16例疑似布氏杆菌的检测分析

中国流行的主要是羊、牛、猪3种布氏杆菌,其中以羊布氏杆菌病最为多见.人类与病畜接触或饮用病畜肉、乳与乳制品及经呼吸道吸入被病菌污染的环境空气可引起感染,表现为反复波浪式热型,逐渐转为慢性.2011年1-7月在住院患者中共检出16例疑似布氏杆菌病例,笔者对此做以下分析.     

布氏杆菌病疫苗的应用和研究现状

布氏杆菌病是由布氏杆菌引起的一种重要的人兽共患病。布氏杆菌具有宿主广泛、传染性强以及感染后根治困难等特点,对畜牧业和人类健康均构成严重威胁,疫苗免疫是预防和控制布氏杆菌病的主要措施。迄今国内外已有多个弱毒活疫苗在使用,但均存在一定的缺陷,因此研究更理想的疫苗一直是控制布氏杆菌病的重点。目前除了常规诱变筛选新的弱毒株外,人们正通过基因工程技术构建重组弱毒疫苗、DNA疫苗以及亚单位疫苗。本文简述了布氏杆菌病疫苗的应用及新型疫苗的研究现状。     

布氏杆菌病12例临床特点与分析

目的 分析布氏杆菌病的临床特点,以便于及时诊断与治疗.方法 回顾性分析大连市第六人民医院自2010年1月至2011年12月收治的12例布氏杆菌病病例.结果 布氏杆菌病一年四季均可发病,但多发生于春、夏、秋季,传播途径复杂,有明显的职业特点.临床表现不典型,病程初期易误诊.利福平联合多西环素治疗有效,症状重者加用喹诺酮药物疗效肯定.结论 掌握布氏杆菌临床特点及流行病学特征,可提高诊断率,合理规范抗菌治疗.     

鹅源鸭疫里默氏杆菌的分离、鉴定与生物学特性研究

【目的】从疑似鸭疫里默氏杆菌病的病死雏鹅分离病原菌进行鉴定。【方法】根据细菌培养特性、生化特性、动物试验、血清型鉴定及分子生物学特性对分离菌株进行鉴定。【结果】分离菌株为革兰氏阴性菌,不发酵糖类和醇类,尿素酶试验和氧化酶还原试验为阳性,致病,不同分离株的16S rRNA基因经多重序列比对分析,结果显示鹅源分离株与鸭源鸭疫里默氏杆菌处于同一进化支上,与鸡源鸭疫里默氏杆菌进化关系稍远,血清型鉴定为1型。【结论】分离菌株为血清1型的鸭疫里默氏杆菌,对鸭和鹅均有高致病性,自家疫苗能够较好地保护雏鹅。     

用MLVA技术和多重PCR对犬种布氏菌基因分型

目的：对犬种布氏菌的遗传关系进行不同分子分型方法的对比研究,为犬布病分子流行病溯源提供有效方法。方法：采用多重PCR和多位点可变数量串联重复序列分析（MLVA）方法对24株犬种布氏菌的遗传关系进行比较研究。结果：多重PCR只鉴定出1株犬种布氏菌,其余23株均鉴定为猪种鲁氏菌,但不能鉴定型别;MLVA方法对已鉴定为猪种的布氏菌仍可再细分为型,87%（20/23）为猪3型,13%（3/20）为猪1型。结论：MLVA可以对布氏菌种（生物型）进行基因分型鉴定,可以作为传统表型鉴定方法的补充。     

