ompA gene amplification, including seven parrot and a lovesickness bird.Inthis experiment,the infection rate of parrot, pigeon and other type avian was1.64%,0%and0.69%.Seven were successfully cultured in Hep-2and Vero cell and theachievement ratio was87.5%.The inclusion-forming units(IFU) of Hep-2was greaterthan Vero cell in primary culture.After tert-cultivation, the consequence wasopposite.Seven A genotype were displayed by phyletic evolution tree,the last onewasn’t typed temporarily.The similarity of seven clinical strains was92%～99%byBLAST.E9was unknown by contrasting with other strains which were known inGenebank.Conclusion：(1) Seven avian sample were successfully segregation.(2) The parrot was the main host to C.psittaci in Hengyang area,the genotype A wasmain and some variation was existing.