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1 Journal of Microbes and Infection, December 2008, Vol 3, No , 3 1, 3, 4 ( BCG) H37Ra, BCG H37Ra CFU CFU CFU/ ml, 6, 14 d,, HE, BCG H37Ra, BCG H37Ra,,, BCG,, BCG H37Ra,, CFU/ ml, BCG ; ; ; ; An examination of a rabbit model of liquefaction induced by Mycobacterium WANG Ming-zhu, SHI Da-zhong, Yang Ai-jun, JIAO Zhi-gang, Zhu Bing-dong, ZHANG Ying ( Lanzhou Center for Tuberculosis Research/Institute of Pathogenic Biology, Lanzhou University, Lanzhou , China) Abstract Objective In order to investigate the mechanism of tuberculosis pathology, in particular the mechanism of liquefaction, studies are conducted to establish a tuberculosis pathology model in rabbit skin using Mycobacterium bovis attenuated strain BCG, Mycobacterium tuberculosis avirulent strain H37Ra, and Mycobacterium smegmatis( M. smegmatis). Methods BCG, H37Ra, and M. smegmatis were injected separately into the flank of New Zealand white rabbits by intradermal route twice every one and half months. Fourteen days after the last injection, lesions were removed for further histological analysis. Results Apparent liquefaction and ulceration were produced in the skin of rabbits injected with high dose( CFU/ml) of BCG, H37Ra, and M. smegmatis, with ulceration happening around 10 days after the first injection and three days after second immunization. The inflammation induced by BCG was stronger than the lesion induced by H37Ra, and the latter was stronger than that induced by M. smegmatis. Moderate( CFU/ ml) and low doses( CFU/ ml) of H37Ra and M. smegmatis did not induce obvious lesions, while the moderate and low doses of BCG induced granulomas. Conclusion High dose of BCG, H37Ra and M. smegmatis could induce caseous necrosis and liquefaction in rabbit skin. These results demonstrate the usefulness of this model for further pathogenesis research. Key words Tuberculosis; Mycobacterium; Pathology; New Zealand rabbits; Animal model, [ 1],,, : 863 ( : 2006AA02Z420) : 1., ; 2., ; 3., ; 4., [ 2 ],,,, ( Bacillus Calmette-Gu rin, BCG) ( ) H37Ra ( Mycobacterium smegmatis), 3 :, bdzhu@ lzu. edu. cn;, yzhang@ jhsph. edu

2 Journal of Microbes and Infection, December 2008, Vol 3, No ( 1) :,,, 18, kg, 100 g/ d,, 1, ( 2) : BCG, H37Ra, OADC Middlebrook 7H10( BD ), 37, OADC Middlebrook 7H9, 10%, - 70, CFU/ml CFU/ ml 2. CFU/ ml 5 ( 1) : 1, BCG H37Ra ( CFU/ml), ( CFU/ ml) ( CFU/ ml) 3, 2, ( ) 3 ( 2) : CFU/ ml CFU/ml CFU/ ml 0. 1 ml, d, ( 3) :, 10% HE ( 4) :,, 1. H37Ra BCG,,,, 2., 1, ( CFU/ ml) H37Ra BCG 1 BCG 1 5 d,, 11 d ; H37Ra BCG,, 12 d ;,, 13 d,,,, 35 d ( CFU/ ml) ( CFU/ ml) 1,,,, H37Ra BCG : BCG 1 2 d, 3 d ; H37Ra 3 d ; 6 d 1 Fig 1. Pathologic changes of rabbit skin lesions induced by different mycobacteria after prime and boosting immunity, respectively For the prime injection, high dose a bacteria induced granuloma 1-5 days after injection, and the injected area formed inflammation with edema. Edema decreased after a few days. Inflammation reached peak after about days and then liquefied. Necrotic foci liquefied and discharged its contents by rupturing through the epidermis. At 35 days, the lesions epithelialized and healed. For the boosting immunity, the inflammation reached peak about 3-7 days after the last injection, then liquefied and discharged its contents. These lesions healed at 32 days. The inflammation reaction, granuloma, liquefaction and ulceration induced by BCG were more severe than that induced by H37 Ra and M. smegmatis. BCG-p: BCG prime injection; BCG-b: BCG boosting injection; H37Rap: H37Ra prime injection; H37Ra -b: H37Ra boosting injection; M. s-p: M. smegmatis prime injection; M. s-b: M. smegmatis boosting injection

余中低剂量组均没有观察到明显的病理改变图3 不同分枝杆菌所引起的液化坏死组织病理改变 ( HE 染色) Fig 3. Pathology of rabbit skin lesions induced by BCG ( A, B), H37Ra ( C, D), and M.