牛源多杀性巴氏杆菌的分离与初步鉴定

【目的】本研究旨在对引起犊牛呼吸道综合征的多杀性巴氏杆菌进行分离鉴定,分析其亲缘关系和毒力基因的分布情况。【方法】收集2017年8月至2018年4月疑似患有犊牛呼吸道综合征的病牛鼻拭子进行细菌分离培养,对菌落形态和染色疑似巴氏杆菌的菌株进行16S rRNA测序和血清型鉴定,选择巴氏杆菌7类23种毒力基因,筛查临床分离株的毒力基因的分布。【结果】从8个省份的237份病料中分离出31株多杀性巴氏杆菌,分离率为13.1%。16S rRNA测序分析表明31株A型多杀性巴氏杆菌属于同一亚群,其序列同源性与中国分离株HB01以及国外分离株USDA-ARS-USMARC-60712、USDA-ARS-USMARC-60214、ATCC 43137以及36950亲缘关系较近。对分离出的31株A型多杀性巴氏杆菌的7类共23种毒力基因鉴定,结果显示31株多杀性巴氏杆菌所携带的毒力因子大多分布在17–19个,且集中度较高。【结论】A型多杀性巴氏杆菌为犊牛呼吸道综合症的主要流行血清型,通过对多杀性巴氏杆菌的临床分离株进化树和毒力基因分析,内蒙古、黑龙江、新疆、山西以及河北的7株分离株进化来源于同一分支,且均缺失毒力基因tadD和hgbA及携带毒力基因hsf-1,提示着其亲缘关系可能与其携带的特定毒力基因存在一定相关性。该研究为犊牛呼吸道综合征的病原学调查和多杀性巴氏杆菌流行病学调查提供了参考数据。     

204例布氏杆菌病实验室检测结果分析

目的通过实验室检测,发现布氏杆菌感染者,掌握疫情动态,为预测布氏杆菌病流行趋势、制订防治对策提供依据。方法根据布氏杆菌病实验室诊断标准,利用虎红平板凝集试验(RBPT)和试管凝集试验(SAT)相结合的试验方法,对布氏杆菌病疑似病例和有接触史等的人员进行布氏杆菌抗体检测。结果在470例监测对象中,布氏杆菌抗体阳性病例204例,阳性率43.4%。在阳性病例中,男女性别比为1.62∶1;40~69岁年龄组占75.49%;畜牧养殖人员占40.69%,屠宰作业者占32.35%。结论应加强对养殖业和畜牧业人员的预防控制工作,提高其自我防护意识。     

自病人气管灌洗物中分离出一株嗜肺性军团杆菌

本文报道了用自制的蓝藻军团杆菌选择性培养基从病人气管灌洗物中分离到一株嗜肺性军团杆菌(Legionella pneumophila)并与BCYE及硝酸铁琼脂平板等培养基作了比较,发现该菌在蓝藻军团杆菌选择性培养基上生长最好。生化、动物试验;细菌学、血清学的鉴定和诊断及临床治疗效果观察均与军团病杆菌(LDB)的鉴定标准相符合,证明经分离得的是一株嗜肺性军团杆菌血清6型菌株。     

带有氯霉素抗性基因标记的重组布氏杆菌S2的构建及其生物学特性

无法区分免疫接种和自然感染是限制布氏杆菌疫苗应用的主要原因。本研究通过基因同源重组技术,用氯霉素抗性基因(Cmr)替代布氏杆菌弱毒S2株的WbkC基因,筛选获得重组布氏杆菌rS2-WbkC株。试验发现,重组菌rS2-WbkC由原先的光滑型转变成粗糙型。rS2-WbkC在TSA培养基上传25代后仍能稳定表达氯霉素抗性蛋白。小鼠动物试验模型表明,rS2-WbkC与S2有相似的保护性,但rS2比S2具有更高的安全性。rS2-WbkC免疫小鼠后,其抗血清可通过平板凝集试验与S2免疫的血清相区分。本研究为布氏杆菌标记疫苗的研制提供了技术平台。     

ARDRA联合RAPD对不动杆菌基因型鉴定的研究

收集多重耐药的不动杆菌10株,以标准参照株作对照,采用扩增核糖体DNA限制性酶切(ARDRA)DNA指纹技术联合随机扩增多态性DNA(RAPD)技术对其基因亚型进行分析;以非加权组间平均法(UPG-MA)进行聚类分析。该法可以有效地鉴定不动杆菌基因亚型;并从10株不动杆菌中鉴定出1株琼氏不动杆菌及9株鲍曼不动杆菌。ARDRA联合RAPD基因指纹分型技术有良好的互补性,可准确鉴定不动杆菌基因型。     

Isolation of Brucella melitensis biovar 3 from a chamois (Rupicapra rupicapra) in the southern French Alps

Systemic brucellosis caused by Brucella melitensis biovar 3 was identified in a chamois (Rupicapra rupicapra) near the Parc National des Ecrins in the southern French Alps (France). Clinical signs included orchiepididymitis, polyarthritis, blindness and various neurological signs; necropsy findings included numerous calcified foci in testis, epididymis, kidney, subcutaneous connective tissue and brain. Brucella sp. were identified in brain by indirect immunofluorescence and B. melitensis biovar 3 was isolated from testis, kidney, eye, lung and joints. This report describes the first case of brucellosis and Brucella sp. isolation in chamois in France and the first case of B. melitensis isolation in this host species.     

