兔支气管败血波氏杆菌双夹心ELISA检测方法的建立

为建立特异性好、敏感性高、稳定可靠的兔支气管败血波氏杆菌(Bb)双夹心ELISA检测方法,制备了兔抗Bb免疫血清和小鼠抗Bb腹水单抗(Bb McAb),并将它们提纯。用方阵法筛选最佳反应浓度,用特异性试验、敏感性试验和重复性试验对该方法进行鉴定,并与PCR检测方法比较。结果表明,兔抗Bb IgG的最佳稀释度为1∶6 400,浓度为2.5 mg/L;单抗的最佳稀释度为1∶800,浓度为5.0 mg/L。特异性试验和重复性试验结果表明,该方法特异性强、重复性好,与兔常见病菌大肠埃希氏菌、多杀性巴氏杆菌和产气荚膜梭菌均不发生反应,同时与PCR检测方法的符合率为100%。     

PCR检测兔支气管败血波氏杆菌方法的建立及应用

为建立准确、快速检测兔支气管败血波氏杆菌(Bordetella bronchiseptica)的PCR方法,根据Gen Bank中波氏杆菌ptxA基因设计1对特异性引物,用建立的PCR方法扩增兔波氏杆菌,可扩增出870 bp的目的片段。该方法特异性好,仅对波氏杆菌有特异性的扩增,而对大肠埃希菌、无乳链球菌、金黄色葡萄球菌、多杀性巴氏杆菌等病原菌的扩增结果呈阴性;敏感性较高,最低检测限可达6.8×10~(-7)μg/m L;用建立的PCR方法检测临床疑似感染支气管败血波氏杆菌病兔的脏器及鼻拭子样品,可扩增出目的条带,且与细菌分离的结果一致。可见,建立的检测方法敏感、特异,可用于临床上兔波氏杆菌病的快速诊断及鉴定。     

Serum C-reactive protein and immune responses in dogs inoculated withBordetella bronchiseptica (phase I cells)

Eight Beagle dogs were inoculated intrabronchially with 5×10⁹ live, avirulent cells ofBordetella bronchiseptica L-414 strain (phase I cells) (B. bronchiseptica) to investigate the serum levels of their C-reactive protein, the white blood cell counts, the antibody responses toB. bronchiseptica in the sera and tracheal secretions, and the effects of prednisolone given to four of the dogs on C-reactive protein (CRP), white blood cells (WBC) and immune responses. In two Beagle dogs inoculated intrabronchially with sterile physiological saline, the concentrations of CRP and the WBC counts did not increase. CRP was markedly increased one day after inoculation in the dogs inoculated withB. bronchiseptica to 385.0–720.0 µg/ml (mean 498±132 µg/ml) in the group given theB. bronchiseptica inoculation only, and to 372.0–649.0 µg/ml (mean 551±106 µg/ml) in the group treated with prednisolone following inoculation ofB. bronchiseptica, as determined by an enzyme-linked immunosorbent assay (ELISA). The CRP levels were 23–95 times the pre-inoculation values, which indicated that prednisolone had no effect on the production of CRP. In the prednisolone-treated group, the WBC count increased and stayed at an increased level for approximately 12 days. An indirect fluorescent antibody test led to the detection of anti-B. bronchiseptica IgM and IgG antibodies in the sera from 5 days afterB. bronchiseptica inoculation and S-IgA and IgG anti-B. bronchiseptica antibodies in the tracheal secretions on the day after the challenge exposure toB. bronchiseptica. The increase in CRP after challenge exposure toB. bronchiseptica was significantly (p<0.05) smaller than that found after the first inoculation ofB. bronchiseptica.Abbreviations CRP C-reactive protein - ELISA enzyme-linked immunosorbent assay - FHA filamentous haemagglutinin - IFA indirect fluorescent antibody - WBC white blood cell(s)     

Concentrations of C-reactive Protein in Normal Monkeys (Macaca irus) and in Monkeys Inoculated with Bordetella bronchiseptica R-5 and Measles Virus

The concentrations of C-reactive protein (CRP) in serum from normal crab-eating monkeys (Macaca irus) were measured by means of a monkey-specific turbidimetric immunoassay (TIA), and the changes in the serum CRP concentrations in crab-eating monkeys inoculated with Bordetella bronchiseptica R-5 and measles virus (Ichinose or NK 3 strain) were also examined. The CRP concentrations in sera from 54 normal crab-eating monkeys ranged from 0 to 8.3 g/ml (mean 2.2±1.9). No significant difference was found in the CRP concentrations between males and females (p>0.05). The concentrations of CRP in the sera from four crab-eating monkeys inoculated intrabronchially with 10⁹ live B. bronchiseptica increased gradually to a peak at 2 days after inoculation. The peak concentrations of CRP were from 102.4 to 313.2 g/ml, 54–96 times the preinoculative values of 1.9–5.6 g/ml. When the same four crab-eating monkeys were inoculated intrabronchially with measles virus 34 days after inoculation of B. bronchiseptica, the serum CRP concentrations did not increase. Monitoring of CRP is useful for assessing monkeys with acute B. bronchiseptica infection and will probably be of value in the diagnosis of other bacterial infections.     

