Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
SHORT REPORT
Open Access
MLVA and MLST typing of Brucella from Qinghai, China Jun-Ying Ma1†, Hu Wang1†, Xue-Fei Zhang1, Li-Qing Xu1, Gui-Ying Hu1, Hai Jiang2, Fang Zhao3, Hong-Yan Zhao2, Dong-Ri Piao2, Yu-Min Qin1, Bu-Yun Cui2* and Gong-Hua Lin3*
Abstract Background: The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection. Methods: A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates. Findings: Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species. Conclusions: There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans. Keywords: Brucella, Molecular identification, Genotype, Zoonotic host
Multilingual abstracts Please see Additional file 1 for translations of the abstract into the six official working languages of the United Nations. Introduction Brucellosis is one of the most widespread and severe zoonotic diseases across the world. This disease affects mammals and is transmitted to humans by animals * Correspondence: [email protected]; [email protected] † Equal contributors 2 State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China 3 Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai, China Full list of author information is available at the end of the article
through direct contact with contaminated animal fluids or indirectly through consumption of unpasteurized milk products [1]. Brucellosis in domestic mammals and humans is the cause of huge economic burden and medical challenges globally, especially in poor regions [2–5]. The causal agent of brucellosis, Brucella genus, currently contains 10 species: B. abortus, B. suis, B. melitensis, B. ovis, B. canis, B. neotomae, B. ceti, B. pinnipedialis, B. microti, and B. inopinata. Some species are further subdivided into biovars, i.e., B. melitensis bv.1 to 3, B. abortus bv. 1 to 6 and 9, and B. suis bv. 1 to 5 [6]. Most species with a preferential host and three other species (B. abortus, B. melitensis, and B. suis) remain the principal causes of zoonotic potential [1].
© 2016 Ma et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Page 2 of 8
Since different species or biovars within a species of Brucella have different epidemiological significances, in order to effectively prevent this disease, it is important to identify the species or strain of Brucella that causes infection. Most previous molecular subtyping tools and “classical biotyping” methods lack sufficient discriminatory power for epidemiological investigations. Recently, two molecular typing methods called MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) have emerged as useful tools for identifying and genotyping Brucella isolates [3, 7–9]. The Qinghai-Tibetan Plateau (QTP) is the largest and highest plateau on Earth, with an area of 2.5 × 106 km2 and an average elevation of 4,000 m above sea level [10]. The QTP is an extensive pastoral and semi-pastoral area, and owing to poverty and bad hygiene, Brucella is highly endemic to this region among yaks (Bos grunniens) [11, 12] and humans [13]. It should be mentioned that, probably due to technical difficulties, most previous studies on Brucella in the QTP merely examined infection surveillance, without paying adequate attention to the study of taxonomic identification of Brucella species. In the present study we collected a considerable number of Brucella samples from Qinghai Province, (Table 1) which is located in the northeastern corner of the QTP and constitutes >25 % of the area of the plateau Table 1 Geographic distribution of Brucella samples in Qinghai, China Locality ID
County
Sample ID
Sample size
1
Datong
Ma10, Ma50
2
2
Dulan
Ma37, Ma51, Ma52, Ma53, Ma54, Ma55, Ma56, Ma57, Ma58
9
3
Gangcha Ma17
1
4
Golmud
Ma13, Ma16, Ma18, Ma19, Ma20, Ma21, Ma26,Ma27, Ma28, Ma29, Ma31, Ma32, Ma33
13
5
Gonghe
Ma03, Ma12, Ma22, Ma24, Ma25, Ma30, Ma34, Ma35
8
6
Qilian
Ma39, Ma40, Ma41, Ma42, Ma43, Ma44, Ma45, Ma46
8
7
Qumalai Ma23
1
8
Tianjun
Ma04, Ma38
2
9
Tongde
Ma36, Ma59, Ma60, Ma61, Ma62, Ma63, Ma64
7
10
Xinghai
Ma01, Ma02, Ma05, Ma06, Ma08, Ma09, 7 Ma15
11
Xining
Ma07, Ma14, Ma65
3
12
Zeku
Ma11
1
13
Zhiduo
Ma47, Ma48, Ma49
3
(Fig. 1). We used both MLVA as well as MLST methods to type these samples and determine the composition characteristics of Brucella in the plateau area.
Methods Ethics statement
This study is a retrospective investigation of our historical collection with modern typing methods. Therefore, the study does not involve the collection or reporting of patient data. No animal work was carried out with the obtained results. Bacterial strains and DNA preparation
A total of 65 isolates were examined: 31 recovered from human, 15 from sheep (Ovis aries), 11 from blue sheep (Pseudois nayaur), 6 from yaks (Bos mutus grunniens), and 1 each from cattle (Bos primigenius taurus) and Tibetan gazelle (Procapra picticaudata). Bacterial strains were cultured on Trypticase soy agar containing 5 % sheep blood (BD Diagnostic Systems, China Ltd., China) at 37 °C for 48 h. Total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) as per the manufacturer’s recommended protocol. MLVA genotyping
MLVA was performed as described earlier [9]. Briefly, 16 primer pairs were designed and classified as belonging to one of the three panels: panel 1 (bruce06, bruce08, bruce11, bruce12, bruce42, bruce43, bruce45, and bruce55), panel 2A (bruce18, bruce19, and bruce21), and panel 2B (bruce04, bruce07, bruce09, bruce16, and bruce30). PCR amplifications were performed in 40 μL reaction volumes, 5 μL of these were loaded in to 2 % (panel 1) or 3 % (panels 2A and 2B) agarose gels containing 0.5 μg/ml of ethidium bromide, visualized under UV light, and photographed. Band intensities were estimated using BioNumerics version 5.1 (Applied Maths, Belgium) and were then converted to repeat units by following the published allele numbering system [7]. In order to improve the genotyping accuracy, PCR products were also directly sequenced after purification. The sequences were aligned and the numbers of repeat units were checked in MEGA version 5 [14] using B. melitensis 16 M genome sequence as a reference (GenBank accession number NC_003317.1 and NC_003318.1). Clustering analysis was based on the categorical coefficient and unweighted pair group method using arithmetic averages (UPGMA) method provided in BioNumerics. The resulting genotypes were compared using the web-based Brucella2012 MLVA database (http://mlva.u-psud.fr/).
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Page 3 of 8
Fig. 1 Geographic distribution of Brucella samples in Qinghai, China. QTP, Qinghai-Tibet Plateau; the dotted counties (NO. 1 ~ 13) correspond to the 13 counties (Locality ID 1 ~ 13) in Table 1. The pie charts showed the composition (the relative pie size corresponds to the number of strains) of the four branches (A, B, C, and E) of B. melitensis
MLST genotyping
Nine distinct genomic loci, including seven housekeeping genes (gap, aroA, glk, dnaK, gyrB, trpE, and cobQ), one outer membrane protein (omp25), and one intergenic fragment (int-hyp), were selected for MLST genotyping [15]. Similar to the MLVA process described, PCR amplifications were performed in 40 μL reaction volumes using primer sequences as previously described [3]. Sequences obtained from purified PCR products were aligned using MEGA program and verified by visualizing. MLST sequences (GenBank accession number AM694191 ~ AM695630) of the strains described by Whatmore et al. [3] were downloaded and a local BLAST database was built using makeblastdb program of BLAST+ program package version 2.2.31 [16]. The sequences were matched with the local database using blastn program of the BLAST+ program package. The BLAST results were parsed using BLAST Parser program version 1.2.6 [17]. Distinct alleles identified at the nine selected loci were each given a numerical designation according to sequence of defined alleles. If the sequence was different from those defined previously, it was designated as a new allele. Each sequence type over all loci (ST) was predicted using web-based MLST service (BrucellaBase, http://59.99.226.203/brucellabase/mlst.html). Phylogenetic
relationships of combined sequences were inferred using the Neighbor-Joining method in BrucellaBase. We could not identify biovars within strains, because it was difficult to distinguish among them using either MLVA16 or MLST markers [3, 18, 19].
