一种改良的布鲁氏菌病抗体微量凝集检测方法的建立与评价

目的 建立一种微量的布鲁氏菌病抗体凝集检测方法,用于布鲁氏菌病高通量检测。方法 依据我国《布鲁氏菌病诊断标准》(WS269-2007)规定的病人诊断标准,用试管凝集试验做诊断标准。通过优化微量凝集试验抗原浓度,对142份疑似病人血清同时做试管凝集和微量凝集试验,进行效果评价。结果 最优的微量凝集抗原浓度为1:10,建立微量凝集法具有较高的敏感度和特异度,分别为98.9%和92.3%;和常规试管凝集法相比,两种方法符合率为96.5%。常规试管凝集检测得到21份阴性样本,22份可疑样本(1:50),99份阳性样本(>1:100)。微量凝集检测得到30份阴性样本、17份可疑样本(1:50)、95份阳性样本(>1:100)。两种试验方法,结果相同的占70.4%(100/142),经统计学检验差异无统计学意义(χ²=0.8,P>0.05)。结论 建立一种可显色的布鲁氏菌病抗体微量凝集检测方法,可以在96孔V型板上同时检测24份标本,且结果易判读,更适宜基层防疫人员现场检测,最终为疫情处置提供强有力的技术支持。     

2001-2015年锦州市布鲁氏菌病疫情流行病学分析

目的描述2001-2015年锦州市布病疫情流行病学特征和变化趋势,探讨下一步的防控重点。方法用描述流行病学方法对锦州市布病疫情进行分析,对试管凝集试验阳性率与发病率进行Pearson相关性分析。结果 2001-2015年锦州市布病发病率为0.45/10万-16.53/10万。发病时间集中在3-7月,占总数的66.89%。2003-2015年共采集重点人群血清5 904份,阳性率为5.01%。2007-2015年从136份血培养标本中分离出布鲁氏菌63株,其中羊种3型48株、羊种1型12株,羊种变异2株、犬种1株。结论锦州市近年布病疫情呈明显上升趋势,其优势菌株主要是羊种3型。对犬的布鲁氏菌携带情况应予以一定的关注,及时淘汰病犬。     

Influence of brucellosis history on serological diagnosis and evolution of patients with acute brucellosis

Serological diagnosis of human brucellosis is problematic in endemic brucellosis regions and with patients having a history of brucellosis. The aim of this study is to ascertain the serologic and evolutionary behavior of the tests of serum agglutination, Coombs anti-Brucella, immunocapture-agglutination, enzyme-linked immunosorbent assay (ELISA) IgG, IgA, IgM and ELISA-IgG avidity against Brucella lipopolysaccharide (S-LPS), in patients with acute brucellosis based on whether or not a history of brucellosis exists. Titers and seropositivity in all the tests assayed were higher in the patients having brucellosis history (from 90.9% in ELISA-IgM to 100% in ELISA-IgG) than in the patients lacking such history (from 79.3% in ELISA-IgM to 86.2% in Coombs, immunocapture-agglutination, and ELISA-IgG). IgG S-LPS avidity results in patients with brucellosis history were significantly higher (always over 84%) than in patients without brucellosis history (from 48.0% in the initial sera to 81% ten months later) (p<0.001). The titers of antibodies against Brucella in the initial sera and ELISA-IgG avidity against S-LPS may allow distinguishing patients with brucellosis caused by primary infection in the initial stages of the disease from patients seropositive due to prior infections from Brucella.     

Characteristics of B. melitensis versus B. abortus bacteraemias

OBJECTIVE: To determine the epidemiological and the clinical characteristics of bacteremic brucellosis. METHODS: A prospective study, performed in the First Infectious Diseases Clinic of Ankara Numune Education and Research Hospital. All the patients had positive culture result for Brucella spp. RESULTS: Fifty-four acute bacteremic brucellosis cases were included. The majority of patients (76%) were from rural Anatolia. Brucella melitensis serotypes were more common than Brucella abortus (83% versus 17%). Fever and arthralgia were the most common symptoms. The number of patients with back pain and arthralgia was higher in B. abortus infected group (p = 0.014 and p = 0.009). CONCLUSIONS: B. melitensis is the most common subtype of Brucella infection in Turkey. The infections with B. abortus spp. are not less severe than the infections with B. melitensis.     