Immunoproteomic Analysis of Bordetella bronchiseptica Outer Membrane Proteins and Identification of New Immunogenic Proteins

Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted from B. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry（MALDI-TOF-MS）, a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins for B. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB（B. bronchiseptica strain isolated from a infected rabbit）. The mortality of mice was 80% compared to 100% of positive controls. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.     

猪支气管败血波氏杆菌PCR诊断方法的建立及应用

根据支气管败血波氏杆菌（Bordetella bronchiseptica）鞭毛蛋白基因的上游序列设计了1对引物flal和fla2，扩增出大小为237bp的目的基因片段，建立了快速检测支气管败血波氏杆菌的PCR方法。特异性和敏感性试验表明，该方法对大肠埃希氏菌、金黄色葡萄球菌和D型多杀性巴氏杆菌均无交叉性反应；最低能够检出112．5pg的模板DNA。用该PCR法检测了从延边州采集的48份表现不同临床症状的猪鼻黏液；结果，检出支气管败血波氏杆菌阳性42例，阳性率为87．5％。同时与传统的细菌检查法、微量凝集法进行了比较；结果显示，该PCR法的检出率是细菌检查法的5．25倍，是微量凝集法的1．75倍。     

兔支气管败血波氏杆菌PRN基因缺失突变株的构建及特性研究

利用自杀性质粒构建兔支气管败血波氏杆菌百日咳黏附素(PRN)缺失突变株以研究PRN在支气管败血波氏杆菌(Bb)致病机理中的作用,同时为支气管败血波氏杆菌病减毒活疫苗的研究提供理论依据.PCR扩增出PRN1(PRN上游基因)和PRN2(PRN下游基因)2个目的基因片段,运用基因重组技术将庆大霉素抗性基因(GM)连接到PRN1和PRN2之间,将连接好的基因片段克隆到pMEG-375自杀性载体中,构建自杀性载体pMEG375-PRN1-GM-PRN2,将其转化到宿主菌SM-10中,通过宿主菌SM-10与受体菌Bb固相滤膜交配,自杀性载体转移到受体菌,根据同源重组原理,抗性筛选得到基因缺失突变株,命名为Bb(△PRN).对突变株Bb(△PRN)与野生株WT进行了遗传稳定性、生长特性、溶血特性、细胞黏附特性、毒力、免疫保护性等比较研究.结果表明:Bb(△PRN)具有遗传稳定性;与野生株相比,突变株生长速度较慢,毒力有所下降,溶血活性及对Hep-2细胞的黏附能力没有明显变化;小鼠免疫原性试验结果显示,突变株免疫小鼠后可以产生强有力的免疫力,能够抵抗野生株的攻击.Bb(△PRN)突变株构建成功并具有良好的免疫原性,为支气管败血波氏杆菌病减毒活疫苗的研究奠定了基础.     

AA肉种鸡胚胎性疾病的病原学研究及主要病原的PCR 检测

2004年6月份以来，山东某一大型AA父母代肉种鸡场孵化场出现胚胎死亡、弱雏增多、孵出的雏鸡30日龄左右免疫应答不良、生长缓慢等症状。为了探明其病因，作者通过流行病学调查和病原学检查，结果表明造成该病的主要原因是由禽波氏杆菌(Bordetella avium)、沙门氏菌(Salmonella)、大肠杆菌(Escherichia coli)、支原体(滑液囊和鸡败血支原体)及鸡传染性贫血病病毒（CIAV）和病毒性关节炎病毒（VAV）共感染种鸡后，由种蛋垂直传播到鸡胚、雏鸡造成的。同时对其主要病原禽波氏杆菌进行了16S rRNA基因的扩增，结果扩增出783 bp的目的条带; 通过PCR对CIAV VP3基因及VAV S1基因进行扩增，结果分别扩增出582和532 bp的目的条带，其PCR产物均分别能与相应的探针杂交，呈现阳性反应。     

支气管败血波氏杆菌的鉴定及药敏试验

从猪萎缩性鼻炎(AR)临床症状猪群分离出34株疑似支气管败血波氏杆菌(Bb),对分离菌进行形态结构、染色特性、培养性状和生化特性等方面的测定,结果这些分离菌均符合Bb的生物学特征;对BbFlaA基因进行PCR扩增,所有分离菌均能扩增出特异性DNA条带,与传统方法的鉴定结果完全一致,且最小检出量为0.64pg;而猪鼻腔和肺组织中常见的多杀性巴氏杆菌、金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、变形杆菌及枯草芽孢杆菌均未出现任何DNA条带。药敏试验显示,这些分离物对多黏霉素B、丙氟哌酸、氟哌酸、卡那霉素和庆大霉素均表现高度敏感。     

v猪场支气管败血波氏杆菌鼻腔带茵和肺部感染调查分析

对珠三角三个曾发生萎缩性鼻炎,现已临床控制两年以上的大型猪场的健康母猪、乳猪和保育猪鼻腔取样81份,分离支气管败血波氏杆菌,结果21份阳性;对珠三角地区16个猪场送检病猪,取有炎症病变的肺184份,分离波氏杆菌,结果13份阳性。