一株中等毒力牛种布鲁氏菌的鉴定和毒力测定

doi:10.3864/j.issn.0578-1752.2014.13.017

摘要/Abstract

摘要： 【目的】对初步鉴定的牛种布鲁氏菌分离株（B. abortus 343）进行全面的生物学特性检定，为深入研究布鲁氏菌病提供参考菌株。【方法】将B. abortus 343划线培养及梯度稀释，使其形成单个菌落，观察菌落形态。挑取单个菌落进行革兰氏染色和柯氏染色，观察其染色特点；分别接种1.5×106 CFU到含硫瑾（1﹕25 000）或含碱性复红（1﹕25 000）的TSA平板上，观察其生长状态；将接种有B. abortus 343的TSA平板分别置于普通培养箱和CO2培养箱37℃培养72 h，观察其对CO2的依赖性；通过醋酸铅试纸条测定B. abortus 343代谢过程中是否释放H2S。通过平板凝集试验测定布鲁氏菌单相特异性血清（牛种布鲁氏菌单因子血清A、羊种布鲁氏菌单因子血清M 和布鲁氏菌粗糙型血清R ）与B. abortus 343抗原的反应性；利用布鲁氏菌AMOS-PCR种属分型等方法对B. abortus 343进行了PCR种属特性鉴定；将B. abortus 343免疫小鼠，分别测定其抗血清与光滑型和粗糙型抗原的反应性；通过小鼠和豚鼠感染试验，全面评价该分离株的毒力；分别以1×105 CFU感染6周龄Balb/c小鼠，测定B. abortus 343在小鼠体内存活时间；以1×109 CFU感染Hartley豚鼠，2周后测定试验豚鼠每克脾脏含菌量；分别以10 000、1 000、100、25 CFU/只4种不同剂量感染豚鼠，初步测定分离株对豚鼠的最小感染量（MID），在此基础上，进一步以40、60和90 CFU/只测定MID。【结果】分离株B. abortus 343 为光滑型牛种布鲁氏菌，菌落逆光观察微带蓝绿色乳光；革兰氏染色为阴性，柯氏染色为红色，H2S试验阳性。该菌能在含硫瑾和碱性复红的培养基上生长，不依赖于CO2。B. abortus 343抗原能与A因子血清呈明显凝集反应，免疫小鼠后能产生特异性抗体。以1×105 CFU感染6周龄Balb/c小鼠，可在小鼠体内存活29周；以1×109 CFU感染350—400 g雌性豚鼠，14 d后豚鼠每克脾脏含菌量2.4×105—1.2×106；以1×105 CFU感染豚鼠1个月后，所有试验豚鼠均能产生特异性光滑型抗体，试管凝集效价为320—1 280；B. abortus 343对豚鼠的最小感染量约为40 CFU。【结论】鉴定了一株中等毒力牛种布鲁氏菌（B. abortus 343），为深入研究布鲁氏菌病提供了参考菌株，丰富了布鲁氏菌菌种资源。

关键词: 布鲁氏菌 , AMOS-PCR , 最小感染量 , 毒力

Abstract: 【Objective】The objective of this study is to explore the biological characteristics and assess the virulence of a B. abortus isolate (strain 343).【Method】The culture of B. abortus 343 was diluted and cultivated to single clone on Tryptic Soy Agar (TSA) for morphological observation, and it was then stained with Gram’s and Koch’s methods. Biochemical characteristics tests (including the growth ability test on thionine and alkaline azaleine, the dependence on CO2, and H2S release test) , single phase specific serum (B. abortus specific anti-serum A, B. melitensis specific anti-serum M and Brucella rough-type specific anti-serum R) agglutination tests and AMOS-PCR were used to identify its comprehensive characteristics. Mice and guinea pigs were infected with a certain dose of B. abortus 343 to determine the virulence of the isolate. The survival time of B. abortus in mice, the isolated number per gram of the spleen from the infected guinea pigs, and the minimum infecting dose (MID) for guinea pigs were tested.【Result】B. abortus 343 is a smooth-type, Gram-negative, Koch's staining red strain, which can reflect glaucous radiance from its surface culture. It releases H2S during its metabolism, and grows on TSA containing thionine or alkaline azaleine, and the growth of which was not dependent on CO2. The antigen prepared from B. abortus 343 was positive with A-type serum in plate agglutination test, and it could induce the immunized mice to produce the specific antibody. The bacteria were isolated from the spleen of the tested mice 29 weeks post infection with a dose of 1×105 CFU. While infected with a dose of 1×109 CFU to the female guinea pigs, 2.4×105 CFU - 1.2×106 CFU could be cultured from spleen per gram 14 days post infection. It could induce specific smooth-type antibody from all guinea pigs infected with a dose of 1×105 CFU, and 1 month post infection the titers of serum were high from 320 to 1 280 in agglutination test. To guinea pigs, the MID is about 40 CFU. 【Conclusion】A middle virulence B. abortus strain, which can be used as a reference strain in the further study was identified. As a new isolate, the detailed identified B. abortus 343 strain is a good supplement to the brucella resources.

