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Molecular Characterization and Drug Susceptibility of Mycobacterium Bovis Isolated from Cattle in Xinjiang, China.

Byline: Xi-Chao. Ou, Fang. Xu, Yang. Zhou, Li-Li. Tian, Qiao-Ying. Zeng, Wei-Xing. Fan, Yan-Lin. Zhao

To the Editor: Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis . It not only seriously influences the raising of livestock but also threatens the health of human beings and other economically important animals. Therefore, the prevention and control of M. bovis has great significance for public and animal health. Spoligotyping is the most widely used method for genotyping of M. bovis and investigates transmission across different geographical regions. However, its discriminatory power varies widely among countries. Variable number of tandem repeats (VNTR) analysis has emerged as an alternative method for genotyping of bacterial species isolates. Despite the similar biological characteristics of M. bovis and Mycobacterium tuberculosis , the two neighboring species show different drug-resistance profiles. Overall, M. bovis is naturally resistant to pyrazinamide and usually susceptible to most antibiotics used to treat human TB. In China, the excessive use or abuse of veterinary antibiotics is common to prevent bacterial infections among livestock, especially among self-employed, cattle-breeding households. Thus, it is interesting to investigate whether the overuse of antibiotics is affecting the drug susceptibility of M. bovis in China, where knowledge of drug susceptibility is limited. This study presented a version of mycobacterial interspersed repetitive units (MIRU)-VNTR analysis based on three triplex polymerase chain reactions with automatic allelic assignation of the products analyzed in capillary electrophoresis (CE) to find which loci are most discriminative in the M. bovis of China, and assessed whether extensive transmission of M. bovis has occurred among cattle in Xinjiang. It also evaluated the susceptibility of M. bovis isolates to two first-line tuberculosis drugs.

It was found that single intradermal comparative cervical tuberculin and interferon-a tests were both positive for 44 of 341 cattles from five regions within the Xinjiang in April 2014. Twenty-six mycobacterium strains were isolated from these 44 cattles. A commercially available DNA strip assay (GenoType MTBC; Hain Lifescience GmbH, Nehren, Germany) was used to identify M. bovis . Molecular species identification confirmed that all 26 isolates belonged to M. bovis , indicating an infection rate of 7.6% (26/341).

CE-VNTR analysis found that the 26 strains could be classified into 15 different MIRU-VNTR types, whereas spoligotyping identified eight different types [Figure 1]. The results of spoligotyping showed four unique genotypes containing only one strain, and the largest genotype contained 10 strains that were isolated from the same region (region 3). Among 15 VNTR patterns, 10 unique cases and 15 clustering cases belonging to 5 clusters (cluster sizes ranging from 2 to 8 cases) were identified. This study further analyzed results from regions with cattles that were infected with the clustered strains. Interestingly, the 8 cases in the largest cluster were all isolated from region 3. The other four clusters were also from the same region.{Figure 1}

Special repeat numbers of three loci (MIRU-4, VNTR 1955, and MIRU 39) were discovered [Table 1]. Five loci (5/24) used in the regular MIRU-VNTR method showed no polymorphism (MIRU02, MIRU 10, MIRU23, MIRU40, VNTR53). Two loci (ETR-D and VNTR 1955) were defined as highly discriminative and eight loci (ETR-B, VNTR 52, QUB11b, QUB26, VNTR49, MIRU16, ETR-A, MIRU 24) as moderately discriminative [Table 1].{Table 1}

Drug susceptibility of M. bovis isolates was determined by both GenoType MTBDRplus assay and a conventional proportion method. Pyrazinamide susceptibility was determined with a BACTEC MGIT 960 system. All 26 strains were sensitive to isoniazid and rifampin but resistant to pyrazinamide as assessed by BACTEC MGIT 960 system.

In this study, the prevalence of M. bovis infection in cattle was higher than that reported in previous studies in Xinjiang.[1] This might be due to an outbreak of M. bovis infection in Xinjiang. Based on CE-VNTR testing, 15 of 26 strains clustered into five groups of indistinguishable strains. All clusters were small, comprising two cases, with the exception of the largest cluster, which had eight cases. In addition, all five clusters were isolated from the same region. This might reflect recent cattle-to-cattle transmission among clustered cases because the indistinguishable cases came from the same region, and this transmission likely contributed to the subsequent outbreak.

