基于比较基因组学方法的世界范围的犬布鲁氏菌系统发育群研究 *

犬布鲁氏菌病是一种世界范围内导致家畜流产和人波状热的人畜共患病, 最近随着相关宿主动物如犬类饲养数量的增多,家庭儿童的感染案例报道屡见不鲜,所以研究犬布鲁氏菌具有重要公共卫生意义.使用了全球的91个菌株,利用贝叶斯方法对菌株分群,基于core-SNPs和分子钟模型构建进化树进行种群结构和时空分布分析,使用COG功能聚类进行功能差异研究.发现的4个系统发育群(PG)与它们的地理来源显著相关,PG1~3中,存在亚洲到非洲及欧美间的传播;PG4定殖于北美,且耐药基因逐渐缺失.功能基因在4个PG中的存在/缺失谱不同,PG3功能最完整,其他PG各具独特的功能基因缺失,特别是PG2大多缺失ABC型转运系统组分.掌握犬布鲁氏菌在全球的分布和传播,理解其基因组变异有助于开发新的诊断和疫苗靶点,以对抗由犬布鲁氏菌引起的人类流行病.     

基于比较基因组学方法的世界范围的犬布鲁氏菌系统发育群研究

犬布鲁氏菌病是一种世界范围内导致家畜流产和人波状热的人畜共患病,最近随着相关宿主动物如犬类饲养数量的增多,家庭儿童的感染案例报道屡见不鲜,所以研究犬布鲁氏菌具有重要公共卫生意义。使用了全球的91个菌株,利用贝叶斯方法对菌株分群,基于core-SNPs和分子钟模型构建进化树进行种群结构和时空分布分析,使用COG功能聚类进行功能差异研究。发现的4个系统发育群(PG)与它们的地理来源显著相关,PG1～3中,存在亚洲到非洲及欧美间的传播; PG4定殖于北美,且耐药基因逐渐缺失。功能基因在4个PG中的存在/缺失谱不同,PG3功能最完整,其他PG各具独特的功能基因缺失,特别是PG2大多缺失ABC型转运系统组分。掌握犬布鲁氏菌在全球的分布和传播,理解其基因组变异有助于开发新的诊断和疫苗靶点,以对抗由犬布鲁氏菌引起的人类流行病。     

蛋白质组学技术在布鲁氏菌病研究中的应用及发展

布鲁氏菌病是由布鲁氏菌引起的重大人畜共患病之一,给我国养殖业发展和公共安全带来严重危害。有效控制和逐步消灭布鲁氏菌病对于公共卫生安全和养殖业的发展具有重要意义。然而,由于布鲁氏菌为胞内寄生菌且结构复杂,其致病机制和相关毒力因子仍不十分清楚;加之我国现有的布鲁氏菌疫苗均为光滑型疫苗,其诱导产生的抗体与自然感染在临床诊断上存在着干扰,给种群净化带来严重困难。虽然已有许多研究通过多种技术尝试解决上述问题,但进展多较为缓慢。蛋白质组学作为研究蛋白质组成和变化规律的新兴学科,随着其研究手段的逐步发展和完善,通过蛋白质组学的手段揭示布鲁氏菌的致病机理、免疫机制等的研究逐渐增多,对于解决布鲁氏菌带来的上述问题提供了崭新的思路。本文结合实验室自身研究,主要从蛋白质组学对布鲁氏菌特异性蛋白的挖掘和对鉴别诊断的意义等方面做一简要阐述。     

宠物犬和猫流感病毒感染

近十年来犬和猫流感病毒感染报道迅速增多,不仅威胁到犬和猫的健康,也对公共卫生造成了影响。自2004年首次发生H3N8亚型流感病毒感染赛犬事件以来,犬流感一直在美国的赛犬和宠物犬中流行。在韩国和我国南方的犬群中出现了因H3N2亚型禽流感感染引起的肺炎病例。亚洲和欧洲均报道了猫H5N1亚型高致病性禽流感致死性感染病例,还通过实验研究发现H5N1亚型流感病毒可在猫与猫之间水平传播。这些现象预示着流感病毒进一步获得了感染哺乳动物的能力,其公共卫生意义需引起关注。为此,本文对犬和猫流感病毒感染的流行病学、临床症状、发病机制、诊断和防控措施进行了综述。     

