Division of Bacterial Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 15000, PR China.
Res Vet Sci. 2011 Jun;90(3):385-91. doi: 10.1016/j.rvsc.2010.07.020. Epub 2010 Aug 24.
We studied throat swabs and corresponding serum samples collected from 1067 protein purified derivative (PPD)-tuberculin skin test (TST) positive cattle from different regions of China. The 1067 throat swabs were inoculated onto modified Löwenstein-Jensen medium for the isolation and culture of Mycobacteria. Acid-fast bacilli were identified using traditional biochemical methods, polymerase chain reaction (PCR) amplification and multiplex PCR. They were distinguished as Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains. An indirect Enzyme-Linked Immunosorbent Assay (ELISA) was applied to detect specific antibodies against bovine TB (bTB). Correlations among the ELISA, bacteriology and TST were analyzed and compared. Spoligotyping and variable number tandem repeats-mycobacterial interspersed repetitive unit (VNTR-MIRU) analysis were used to genotype the MTBC. In total, 111 strains of Mycobacteria were cultured from the 1067 throat swab samples, including 43 stains of MTBC (14 strains of Mycobacterium bovis and 29 of Mycobacterium tuberculosis) and 68 strains of NTM. Thirty-eight MTBC strains and four NTM strains were isolated from 72 throat swab samples that the ELISA determined were antibody positive; five MTBC strains and 64 NTM strains were isolated from 995 throat swab samples that were antibody negative on the ELISA. The positive isolation rates of MTBC and NTM were 38.7% (43/111) and 61.3% (68/111), respectively. The concordance rate of cultured MTBC with a positive result on the indirect ELISA for antibody was 52.8% (38/72), which was much higher than the positive rate for TST (4.0%; 43/1067). Genotyping of the 43 strains of MTBC isolated, using spoligotyping and VNTR-MIRU, showed that the 43 isolates had 26 genotypes; 16 strains had a unique genotype. Two groups of six strains and two strains, respectively, showed the same spoligotyping pattern, and belonged to the Beijing family and Beijing-like family, respectively. Combined application of spoligotyping and VNTR-MIRU typing would improve the molecular epidemiological investigation and monitoring of the etiology of bTB in China.
我们研究了从中国不同地区的 1067 例蛋白纯化衍生物（PPD）-结核菌素皮肤试验（TST）阳性牛采集的咽拭子和相应的血清样本。将 1067 份咽拭子接种到改良 Löwenstein-Jensen 培养基上，用于分离和培养分枝杆菌。使用传统的生化方法、聚合酶链反应（PCR）扩增和多重 PCR 鉴定抗酸杆菌。它们被鉴定为结核分枝杆菌复合群（MTBC）和非结核分枝杆菌（NTM）菌株。应用间接酶联免疫吸附试验（ELISA）检测针对牛结核病（bTB）的特异性抗体。分析和比较了 ELISA、细菌学和 TST 之间的相关性。采用 spoligotyping 和可变数串联重复-分枝杆菌插入重复单位（VNTR-MIRU）分析对 MTBC 进行基因分型。从 1067 份咽拭子样本中培养出 111 株分枝杆菌，包括 43 株 MTBC（14 株牛分枝杆菌和 29 株结核分枝杆菌）和 68 株 NTM。从 72 份 ELISA 检测为抗体阳性的咽拭子样本中分离出 38 株 MTBC 菌株和 4 株 NTM 菌株；从 995 份 ELISA 检测为抗体阴性的咽拭子样本中分离出 5 株 MTBC 菌株和 64 株 NTM 菌株。MTBC 和 NTM 的阳性分离率分别为 38.7%（43/111）和 61.3%（68/111）。间接 ELISA 检测抗体阳性的 MTBC 培养阳性符合率为 52.8%（38/72），明显高于 TST 的阳性率（4.0%；43/1067）。对 43 株分离出的 MTBC 进行 spoligotyping 和 VNTR-MIRU 基因分型显示，43 株分离株有 26 种基因型；16 株具有独特的基因型。两组 6 株和 2 株分别表现出相同的 spoligotyping 模式，分别属于北京家族和北京样家族。 spoligotyping 和 VNTR-MIRU 联合应用将提高中国牛结核病病因的分子流行病学调查和监测。
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