Complete Genome Sequence of Brucella melitensis 133, an Isolate of Biovar 1 of Sequence Type 32

Brucellosis is highly epidemic in China. Of the six classical species, Brucella melitensis and biovar 1 are the most represented species and biovar that cause human brucellosis in China. Here, we report the genome sequence of Brucella melitensis strain 133, a strain of biovar 1 of sequence type 32.     

Experimental Brucella suis biovar 4 infection in a moose

A moose (Alces alces gigas) was inoculated with Brucella suis biovar 4 to better understand the effects of brucellosis in this species. Serum antibody titers increased rapidly and peaked within 21 to 56 days. Fever, leukocytosis, recumbency, anorexia and depression were observed starting 42 days post inoculation. Brucella suis biovar 4 was isolated from blood, lymph nodes, liver and spleen.     

Genetic Polymorphism Characteristics of Brucella canis Isolated in China

In China, brucellosis is an endemic disease typically caused by Brucella melitensis infection (biovars 1 and 3). Brucella canis infection in dogs has not traditionally recognized as a major problem. In recent years however, brucellosis resulting from Brucella canis infection has also been reported, suggesting that infections from this species may be increasing. Data concerning the epidemiology of brucellosis resulting from Brucella canis infection is limited. Therefore, the purpose of this study was to assess the diversity among Chinese Brucella canis strains for epidemiological purposes. First, we employed a 16-marker VNTR assay (Brucella MLVA-16) to assess the diversity and epidemiological relationship of 29 Brucella canis isolates from diverse locations throughout China with 38 isolates from other countries. MLVA-16 analysis separated the 67 Brucella canis isolates into 57 genotypes that grouped into five clusters with genetic similarity coefficients ranging from 67.73 to 100%. Moreover, this analysis revealed a new genotype (2-3-9-11-3-1-5-1:118), which was present in two isolates recovered from Guangxi in 1986 and 1987. Second, multiplex PCR and sequencing analysis were used to determine whether the 29 Chinese Brucella canis isolates had the characteristic BMEI1435 gene deletion. Only two isolates had this deletion. Third, amplification of the omp25 gene revealed that 26 isolates from China had a T545C mutation. Collectively, this study reveals that considerable diversity exists among Brucella canis isolates in China and provides resources for studying the genetic variation and microevolution of Brucella.     

Complete Genome Sequence of Brucella abortus 134, a Biovar 1 Strain Isolated from Human

Brucella abortus is one of the common pathogens causing brucellosis in China. Here, we report the genome sequence of B. abortus strain 134, a strain isolated from a human patient and belonging to biovar 1, the most highly represented biovar among B. abortus strains in China.     

Intraspecies biodiversity of the genetically homologous species Brucella microti

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk, leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a, and omp2b gene sequences and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb, whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by phage R/C. B. microti exhibited high metabolic activities similar to those of closely related soil organisms, such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be a matter of gene regulation rather than a matter of gene configuration.     

