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为探究中国不同地区来源的乳房链球菌（Streptococcus uberis，S.uberis）之间的多样性和遗传进化关系，本研究以部分地区分离到的38株乳房链球菌为研究对象，通过对其最小抑菌浓度（minimun inhibitory concentration，MIC）测定和全基因组框架测序，获取38株菌耐药性、耐药基因及毒力基因等相关信息；通过多位点序列分型技术（multilocus sequence typing，MLST）分析了7个管家基因的等位基因谱、序列型（sequence type，ST）和克隆复合体（clonal complexes，CCs），并构建系统进化树。MIC试验共采用15种抗生素，除头孢噻呋和氟苯尼考外，38株乳房链球菌对其中13种抗生素具有不同程度耐药，且江苏地区的耐药性明显强于新疆地区；耐药和毒力基因比对后发现，仅24株菌携带耐药基因，剩余14株则不携带耐药基因且属新疆地区分离株，与MIC试验结果相符；15种毒力基因中，除cfu、hasA/B和lbp外，余下12种毒力基因的携带率均高达100%。MLST结果表明，38株乳房链球菌共属于17个ST型，其中有15个ST型从未报道，仅supan01 1株菌属于ST-5 CCs。进化树结果揭示，38株分离株之间的亲缘性较远，分布呈地区依赖性，且中国部分地区当前流行的乳房链球菌与西方国家流行的菌株差异较大。本研究丰富了乳房链球菌的MLST分型数据库，为了解中国乳房链球菌遗传特性和分布特点提供了参考依据。 相似文献
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《中国预防兽医学报》2017,(11)
为了解人源、牛源及兔源无乳链球菌(S.agalactiae)的基因型差异及分子分型的总体结构特征,本实验对30株2012年~2015年分离于人、牛及兔的S.agalactiae在cfb基因鉴定的基础上,通过荚膜多糖分子血清分型(CPS)、多位点序列分型(MLST)和脉冲场凝胶电泳(PFGE)进行基因型的确定和分子特征差异性比较。CPS结果显示,牛源和兔源S.agalactiae均为Ia血清型,而人源无乳链球菌包括Ia和III型两种血清型,以Ia血清型为主要血清类型;MLST分型获得8个STs序列型(ST7、ST10、ST19、ST61、ST103、ST199、ST486、ST651)和6个克隆群(CC7、CC10、CC19、CC23、CC61、CC103),其中人源S.agalactiae拥有最多的STs和克隆群分型;PFGE聚类分析可以将其分为18个PFGE带型,且不同动物源性S.agalactiae之间基因分型差异性较大。血清分型、STs序列型、PFGE带型与宿主的来源之间无明显的相关性。不同来源菌株可以同属于血清型Ia,且所有Ia血清型菌株可以分布于不同的STs序列型和PFGE带型中。但分型为Ia/ST651的人源S.agalactiae在MLST的克隆分群中是分型为Ia/ST103牛源S.agalactiae的克隆衍生物,同属克隆群CC103,Ia/ST651只是Ia/ST103单一位点变化的产物,从分子水平的相似性推测二者存在交叉感染的可能性。本研究为奶牛乳房炎的有效预防和治疗提供依据。 相似文献
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《中国动物传染病学报》2019,(5)
从石河子及附近地区规模化牛场死于肺炎的犊牛肺脏中共分离8株多杀性巴氏杆菌(Pasteurella multocida,Pm),为了确定分离菌株的血清型和基因型,分别进行了多重荚膜特异基因PCR检测、脂多糖多重PCR(LPS-m PCR)分型和多位点序列分型(MLST)鉴定和分析。结果显示8株分离菌中,7株为荚膜A型,1株为荚膜B型;LPS-m PCR结果显示其中7株菌为L2,1株为L3基因型;MLST技术将8株细菌中的7株分为ST1型,1株分为ST44型,且两者亲缘关系较近。结果表明8株Pm以荚膜血清A型为主,根据LPS基因特异性将其分为2个基因型,基于管家基因的MLST将8株Pm分为两种ST型。 相似文献
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为了解我国猪肺炎支原体种群结构和进化关系，2018—2019年采集山东、广西、河南、江西、云南、天津、湖南等地猪组织样品1 242份，进行猪肺炎支原体荧光定量PCR和巢式PCR检测，并对测序确认的87份阳性样品，采用adk、rpoB、tpiA 3个管家基因作为分型参考进行多位点序列分型（MLST），并对3个管家基因均扩增成功的阳性样品进行p146基因分型，分析我国猪肺炎支原体种群结构，并结合pubmlst数据库公布的151个序列类型（ST）进行eBURST分析。结果显示：有24份阳性样品的3个管家基因全部扩增且测序成功，共分为10个ST，均为国内外首次报道；10个ST分为3个克隆复合体（CC）和4个独特型，其中ST128占41.7%（10/24），样品来自江苏、河南和广西，为本次检测的主要流行型。结果表明，我国猪肺炎支原体分布较广，具有一定的基因型独特性。本研究为我国猪肺炎支原体的流行病学数据库研究提供了新资料，有助于掌握国内猪肺炎支原体基因型分布情况，进一步完善了猪肺炎支原体流行病学数据库。 相似文献
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多重PCR检测奶牛乳腺炎金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母菌方法的建立与应用 总被引：2，自引：0，他引：2
参照GenBank发表的序列，在金黄色葡萄球菌、无乳链球菌和停乳链球菌16SrRNA与23SrRNA之间的区域设计了3对引物，参照念珠菌和隐球菌的18SrRNA的序列设计1对引物，建立了检测金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母真菌4种乳腺炎主要致病菌的多重PCR方法。参照Skladny的方法制备模拟了细菌感染l临床标本。结果表明：本试验建立的多重PCR方法具有较好的特异性，多重PCR方法检测乳样中的金黄色葡萄球菌的细菌最小浓度为10^4CFU／mL，检测无乳链球菌、停乳链球菌和酵母真菌的细菌最小浓度分别为10^4CFU／mL、10^3CFU／mL和10^3CFU／mL。通过对采自临床型乳腺炎（46个）和隐性乳腺炎（167个）动物共计213个乳样分别用传统细菌学培养法和多重PCR方法进行检测，多重PCR对金黄色葡萄球菌和酵母真菌的检测具有更高的检出率（P〈0．01），但该方法对无乳链球菌和停乳链球菌的检出率与培养法差异不显著（P〉0．05）。 相似文献
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停乳链球菌是引起奶牛乳房炎的主要病原菌之一，由其引发的奶牛乳房炎占总发病率的10%～50%。由于该菌具有传染性病原体和环境性病原体的双重特性，因此，该菌在奶牛乳房炎感染和传播过程中扮演着重要角色。牛源停乳链球菌的检测技术包括传统的生化鉴定和分子生物学技术，分子生物学技术包括PCR技术、16S rRNA基因序列测序、基因芯片、环介导恒温扩增法等，不同的检测方法均有其优缺点。牛源停乳链球菌致病性及致病机制与毒力基因相关，如黏附性基因、酶相关基因、生物膜相关基因等。停乳链球菌对常见的抗菌药耐药严重，且出现多重耐药性，耐药机制主要是停乳链球菌携带耐药基因所致。停乳链球菌疫苗有灭活疫苗、重组亚单位疫苗、蛋白疫苗等，但仍处于研究阶段。本文对牛源停乳链球菌分离鉴定技术、致病性及致病机制、耐药性及耐药机制、疫苗研发等方面进行综述，以期为防控停乳链球菌引起的乳房炎提供指导。 相似文献