犬种布鲁氏菌的遗传多样性分析

【背景】犬种布鲁氏菌是犬种布病的病原菌,主要导致犬流产和繁殖障碍。虽然犬种布鲁氏菌感染人群的病例极为少见,但是犬种布鲁氏菌对人的安全风险仍存在争议。目前,我国犬种布病的流行病学特征及犬种布鲁氏菌的遗传多样性的研究相对缺乏。开展犬种布病的流行特征及遗传多样性调查对加强犬种布病的监测防控具有重要意义。【目的】对犬种布病的流行病学特征和犬种布鲁氏菌的遗传多态性进行调查,为犬种布病的防控提供参考。【方法】采用常规鉴定方法和BCSS-PCR对63株试验菌株进行鉴定。采用HGDI (Hunter and Gaston diversity index)多态性指数调查犬种布鲁氏菌的遗传多态性,用MLVA方法基于BioNumerics5.0软件对菌株进行聚类分析,揭示犬种布病的流行病学特点。此外,基于MLVA-11采用goeBURST软件构建犬种布鲁氏菌的最小生成树(Minimum spanning tree,MST),阐述我国犬种布鲁氏菌的地理起源特征。【结果】常规鉴定方法和BCSS-PCR扩增结果显示63株试验菌株全部为犬种布鲁氏菌。BCSS-PCR与常规鉴定方法的符合率为100%,BCSS-PCR的分析敏感性为10-3 (即50 pg/μL犬种布鲁氏菌DNA)。我国犬种布鲁氏菌具有较高的遗传多样性,基于HGDI分析表明Panel 2B的5个位点具有较高的变异度,等位基因型由高到底依次为bruce09(11) bruce07(8)bruce16(7)bruce04(6)bruce30(5)。MLVA聚类分析表明北京地区出现了3次较小规模的犬种布病暴发流行,其余地区均为零星散发。我国犬种布鲁氏菌可分为5个地理集群,以MLVA-11基因26型克隆群为主导种群,该种群与来自美国、希腊、加拿大、法国、罗马尼亚和韩国等国家的菌株具有共同的地理起源,其余4个种群为中国特有。【结论】我国犬种布鲁氏菌呈现高度的遗传多样性并有广泛的地理来源,表现为输入性和中国特有血统共存的起源进化特征。     

运用免疫蛋白质组学方法筛选羊种布鲁氏菌免疫反应性蛋白

布鲁氏菌是布鲁氏菌病的病原体,能引起人类慢性感染,造成家畜流产和不孕.在不同种布鲁氏菌中,羊种布鲁氏菌毒力最强,在中国布鲁氏菌病流行中占主导地位.目前,还没有安全的用于预防人类布鲁氏菌感染的疫苗.用于预防家畜布鲁氏菌感染的弱毒活疫苗产生的抗体能够干扰对免疫动物感染布鲁氏菌野毒的诊断,从而对根除布鲁氏菌病的计划实施有负面影响.然而,羊种布鲁氏菌免疫蛋白质谱目前还不完善.为了研制更安全、有效且能区分野毒感染的疫苗,本研究应用免疫蛋白质组学技术筛选羊种布鲁氏菌疫苗株M5的免疫反应性蛋白.经二维电泳结合免疫杂交技术筛选到88个具有免疫反应性的蛋白质点,后经质谱鉴定其归属于61个蛋白,包括许多新的免疫反应性蛋白,如延伸因子G,F0F1ATP合成酶亚单位？？,OMP1.本研究为布鲁氏菌病疫苗研制提供了许多免疫反应性候选蛋白.     