Findings MLVA results
Using panel 1 markers, the present population clustered into eight known genotypes: 42 (1-5-3-13-2-2-3-2; N = 51), 43 (1-5-3-13-3-2-3-2; N = 6), 47 (3-4-2-13-4-2-3-3; N = 2), 28 (4-5-4-12-2-2-3-3; N = 2), 63 (1-5-3-13-2-3-3-2; N = 1), 36 (4-5-3-12-2-2-3-1; N = 1), 112 (4-5-3-12-2-2-3-3; N = 1), and 6 (2-3-6-10-4-1-5-2; N = 1). The Clustering analysis showed that the 65 isolates formed six main clusters (A ~ F). Cluster A had two genotypes (42 and 63); cluster B, C, E and F had a single genotype 42, 43, 47 and 6, respectively; cluster D had three genotypes (112, 36, and 28) (Fig. 2). According to Brucella2012 MLVA database and based on panel 1 markers we identified our samples as containing three species: B. melitensis (genotype 42, 43 and 47; cluster A, B, C and E), B. abortus (genotype 28, 36 and 112; cluster D) and B. suis (genotype 6; cluster F). Based on a previous study [20], the B. melitensis strains were sub-grouped into “East Mediterranean” group (genotype 42 and 43) and “American” group
bruce06
bruce08
bruce11
bruce12
bruce42
bruce43
bruce45
bruce55
bruce18
bruce19
bruce21
bruce04
bruce07
bruce09
bruce16
bruce30
panel 1
MLVA-16
Year
3
13
2
2
3
2
4
41
8
6
4
3
7
5
42
Bm01
blue sheep
80345
1980
Ma38
1
5
3
13
2
2
3
2
4
41
8
6
4
3
7
5
42
Bm01
Tibetan gazelle 80346
1980
Ma58
1
5
3
13
2
2
3
2
4
41
8
6
4
3
7
5
42
Bm01
blue sheep
86032
1986
Ma17
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
5
42
Bm02
human
79125
1979
Ma43
1
5
3
13
2
2
3
2
4
41
8
6
4
3
7
6
42
Bm03
sheep
80351
1980
Ma57
1
5
3
13
2
2
3
2
4
41
8
5
4
6
7
5
42
Bm04
blue sheep
86031
1986
Ma25
1
5
3
13
2
2
3
2
4
41
8
5
4
3
8
6
42
Bm05
human
79133
1979
Ma52
1
5
3
13
2
2
3
2
3
41
8
5
4
3
8
5
42
Bm06
yak
80361
1980
Ma53
1
5
3
13
2
2
3
2
3
41
8
5
4
3
8
5
42
Bm06
yak
80362
1980
Ma59
1
5
3
13
2
2
3
2
3
41
8
5
4
3
8
5
42
Bm06
blue sheep
88036
1988
Ma19
1
5
3
13
2
2
3
2
3
41
8
5
4
3
7
5
42
Bm07
human
79127
1979
Ma61
1
5
3
13
2
2
3
2
3
41
8
5
4
3
7
5
42
Bm07
blue sheep
88038
1988
Ma15
1
5
3
13
2
2
3
2
3
41
8
5
4
3
9
5
42
Bm08
human
79123
1979
Ma05
1
5
3
13
2
2
3
2
3
41
8
5
4
3
9
5
42
Bm08
human
65063
1965
Ma54
1
5
3
13
2
2
3
2
3
41
8
5
4
3
9
5
42
Bm08
yak
80363
1980
Ma01
1
5
3
13
2
2
3
2
3
41
8
5
4
3
11
5
42
Bm09
sheep
64017
1964
Ma06
1
5
3
13
2
2
3
2
3
41
8
5
4
3
11
5
42
Bm09
human
65064
1965
Ma39
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
6
42
Bm10
sheep
80347
1980
Ma40
1
5
3
13
2
2
3
2
4
41
8
4
4
3
9
6
42
Bm11
sheep
80348
1980
Ma22
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
79130
1979
Ma24
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
79132
1979
Ma03
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
65050
1965
Ma30
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
79138
1979
Ma12
1
5
3
13
2
2
3
2
4
41
8
4
4
3
10
5
42
Bm13
sheep
73102
1973
Ma09
1
5
3
13
2
2
3
2
4
41
8
4
4
3
3
7
42
Bm14
human
66103
1966
Ma04
1
5
3
13
2
2
3
2
3
41
8
4
4
3
6
5
42
Bm15
human
65051
1965
Ma62
1
5
3
13
2
2
3
2
3
41
8
4
4
3
6
5
42
Bm15
blue sheep
88039
1988
Ma34
1
5
3
13
2
2
3
2
4
41
8
4
4
3
6
5
42
Bm16
human
79143
1979
Ma64
1
5
3
13
2
2
3
2
3
41
8
4
4
8
6
5
42
Bm17
blue sheep
88041
1988
Ma11
1
5
3
13
2
3
3
2
4
41
8
7
4
3
4
5
63
Bm18
sheep
73101
1973
Ma16
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
human
79124
1979
Ma18
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
human
79126
1979
Ma20
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
human
79128
1979
Ma42
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
sheep
80350
1980
Ma13
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79121
1979
Ma21
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79129
1979
Ma28
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79136
1979
Ma31
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79139
1979
Ma41
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
sheep
80349
1980
Ma33
1
5
3
13
2
2
3
2
4
36
8
5
4
3
10
7
42
Bm21
human
79142
1979
Ma36
1
5
3
13
2
2
3
2
4
36
8
5
4
3
15
7
42
Bm22
human
79145
1979
Ma50
1
5
3
13
2
2
3
2
4
36
8
5
4
3
11
7
42
Bm23
yak
80358
1980
Ma44
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
7
42
Bm24
sheep
80352
1980
Ma45
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
7
42
Bm24
sheep
80353
1980
Ma46
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
7
42
Bm24
sheep
80354
1980
Ma02
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
sheep
64018
1964
Ma29
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
human
79137
1979
Ma32
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
human
79141
1979
Ma60
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
blue sheep
88037
1988
Ma27
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
6
42
Bm26
human
79135
1979
Ma35
1
5
3
13
2
2
3
2
4
36
8
5
4
3
9
8
42
Bm27
human
79144
1979
Ma26
1
5
3
13
2
2
3
2
4
36
8
6
4
3
12
7
42
Bm28
human
79134
1979
Ma47
1
5
3
13
3
2
3
2
4
41
5
4
4
6
6
4
43
Bm29
sheep
80355
1980
Ma48
1
5
3
13
3
2
3
2
4
41
5
4
4
6
6
4
43
Bm29
sheep
80356
1980
Ma14
1
5
3
13
3
2
3
2
4
41
5
8
4
4
6
4
43
Bm30
human
79122
1979
Ma07
1
5
3
13
3
2
3
2
4
41
5
8
4
4
6
4
43
Bm30
human
65066
1965
Ma23
1
5
3
13
3
2
3
2
4
41
5
4
4
7
5
4
43
Bm31
human
79131
1979
Ma49
1
5
3
13
3
2
3
2
4
41
5
6
4
6
4
4
43
Bm32
sheep
80357
1980
Ma10
4
5
3
12
2
2
3
3
8
43
8
4
4
3
3
3
112
Ba01
yak
73096
1973
D
Ma65
4
5
3
12
2
2
3
1
6
43
8
4
6
7
3
3
36
Ba02
catle
80359
1980
B. a.
Ma51
4
5
4
12
2
2
3
3
6
43
8
3
6
3
3
5
28
Ba03
yak
80360
1980
Ma08
4
5
4
12
2
2
3
3
6
43
8
3
6
3
3
5
28
Ba03
human
65079
1965
Ma55
3
4
2
13
4
2
3
3
5
36
6
2
5
7
3
5
47
Bm33
blue sheep
86029
1986
Ma56
3
4
2
13
4
2
3
3
5
36
6
2
5
8
3
7
47
Bm34
blue sheep
86030
1986
Ma63
2
3
6
10
4
1
5
2
4
40
9
6
6
7
5
3
6
Bs01
blue sheep
88040
1988
80
90
Isolate
5
A B. m.