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A multilocus sequence typing (MLST) scheme was established and evaluated for Mycoplasma hyopneumoniae, the etiologic agent of enzootic pneumonia in swine with the aim of defining strains. Putative target genes were selected by genome sequence comparisons. Out of 12 housekeeping genes chosen and experimentally validated, the 7 genes efp, metG, pgiB, recA, adk, rpoB, and tpiA were finally used to establish the MLST scheme. Their usefulness was assessed individually and in combination using a set of well-defined field samples and strains of M. hyopneumoniae. A reduction to the three targets showing highest variation (adk, rpoB, and tpiA) was possible resulting in the same number of sequence types as using the seven targets. The established MLST approach was compared with the recently described typing method using the serine-rich repeat motif-encoding region of the p146 gene. There was coherence between the two methods, but MLST resulted in a slightly higher resolution. Farms recognized to be affected by enzootic pneumonia were always associated with a single M. hyopneumoniae clone, which in most cases differed from farm to farm. However, farms in close geographic or operational contact showed identical clones as defined by MLST typing. Population analysis showed that recombination in M. hyopneumoniae occurs and that strains are very diverse with only limited clonality observed. Elaborate classical MLST schemes using multiple targets for M. hyopneumoniae might therefore be of limited value. In contrast, MLST typing of M. hyopneumoniae using the three genes adk, rpoB, and tpiA seems to be sufficient for epidemiological investigations by direct amplification of target genes from lysate of clinical material without prior cultivation. 相似文献
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《Veterinary microbiology》2015,175(1):92-98
Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and the bacterium most frequently isolated from the polymicrobial syndrome known as bovine respiratory disease complex. Recently, M. bovis has emerged as a significant health problem in bison, causing necrotic pharyngitis, pneumonia, dystocia and abortion. Whether isolates from cattle and bison comprise genetically distinct populations is unknown. This study describes the development of a highly discriminatory multilocus sequencing typing (MLST) method for M. bovis and its use to investigate the population structure of the bacterium. Genome sequences from six M. bovis isolates were used for selection of gene targets. Seven of 44 housekeeping genes initially evaluated were selected as targets on the basis of sequence variability and distribution within the genome. For each gene target sequence, four to seven alleles could be distinguished that collectively define 32 sequence types (STs) from a collection of 94 cattle isolates and 42 bison isolates. A phylogeny based on concatenated target gene sequences of each isolate revealed that bison isolates are genetically distinct from strains that infect cattle, suggesting recent disease outbreaks in bison may be due to the emergence of unique genetic variants. No correlation was found between ST and disease presentation or geographic origin. MLST data reported here were used to populate a newly created and publicly available, curated database to which researchers can contribute. The MLST scheme and database provide novel tools for exploring the population structure of M. bovis and tracking the evolution and spread of strains. 相似文献