Key words: Brucella , AMOS-PCR , minimum infecting dose (MID) , virulence

丁家波1, 王芳1, 杨宏军2, 王楠1, 朱良全1, 顾进华1, 张广川1, 王海光1, 赵鹏3, 程君生1, 毛开荣1, 冯宇3. 一株中等毒力牛种布鲁氏菌的鉴定和毒力测定[J]. 中国农业科学, 2014, 47(13): 2652-2658.

DING Jia-Bo-1, WANG Fang-1, YANG Hong-Jun-2, WANG Nan-1, ZHU Liang-Quan-1, GU Jin-Hua-1, ZHANG Guang-Chuan-1, WANG Hai-Guang-1, ZHAO Peng-3, CHENG Jun-Sheng-1, MAO Kai-Rong-1, FENG Yu-3. Identification and Virulence Assessment for a Strain of Brucella abortus[J]. Scientia Agricultura Sinica, 2014, 47(13): 2652-2658.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.13.017

https://www.chinaagrisci.com/CN/Y2014/V47/I13/2652

参考文献

[1]Ariza J, Corredoira J, Pallares R, Viladrich P F, Rufi G, Pujol M, Gudiol F. Characteristics of and risk factors for relapse of brucellosis in humans. Clinical Infectious Diseases, 1995, 20(5):1241-1249.

[2]Moriyón I, Grilló M J, Monreal D, González D, Marín C, López-Goñi I, Mainar-Jaime R C, Moreno E, Blasco J M. Rough vaccines in animal brucellosis: structural and genetic basis and present status. Veterinary Research, 2004, 35: 1-38.

[3]Avila-Calderón E D, Lopez-Merino A, Sriranganathan N, Boyle S M, Contreras-Rodríguez A. A history of the development of Brucella vaccines. BioMed Research International, 2013, 743509. doi: 10.1155/2013/743509.

[4]Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos E V. The new global map of human brucellosis. Lancet Infectious Disease, 2006, 6(2):91-99.

[5]易新萍, 马晓菁, 闫晶华, 谷文喜, 刘丽娅, 叶锋, 吐尔洪•努尔, 钟旗. 牛乳样品中布鲁氏菌的分离和鉴定. 中国预防兽医学报， 2013,35(1):28-30.

Yi X P, Ma X Q, Yan J H, Gu W X, Liu L Y, Ye F, Tu E H, Zhong Q. Isolation and identification of Brucella in raw milk. Chinese Journal of Preventive Veterinary Medicine, 2013,35(1):28-30. (in Chinese)

[6]孙永, 胡守奎, 胡万富, 张永根. 安徽省首次分离培养布鲁氏菌方法的研究. 安徽预防医学杂志, 2013, 19(2): 74-82.

Sun Y, Hu S K, Hu W L, Zhang Y G. First isolation and laboratory diagnosis of brucella in Anhui province. Anhui Journal of Preventive Medicine, 2013, 19(2): 74-82. (in Chinese)

[7]陈强, 王立成, 费东亮, 苏禹刚. 锦州地区奶牛布鲁氏菌的分离鉴定. 中国牛业科学, 2013, 39(2): 40-41.

Chen Q, Wang L C, Fei D L, Su Y G. Isolation and identification of brucella of dairy cow in Jinzhou. China Cattle Science, 2013, 39(2): 40-41. (in Chinese)

[8]刘丽娅, 易新萍, 谷文喜, 马晓菁, 李博, 吐尔洪•努尔, 叶锋, 钟旗. 牛种布鲁氏菌生物Ⅰ型的分离与鉴定. 草食家畜, 2013, 155(2): 70-74.

Liu L Y, Yi X P, Gu W X, Ma X Q, Li B, Tu E H, Ye F, Zhong Q. Isolation and identification of Brucella Abortus I. Grass-Feeding Livestock, 2013, 155(2): 70-74. (in Chinese)

[9]陈军, 崔步云, 赵鸿雁, 朴东日, 姜海, 邰新平, 田国忠. 应用多重PCR鉴别布鲁氏菌种及猪种5个生物型研究. 中国病原生物学杂志, 2013, 8(2): 126-130.

Chen J, Cui B Y, Zhao H Y, Piao D R, Jiang H, Tai X P, Tian G Z. Evaluation of the ability of two multiplex PCR assays (Bruce-ladder and Suis-ladder) to distinguish six Brucella species and Brucella suis at the biovar level. Journal of Pathogen Biology, 2013, 8(2): 126-130. (in Chinese)

[10]Caswell C C, Baumgartner J E, Martin D W, Roop R M 2nd. Characterization of the organic hydroperoxide resistance system of Brucella abortus 2308. Journal of Bacteriology, 2012, 194(18): 5065-5072.