The QUB 11b, QUB 26, and ETR-A loci used for CE-VNTR showed more polymorphism than did other loci. A new combination of 10 MIRU-VNTR loci (ETR-D, VNTR 1955, ETR-B, VNTR 52, QUB11b, QUB26, VNTR49, MIRU16, ETR-A, MIRU 24) was most appropriate for first-line typing of M. bovis clones from China. It was interesting to note that the copy numbers of tandem repeat in the loci of MIRU 4, VNTR 1955, and MIRU 39 were different from those in previous studies.[2],[3],[4] Few genotypes were identified, possibly because of the limited number of cattle in this study.

There is increasing concern over the observation of drug-resistant isolates of M. bovis and M. tuberculosis. [5] The main control of M. bovis is through a tuberculin test and slaughter strategy, but because of the financial burden of this strategy, uncontrolled and unregistered use of antibiotics, mainly isoniazide, is frequently occurring for treatment of cattle with signs of infection.[6] Although monotherapy, mainly with isoniazid, might result in the generation of drug-resistant M. bovis , this study did not find any strains resistant to isoniazid and rifampin.

In conclusion, CE-VNTR method using 10 loci was discriminative for M. bovis in Xinjiang, and there was evidence of recent zoonotic transmission in the farms of Xinjiang. Future studies are needed to definitively assess the prevalence of drug-resistant M. bovis in China.

Financial support and sponsorship

This study was supported by grants from the Chinese National Important Special Fund for Control and Therapy of the Serious Diseases (No. 2014ZX10003002) and the National Basic Research Program of China (No. 2014CB744403).

Conflicts of interest

There are no conflicts of interest.


1. Du Y, Qi Y, Yu L, Lin J, Liu S, Ni H, et al. Molecular characterization of Mycobacterium tuberculosis complex (MTBC) isolated from cattle in Northeast and Northwest China. Res Vet Sci 2011;90:385-91. doi: 10.1016/j.rvsc.2010.07.020.

2. Boniotti MB, Goria M, Loda D, Garrone A, Benedetto A, Mondo A, et al. Molecular typing of Mycobacterium bovis strains isolated in Italy from 2000 to 2006 and evaluation of variable-number tandem repeats for geographically optimized genotyping. J Clin Microbiol 2009;47:636-44. doi: 10.1128/JCM.01192-08.

3. Allix C, Walravens K, Saegerman C, Godfroid J, Supply P, Fauville-Dufaux M, et al. Evaluation of the epidemiological relevance of variable-number tandem-repeat genotyping of Mycobacterium bovis and comparison of the method with IS6110 restriction fragment length polymorphism analysis and spoligotyping. J Clin Microbiol 2006;44:1951-62. doi: 10.1128/JCM.01775-05.

4. Martinez LR, Harris B, Black WC 4th, Meyer RM, Brennan PJ, Vissa VD, et al. Genotyping North American animal Mycobacterium bovis isolates using multilocus variable number tandem repeat analysis. J Vet Diagn Invest 2008;20:707-15. doi: 10.1177/104063870802000601.

5. Yuen CM, Kurbatova EV, Click ES, Cavanaugh JS, Cegielski JP. Association between Mycobacterium tuberculosis complex phylogenetic lineage and acquired drug resistance. PLoS One 2013;8:e83006. doi: 10.1371/journal.pone.0083006.

6. Parreiras PM, Lobato FC, Alencar AP, Figueiredo TD, Gomes HM, Boechat N, et al. Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques. Mem Inst Oswaldo Cruz 2004;99:749-52. doi: /S0074-02762004000700015.
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Title Annotation:Correspondence
Author:Ou, Xi-Chao; Xu, Fang; Zhou, Yang; Tian, Li-Li; Zeng, Qiao-Ying; Fan, Wei-Xing; Zhao, Yan-Lin
Publication:Chinese Medical Journal
Article Type:Letter to the editor
Date:Dec 20, 2018
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