鹦鹉嗜衣原体禽鸟株的分离鉴定及小鼠呼吸道感染模型的建立

Isolation of Chlamydophila psittaci from avian samples and establishment of a respiratory infection murine model

摘要:

摘要：【目的】优化鹦鹉热嗜衣原体（Chlamydophila psittaci，C. psittaci，Cps）禽鸟株分离培养技术，建立Cps呼吸道感染的小鼠模型。【方法】从禽鸟肝组织中抽提总DNA，PCR法体外扩增Cps ompA基因，初步鉴定Cps阳性标本；同时将阳性标本的肝组织匀浆液接种到HeLa和Vero细胞中培养，Giemsa和免疫荧光染色法鉴定衣原体包涵体。将临床株扩大培养后，用2×104 、2×105 、2×106 IFU三个剂量鼻内接种小鼠，分别于感染后5d和10d处死小鼠，显微镜观察受染小鼠各脏器病理变化。【结果】采用PCR扩增Cps ompA基因，从100只禽鸟标本中检测到阳性Cps标本6例（6%），并成功地在HeLa及Vero细胞中培养出3例，且Vero细胞内的衣原体包涵体体积大，结构致密，对衣原体感染引起的宿主细胞溶解耐受性较HeLa细胞强，更适合用于Cps的分离培养及体外研究。成功建立Cps的鼠呼吸道感染模型，105IFU 是建立Cps呼吸道感染小鼠模型的适宜菌量。【结论】优化了Cps临床株的分离培养技术，成功建立了Cps呼吸道感染小鼠模型，为Cps流行病学调查及研究衣原体的致病性和致病机制奠定基础。

Abstract:

Abstract:[Objective] To optimize the isolation and culture technique of Chlamydophila psittaci avian strains and to establish an animal model infected with C. psittaci. [Methods] C. psittaci ompA gene was amplified from DNA extracted from bird livers by polymerase chain reactions (PCR). For the PCR positive avian samples, the liver tissues were homogenized and used to incubated with HeLa or Vero cell monolayers for 72 h in different dilutions，and chlamydia inclusion bodies were detected by immunofluorescence or Giemsa staining. Different dose of the avian strains(2×104, 2×105, 2×106 IFUs) were used to attack C57BL/6 mice by intranasal injection，mice were sacrificed on day 5 or day 10 after infection, and the histopathology changes were analyzed by H&E and immunohistochemistry staining in different organs. [Results] Six of one hundred avian samples were positive by C.psittaci ompA gene amplification, and three were positive by cell culture. The C.psittaci avian strains were cultured in Vero or HeLa cells. Vero cells showed stronger tolerance of cytolysis after chlamydia infection and chlamydia inclusion bodies were larger and more dense. Successfully establish a murine model of intranasal infection with C.psittaci, and 2×105 IFU is the suitable amount of organisms to induce respiratory chlamydia infection．[Conclusion] The isolation and culture condition was optimized for C.psittaci avian strains, and a murine model of respiratory tract infection by C.psittaci was successfully established, which can be applied to the clincal diagnosis of C.psittaci and epidemiological or pathogenetic study.