在新疆地区首次检出犬种布鲁氏菌

犬种布氏菌不仅是犬种布病的病原菌,也能在人体中和其他动物间引起感染。 1966年Carmichael等首次报道,在美国从200例流产的Beagle狗中首先分离到犬种布氏菌。此后,相继在德国、巴西、墨西哥和阿根廷等国都从人和犬中分离到本菌。在国内尚德秋氏等于1984年报道在上海第一医学院实验动物部的犬中分离到两株犬种布氏菌。     

Use of the Brucella IgM and IgG flow assays in the serodiagnosis of human brucellosis in an area endemic for brucellosis

The clinical utility of two complementary tests for brucellosis, the Brucella IgM and IgG flow assays, was evaluated in a hospital in eastern Turkey. The results show that the flow assays are convenient diagnostic tests for use in endemic areas. A positive result in the flow assays was obtained in 91% and 97% of the admission sera from adult and pediatric patients with brucellosis, respectively, and the sensitivity at admission was 100% for culture-confirmed brucellosis. The assay system performed equally well in diagnosing patients at different stages of illness including patients with acute, subacute, or chronic disease and with relapse. The results of the flow assays correlated well with those of a serum agglutination test at a cut-off > or =1:160. The agreement was 92%. Application of the flow assays on serum samples collected during a village survey for brucellosis after an outbreak demonstrated their diagnostic potential as field tests.     

Investigation of the spread of Brucella canis via the U.S. interstate dog trade.

OBJECTIVES: The aim of this study was to illustrate and help address a growing need for regulatory or molecular tools to track and control the spread of canine brucellosis. Our study objectives were to first characterize Brucella canis outbreaks in Wisconsin kennels in the context of the dog trade in the USA, and then to identify a molecular technique that may be useful for strain differentiation of B. canis isolates. METHODS: Wisconsin Veterinary Diagnostic Laboratory (WVDL) B. canis serology data from 1995 to 2005 were reviewed, three canine brucellosis outbreaks in Wisconsin dog kennels were investigated, and eight B. canis isolates recovered from Wisconsin outbreaks and kennels in Missouri and Arkansas and four isolates received from outside sources were subjected to ribotyping, pulsed-field gel electrophoresis (PFGE), outer membrane protein analysis (OMPA), and cellular fatty acid profiling (CFAP). RESULTS: WVDL has received increasing numbers of B. canis positive samples from Wisconsin kennels, and Wisconsin outbreaks are associated with the interstate dog trade. All of the B. canis isolates we examined were genetically homogenous and as such could not be differentiated by ribotyping, PFGE and OMPA. However, dendrogram analysis of CFAP divided the isolates into two groups, indicating that CFAP methyl ester analysis has discriminatory power. CONCLUSIONS: CFAP methyl ester analysis has promise as a tool for epidemiological tracing of B. canis outbreaks and will be useful in comparison studies as isolation of B. canis continues to expand globally.     

布鲁菌病血清DNA定量检测的临床意义

目的：分析布鲁菌病血清DNA定量检测与布鲁菌病临床表现的相关性,从而探讨其对布鲁菌病的诊断价值。方法选择2010年5月-2011年6月内蒙古自治区100例布鲁菌血清凝集试验阳性（玻片凝集试验＋~＋＋＋＋）的患者。采用荧光定量PCR法测定血清布鲁菌DNA含量,分析血清布鲁菌DNA检测结果与临床表现的相关性。结果100例布鲁菌血清凝集试验阳性患者中,23例有明显的临床症状,包括发热（91.3%）、全身关节疼痛（78.3%）、乏力（87.0%）、多汗（65.2%）等全身不适症状,其中的20例血清布鲁菌DNA定量值明显升高[范围（1.30～9.27）×106 copies/ml,平均（2.00±7.87）×106 copies/ml],检测时间仅3 h,灵敏度、特异度和准确度分别达到86.96%、97.40%和95.00%。结论血清布鲁菌DNA定量检测可以为布鲁菌病的诊断提供准确、快速和可靠的实验室确诊依据,用以区分布鲁菌现症感染和既往感染,为布鲁菌病的及时治疗提供客观依据。     