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Avian pathogenic Escherichia coli (APEC) causes economically significant infections in poultry. The genetic diversity of APEC and phylogenetic relationships within and between APEC and other pathogenic E. coli are not yet well understood. We used multilocus sequence typing (MLST), PCR-based phylogrouping and virulence genotyping to analyse 75 avian E. coli strains, including 55 isolated from outbreaks of colisepticaemia and 20 from healthy chickens. Isolates were collected from 42 commercial layer and broiler chicken farms in Sri Lanka. MLST identified 61 sequence types (ST) with 44 being novel. The most frequent ST, ST48, was represented by only six isolates followed by ST117 with four isolates. Phylogenetic clusters based on MLST sequences were mostly comparable to phylogrouping by PCR and MLST further differentiated phylogroups B1 and D into two subgroups. Genotyping of 16 APEC associated virulence genes found that 27 of the clinical isolates and one isolate from a healthy chicken belonged to highly virulent genotype according to previously established classification schemes. We found that a combination of four genes, ompT, hlyF, iroN and papC, gave a comparable prediction to that of using five and nine genes by other studies. Four STs (ST10, ST48, ST117 and ST2016) contained APEC isolates from this study and human UPEC isolates reported by others, suggesting that these STs are potentially zoonotic. Our results enhanced the understanding of APEC population structure and virulence association. 相似文献
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Daisuke Takamatsu Hiroto Nishino Tomono Ishiji Jun Ishii Makoto Osaki Nahuel Fittipaldi Marcelo Gottschalk Prasit Tharavichitkul Shinji Takai Tsutomu Sekizaki 《Veterinary microbiology》2009,138(1-2):132-139
Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis (srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A–L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection. 相似文献
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This study was aimed at determining the genetic diversity of Campylobacter jejuni from healthy ruminants and poultry, and study by multilocus sequence typing (MLST) their links to human isolates in Spain. MLST analysis of 160 animal isolates generated 45 sequence types (STs, nine of them new to this study), that clustered into 18 clonal complexes (CC) and nine singletons. The 71 isolates from humans generated 28 STs (13 CC plus four singletons). Only 11 STs and nine CCs were shared by humans and animals (particularly from dairy cattle and sheep), mainly corresponding to sporadic cases rather than outbreaks, probably as an adaptation of the general human population to the types commonly circulating in livestock. PCR analysis of the distribution of four virulence-associated genes detected the cdtABC gene cluster in all 160 isolates but with a 700-bp deletion in four of them, and amplified the virB11, cgtB and wlaN genes in 4.7%, 21.3% and 21.9% respectively. A subset of 87 C. jejuni animal isolates analysed using flaA PCR-RFLP, MLST and pulsed-field gel electrophoresis generated 31, 38 and 55 types respectively. The combined typing approach used provided reliable inter-strain relationships, confirming the co-existence of several strains in some farms, but also identifying identical genotypes sampled over a wide temporal span in different environments and hosts. Typing results confirmed a high genetic diversity of C. jejuni in our region and suggested that ruminants are also important sources for human infection. MLST data provided will help to obtain a more comprehensive image of the population structure of C. jejuni and establish reliable source attribution schemes. 相似文献
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Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus 总被引：1，自引：0，他引：1
Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected. 相似文献