Evaluation of Brucella MLVA typing for human brucellosis

Human brucellosis is still the most common bacterial zoonosis worldwide. Neither well-known molecular tools nor the classical biotyping methods are satisfactory for subtyping of Brucella spp. Loci containing Variable Number of Tandem Repeats (VNTRs) have recently proved their usefulness in typing strains from animal origin despite the high genetic homogeneity within the genus Brucella (DNA-DNA homology >90%). The aim of this study was to evaluate MLVA (Multiple Locus VNTR Analysis) for diagnostic and epidemiological use in human brucellosis. One hundred and twenty-eight B. melitensis isolates of all three biovars were typed using eight minisatellite (panel 1) and eight microsatellite (panel 2) markers. One hundred and ten different genotypes were identified. The MLVA clustering pattern correlated with the geographic origin of the strains. Brucella strains isolated from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. Fuchsin sensitive B. melitensis strains were found in closely related clusters giving evidence for an association between VNTRs and some phenotypic characteristics. However, the validity of biovars established by classical microbiological methods could not be confirmed by MLVA clustering. The original data can be queried on the genotyping web page at http://bacterial-genotyping.igmors.u-psud.fr. The MLVA assay is rapid, highly discriminatory, and reproducible within human Brucella isolates. MLVA can significantly contribute to epidemiological trace-back analysis of Brucella infections and may advance surveillance and control of human brucellosis.     

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

In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the ‘East Mediterranean’ group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the ‘Americas’ group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.     

Human Brucellosis in Maghreb: Existence of a Lineage Related to Socio-Historical Connections with Europe

Despite control/eradication programs, brucellosis, major worldwide zoonosis due to the Brucella genus, is endemic in Northern Africa and remains a major public health problem in the Maghreb region (Algeria/Morocco/Tunisia). Brucella melitensis biovar 3 is mostly involved in human infections and infects mainly small ruminants. Human and animal brucellosis occurrence in the Maghreb seems still underestimated and its epidemiological situation remains hazy. This study summarizes official data, regarding Brucella melitensis infections in Algeria, from 1989 to 2012, with the purpose to provide appropriate insights concerning the epidemiological situation of human and small ruminant brucellosis in Maghreb. Algeria and Europe are closely linked for historical and economical reasons. These historical connections raise the question of their possible impact on the genetic variability of Brucella strains circulating in the Maghreb. Other purpose of this study was to assess the genetic diversity among Maghreb B. melitensis biovar 3 strains, and to investigate their possible epidemiological relationship with European strains, especially with French strains. A total of 90 B. melitensis biovar 3 Maghreb strains isolated over a 25 year-period (1989–2014), mainly from humans, were analysed by MLVA-16. The obtained results were compared with genotypes of European B. melitensis biovar 3 strains. Molecular assays showed that Algerian strains were mainly distributed into two distinct clusters, one Algerian cluster related to European sub-cluster. These results led to suggest the existence of a lineage resulting from socio-historical connections between Algeria and Europe that might have evolved distinctly from the Maghreb autochthonous group. This study provides insights regarding the epidemiological situation of human brucellosis in the Maghreb and is the first molecular investigation regarding B. melitensis biovar 3 strains circulating in the Maghreb.     

Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus

A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals such as seals, porpoises, dolphins and a minke whale. These strains were identified as brucellae by conventional typing tests. However, their overall characteristics were not assimilable to those of any of the six currently recognized Brucella species and it was suggested that they comprise a new nomen species to be called Brucella maris. In the present study we analysed DNA polymorphism at the omp2 locus of 33 marine mammal Brucella strains isolated from seals, dolphins, porpoises and an otter. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing showed that strains isolated from dolphins and porpoises carry two omp2b gene copies instead of one omp2a and one omp2b gene copy or two similar omp2a gene copies reported in the currently recognized species. This observation was also recently made for a minke whale Brucella isolate. The otter and all seal isolates except one were shown to carry one omp2a and one omp2b gene copy as encountered in isolates from terrestrial mammals. By PCR-RFLP of the omp2b gene, a specific marker was detected grouping the marine mammal Brucella isolates. Although marine mammal Brucella isolates may represent a separate group from terrestrial mammal isolates based on omp2b sequence constructed phylogenetic trees, the divergence found between their omp2b and also between their omp2a nucleotide sequences indicates that they form a more heterogeneous group than isolates from terrestrial mammals. Therefore, grouping the marine mammal Brucella isolates into one species Brucella maris seems inappropriate unless the currently recognized Brucella species are grouped. With respect to the current classification of brucellae according to the preferential host, brucellae isolated from such diverse marine mammal species as seals and dolphins could actually comprise more than one species, and at least two new species, B. pinnipediae and B. cetaceae, could be compatible with the classical criteria of host preferentialism and DNA polymorphism at their omp2 locus.