ELISA补体结合试验(COMPELISA)检测布鲁氏菌抗体的研究

我们参考Robertson氏介绍的方法,建立了ELISA补体结合试验(COMPELISA)、并以此方法检查了非布鲁氏菌病人血清120份、布鲁氏菌病人血清100份。结果表明此法敏感性比补体结合反应高得多,按Vecchio氏介绍的评价力法,COMPELISA试验诊断布鲁氏菌病的敏感性为96%,特异性100%。 此外,证实此试验结果的重现性较好。     

Brucellosis: an unusual diagnosis for a seronegative patient with abscesses, osteomyelitis, and ulcerative colitis.

A 16-year-old girl developed multiple subcutaneous abscesses, osteomyelitis, and severe colitis. On the patient's second admission, a single blood culture--and, subsequently, a specimen of pus--yielded Brucella melitensis biovar 1. A second set of serologic tests, including the rose bengal test, the standard tube agglutination test, the CF test, and Coombs' test, were all negative for Brucella on the patient's second admission and 1 month later. However, a lymphocyte proliferation assay with extracted antigen of Brucella was markedly positive. Thus, this case illustrates that patients with B. melitensis infection may have a unique clinical presentation and that the lymphocyte proliferation assay is an important diagnostic tool for patients whose serologic test results are negative but for whom brucellosis is suspected.     

Late-onset herpes simplex virus-associated interstitial pneumonia after allogeneic bone marrow transplantation

We report a unique case of brucellosis transmitted by BMT. An 8-year-old boy with the diagnosis of Fanconi's anemia received an allogeneic BMT from his HLA-identical sibling. Routine culture from the infused marrow suspension grew Brucella abortus on day +4 post BMT. Spiking fevers occurred on days +2 and +16. The first febrile episode responded to broad-spectrum antibiotic therapy. However, the second episode did not. B. abortus was isolated from blood cultures taken during the second febrile episode. The Brucella agglutination titer was negative. Antibiotic therapy with oral doxycycline and i.v. gentamycin was successful with no recurrence of infection during 13 months of follow-up. The donor's blood culture was also positive for B. abortus and Brucella antibodies were detectable at 1:320 titer when he presented with fever and hepatosplenomegaly on day +32. We emphasize the need to consider brucellosis in patients undergoing BMT. We suggest that donor and recipient be evaluated for brucellosis especially in countries where the incidence of this infection is relatively high.     

2020年陕西省一起布鲁氏菌病暴发疫情的调查分析

目的 调查2020年陕西省泾阳县一起家庭布鲁氏菌病(布病)暴发疫情的原因,明确传染源和传播途径,为阻断疫情传播提供科学依据。方法 对报告病例开展布病流行病学调查,并对病例和牲畜进行布病检测和布鲁氏菌分离培养鉴定种型,对结果进行描述和分析。结果 共发现7例确诊病例和6例隐性感染者;确诊病例发病时间为3月18日至5月7日,临床表现以发热为主;经分析,病例发病与食用牛肉无关联(OR=0.48,95%CI:0.08~2.85),与去过首发病例家有关(OR=141.00, 95%CI:17.15~1 159.28);13例病例、2只羊和2只公犬布鲁氏菌抗体检测阳性,血清抗体滴度从1∶100(++)到1∶800(++++)以上;从2例病例、2只羊和1只公犬血液中共分离到5株布鲁氏菌,均为羊种3型,MLVA-16基因型均为(1-5-3-13-2-2-3-2-4-41-8-6-4-3-4-5)。结论 该起布病家庭暴发疫情的原因为公犬叼食了布病阳性羊只的流产物而感染布病,继而人通过与病犬直接接触以及接触被病犬污染的水等生活物质而发病。     

Changes in IgM and IgG antibody concentrations in brucellosis over time: importance for diagnosis and follow-up

Changes in concentrations of IgM and IgG antibodies to Brucella were monitored for at least 13 mo by an enzyme-linked immunosorbent assay (ELISA) in 52 patients with culture-positive brucellosis. Two main patterns were observed. After an initial peak, 29 patients (56%) had a steady drop in their IgG levels, whereas 23 (44%) had more than one peak over time. All patients with a chronic form of brucellosis or a relapse were in the second group. In most cases, Brucella antibodies, although falling to low levels, remained measurable. Cutoff levels for IgM and IgG were calculated after considering serum antibody concentrations in people who had recovered from an infection. A separate normal range was established for occupationally exposed workers. On admission, sera from all patients contained Brucella antibody levels greater than established cutoff levels. Our results show that ELISA is an excellent method for diagnosis and follow-up of brucellosis.     

