绵羊肺炎支原体小鼠感染模型的建立

Establishment of the Mice Model Infected with Mycoplasma ovipneumoniae

旨在探索小鼠作为绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)实验室感染模型的可行性,筛选建立Mo感染小鼠模型的最佳方法。将60只BALB/c小鼠随机分为对照组、Mo感染组1、感染组2、感染组3和感染组4(n=12)。Mo感染组1小鼠分别滴鼻30μL和腹腔注射25μL 10^(8 )CCU·mL^(-1) Mo NJ01株菌液各1次,Mo感染组2和3小鼠分别滴鼻2和3次30μL 10^(8 )CCU·mL^(-1) Mo NJ01株菌液,Mo感染组4小鼠喉头喷雾50μL 10^(8 )CCU·mL^(-1) Mo NJ01株菌液2次,对照组用正常培养基处理。分别于感染后第0、7和14天称量小鼠体重。感染后第14天处死所有小鼠,采集血液和肺组织。通过HE染色法进行肺组织病理学检查、剖检进行肺组织病理评分、qPCR方法检测肺组织中Mo的载荷量和ELISA检测血清Mo IgG水平,确定小鼠感染模型是否建立成功及最佳建立方法。Mo感染组2和组4中小鼠体重第14天时分别比对照组显著降低17.2%(P<0.05)和21.6%(P<0.05);感染第13天,感染组2和组4小鼠出现死亡;感染后第14天剖检,Mo感染组2和组4小鼠肺部有炎症,肺病变平均评分分别为3.5和3.3,均显著高于Mo感染组1(P<0.05)和组3(P<0.05),而对照组小鼠无病变;组织病理学检查发现,Mo感染组小鼠肺可见不同程度的间质性肺炎,肺泡腔内有炎细胞浸润;对照组小鼠肺组织结构正常、完整,肺泡内未见明显细胞浸润。小鼠肺组织中Mo DNA拷贝数在Mo感染组1和组3中分别为10^(2.56)和10^(3.21)拷贝·g^(-1);感染组2和组4分别为10^(3.84)和10^(3.77)拷贝·g^(-1),且显著高于Mo感染组1(P<0.05)和组3(P<0.05),对照组为阴性。小鼠血清Mo抗体OD_(450 nm)值在Mo感染组1、组2、组3和组4分别为0.63、1.05、0.81和0.99,均显著高于对照组(P<0.05),且组2和组4均显著高于1组(P<0.05)。本研究通过1×10^(8 )CCU·mL^(-1)的Mo NJ01株菌液滴鼻2次或喉头喷雾2次均可成功建立Mo感染小鼠模型。为绵羊肺炎支原体的致病机制及防治策略研究奠定基础。

The present study aimed to evaluate mice as a candidate of the experimental model of Mycoplasma ovipneumoniae(Mo)infection,and screen the optimal method of Mo infection mice model.Sixty healthy BALB/c mice were randomly divided into control group,Mo infection group 1,infection group 2,infection group 3 and infection group 4(n=12).Mice in Mo infection group 1 were challenged once with 30μL and 25μL 10^(8) CCU·mL^(-1) NJ01 strain cells by nasal and intraperitoneal drip,respectively.Mice in Mo infection group 2 and 3 were intranasally inoculated twice and three times 30μL 10^(8) CCU·mL^(-1),respectively.Mice in Mo infection group 4 were inoculated twice 50μL 10^(8) CCU·mL^(-1) by throat spry.The control group were inoculated with the normal liquid culture.The body weight was respectively determined on the 0,7th and 14th day post-infection(DPI).Mice in five groups were killed on the 14th DPI,blood and lung tissue samples were collected.To determine whether the infection model was established successfully and evaluate the optimal establishment method of M.ovipneumoniae infection model,the histopathological examination of lung tissue were observed by HE staining and histopathologic score,the payload of Mo in lung tissue was detected by quantitative PCR,and Mo IgG level in serum was determined by ELISA.On the 14th DPI,compared with control group,the body weight of mice in infection group 2 and group 4 significantly reduced 17.2%(P<0.05)and 21.6%(P<0.05).A mice died on the 13th DPI in infection group 2 and group,4 respectively.On the 14th DPI,mice had inflammation in lung and lung histopathologic score was 3.5 and 3.3 in infection group 2 and group 4,respectively,which were significantly higher than both infection group 1(P<0.05)and 3 group(P<0.05).No lung lesion was found in control mice.Histopathological examination showed that different degrees of interstitial pneumonia were observed in the lungs of the Mo infection groups.Inflammatory cell infiltration was seen in alveolar cavity.In the control group,the lung tissue structure was normal and intact,and there was no obvious inflammatory cell in alveolar cavity.The DNA copy numbers of Mo in infection group 1 and group 3 were 10^(2.56) and 10^(3.21) copies·g^(-1),respectively.The infection group 2 and group 4 were 10^(3.84) and 10^(3.77) copies·g^(-1) respectively.They were significantly higher than both group 1(P<0.05)and group 3(P<0.05).Control group was negative.The serum antibody OD_(450 nm) of Mo infection mice were 0.63,1.05,0.81 and 0.99 in infection group 1 group 2,group 3 and group 4,respectively,which were all significantly higher than control group(P<0.05).Mo antibody level in infection group 2 and group 4 were significantly higher than infection group 1(P<0.05).Mice model infected M.ovipneumoniae were successfully developed in this experiment by nasal drip and throat spray 1×10^(8) CCU·mL^(-1) for continued two times Mo which can provide important basis for investigating the pathogenesis and therapy of mycoplasma pneumonia of sheep.