3 210 微生物与感染 2008 年 12 月第 3 卷第 4 期 Journal of Microbes and Infection, December 2008, Vol 3, No. 4 疫相比, 各组皮肤肿块炎症反应明显, 液化时间提前组织液化突破表皮, 溃疡形成, 坏死液化组织流出, 肉芽肿体积缩小 ( 图 2A) 免疫后 32 d 左右病灶恢复部分家兔病变组织液化明显, 维持较长时间( 图 2B) BCG 中低剂量组再次免疫可诱导小结节形成, 炎症反应轻, 不发生液化溃疡其余中低剂量组均没有观察到明显的病理改变图3 不同分枝杆菌所引起的液化坏死组织病理改变 ( HE 染色) Fig 3. Pathology of rabbit skin lesions induced by BCG ( A, B), H37Ra ( C, D), and M. smegmatis ( E, F) ( HE stain) 图2 BCG 诱导的家兔皮肤脓肿液化 In the left figure, the arrow indicates the liquefaction area ( 100 ). The BCG -induced ulcer and liquefaction in rabbit skin red square zoom was amplified in the right figure. In the right figure ( 400 BCG ( 10 5 CFU/ ml) was intradermally injected into rabbit skin and then ), infiltration of lymphocytes, fibroblasts and macrophages could be Fig 2. boosted with the same dose of BCG one and half months later. Ulcer or seen liquefaction could be observed several days after the last injection. A: Ulcer that occurred 5 days after the last injection; B: An abscess in rabbit skin 10 days after the last injection. After the development of an incomplete ulcer ( arrowhead indicated ), which happened 5 days after injection, the abscess continued to develop 4. 细菌学检查 H37Ra BCG 耻垢分枝杆菌高剂量组新西兰兔皮肤组织脓液细菌抗酸染色阳性讨论结核病是一种由迟发型变态反应介导的慢性肉 3. 新西兰兔皮肤组织病理改变新西兰兔经芽肿性疾病, 其特征性病理改变是结核性肉芽肿形皮内注射接种高剂量 BCG H37Ra 耻垢分枝杆菌 3 成伴干酪样坏死和液化种不同菌株形成的病理结构类似( 图 3), 显微镜下结核病向恶性方向发展的关键环节, 液化组织可为均可见典型的结核液化坏死病灶: 病灶中央可见中结核杆菌生长提供丰富的培养基, 排出的液化组织性粒细胞脓液和液化坏死组织外周可见大量淋中含有结核杆菌, 会播散引起其他部位感染, 也会污巴细胞单核细胞渗出, 上皮样细胞巨噬细胞和朗染环境, 导致人群感染格汉巨细胞增生, 偶见嗜酸性细胞浸润病理已有一些研究基础, 然而坏死组织液化的发生 [ 3] [ 2] 坏死组织液化是人肺虽然对于肺结核的免疫机制和影响因素仍不明确

4 Journal of Microbes and Infection, December 2008, Vol 3, No , [ 4 ],, ;,, [ 5, 6 ],, [ 7] BCG, H37Ra, H37Rv, 1940, [ 7 ],,,,, BCG H37Ra,, 3, ( CFU/ ml), Sugisaki [ 8 ] BCG,, 1, BCG, H37Ra, BCG, 11, 3 ( 1 2) ; H37Ra, 12, 3 ;, 13 d, 6 BCG > H37Ra > [ 9, 10] 3, [ 11 ], CFU/ml CFU/ ml, 24 h, ; ( CFU/ ml),,,, [ 1 2],, BCG H37Ra CFU/ml 3, 3, BCG, H37Ra, 1.,,,...,, 2001, Converse PJ, Dannenberg AM, Estep JE, et al. Cavitary tuberculosis produced in rabbits by aerosolized virulent tubercle bacilli. Infect Immun, 1996, 64: Sandor M, Weinstock JV, Wynn TA. Granulomas in schistosome and mycobacterial infections: a model of local immume responses. Trends Immunol, 2003, 24: Dannenberg AM, Collins FM. Progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products. Tuberculosis ( Edinb), 2001, 81: Orme IM, Roberts AD, Furney SK, et al. Animal and cellculture models for the study of mycobacterial infections and treatment. Eur J Clin Microbiol Infect Dis, 1994, 13: Smith DW, Wiegeshaus EH. What animal models can teach us about the pathogenesis of tuberculosis in humans. Rev Infect Dis, 1989, 11: S385 S Zheng H, Lu L, Wang B, et al. Genetic basis of virulence attenuation revealed by comparative genomic analysis of Mycobacterium tuberculosis strain H37Ra versus H37Rv. PLoS ONE, 2008, 3: e Sugisaki K, Dannenberg AM, Abe Y, et al. Nonspecific and immune-specific up-regulation of cytokines in rabbit dermal tuberculous ( BCG) lesions. J Leukoc Biol, 1998, 63: Dannenberg AM. Immunopathogenesis of pulmonary tuberculosis. Hosp Pract, 1993, 28: ( 218 )

5 ( 211 ) 10. Dannenberg AM. Pathogenesis of pulmonary Mycobacterium bovis infection: basic principles established by the rabbit model. Tuberculosis ( Edinb), 2001, 81: Ando M. Macrophage activation in tuberculin reactions of rabbits with primary BCG infection and reinfection. J Reticuloendothel Soc, 1973, 14: Dannenberg AM. Cellular hypersensitivity and cellular immunity in the pathogenesis of tuberculosis: specificity, systemic and local nature, and associated macrophage enzymes. Bacteriol Rev, 1968, 32: ( : )

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