B B. m.
C B. m.
E
F
B. m.
100
Host
1
70
ID
60
40
Page 4 of 8
Ma37
50
30
20
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
B. s. Fig. 2 Dendrogram based on the MLVA genotyping assay showing relationships of the 65 Brucella isolates. ID: serial number for the 65 isolates; MLVA type: panel 1 and MLVA-16 genotypes; Host: the hosts from which the bacteria was isolated; Isolate: isolate name in the laboratory in which the DNA extraction was done; Year: the years when the strains were collected
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
(genotype 47). Considering all three MLVA panels, 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes were identified for B. melitensis, B. abortus and B. suis isolates respectively (Fig. 2). No genotypes completely matched any of the genotypes in the Brucella2012 MLVA database. MLST results
A total of five known MLST genotypes were identified: ST7 (3-5-3-2-1-5-2-10-2; N = 2), ST8 (3-2-3-2-1-5-3-8-2; N = 57), ST2 (2-1-2-2-1-3-1-1-1; N = 2), ST5 (2-1-1-2-1-41-1-1; N = 2), ST14 (1-6-4-1-4-3-5-2-1; N = 1). The Neighbor-Joining clustering showed that the 65 isolates formed four main clusters – a, d, e and f. Of these, clusters a, e and f correspond to genotypes ST8, ST7 and ST14 respectively while cluster d corresponds to two genotypes, ST2 and ST5. According to Whatmore et al. [3], genotypes ST7 and ST8 belong to B. melitensis, ST2 and ST5 belong to B. abortus, and ST14 belongs to B. suis. The gyrB sequence of strain Ma06 was different from any of known alleles (allele 1 ~ 6). Its sequence closely matched that of allele 1 (identical except for a dinucleotide change of GC to AA on site 405 ~ 406 relative to the gyrB allele 1 sequence or on 2504 ~ 2505 sites relative to the ST8 sequence). Considering all MLST alleles, the strain Ma06 could be viewed as a mutant variant (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2).
Discussion In this study we used both MLVA and MLST methods to identify Brucella species in the east of the QinghaiTibet Plateau. Although there were some minor incongruences for e.g., in clustering results (Fig. 2 and Fig. 3), both methods showed a consistent conclusion that there were at least three Brucella species in the sample analyzed. Of the 65 isolates 60, 4, and 1 were respectively identified to B. melitensis, B. abortus, and B. suis, indicating that B. melitensis was a predominant species on the plateau. Interestingly however, when searching in the Brucella2012 MLVA database none of the genotypes found in our study were identical to any of the genotypes in the database. Based on MLST the ST8 seems to be the main ST type in the QTP region. Moreover, there was also a unique genotype (strain Ma06) which has not been reported anywhere before. These results demonstrated that the Brucella population found in the QTP region was very different from that in other regions. We attribute this to relatively isolated and special environment of the plateau. Extremely high altitudes make it difficult for lowland livestock breeds or wild animals to survive, thereby favoring only the endemic ones. Hence, there was a very limited breeds exchange between QTP and other regions, which may have consequently formed a unique local Brucella population.
Page 5 of 8
Based on MLVA-16, ten genotypes were shared by two or more host species. To elaborate further, four genotypes (Bm09, Bm19, Bm20, and Bm25) were shared between humans and sheep, and two (Bm08 and Ba03) were shared between humans and yaks (Fig. 2). Based on MLST, the ST8 type was also shared between humans and four other animals (sheep, yaks, blue sheep and Tibetan gazelle); the ST5 type was shared between humans and yaks (Fig. 3). Sheep and yaks are the main livestock on QTP and it is therefore not surprising that these animals pose a heavy zoonotic disease threat to humans. It should be noted that, all sheep and four out of six yaks were infected by B. melitensis, indicating that this bacterial species should be paid special attention to in both host species. It is well known that different bacterial species need different vaccine strains. For instance, there are three vaccine strains M5, S19 and S2, which are used to immunize animals in China for B. melitensis, B. abortus, and B. suis, respectively [21]. Because yaks could be infected by either B. melitensis or B. abortus, we suggest based on our study that both the B. melitensis type (M5) and B. abortus type vaccines (S19) should be tried in this animal. The blue sheep were infected by all three Brucella species including B. suis, which is generally carried by pigs [1, 22]. More importantly, this animal shared Brucella genotypes with human (Bm07, Bm15 and Bm25), yaks (Bm06), and Tibetan gazelle (Bm01), indicating this animal is an important natural repertory for Brucella. Since there is obvious spatial and diet overlap between blue sheep and domestic livestock [23], we suggest that this animal is also an important infection source of Brucella in humans. Additionally, due to high genetic diversity of B. melitensis, we also discuss the geographic distribution features of the strains. Figure 1 showed that 7 counties (locality ID: 2, 3, 5, 8, 9, 10, and 12) were dominated by A branch strains; 3 counties (locality ID: 1, 4, and 6) were dominated by B branch strains; 3 counties (locality ID: 7, 11, and 13) contained only C branch strains; and the E branch genotypes appeared only in 1 county (locality ID: 2). These results showed that the B. melitensis prevalence in the QTP region might have a considerable regional characteristic i.e., there might be genetic variations among different regions. Our results for the first time elucidated the composition of species and genotypes of Brucella samples in the QTP region. We also presented the host- as well as geographic distributions of the species and genotypes. These results may have some implications for the future disease control programmes in QTP. First, since B. melitensis was the predominant species in the study area, special attention should be paid on this species in brucellosis control programs such as livestock vaccination.
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
omp25
int-hyp
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 4
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 3 3 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 1 1 1 1 5
8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 10 10 1 1 1 1 2
2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8? 2 7 2 7 1 5 1 5 1 2 1 2 1 14
ST
cobQ
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 2 2 4
trpE
f B.s.
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 1 1 1 1 6
dnaK
d B.a.
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 1
gyrB
e
B.m.
glk
0.0002
gap
a B.m.