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Multilocus-sequence typing (MLST) was used to analyse Streptococcus uberis isolates from a single herd associated with long duration (50-260 days) and rapidly cleared (less than 1 month) bovine intramammary infections to determine whether the bacterial type had any impact on the duration of infection. Most chronic infections (24 of 33) were due to continuous infection of the mammary quarter with the same sequence type, and infections were found to persist for many months. The remaining quarters were re-infected with a different sequence type within a single lactation. No particular sequence type or clonal complex (lineage) was associated with persisting infections, indicating that the outcome of intramammary infections with S. uberis is more likely to be dependent on host factors than on inter-strain differences. Analysis of these strains alongside others obtained from the same herd at a later date revealed the shift in the predominant genotypes with time. 相似文献
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Bibek Ranjan Shome Mani Bhuvana Susweta Das Mitra Natesan Krithiga Rajeswari Shome Dhanikachalam Velu Apala Banerjee Sukhadeo B. Barbuddhe Krishnamshetty Prabhudas Habibar Rahman 《Tropical animal health and production》2012,44(8):1981-1992
Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A–D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India. 相似文献
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Background
Streptococcus equi ssp. equi is the causative agent of ‘Strangles’ in horses. This is a debilitating condition leading to economic loss, yard closures and cancellation of equestrian events. There are multiple genotypes of S. equi ssp. equi which can cause disease, but to date there has been no systematic study of strains which are prevalent in Ireland. This study identified and classified Streptococcus equi ssp. equi strains isolated from within the Irish equine industry.Results
Two hundred veterinary isolates were subjected to SLST (single locus sequence typing) based on an internal sequence from the seM gene of Streptococcus equi ssp equi. Of the 171 samples which successfully gave an amplicon, 162 samples (137 Irish and 24 UK strains) gave robust DNA sequence information. Analysis of the sequences allowed division of the isolates into 19 groups, 13 of which contain at least 2 isolates and 6 groups containing single isolates. There were 19 positions where a DNA SNP (single nucleotide polymorphism) occurs, and one 3 bp insertion. All groups had multiple (2–8) SNPs. Of the SNPs 17 would result in an amino acid change in the encoded protein. Interestingly, the single isolate EI8, which has 6 SNPs, has the three base pair insertion which is not seen in any other isolate, this would result in the insertion of an Ile residue at position 62 in that protein sequence. Comparison of the relevant region in the determined sequences with the UK Streptococcus equi seM MLST database showed that Group B (15 isolates) and Group I (2 isolates), as well as the individual isolates EI3 and EI8, are unique to Ireland, and some groups are most likely of UK origin (Groups F and M), but many more probably passed back and forth between the two countries.Conclusions
The strains occurring in Ireland are not clonal and there is a considerable degree of sequence variation seen in the seM gene. There are two major clades causing infection in Ireland and these strains are also common in the UK. 相似文献20.
为了探明2018年3月江苏省某规模化猪场发病猪的病原,本研究对发病猪进行采样和细菌分离,并对细菌进行了PCR鉴定、多位点序列分型(MLST)及小鼠致病力分析。结果发现:在该场4份发病猪组织中分离到猪链球菌2型(SS2)、猪链球菌9型(SS9)菌株各2株,MLST分析显示分离的SS2菌株属于ST7,SS9菌株属于ST243,SS2、SS9分离菌株的毒力基因表现型分别为mrp^+epf^+sly^+orf2^+sao^+fbps^+gdh^+和mrp^-epf^-sly^-orf2^+sao^-fbps^+gdh^+。动物试验显示其中3株猪链球菌分离菌株对BALB/c小鼠有较强致病力。研究结果为该养殖场猪链球菌感染防控措施的制定提供了重要参考。 相似文献