40株地方犬种布氏菌的鉴定

我们从5个地区犬体分离40株犬种布氏菌(B.canis),在常规鉴定试验中发现,40株犬种菌有13株不被碱性复红抑制。这些菌种多数与粗糙型血清产生较高的凝集效价;所有菌种能被犬种噬菌体R/C株裂解,但对其它噬菌体不敏感,其中309号菌株为溶源菌,从中分离到噬菌体,命名Bj-309株,它能裂解所有的犬种布氏菌,但不裂解其它种布氏菌,用DNA中G C mol％法,检查了9株菌,其值为56.6～58.4,与标准犬种菌基本一致。电镜观察形态,符合于布氏菌的特征。扫描电镜见有不规则凹陷,超微切片除见有明显的脆膜和肽聚糖层外,还常见乳头状突起。     

Brucellosis--cause of abortion in sheep and its public health significance

Present study was undertaken to determine the association of brucellosis with abortions occurring naturally in sheep at an organized local sheep breeding farm. A total of 15 strains of Brucella melitensis biovar I were isolated from the abortion material. Serologically the aborted ewes were positive for brucellosis by one or more tests. During acute infection (abortion), standard tube agglutination test (SAT) detected more positive reactors (70.7%) while counter immunoelectrophoresis (CIE) detected more positive reactors (33.9%) in chronic infection (in-contact and apparently healthy sheep). Personnel handling the abortion material at the farm were found positive clinically as well as serologically for brucellosis. These observations suggest the zoonotic importance of brucellosis.     

The Antibody Response to Brucella: Immunoglobulin Response Measured by Enzyme-linked lmmunosorbent Assay and Conventional Tests

Abstract: The antibody response to Brucella: immunoglobulin response measured by enzymelinked immunosorbent assay and conventional tests. G. L. Gilbert and L. A. Hawes, Aust. N.Z. J. Med ., 1981, 11, pp 40–45.
An enzyme-linked immunosorbent assay was adapted to measure total and individual classes of brucella antibody. The results were compared with those of conventional tests for brucella antibody on the sera of a number of healthy seropositive abattoir workers and several patients with either acute or suspected chronic brucellosis. IgG was the class of brucella specific immunoglobulin most commonly detected in all groups. IgM was present in the sera of 40% of seropositive abattoir workers, all but one of the patients with recent acute brucellosis or seroconversion and none of those with suspected chronic brucellosis. Many of the abattoir workers' sera which contained brucella specific IgM gave negative results in the direct agglutination test. The presence of brucella specific lgM in the sera of these men was, in most cases, associated with no past history of acute brucellosis and a relatively short period of employment in the abattoir. It is suggested that the presence of brucella specific IgM in the serum of a person occupationally exposed to 6. abortus, probably indicates a relatively recent primary infection, either symptomatic or sub clinical and has no prognostic significance. Repeated or prolonged exposure is associated with IgG brucella antibodies, often in high titre, irrespective of symptoms. It was not possible, on the basis of any serological tests performed in this study to distinguish healthy people exposed to brucella from those with symptoms consistent with chronic brucellosis     

双抗原夹心酶联免疫试验检测急性布鲁杆菌病患者抗体真实性观察

目的观察双抗原夹心酶联免疫试验(DAgS-ELISA)检测急性布鲁杆菌病(布病)患者的真实性,即灵敏度和特异度。方法以试管凝集试验(SAT)法为金标准,用DAgS-ELISA与虎红平板凝集试验(RBPT)、补体结合试验(CFT)以及间接酶联免疫试验(I-ELISA)对同一布病患者人群和对照组人群血清标本进行检测比较。结果真实性比较,与金标准SAT的符合率分别为:DAgS-ELISA 96.49%,RBPT 94.74%,I-ELISA 85.09%,CFT 72.81%;诊断效能比较,阳性和阴性预测值DAgS-ELISA分别为93.48%、98.53%,RBPT分别为88.00%、100%,CFT分别为100%、69.30%,I-ELISA分别为78.72%、89.55%。结论DAgS-ELISA检测急性布病患者的敏感性和特异性优于其他方法。     