ID Ma01 Ma02 Ma03 Ma04 Ma05 Ma07 Ma09 M a 11 Ma12 Ma13 Ma14 Ma15 Ma16 Ma17 Ma18 Ma19 Ma20 Ma21 Ma22 Ma23 Ma24 Ma25 Ma26 Ma27 Ma28 Ma29 Ma30 Ma31 Ma32 Ma33 Ma34 Ma35 Ma36 Ma37 Ma38 Ma39 Ma40 Ma41 Ma42 Ma43 Ma44 Ma45 Ma46 Ma47 Ma48 Ma49 Ma50 Ma52 Ma53 Ma54 Ma57 Ma58 Ma59 Ma60 Ma61 Ma62 Ma64 Ma06 Ma55 Ma56 Ma08 Ma51 Ma10 Ma65 Ma63
aroA
Page 6 of 8
Host sheep sheep human human human human human sheep sheep human human human human human human human human human human human human human human human human human human human human human human human human blue sheep Ti b e t a n g a z e l l e sheep sheep sheep sheep sheep sheep sheep sheep sheep sheep sheep yak yak yak yak blue sheep blue sheep blue sheep blue sheep blue sheep blue sheep blue sheep human blue sheep blue sheep human yak yak cattle blue sheep
Fig. 3 Dendrogram based on the MLST genotyping assay showing relationships of the 65 Brucella isolates. ID: serial number for the 65 isolates; ST: MLST genotypes; Host: the hosts from which the bacteria was isolated. (Also see Fig. 2 for additional information)
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Second, the genotypes of Brucella in the QTP region were very different from other regions, but whether the genetic difference may result in different vaccination efficiencies should be elucidated in the further studies. Third, since there were extensive genotype-sharing events between humans and animals, brucellosis surveillance should be regularly executed on both livestock (sheep and yaks) and wild ungulates (blue sheep). It should be noted that, as mentioned above, none of the genotypes found in our study were identical to any of the genotypes in the Brucella2012 database, in order to prevent over-interpreting our results, we did not compare our strains with those from other studies. This might limit our understanding of genetic relationships between the Brucella populations from QTP and from other regions. Moreover, because of imbalance of sample sizes among different counties and among different hosts, in this study, we were not able to do a detail epidemiological investigation. We suggest that the accumulation of more samples will enable us to further elucidate the genetic characteristics of Brucella species in QTP.
Conclusions Our study drew three main conclusions: (i) there were at least three Brucella (B. melitensis, B. abortus and B. suis) species in the east of QTP and B. melitensis was the predominant species in the area studied; (ii) the Brucella population in the QTP region was very different from other regions probably due to the unique geographical characteristics e.g., extremely high altitude in QTP; and (iii) there were extensive genotype-sharing events between humans and animals; sheep, yaks and blue sheep were important zoonotic hosts of brucellosis to humans in the area studied. Additional file Additional file 1: Multilingual abstracts in the six official working languages of the United Nations. (PDF 350 kb) Abbreviations MLST: multi locus sequence typing; MLVA: multi-locus variable-number tandem-repeat analysis; QTP: Qinghai-Tibet Plateau. Competing interests The authors declare that they have no competing interests. Authors’ contributions JYM, HW, BYC and GHL contributed to the design of the study. JYM, BYC and GHL collected the data and wrote the paper. JYM, XFZ, LQX, GYH, HJ, FZ, HYZ, DRP, YMQ and GHL analyzed the data. All authors read and approved the final paper. Acknowledgments This study was supported by the Science and Technology Plan of Qinghai Province (NO. 2013-Z-749) and the Youth Innovation Promotion Association of Chinese Academy of Sciences (NO. 2015352).
Page 7 of 8
Author details 1 Qinghai Institute for Endemic Disease Prevention and Control, Xining 811602, Qinghai, China. 2State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 3Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai, China. Received: 21 October 2015 Accepted: 23 March 2016
References 1. Atluri VL, Xavier MN, de Jong MF, den Hartigh AB, Tsolis RM. Interactions of the human pathogenic Brucella species with their hosts. Annu Rev Microbiol. 2011;65:523–41. 2. Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV. The new global map of human Brucellosis. Lancet Infect Dis. 2006;6:91–9. 3. Whatmore AM, Perrett LL, MacMillan AP. Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol. 2007;7:34. 4. Kansiime C, Rutebemberwa E, Asiimwe BB, Makumbi F, Bazira J, Mugisha A. Annual trends of human brucellosis in pastoralist communities of southwestern Uganda: a retrospective ten-year study. Infec Dis Poverty. 2015;4:39. 5. Ducrotoy MJ, Ammary K, Lbacha HA, Zouagui Z, Mick V, Prevost L, Bryssinckx W, Welburn SC, Benkirane A. Narrative overview of animal and human brucellosis in Morocco: intensification of livestock production as a driver for emergence? Infec Dis Poverty. 2015;4:57. 6. Garin-Bastuji B, Mick V, Le Carrou G, Allix S, Perrett LL, Dawson CE, et al. Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches. Appl Environ Microbiol. 2014; 80(5):1570–9. 7. Le Flèche P, Jacques I, Grayon M, Al Dahouk S, Bouchon P, Denoeud F, et al. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol. 2006;6:9. 8. Chen Y, Zhen Q, Wang Y, Xu J, Sun Y, Li T, et al. Development of an extended multilocus sequence typing for genotyping of Brucella isolates. J Microbiol Methods. 2011;86(2):252–4. 9. Di D, Cui B, Wang H, Zhao H, Piao D, Tian L, et al. Genetic polymorphism characteristics of Brucella canis isolated in China. PLoS One. 2014;9(1):e84862. 10. Wu T. The Qinghai-Tibetan plateau: how high do Tibetans live? High Alt Med Biol. 2001;2:489–99. 11. Wiener G, Han J, Long R. The yak. 2nd ed. Bangkok: RAP publication; 2003. 12. Lang X, Qu H, Bu Z, Yang Y, Sun C, Li X, et al. Seroprevalence of Brucella infection in yaks (Bos grunniens) on the Qinghai-Tibet plateau of China. Trop Anim Health Prod. 2011;43:305–6. 13. Xue H. Analysis on infection and the related factor of brucellosis in high-risk population in Qinghai Province of 2006 to 2010. Taiyuan, Shanxi Province, China: Master thesis of Shanxi Medical University; 2012. 14. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731–9. 15. Chen Y, Ke Y, Wang Y, Yuan X, Zhou X, Jiang H, et al. Changes of predominant species/biovars and sequence types of Brucella isolates, Inner Mongolia. China BMC Infect Dis. 2013;13:514. 16. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421. 17. Marco C, Roberta B. BLAST Parser. BITS Conference. Societa di Bioinformatica Italiana. Bologna. Societa di Bioinformatica Italiana Bologna Italy. 2006;5:28–9. 18. Jiang H, Fan M, Chen J, Mi J, Yu R, Zhao H, et al. MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolates. BMC Microbiol. 2011;11:256. 19. Jiang H, Wang H, Xu L, Hu G, Ma J, Xiao P, et al. MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identification of Brucella suis vaccine strain s2 from cattle in China. PLoS One. 2013;8(10):e76332.
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Page 8 of 8
20. Al Dahouk S, Le Flèche P, Nöckler P, Jacques I, Grayon M, Scholz HC, et al. Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Methods. 2007;69:137–45. 21. Shang D, Xiao D, Yin J. Epidemiology and control of brucellosis in China. Vet Microbiol. 2002;90:165–82. 22. Burniston S, Okello AL, Khamlome B, Inthavong P, Gilbert J, Blacksell SD, Allen J, Welburn SC. Cultural drivers and health-seeking behaviours that impact on the transmission of pig-associated zoonoses in Lao People’s Democratic Republic. Infec Dis Poverty. 2015;4:11. 23. Mishra C, Van Wieren SE, Ketner P, Heitkonig IMA, Prins HHT. Competition between domestic livestock and wild bharal Pseudois nayaur in the Indian Trans-Himalaya. J Appl Ecol. 2004;41(2):344–54. doi:10.1186/s40249-016-0123-z Cite this article as: Ma et al.: MLVA and MLST typing of Brucella from Qinghai, China. Infectious Diseases of Poverty 2016 4:.
Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit
SHORT REPORT
Open Access
MLVA and MLST typing of Brucella from Qinghai, China Jun-Ying Ma1†, Hu Wang1†, Xue-Fei Zhang1, Li-Qing Xu1, Gui-Ying Hu1, Hai Jiang2, Fang Zhao3, Hong-Yan Zhao2, Dong-Ri Piao2, Yu-Min Qin1, Bu-Yun Cui2* and Gong-Hua Lin3*
Abstract Background: The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection. Methods: A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates. Findings: Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species. Conclusions: There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans. Keywords: Brucella, Molecular identification, Genotype, Zoonotic host
Multilingual abstracts Please see Additional file 1 for translations of the abstract into the six official working languages of the United Nations. Introduction Brucellosis is one of the most widespread and severe zoonotic diseases across the world. This disease affects mammals and is transmitted to humans by animals * Correspondence: [email protected]; [email protected] † Equal contributors 2 State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China 3 Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai, China Full list of author information is available at the end of the article
through direct contact with contaminated animal fluids or indirectly through consumption of unpasteurized milk products [1]. Brucellosis in domestic mammals and humans is the cause of huge economic burden and medical challenges globally, especially in poor regions [2–5]. The causal agent of brucellosis, Brucella genus, currently contains 10 species: B. abortus, B. suis, B. melitensis, B. ovis, B. canis, B. neotomae, B. ceti, B. pinnipedialis, B. microti, and B. inopinata. Some species are further subdivided into biovars, i.e., B. melitensis bv.1 to 3, B. abortus bv. 1 to 6 and 9, and B. suis bv. 1 to 5 [6]. Most species with a preferential host and three other species (B. abortus, B. melitensis, and B. suis) remain the principal causes of zoonotic potential [1].
© 2016 Ma et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Page 2 of 8
Since different species or biovars within a species of Brucella have different epidemiological significances, in order to effectively prevent this disease, it is important to identify the species or strain of Brucella that causes infection. Most previous molecular subtyping tools and “classical biotyping” methods lack sufficient discriminatory power for epidemiological investigations. Recently, two molecular typing methods called MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) have emerged as useful tools for identifying and genotyping Brucella isolates [3, 7–9]. The Qinghai-Tibetan Plateau (QTP) is the largest and highest plateau on Earth, with an area of 2.5 × 106 km2 and an average elevation of 4,000 m above sea level [10]. The QTP is an extensive pastoral and semi-pastoral area, and owing to poverty and bad hygiene, Brucella is highly endemic to this region among yaks (Bos grunniens) [11, 12] and humans [13]. It should be mentioned that, probably due to technical difficulties, most previous studies on Brucella in the QTP merely examined infection surveillance, without paying adequate attention to the study of taxonomic identification of Brucella species. In the present study we collected a considerable number of Brucella samples from Qinghai Province, (Table 1) which is located in the northeastern corner of the QTP and constitutes >25 % of the area of the plateau Table 1 Geographic distribution of Brucella samples in Qinghai, China Locality ID
County
Sample ID
Sample size
1
Datong
Ma10, Ma50
2
2
Dulan
Ma37, Ma51, Ma52, Ma53, Ma54, Ma55, Ma56, Ma57, Ma58
9
3
Gangcha Ma17
1
4
Golmud
Ma13, Ma16, Ma18, Ma19, Ma20, Ma21, Ma26,Ma27, Ma28, Ma29, Ma31, Ma32, Ma33
13
5
Gonghe
Ma03, Ma12, Ma22, Ma24, Ma25, Ma30, Ma34, Ma35
8
6
Qilian
Ma39, Ma40, Ma41, Ma42, Ma43, Ma44, Ma45, Ma46
8
7
Qumalai Ma23
1
8
Tianjun
Ma04, Ma38
2
9
Tongde
Ma36, Ma59, Ma60, Ma61, Ma62, Ma63, Ma64
7
10
Xinghai
Ma01, Ma02, Ma05, Ma06, Ma08, Ma09, 7 Ma15
11
Xining
Ma07, Ma14, Ma65
3
12
Zeku
Ma11
1
13
Zhiduo
Ma47, Ma48, Ma49
3
(Fig. 1). We used both MLVA as well as MLST methods to type these samples and determine the composition characteristics of Brucella in the plateau area.
Methods Ethics statement
This study is a retrospective investigation of our historical collection with modern typing methods. Therefore, the study does not involve the collection or reporting of patient data. No animal work was carried out with the obtained results. Bacterial strains and DNA preparation
A total of 65 isolates were examined: 31 recovered from human, 15 from sheep (Ovis aries), 11 from blue sheep (Pseudois nayaur), 6 from yaks (Bos mutus grunniens), and 1 each from cattle (Bos primigenius taurus) and Tibetan gazelle (Procapra picticaudata). Bacterial strains were cultured on Trypticase soy agar containing 5 % sheep blood (BD Diagnostic Systems, China Ltd., China) at 37 °C for 48 h. Total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) as per the manufacturer’s recommended protocol. MLVA genotyping
MLVA was performed as described earlier [9]. Briefly, 16 primer pairs were designed and classified as belonging to one of the three panels: panel 1 (bruce06, bruce08, bruce11, bruce12, bruce42, bruce43, bruce45, and bruce55), panel 2A (bruce18, bruce19, and bruce21), and panel 2B (bruce04, bruce07, bruce09, bruce16, and bruce30). PCR amplifications were performed in 40 μL reaction volumes, 5 μL of these were loaded in to 2 % (panel 1) or 3 % (panels 2A and 2B) agarose gels containing 0.5 μg/ml of ethidium bromide, visualized under UV light, and photographed. Band intensities were estimated using BioNumerics version 5.1 (Applied Maths, Belgium) and were then converted to repeat units by following the published allele numbering system [7]. In order to improve the genotyping accuracy, PCR products were also directly sequenced after purification. The sequences were aligned and the numbers of repeat units were checked in MEGA version 5 [14] using B. melitensis 16 M genome sequence as a reference (GenBank accession number NC_003317.1 and NC_003318.1). Clustering analysis was based on the categorical coefficient and unweighted pair group method using arithmetic averages (UPGMA) method provided in BioNumerics. The resulting genotypes were compared using the web-based Brucella2012 MLVA database (http://mlva.u-psud.fr/).
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Page 3 of 8
Fig. 1 Geographic distribution of Brucella samples in Qinghai, China. QTP, Qinghai-Tibet Plateau; the dotted counties (NO. 1 ~ 13) correspond to the 13 counties (Locality ID 1 ~ 13) in Table 1. The pie charts showed the composition (the relative pie size corresponds to the number of strains) of the four branches (A, B, C, and E) of B. melitensis
MLST genotyping
Nine distinct genomic loci, including seven housekeeping genes (gap, aroA, glk, dnaK, gyrB, trpE, and cobQ), one outer membrane protein (omp25), and one intergenic fragment (int-hyp), were selected for MLST genotyping [15]. Similar to the MLVA process described, PCR amplifications were performed in 40 μL reaction volumes using primer sequences as previously described [3]. Sequences obtained from purified PCR products were aligned using MEGA program and verified by visualizing. MLST sequences (GenBank accession number AM694191 ~ AM695630) of the strains described by Whatmore et al. [3] were downloaded and a local BLAST database was built using makeblastdb program of BLAST+ program package version 2.2.31 [16]. The sequences were matched with the local database using blastn program of the BLAST+ program package. The BLAST results were parsed using BLAST Parser program version 1.2.6 [17]. Distinct alleles identified at the nine selected loci were each given a numerical designation according to sequence of defined alleles. If the sequence was different from those defined previously, it was designated as a new allele. Each sequence type over all loci (ST) was predicted using web-based MLST service (BrucellaBase, http://59.99.226.203/brucellabase/mlst.html). Phylogenetic
relationships of combined sequences were inferred using the Neighbor-Joining method in BrucellaBase. We could not identify biovars within strains, because it was difficult to distinguish among them using either MLVA16 or MLST markers [3, 18, 19].
