结核分枝杆菌潜伏感染豚鼠模型的建立和验证

Establishment and validation of a guinea pig model of latent Mycobacterium tuberculosis infection

目的：建立结核分枝杆菌（M ycobacterium tuberclu osis，Mtb）潜伏感染治疗用疫苗的豚鼠评价模型。方法豚鼠皮下攻毒5．0×103 CFU Mtb，于不同时间点用0．5μg重组结核杆菌ESAT6-CFP10蛋白对豚鼠皮试，观察皮试阳转情况。攻毒2周后，模型组豚鼠采用5 mg异烟肼灌胃治疗，每周3次，持续4周；对照组豚鼠不做化疗。在Mtb感染后的第6周，分别取未经异烟肼治疗的豚鼠和经异烟肼治疗的豚鼠解剖，比较豚鼠的肝、脾和肺的脏器综合病变指数以及脾活菌数（ log10 CFU）。根据上述结果建立豚鼠的潜伏感染模型，并用两个参考疫苗对该模型进行验证。结果豚鼠攻毒Mtb 2周后，EC皮试反应全部转阳，皮试反应大小为（19．9±3．0） mm。模型组豚鼠经4周异烟肼治疗后，脏器综合病变指数为0，脾未分离出活菌数。对照组豚鼠不经化疗，脏器综合病变指数为38．8±16．5，脾活菌数为（5．1±0．3）log10 CFU。停药后，模型组豚鼠结核病自然复发，在两次参考疫苗的验证中，脏器综合病变指数分别为48．5±23．9和51．30±23．41，脾活菌数分别为（4．5±1．3）和（4．2±1．1） log10 CFU，肺部活菌数分别为（4．1±1．2）和（3．4±1．3） log10 CFU。结论 Mtb攻毒豚鼠后，异烟肼化疗可抑制Mtb增殖，成功建立豚鼠的Mtb潜伏感染模型。该模型重复性好，可用于潜伏感染治疗用疫苗的评价。

Objective To establish a guinea pig model of latent Mycobacterium tuberculosis infec-tion for evaluating the effects of therapeutic vaccines .Methods Guinea pigs were subcutaneously inocula-ted with 5.0×103 CFU Mtb.The skin test was performed with 0.5μg recombinant ESAT6-CFP10 protein to detect positive conversion rates at different time points .Two weeks after Mtb inoculation , guinea pigs in model group received 5 mg isoniazid treatment （ three times a week for four weeks ） by oral gavage , while those in control group received normal saline .At the sixth week after Mtb infection , guinea pigs with and without isoniazid treatment were dissected for pathology examination .The pathological scores of liver , spleen and lung, as well as bacteria loads in spleen were compared between two groups .The established guinea pig model of latent infection was then validated by testing two reference vaccines （ AEC/BC02 and AEC/BC03 ） . Results Two weeks after Mtb inoculation , all guinea pigs showed positive EC skin test with induration area of （19.9±3.0） mm.Upon four weeks of isoniazid treatment , the guinea pigs in model group showed no pathological changes with zero scores in the examined organs .No bacterium was detected in spleen of ani-mals from model group.However, the total pathological score was 38.8±16.5 and bacteria load in spleen was （5.1±0.3） Log10 CFU with the guinea pigs from control group .Natural recurrence of tuberculosis in model group was observed after drug withdrawal .The total pathological scores were 48.5±23.9 and 51.3± 23.41.The bacterial loads in spleen were （4.5±1.3） and （4.2±1.1） Log10 CFU and bacterial loads in lung were （4.1±1.2） and （3.4±1.3） Log10 CFU respectively as verified with reference vaccines of AEC /BC02 and AEC/BC03.Conclusion Isoniazid treatment inhibited the proliferation of inoculated Mtb in guinea pigs.A guinea pig model of latent Mycobacterium tuberculosis infection is successfully established with an advantage of good repeatability .Therefore, it can be used to evaluate the effects of therapeutic vaccines on latent Mycobacterium tuberculosis infection.