牛布鲁氏菌病原快速检测技术研究

由布鲁氏菌所引起的布鲁氏菌病,主要特征为发热、流产,是世界范围之内人畜共患慢性细菌传染病,对多种动物的健康带来了严重的影响,同时也影响着人们的生命健康和生产生活。布鲁氏菌能够感染多种家畜、野生动物,也可以感染人类,给国家、养殖户以及生产者带来了严重的损失,还会引起一系列公共卫生的问题。基于此,研究布鲁氏菌检查办法,能够为该病的预防和治疗提供针对性的解决信息。本研究主要阐述了免疫学ELASA和PCR等布鲁氏菌检测技术,这些快速检测技术均能够准确的检测出布鲁氏菌。     

布鲁氏菌感染实验室诊断研究进展

布鲁氏菌（Brucella,简称布氏菌）是一种重要的人兽共患病原菌,布鲁氏菌病常发生在牛、羊、猪等家畜中,主要通过接触传播给人类。人类在感染该菌的早期并无特异性症状,其临床表现多样,若不能及时诊断并予以干预,可导致严重关节疼痛和菌血症,甚至引起死亡。布鲁氏菌病一旦广泛发病,不但对畜牧业造成巨大经济损失,还会严重威胁公共安全,因此,布鲁氏菌的快速准确诊断对防治疾病的发展具有重要意义。目前,针对该病原体常见的实验室检测方法包括微生物检测法、免疫学检测法、分子生物学检测法等。本文将就人感染布鲁氏菌的实验室诊断研究进展作以简要综述。     

大熊猫布鲁氏菌病、弓形虫病以及心丝虫病的血清学调查研究

为摸清大熊猫布鲁氏菌病、弓形虫病以及心丝虫病的血清学感染资料,采用虎红平板凝集试验、试管凝集试验以及外膜蛋白BCSP3基因PCR扩增检测布鲁氏菌病;同时采用弓形虫间接血凝试验和犬心丝虫抗原快速诊断对样品进行检测。结果表明,6只大熊猫RBPT检测为阳性,进一步采用SAT和血液细菌BCSP31基因PCR扩增结果均为阴性,排除了布鲁氏菌感染;大熊猫蜀兰弓形虫抗体检测呈阳性,复查也为阳性,表明存在弓形虫感染;所有大熊猫心丝虫抗原检测结果均为阴性,表明无心丝虫感染。本次检测的3种疾病中,仅发现1只熊猫(蜀兰)存在弓形虫感染,布鲁氏菌病和心丝虫病均为阴性,表明目前成都大熊猫繁育研究基地内大熊猫疾病的预防工作成果显著。     

犬小孢子菌病67例临床观察

犬小孢子菌病为亲动物真菌,感染人类致头皮、头发、皮肤及甲的传染性疾病,还可致头皮深在性感染引起脓癣等。以往均按其发病部位分别归人其他皮肤癣菌病如头癣、体癣等病种内报告。国内少有人将犬小孢子菌所致的病作为一独立的疾病来全面描述。为进一步认识犬小孢子菌及其所致疾病的特征,提高诊断、治疗、预防水平。作者在上海复旦大学华山医院皮肤科进修期间观察了经真菌培养为犬小孢子菌的67例癣菌病患者的真菌学特点和临床表现,并对其特征进行分析研究,现归纳报道如下。     

Evaluation of Micro Complement Fixation Tests for Antibodies Against Group A Streptococcal M and M-Associated Antigens in Rabbit and Human Sera

The microslide gel-diffusion and macro-tube agglutination techniques to detect Brucella canis antibodies in dogs were compared. Sera from dogs experimentally infected with B. canis and a random sample of dog sera with unknown histories of exposure to this organism were examined. The results of the gel-diffusion method employing specific rough Brucella saline-extract antigens of B. canis and Brucella ovis were comparable to those obtained by the tube agglutination test. The easily prepared, stable R antigen in the freeze-dried form offers a convenient, simple, and reliable diagnostic method for the serological detection of canine brucellosis by the gel-diffusion test.     

Complete Genome Sequence of Brucella canis Strain HSK A52141, Isolated from the Blood of an Infected Dog

Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.     