Findings MLVA results
Using panel 1 markers, the present population clustered into eight known genotypes: 42 (1-5-3-13-2-2-3-2; N = 51), 43 (1-5-3-13-3-2-3-2; N = 6), 47 (3-4-2-13-4-2-3-3; N = 2), 28 (4-5-4-12-2-2-3-3; N = 2), 63 (1-5-3-13-2-3-3-2; N = 1), 36 (4-5-3-12-2-2-3-1; N = 1), 112 (4-5-3-12-2-2-3-3; N = 1), and 6 (2-3-6-10-4-1-5-2; N = 1). The Clustering analysis showed that the 65 isolates formed six main clusters (A ~ F). Cluster A had two genotypes (42 and 63); cluster B, C, E and F had a single genotype 42, 43, 47 and 6, respectively; cluster D had three genotypes (112, 36, and 28) (Fig. 2). According to Brucella2012 MLVA database and based on panel 1 markers we identified our samples as containing three species: B. melitensis (genotype 42, 43 and 47; cluster A, B, C and E), B. abortus (genotype 28, 36 and 112; cluster D) and B. suis (genotype 6; cluster F). Based on a previous study [20], the B. melitensis strains were sub-grouped into “East Mediterranean” group (genotype 42 and 43) and “American” group
bruce06
bruce08
bruce11
bruce12
bruce42
bruce43
bruce45
bruce55
bruce18
bruce19
bruce21
bruce04
bruce07
bruce09
bruce16
bruce30
panel 1
MLVA-16
Year
3
13
2
2
3
2
4
41
8
6
4
3
7
5
42
Bm01
blue sheep
80345
1980
Ma38
1
5
3
13
2
2
3
2
4
41
8
6
4
3
7
5
42
Bm01
Tibetan gazelle 80346
1980
Ma58
1
5
3
13
2
2
3
2
4
41
8
6
4
3
7
5
42
Bm01
blue sheep
86032
1986
Ma17
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
5
42
Bm02
human
79125
1979
Ma43
1
5
3
13
2
2
3
2
4
41
8
6
4
3
7
6
42
Bm03
sheep
80351
1980
Ma57
1
5
3
13
2
2
3
2
4
41
8
5
4
6
7
5
42
Bm04
blue sheep
86031
1986
Ma25
1
5
3
13
2
2
3
2
4
41
8
5
4
3
8
6
42
Bm05
human
79133
1979
Ma52
1
5
3
13
2
2
3
2
3
41
8
5
4
3
8
5
42
Bm06
yak
80361
1980
Ma53
1
5
3
13
2
2
3
2
3
41
8
5
4
3
8
5
42
Bm06
yak
80362
1980
Ma59
1
5
3
13
2
2
3
2
3
41
8
5
4
3
8
5
42
Bm06
blue sheep
88036
1988
Ma19
1
5
3
13
2
2
3
2
3
41
8
5
4
3
7
5
42
Bm07
human
79127
1979
Ma61
1
5
3
13
2
2
3
2
3
41
8
5
4
3
7
5
42
Bm07
blue sheep
88038
1988
Ma15
1
5
3
13
2
2
3
2
3
41
8
5
4
3
9
5
42
Bm08
human
79123
1979
Ma05
1
5
3
13
2
2
3
2
3
41
8
5
4
3
9
5
42
Bm08
human
65063
1965
Ma54
1
5
3
13
2
2
3
2
3
41
8
5
4
3
9
5
42
Bm08
yak
80363
1980
Ma01
1
5
3
13
2
2
3
2
3
41
8
5
4
3
11
5
42
Bm09
sheep
64017
1964
Ma06
1
5
3
13
2
2
3
2
3
41
8
5
4
3
11
5
42
Bm09
human
65064
1965
Ma39
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
6
42
Bm10
sheep
80347
1980
Ma40
1
5
3
13
2
2
3
2
4
41
8
4
4
3
9
6
42
Bm11
sheep
80348
1980
Ma22
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
79130
1979
Ma24
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
79132
1979
Ma03
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
65050
1965
Ma30
1
5
3
13
2
2
3
2
4
41
8
4
4
3
5
5
42
Bm12
human
79138
1979
Ma12
1
5
3
13
2
2
3
2
4
41
8
4
4
3
10
5
42
Bm13
sheep
73102
1973
Ma09
1
5
3
13
2
2
3
2
4
41
8
4
4
3
3
7
42
Bm14
human
66103
1966
Ma04
1
5
3
13
2
2
3
2
3
41
8
4
4
3
6
5
42
Bm15
human
65051
1965
Ma62
1
5
3
13
2
2
3
2
3
41
8
4
4
3
6
5
42
Bm15
blue sheep
88039
1988
Ma34
1
5
3
13
2
2
3
2
4
41
8
4
4
3
6
5
42
Bm16
human
79143
1979
Ma64
1
5
3
13
2
2
3
2
3
41
8
4
4
8
6
5
42
Bm17
blue sheep
88041
1988
Ma11
1
5
3
13
2
3
3
2
4
41
8
7
4
3
4
5
63
Bm18
sheep
73101
1973
Ma16
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
human
79124
1979
Ma18
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
human
79126
1979
Ma20
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
human
79128
1979
Ma42
1
5
3
13
2
2
3
2
4
36
8
5
4
3
13
7
42
Bm19
sheep
80350
1980
Ma13
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79121
1979
Ma21
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79129
1979
Ma28
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79136
1979
Ma31
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
human
79139
1979
Ma41
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
7
42
Bm20
sheep
80349
1980
Ma33
1
5
3
13
2
2
3
2
4
36
8
5
4
3
10
7
42
Bm21
human
79142
1979
Ma36
1
5
3
13
2
2
3
2
4
36
8
5
4
3
15
7
42
Bm22
human
79145
1979
Ma50
1
5
3
13
2
2
3
2
4
36
8
5
4
3
11
7
42
Bm23
yak
80358
1980
Ma44
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
7
42
Bm24
sheep
80352
1980
Ma45
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
7
42
Bm24
sheep
80353
1980
Ma46
1
5
3
13
2
2
3
2
4
41
8
5
4
3
7
7
42
Bm24
sheep
80354
1980
Ma02
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
sheep
64018
1964
Ma29
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
human
79137
1979
Ma32
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
human
79141
1979
Ma60
1
5
3
13
2
2
3
2
4
36
8
5
4
3
7
7
42
Bm25
blue sheep
88037
1988
Ma27
1
5
3
13
2
2
3
2
4
36
8
5
4
3
14
6
42
Bm26
human
79135
1979
Ma35
1
5
3
13
2
2
3
2
4
36
8
5
4
3
9
8
42
Bm27
human
79144
1979
Ma26
1
5
3
13
2
2
3
2
4
36
8
6
4
3
12
7
42
Bm28
human
79134
1979
Ma47
1
5
3
13
3
2
3
2
4
41
5
4
4
6
6
4
43
Bm29
sheep
80355
1980
Ma48
1
5
3
13
3
2
3
2
4
41
5
4
4
6
6
4
43
Bm29
sheep
80356
1980
Ma14
1
5
3
13
3
2
3
2
4
41
5
8
4
4
6
4
43
Bm30
human
79122
1979
Ma07
1
5
3
13
3
2
3
2
4
41
5
8
4
4
6
4
43
Bm30
human
65066
1965
Ma23
1
5
3
13
3
2
3
2
4
41
5
4
4
7
5
4
43
Bm31
human
79131
1979
Ma49
1
5
3
13
3
2
3
2
4
41
5
6
4
6
4
4
43
Bm32
sheep
80357
1980
Ma10
4
5
3
12
2
2
3
3
8
43
8
4
4
3
3
3
112
Ba01
yak
73096
1973
D
Ma65
4
5
3
12
2
2
3
1
6
43
8
4
6
7
3
3
36
Ba02
catle
80359
1980
B. a.
Ma51
4
5
4
12
2
2
3
3
6
43
8
3
6
3
3
5
28
Ba03
yak
80360
1980
Ma08
4
5
4
12
2
2
3
3
6
43
8
3
6
3
3
5
28
Ba03
human
65079
1965
Ma55
3
4
2
13
4
2
3
3
5
36
6
2
5
7
3
5
47
Bm33
blue sheep
86029
1986
Ma56
3
4
2
13
4
2
3
3
5
36
6
2
5
8
3
7
47
Bm34
blue sheep
86030
1986
Ma63
2
3
6
10
4
1
5
2
4
40
9
6
6
7
5
3
6
Bs01
blue sheep
88040
1988
80
90
Isolate
5
A B. m.