[11]Gomez G, Pei J, Mwangi W, Adams L G, Rice-Ficht A, Ficht T A. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach. PLoS One, 2013, 8(3): e59751.

[12]Hanna N, Ouahrani-Bettache S, Drake K L, Adams L G, Köhler S, Occhialini A. Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses. BMC Genomics, 2013, 8;14: 459. doi: 10.1186/1471-2164-14-459.

[13]Dorneles E M, de Faria A P, Pauletti R B, Santana J A, Caldeira G A, Heinemann M B, Titze-de-Almeida R, Lage A P. Genetic stability of Brucella abortus S19 and RB51 vaccine strains by multiple locus variable number tandem repeat analysis (MLVA16). Vaccine, 2013, 31(42): 4856-4859.

[14]Noutsios G T, Papi R M, Ekateriniadou L V, Minas A, Kyriakidis D A. Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1. Veterinary Research Communications, 2012, 36(1): 7-20.

[15]周桂兰, 李旭妮, 吴启, 程君生, 王楠, 丁家波, 毛开荣, 韦海涛, 薛水玲, 祝俊杰. 一株犬种布鲁氏菌的分离与鉴定. 中国兽药杂志, 2013, 47(2): 11-13.

Zhou G L , Li X N, Wu Q, Cheng J S, Wang N, Ding J B, Mao K R, Wei H T, Xue S L, Zhu J J. Isolation and identification for a strain of Brucella canis from a poodle. Chinese Journal of Veterinary Science , 2013, 47(2): 11-13. (in Chinese)

[16]丁家波, 张存帅, 彭小兵, 蒋颖, 程君生, 张尔利, 蒋玉文, 毛开荣. 布鲁菌种属鉴定多重PCR方法的建立及初步应用. 中国兽医学报, 2009, 29(5): 594-597.

Ding J B, Zhang C S, Peng X B, Jiang Y, Cheng J S, Zhang E L, Jiang Y W, Mao K R. Development and preliminary application of multi-PCR in differentiating Brucella spp. Chinese Journal of Veterinary Science, 2009, 29(5): 594-597. (in Chinese)

[17]彭小兵, 程君生, 夏业才, 毛开荣, 蒋玉文, 丁家波. 多重PCR方法鉴别牛、羊、猪种布鲁氏菌株. 中国兽药杂志, 2010, 44(12): 12-14.

Peng X B, Cheng J S, Ye C X, Mao K R. Jiang Y W, Ding J B. A multi-PCR assay for differentiating the strains of B. abortus, B. melitensis and B. suis. Chinese Journal of Veterinary Science, 2010, 44(12): 12-14. (in Chinese)

[18]冯忠武. 中国兽医菌种目录. 北京: 中国农业科技出版社, 2002: 22-36.

Feng Z W. CVCC Catalogue of Cultures. Beijing: Chinese Agricultural Press, 2002: 22-36. (in Chinese)

[19]程君生, 彭小兵, 毛开荣, 蒋玉文, 夏业才, 丁家波. 2308、M28、S1330三种不同种布鲁氏菌的毒力测定. 中国兽药杂志, 2010, 44( 12): 29- 31.

Cheng J S, Peng X B, Mao K R, Jiang Y W, Xia Y C, Ding J B. Detection of virulences of B. abortus 2308, B.melitensis M28 and B. suis S1330. Chinese Journal of Veterinary Science, 2010, 44 (12): 29-31. (in Chinese)

[20]程君生, 吴梅花, 赵丽霞, 彭小兵, 丁家波, 王楠, 夏业才, 毛开荣. 三种布鲁氏菌病疫苗株的毒力比较. 中国兽药杂志, 2012, 46(9): 1-3.

Cheng J S, Wu M H, Zhao L X, Peng X B, Ding J B, Wang N, Xia Y C, Mao K R. Comparision of the virulence between three different vaccine strains of brucellosis in mice and guinea pigs. Chinese Journal of Veterinary Science, 2012, 46(9): 1-3. (in Chinese)

[21]中华人民共和国兽用生物制品规程(2000年版). 布鲁氏菌病活疫苗制造及检验规程. 北京: 化学工业出版社, 2000: 154-160.

The Veterinary Biological Products Regulations of the People's Republic of China (2000 edition). The manufacture and test for brucellosis live vaccine. Beijing: Chemical Industry Press, 2000: 154-160. (in Chinese)

[22]Palmer M V, Cheville N F, Jensen A E. Experimental infection of pregnant cattle with vaccine candidate Brucella abortus strain RB51: Pathologic, bacteriologic and serologic findings. Veterinary Pathology, 1996, 33: 682-691.