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.     

Use of enrofloxacin in the treatment of canine brucellosis in a dog kennel (clinical trial)

To date, no totally effective antibiotic for the eradication of canine brucellosis has been found. The purpose of this study was to evaluate the efficacy of enrofloxacin in a kennel infected with Brucella canis. Twelve dogs, 2 males and 10 females (including 1 in estrus, 3 pregnant, and 6 in anestrus) infected with B. canis were given 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Females received additional courses of enrofloxacin during the estral and luteal phases of the subsequent cycles (0-2 cycles). They were repeatedly mated by infected males. A serological follow-up was carried out for 38 months. The clinical, serological and bacteriological findings were recorded. In a trial carried out 14 months after the beginning of this study, all dogs were negative on the Rapid Slide Agglutination Test (RSAT). No abortions were observed. All mated female dogs conceived and gave birth to healthy puppies. Cultures of postpartum vaginal discharges (lochia) were negative for B. canis. Similar to other treatments, although enrofloxacin was not completely efficacious in treating canine brucellosis, it maintained fertility and avoided the recurrence of abortions, transmission of the disease to the puppies and dissemination of microorganisms during parturition. We inferred that enrofloxacin could be used as an alternative drug for the treatment of canine brucellosis.     

Brucella melitensis B115-based complement fixation test to detect antibodies induced by Brucella rough strains

Aims:  To assess the efficiency of a Brucella melitensis B115 rough strain, naturally devoid of anticomplementary activity, used as antigen in a complement fixation test (CFT) to detect antibodies induced by Brucella strains with rough phenotype, such as Brucella abortus RB51, Brucella ovis and Brucella canis .
Methods and Results:  Complement fixation testing was performed on sera from RB51-vaccinated cattle and buffaloes, B. ovis -infected sheep and B. canis -infected dogs using B115, RB51 and the hot saline extract (HSE) as antigens. The B115-based CFT proved highly sensitive and specific in detecting rough antibodies and its efficiency was comparable with that of RB51 and HSE-based CFT.
Conclusions:  Brucella melitensis B115 can be successfully used as an antigen in CFT to detect antibodies induced by Brucella rough strains.
Significance and Impact of the Study:  Brucella melitensis B115 antigen may represent an improvement over Brucella rough strains for Brucella antibody detection by CFT, thus enhancing the efficiency of brucellosis surveillance systems. Owing to the absence of anticomplementary activity, it does not require particular growth conditions or modifications and can be accurately standardized. The B115-based CFT may constitute a suitable supplementary test for the diagnosis of human infections owing to rough Brucellae .     

Canine brucellosis: outbreaks and compliance

Canine infertility has many causes that must be considered during evaluation of abnormal reproductive function. An important infectious agent is Brucella canis. Classically deemed a major reason of abortion, this organism also produces infertility in stud dogs and poses a potential health hazard to dogs and humans. The State of Georgia has, out of necessity, instigated regulations to manage outbreaks and seek compliance by educating the pet owner population about this disease. A review of its etiology, methods of transmission, pathophysiology, clinical signs, diagnosis, serology and culture, pathology, treatment options, and regulated prevention featured by Georgia, are presented.     

布鲁菌病的生物安全及防控

布鲁菌病(brucellosis)是布鲁菌入侵机体引起的传染-变态反应性人兽共患传染病,易被忽视。该病严重威胁人类健康,影响畜牧业、旅游业的发展,阻碍正常国际贸易,还会造成食品安全隐患。布鲁菌可通过消化道、呼吸道及皮肤黏膜破损处等途径传播至人。人群对布鲁菌的易感性无年龄和性别差异,可通过直接或间接接触感染。从事布鲁氏菌研究的实验室操作人员有发生实验室获得性感染的风险,但可通过有效的生物安全防护措施避免感染的发生。布鲁菌病是一种可防可控的疾病,必须早期诊断和正确治疗,防止疾病慢性化。本文对布鲁菌病的生物安全及其防控原则进行介绍。     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.