Simultaneous detection of the genus Brucella by combinatorial PCR

We have developed a combinatorial polymerase chain reaction (PCR) procedure to identify four major species of the genus Brucella simultaneously. Four pairs of primers targeting the genes encoding a cell surface protein (BCSP31) and outer membrane proteins (omp2b, omp2a and omp31) were prepared. PCR using these primers gave rise to specific patterns of amplification for each Brucella spp. examined in this study. B. abortus could be identified when fragments of BCSP31 and omp2b/2a were amplified by B. abortus-specific primers. B. melitensis could be identified by the amplification of fragments of BCSP31, omp2b/2a and omp31 using pair of primers B4/B5, JRF/JPR-ab and omp31. Identification of B. canis could be achieved when the amplicons of omp2b/2a were detected by B. canis-specific primers, as could the identification of BCSP31 and omp31. If specific amplifications occurred using all pairs of primers, the strain was identified as B. suis. Combinatorial PCR reported here thus appeared to be an ideal method of identifying Brucella spp., the causative pathogen of human brucellosis.     

内蒙古羊种布鲁氏菌传播模式调查研究

目的 调查内蒙古羊种布鲁氏菌的传播模式和流行规律。方法 采用AMOS-PCR对60株布鲁氏菌的种型进行鉴定,用Hunter-Gaston Diversity Index(HGDI)评价菌株的遗传多态性特征,采用MLVA-16分型方法对菌株进行基因分型,确定菌株的亲缘关系。结果 60株试验菌全部为羊种布鲁氏菌。MLVA-16对菌株具有极高的分辨力,多态性指数为0.981;Panel 1,Panel 2A和Panel 2B的多态性指数分别为0.264,0.345和0.980;Panel 2B中Bru16位点的多态性指数最高,多态性指数为0.835。60株羊种布鲁氏菌聚为5大类37个基因型,其中15个共享基因型包括38株羊种布鲁氏菌,聚类率为63.3%(38/60),提示病例多为有流行病学关联的暴发流行;另外22株菌表现为独特基因型表明菌株无明显的流行病学相关性。共享基因型GT5包括2株分别分离自羊和骆驼的菌株且有相同的MLVA-16基因型,提示布鲁氏菌在羊和骆驼中循环传播;3个共享基因型(GT11、GT17和GT23)分别包含来自人和羊的菌株并呈现完全相同的MLVA-16基因型,表明羊是人间布病的传染源。GT35由3株分离自羊脾的菌株构成且共享相同的基因型,提示布病在羊中呈暴发流行。类群E由12个来自不同宿主(羊,牛,野生骆驼和人)的菌株构成,共享相同或相似的MLVA-16基因型,揭示了内蒙古羊种布鲁氏菌潜在的传播模式。结论 疫羊是主要传染源,野生动物(骆驼)是贮藏宿主。羊种布鲁氏菌在羊牛(骆驼)和骆驼(羊牛)中相互循环传播,最后传染给人是内蒙古羊种布鲁氏菌的潜在传播模式。     

牛布鲁氏菌病诊断中应用EDTA试验的比较及评价

本文报告了诊断牛布鲁氏菌病一种新的EDTA改良试验、作者用此法对不同来源的牛血清进行检测,结果表明EDTA试验明显地降低了326份健康牛血清中非特异性凝集滴度、但对布鲁氏菌感染的10份病牛血清凝集滴度没有大的改变。不论SAT还是EDTA试验结果完全一致,而且SAT和CFT结果也相符,在SAT单项诊断中可疑和阳性的139头牛中有98头(70.5%)用EDTA试验证实为假阳性反应试验中还将血清中各类Ig进行EDTA敏感性检查,发现对EDTA不稳定的凝集素主要是IgM。本试验操作简便,能提高牛布鲁氏菌病正确诊断率。