B B. m.
C B. m.
E
F
B. m.
100
Host
1
70
ID
60
40
Page 4 of 8
Ma37
50
30
20
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
B. s. Fig. 2 Dendrogram based on the MLVA genotyping assay showing relationships of the 65 Brucella isolates. ID: serial number for the 65 isolates; MLVA type: panel 1 and MLVA-16 genotypes; Host: the hosts from which the bacteria was isolated; Isolate: isolate name in the laboratory in which the DNA extraction was done; Year: the years when the strains were collected
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
(genotype 47). Considering all three MLVA panels, 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes were identified for B. melitensis, B. abortus and B. suis isolates respectively (Fig. 2). No genotypes completely matched any of the genotypes in the Brucella2012 MLVA database. MLST results
A total of five known MLST genotypes were identified: ST7 (3-5-3-2-1-5-2-10-2; N = 2), ST8 (3-2-3-2-1-5-3-8-2; N = 57), ST2 (2-1-2-2-1-3-1-1-1; N = 2), ST5 (2-1-1-2-1-41-1-1; N = 2), ST14 (1-6-4-1-4-3-5-2-1; N = 1). The Neighbor-Joining clustering showed that the 65 isolates formed four main clusters – a, d, e and f. Of these, clusters a, e and f correspond to genotypes ST8, ST7 and ST14 respectively while cluster d corresponds to two genotypes, ST2 and ST5. According to Whatmore et al. [3], genotypes ST7 and ST8 belong to B. melitensis, ST2 and ST5 belong to B. abortus, and ST14 belongs to B. suis. The gyrB sequence of strain Ma06 was different from any of known alleles (allele 1 ~ 6). Its sequence closely matched that of allele 1 (identical except for a dinucleotide change of GC to AA on site 405 ~ 406 relative to the gyrB allele 1 sequence or on 2504 ~ 2505 sites relative to the ST8 sequence). Considering all MLST alleles, the strain Ma06 could be viewed as a mutant variant (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2).
Discussion In this study we used both MLVA and MLST methods to identify Brucella species in the east of the QinghaiTibet Plateau. Although there were some minor incongruences for e.g., in clustering results (Fig. 2 and Fig. 3), both methods showed a consistent conclusion that there were at least three Brucella species in the sample analyzed. Of the 65 isolates 60, 4, and 1 were respectively identified to B. melitensis, B. abortus, and B. suis, indicating that B. melitensis was a predominant species on the plateau. Interestingly however, when searching in the Brucella2012 MLVA database none of the genotypes found in our study were identical to any of the genotypes in the database. Based on MLST the ST8 seems to be the main ST type in the QTP region. Moreover, there was also a unique genotype (strain Ma06) which has not been reported anywhere before. These results demonstrated that the Brucella population found in the QTP region was very different from that in other regions. We attribute this to relatively isolated and special environment of the plateau. Extremely high altitudes make it difficult for lowland livestock breeds or wild animals to survive, thereby favoring only the endemic ones. Hence, there was a very limited breeds exchange between QTP and other regions, which may have consequently formed a unique local Brucella population.
Page 5 of 8
Based on MLVA-16, ten genotypes were shared by two or more host species. To elaborate further, four genotypes (Bm09, Bm19, Bm20, and Bm25) were shared between humans and sheep, and two (Bm08 and Ba03) were shared between humans and yaks (Fig. 2). Based on MLST, the ST8 type was also shared between humans and four other animals (sheep, yaks, blue sheep and Tibetan gazelle); the ST5 type was shared between humans and yaks (Fig. 3). Sheep and yaks are the main livestock on QTP and it is therefore not surprising that these animals pose a heavy zoonotic disease threat to humans. It should be noted that, all sheep and four out of six yaks were infected by B. melitensis, indicating that this bacterial species should be paid special attention to in both host species. It is well known that different bacterial species need different vaccine strains. For instance, there are three vaccine strains M5, S19 and S2, which are used to immunize animals in China for B. melitensis, B. abortus, and B. suis, respectively [21]. Because yaks could be infected by either B. melitensis or B. abortus, we suggest based on our study that both the B. melitensis type (M5) and B. abortus type vaccines (S19) should be tried in this animal. The blue sheep were infected by all three Brucella species including B. suis, which is generally carried by pigs [1, 22]. More importantly, this animal shared Brucella genotypes with human (Bm07, Bm15 and Bm25), yaks (Bm06), and Tibetan gazelle (Bm01), indicating this animal is an important natural repertory for Brucella. Since there is obvious spatial and diet overlap between blue sheep and domestic livestock [23], we suggest that this animal is also an important infection source of Brucella in humans. Additionally, due to high genetic diversity of B. melitensis, we also discuss the geographic distribution features of the strains. Figure 1 showed that 7 counties (locality ID: 2, 3, 5, 8, 9, 10, and 12) were dominated by A branch strains; 3 counties (locality ID: 1, 4, and 6) were dominated by B branch strains; 3 counties (locality ID: 7, 11, and 13) contained only C branch strains; and the E branch genotypes appeared only in 1 county (locality ID: 2). These results showed that the B. melitensis prevalence in the QTP region might have a considerable regional characteristic i.e., there might be genetic variations among different regions. Our results for the first time elucidated the composition of species and genotypes of Brucella samples in the QTP region. We also presented the host- as well as geographic distributions of the species and genotypes. These results may have some implications for the future disease control programmes in QTP. First, since B. melitensis was the predominant species in the study area, special attention should be paid on this species in brucellosis control programs such as livestock vaccination.
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
omp25
int-hyp
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 4
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 3 3 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 1 1 1 1 5
8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 10 10 1 1 1 1 2
2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8 2 8? 2 7 2 7 1 5 1 5 1 2 1 2 1 14
ST
cobQ
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 2 2 4
trpE
f B.s.
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 1 1 1 1 6
dnaK
d B.a.
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 1
gyrB
e
B.m.
glk
0.0002
gap
a B.m.
