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Isolation of Chlamydophila Psittaci from Clinical Samples and the Role of IFN-γ Played in C.psittaci Infection

Author: TangGuoFang
Tutor: WuYiMou
School: Nanhua University
Course: Pathogen Biology
Keywords: Chlamydophila psittaci clinical strain animal model IFN-γ
CLC: S852.67
Type: Master's thesis
Year: 2010
Downloads: 35
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Abstract

Objective:To optimize the isolation and culture technique of Chlamydophila psittaci clinical strains and establish an animal model infected with C.psittaci, which can be applied to the clincal diagnosis of C.psittaci and epidemiological or pathogenetic study. To preliminary study whether IFN-γcan resist C.psittaci serovar A infection, providing experimental evidence for further illustrating the pathogenetic or immune mechanism of Chlamydia infection. .Methods:C.psittaci ompA gene was amplified from DNA extracted from bird livers or lungs by polymerase chain reactions (PCR). The PCR products were identified by enzymes cleavage or sequencing. For the PCR positive clinical samples, the liver tissues were homogenized and used to incubated with HeLa or Vero cell monolayers for 24 h in different dilutions,and chlamydia inclusion bodies were detected by immunofluorescence or Giemsa staining. Different dose of the clinical 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. To study the role of IFN-γ, C. psittaci 6BC-infected HeLa cells were treated with different concentrations of rhIFN-γ(5ng/mL, 25ng/mL, 50ng/mL). Forty-eight hours later, inclusion bodies were calculated and morphology was compared in different groups. For the in vivo experiment, C57BL/6 mice were inoculated intranasal with 2×106 IFU of live C. psittaci 6BC in 50μL SPG, and 10ug rmIFN-γor PBS were injected intraperitoneally at 24 hours prior or after infection. The body weight, activity and survival rate were recorded, and histopathology changes were analyzed by H&E and immunohistochemistry staining in mice livers and lungs after sacrificed. The lung tissues were homogenized, followed by sonication on ice, and the released organisms were titrated on Vero cell monolayers.Results:1) Six of one hundred clinical samples were positive by C.psittaci ompA gene amplification, and three were positive by cell culture.2) Vero cells showed stronger tolerance of cytolysis than HeLa cells. And chlamydia inclusion bodies were larger and more dense in Vero cells than in HeLa cells.3) C.psittaci infected mouse model was successfully established. After intranasally infected with 2×106, 2×105 or 2×104 IFUs of C.psittaci, corresponding clinical symptoms and pathology changes could be seen in all mice group in a dose-dependent manner. The body weight was decreased 4.33±1.03g, 2.83±1.17g, 2.67±0.82g respectively, but only decreased a little bit(0.17±0.75g) in control group. The survival rate was 0%, 33.3% or 100%, and no mice died in control group till day10 after infection.4) The in vitro experiment showed that IFN-γcould resist C.psittaci infection in a dose dependent manner. After incobulated 48h with the same amount of chlamydia and different dose (5ng/m, 25ng/mL, 50ng/mL) of rhIFN-γ, and the inclusion body numbers were significantly lower than those of control groups ((23.8±5.1)×106, (10±3.58)×106, (8.0±2.22)×106, (43.3±11.05)×106, respectively). The inclusion body was out-of-shape, and the size is only one fourth or one fifth of the control group.5) The results in the animal model of mouse infected with C.psittaci also showed that IFN-γcan resist C.psittaci infection and significantly alleviate the symptoms and pathology. And the survival rate was much higher than that of untreated group (75%, 43.75%, 12.5%, respectively) on day 10 after infection. Conclusion:The isolation and culture condition was optimized for C.psittaci clinical strains, and animal model of respiratory tract infection by C.psittaci was successfully established. IFN-γwas found to mediate strong protection against C.psittaci serovar A strain in a dose-dependent manner.

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