ID Ma01 Ma02 Ma03 Ma04 Ma05 Ma07 Ma09 M a 11 Ma12 Ma13 Ma14 Ma15 Ma16 Ma17 Ma18 Ma19 Ma20 Ma21 Ma22 Ma23 Ma24 Ma25 Ma26 Ma27 Ma28 Ma29 Ma30 Ma31 Ma32 Ma33 Ma34 Ma35 Ma36 Ma37 Ma38 Ma39 Ma40 Ma41 Ma42 Ma43 Ma44 Ma45 Ma46 Ma47 Ma48 Ma49 Ma50 Ma52 Ma53 Ma54 Ma57 Ma58 Ma59 Ma60 Ma61 Ma62 Ma64 Ma06 Ma55 Ma56 Ma08 Ma51 Ma10 Ma65 Ma63
aroA
Page 6 of 8
Host sheep sheep human human human human human sheep sheep human human human human human human human human human human human human human human human human human human human human human human human human blue sheep Ti b e t a n g a z e l l e sheep sheep sheep sheep sheep sheep sheep sheep sheep sheep sheep yak yak yak yak blue sheep blue sheep blue sheep blue sheep blue sheep blue sheep blue sheep human blue sheep blue sheep human yak yak cattle blue sheep
Fig. 3 Dendrogram based on the MLST genotyping assay showing relationships of the 65 Brucella isolates. ID: serial number for the 65 isolates; ST: MLST genotypes; Host: the hosts from which the bacteria was isolated. (Also see Fig. 2 for additional information)
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Second, the genotypes of Brucella in the QTP region were very different from other regions, but whether the genetic difference may result in different vaccination efficiencies should be elucidated in the further studies. Third, since there were extensive genotype-sharing events between humans and animals, brucellosis surveillance should be regularly executed on both livestock (sheep and yaks) and wild ungulates (blue sheep). It should be noted that, as mentioned above, none of the genotypes found in our study were identical to any of the genotypes in the Brucella2012 database, in order to prevent over-interpreting our results, we did not compare our strains with those from other studies. This might limit our understanding of genetic relationships between the Brucella populations from QTP and from other regions. Moreover, because of imbalance of sample sizes among different counties and among different hosts, in this study, we were not able to do a detail epidemiological investigation. We suggest that the accumulation of more samples will enable us to further elucidate the genetic characteristics of Brucella species in QTP.
Conclusions Our study drew three main conclusions: (i) there were at least three Brucella (B. melitensis, B. abortus and B. suis) species in the east of QTP and B. melitensis was the predominant species in the area studied; (ii) the Brucella population in the QTP region was very different from other regions probably due to the unique geographical characteristics e.g., extremely high altitude in QTP; and (iii) there were extensive genotype-sharing events between humans and animals; sheep, yaks and blue sheep were important zoonotic hosts of brucellosis to humans in the area studied. Additional file Additional file 1: Multilingual abstracts in the six official working languages of the United Nations. (PDF 350 kb) Abbreviations MLST: multi locus sequence typing; MLVA: multi-locus variable-number tandem-repeat analysis; QTP: Qinghai-Tibet Plateau. Competing interests The authors declare that they have no competing interests. Authors’ contributions JYM, HW, BYC and GHL contributed to the design of the study. JYM, BYC and GHL collected the data and wrote the paper. JYM, XFZ, LQX, GYH, HJ, FZ, HYZ, DRP, YMQ and GHL analyzed the data. All authors read and approved the final paper. Acknowledgments This study was supported by the Science and Technology Plan of Qinghai Province (NO. 2013-Z-749) and the Youth Innovation Promotion Association of Chinese Academy of Sciences (NO. 2015352).
Page 7 of 8
Author details 1 Qinghai Institute for Endemic Disease Prevention and Control, Xining 811602, Qinghai, China. 2State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 3Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai, China. Received: 21 October 2015 Accepted: 23 March 2016
References 1. Atluri VL, Xavier MN, de Jong MF, den Hartigh AB, Tsolis RM. Interactions of the human pathogenic Brucella species with their hosts. Annu Rev Microbiol. 2011;65:523–41. 2. Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV. The new global map of human Brucellosis. Lancet Infect Dis. 2006;6:91–9. 3. Whatmore AM, Perrett LL, MacMillan AP. Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol. 2007;7:34. 4. Kansiime C, Rutebemberwa E, Asiimwe BB, Makumbi F, Bazira J, Mugisha A. Annual trends of human brucellosis in pastoralist communities of southwestern Uganda: a retrospective ten-year study. Infec Dis Poverty. 2015;4:39. 5. Ducrotoy MJ, Ammary K, Lbacha HA, Zouagui Z, Mick V, Prevost L, Bryssinckx W, Welburn SC, Benkirane A. Narrative overview of animal and human brucellosis in Morocco: intensification of livestock production as a driver for emergence? Infec Dis Poverty. 2015;4:57. 6. Garin-Bastuji B, Mick V, Le Carrou G, Allix S, Perrett LL, Dawson CE, et al. Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches. Appl Environ Microbiol. 2014; 80(5):1570–9. 7. Le Flèche P, Jacques I, Grayon M, Al Dahouk S, Bouchon P, Denoeud F, et al. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol. 2006;6:9. 8. Chen Y, Zhen Q, Wang Y, Xu J, Sun Y, Li T, et al. Development of an extended multilocus sequence typing for genotyping of Brucella isolates. J Microbiol Methods. 2011;86(2):252–4. 9. Di D, Cui B, Wang H, Zhao H, Piao D, Tian L, et al. Genetic polymorphism characteristics of Brucella canis isolated in China. PLoS One. 2014;9(1):e84862. 10. Wu T. The Qinghai-Tibetan plateau: how high do Tibetans live? High Alt Med Biol. 2001;2:489–99. 11. Wiener G, Han J, Long R. The yak. 2nd ed. Bangkok: RAP publication; 2003. 12. Lang X, Qu H, Bu Z, Yang Y, Sun C, Li X, et al. Seroprevalence of Brucella infection in yaks (Bos grunniens) on the Qinghai-Tibet plateau of China. Trop Anim Health Prod. 2011;43:305–6. 13. Xue H. Analysis on infection and the related factor of brucellosis in high-risk population in Qinghai Province of 2006 to 2010. Taiyuan, Shanxi Province, China: Master thesis of Shanxi Medical University; 2012. 14. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731–9. 15. Chen Y, Ke Y, Wang Y, Yuan X, Zhou X, Jiang H, et al. Changes of predominant species/biovars and sequence types of Brucella isolates, Inner Mongolia. China BMC Infect Dis. 2013;13:514. 16. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421. 17. Marco C, Roberta B. BLAST Parser. BITS Conference. Societa di Bioinformatica Italiana. Bologna. Societa di Bioinformatica Italiana Bologna Italy. 2006;5:28–9. 18. Jiang H, Fan M, Chen J, Mi J, Yu R, Zhao H, et al. MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolates. BMC Microbiol. 2011;11:256. 19. Jiang H, Wang H, Xu L, Hu G, Ma J, Xiao P, et al. MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identification of Brucella suis vaccine strain s2 from cattle in China. PLoS One. 2013;8(10):e76332.
Ma et al. Infectious Diseases of Poverty 2016, 4: http://www.idpjournal.com/content/4/1/
Page 8 of 8
20. Al Dahouk S, Le Flèche P, Nöckler P, Jacques I, Grayon M, Scholz HC, et al. Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Methods. 2007;69:137–45. 21. Shang D, Xiao D, Yin J. Epidemiology and control of brucellosis in China. Vet Microbiol. 2002;90:165–82. 22. Burniston S, Okello AL, Khamlome B, Inthavong P, Gilbert J, Blacksell SD, Allen J, Welburn SC. Cultural drivers and health-seeking behaviours that impact on the transmission of pig-associated zoonoses in Lao People’s Democratic Republic. Infec Dis Poverty. 2015;4:11. 23. Mishra C, Van Wieren SE, Ketner P, Heitkonig IMA, Prins HHT. Competition between domestic livestock and wild bharal Pseudois nayaur in the Indian Trans-Himalaya. J Appl Ecol. 2004;41(2):344–54. doi:10.1186/s40249-016-0123-z Cite this article as: Ma et al.: MLVA and MLST typing of Brucella from Qinghai, China. Infectious Diseases of Poverty 2016 4:.
Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit
