应用PCR技术检测空肠弯曲菌及结肠弯曲菌的研究

本研究根据空肠弯曲菌及结肠弯曲菌的16s rRNA基因相同保守序列来设计引物,建立了一种PCR检测方法,该方法具有较强的特异性及灵敏性,其对空肠弯曲菌及结肠弯曲菌的最低检测限度为5 CFU/mL,它无需增菌培养过程可以直接对样品进行检验,在对鸡肉样品空肠弯曲菌及结肠弯曲菌的检测中取得了较好的效果.     

多重PCR-变性高效液相色谱检测食品中空肠弯曲菌和结肠弯曲菌

本研究应用多重PCR反应(mPCR)结合变性高效液相色谱(DHPLC)技术建立食品中空肠弯曲菌和结肠弯曲菌的快速检测方法.以编码嗜热弯曲菌属的16S rRNA基因、编码空肠弯曲菌的gyrA基因和编码结肠弯曲菌的cdt真基因为靶基因,选择3对引物,建立并优化了鉴别空肠弯曲菌和结肠弯曲菌的mPCR体系,扩增产物分别为287 bp、159 bp和173 bp.采用22株细菌验证了该mPCR具有特异性.mPCR检测的灵敏度在DNA水平上达到空肠弯曲菌10pg/μL、结肠弯曲菌1 pg/μL;在人工模拟污染样品起始污染浓度为1.5个/mL时,42℃微需氧条件下培养24 h即可被检出.在随机采集的172份冷冻鸡肉类样品中,检出了18份样品为空肠弯曲菌阳性,7份样品为结肠弯曲菌阳性.本研究建立的mPCR-DHPLC方法可特异、灵敏地实现对空肠弯曲菌和结肠弯曲菌的快速检测.     

鸡源空肠弯曲菌和结肠弯曲菌的临床分离及多重PCR鉴定

弯曲菌(Campylobacter)尤其是空肠弯曲菌(C.jejuni)是重要的人畜共患病病原菌,在人可引起急性胃肠炎,感染严重者伴有心内膜炎、肺炎、败血症、血栓静脉炎、脑膜炎、多发性关节炎以及格林-巴利综合征等疾病[1-3].家禽、家畜、宠物等动物是弯曲菌的常见宿主,因此研究动物源弯曲菌具有重要的公共卫生意义.     

Prevalence of Campylobacter jejuni and its resistance to antibiotics in poultry in the Czech Republic

Thermotolerant Campylobacter spp., in particular Campylobacter jejuni, are among the most frequently identified pathogens, found to be causing human gastrointestinal infections in Europe, with the Czech Republic being no exception. The presented work aimed at assessing results of the first nationwide monitoring of prevalence and antibiotic resistance of Campylobacter spp. in broiler flocks in the Czech Republic, including a comparison of antibiotic resistance of C. jejuni isolates collected from poultry and the human community. The monitoring was carried out in poultry slaughterhouses in 2006 and 2007. From broilers, cloacal swabs were collected and examined. The human isolates of C. jejuni were acquired from rectal swabs in community patients with diarrhoeal diseases. Suspected isolates of both animal and human origin were confirmed by the PCR methods. Antibiotic resistance to selected anti-microbial agents was tested by the microdilution method. In the monitored period, the prevalence of thermotolerant Campylobacter spp. in broilers in the Czech Republic reached almost 50%. In 2006, C. jejuni was detected in 46% and Campylobacter coli in 3% of the tested samples. In 2007, C. jejuni was found in 43% and C. coli in 2% of the samples. The results of anti-microbial susceptibility testing of C. jejuni showed higher resistance in animals when compared with humans. The only exception was tetracycline with higher resistance in isolates of human origin. The highest resistance detected was to quinolone antibiotics. Resistance to oxolinic acid was 77% in animal and 60% in human isolates, to ciprofloxacin 72% in isolates from poultry and 55% in those from humans. In ampicillin, 26% of poultry isolates and 16% of human isolates were resistant. Moreover, 9% of animal isolates demonstrated resistance to streptomycin, undetected in human isolates. In erythromycin, resistance was found in 6% of poultry and 1% of human isolates.     

2013-2018年福建省部分规模化猪场猪伪狂犬病病毒野毒感染的流行病学调查

为了解2013-2018年福建省西南地区规模化猪场猪群伪狂犬病病毒(PRV)野毒感染情况,本研究分别采用PCR和ELISA检测方法,对2013-2018年该地区787个规模化猪场送检的1320份疑似发病猪群组织样品、3041场次的猪场67660份猪血清样品进行PRV病原学、血清学检测.结果显示:送检病例猪组织样品PRV...     

鹿布氏杆菌病流行病学调查

为确定我国鹿群的布氏杆菌病的流行情况 ,应用间接酶联免疫吸附试验 (ELISA)对采自国内某地区鹿场的 30 0份血清进行检测 ,结果表明 ,鹿群的布氏杆菌血清抗体阳性率为 14 0 % ,证明鹿群中仍然存在着布氏杆菌病     

Risk factors for Campylobacter spp. colonization in broiler flocks in Iceland

We sampled 1091 Icelandic broiler flocks at slaughter from May 2001 to December 2003 to determine the prevalence of, and investigate risk factors for the presence of, Campylobacter spp. at the flock level. Approximately 15% of the flocks were positive for Campylobacter spp.; most (95%) of the infected flocks being raised during the months of April–September. Based on the data from the latter months, and using multivariable logistic regression with random effects for herd, we found that the odds of a flock being positive for Campylobacter spp. increased with age and flock size. Additionally, vertical ventilation systems were strongly associated with positive flocks (OR = 5.3). After controlling for these variables, we found no evidence of an effect of: year; company; Campylobacter being carried over from one flock to the next; time interval between flocks; using (at the hatcheries) eggs laid on the floor; density of bird housing, or the number of catch lots a flock was divided into for slaughtering purposes on the risk of a Campylobacter-positive flock.     

牛源胎儿弯曲菌PCR检测方法的建立

根据胎儿弯曲菌的16S rRNA基因序列设计引物,以微需氧培养的胎儿弯曲菌标准株菌体裂解物为模板,进行PCR扩增目的片段。同法检测了胎儿弯曲菌的10个参考菌株,结果均为阳性。采用异硫氰酸胍快速提取流产牛阴道分泌物中胎儿弯曲菌的DNA,然后用PCR方法检测,5份样品阳性,该试验可在24 h内完成,比病原分离法快5~7 d;而检测空肠弯曲菌、大肠杆菌、布氏杆菌、葡萄球菌、链球菌等相关病原时,均无特异性扩增产物,表明该检测方法具有快速、特异、实用性强的特点。     

链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌多重PCR检测方法的建立及应用

根据NCBI上已收录的链球菌ef-tu基因、金黄色葡萄球菌nuc基因、沙门菌hut基因和大肠杆菌23SrRNA基因的序列,设计并合成4对特异性引物,通过优化多重PCR的反应条件,建立了能够同时检测4种细菌混合感染的多重PCR诊断方法。特异性分析结果表明,应用该方法可以从链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌以及4种细菌的混合物中扩增出4条大小分别为197、278、495和652bp的特异性条带,其他对照组的检测结果均为阴性;敏感性分析表明,该方法对4种病原菌基因组DNA的检出量分别为链球菌25.6pg、金黄色葡萄球菌33.2pg、沙门菌35.7pg、大肠杆菌52.1pg;人工模拟感染样本检测表明,该方法能从混合感染的病料中特异地检测出4种病原菌。本试验建立的多重PCR方法具有特异性强,敏感度高,稳定性好的特点,可以有效的检测链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌的混合感染。     

规模化猪场猪圆环病毒2型感染的流行病学调查

20 0 1年 11月至 2 0 0 2年 8月 ,对北京、天津、广东、深圳、山东、山西等地 12个规模化猪场猪圆环病毒 2型 (PCV2 )感染发病群猪进行了临床发病情况调查 ,并采用 PCR方法对所收集的 5 5份组织病料进行 PCV2的检测 ,结果表明 ,12个猪场中有 11个猪场发病猪群表现为断奶后多系统衰竭综合征 ,1个猪场表现为皮炎和肾病综合征。由此可见 ,PCV2感染在我国规模化猪场已普遍存在     

Distribution and genetic variability among Campylobacter spp. isolates from different animal species and humans in Switzerland

In Switzerland, a national database with 1028 Campylobacter isolates from poultry, pigs, cats, dogs, cattle, humans, zoo animals and water has been created. The database contains the genetic fingerprint and background information of each Campylobacter isolate. Dominant species could be identified in the different sources with a majority of Campylobacter jejuni in poultry (73%), humans (79%), cattle (95%), zoo animals (40%) and water (100%), of Campylobacter coli in pigs (72%), and of Campylobacter upsaliensis/helveticus in cats and dogs (55%). The comparison of three genotyping methods, amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis and restriction fragment length polymorphism, revealed that AFLP allows discrimination between the different Campylobacter species and is the most appropriate method to distinguish specific strains within the same species. Genotyping analysis demonstrated that the Campylobacter population is heterogeneous among the different sources and that no dominant clone is spread in the country. Genotyping and the resulting database are useful tools to trace back future Campylobacter infections.     

The prevalence and genetic diversity of Campylobacter spp. in domestic 'backyard' poultry in Canterbury, New Zealand

Campylobacteriosis is the most commonly notified illness in New Zealand. Whilst the importance of commercial poultry in campylobacteriosis is well established, little is known about the possible role of chickens kept at home as a direct animal/faecal contact or consumption exposure pathway. The aim of this study was to determine the prevalence and genetic diversity of Campylobacter spp. in domestic backyard chicken flocks in the Canterbury region of New Zealand. Poultry faecal samples were collected from 35 domestic 'backyard' poultry flocks from urban and rural properties around the Canterbury Region of New Zealand. A total of 291 samples were collected and tested for the presence of thermotolerant Campylobacter spp. and positive isolates were analysed using pulsed-field gel electrophoresis (PFGE) using both SmaI and KpnI enzymes. There was a high prevalence of Campylobacter spp. with 86% of flocks testing positive. Campylobacter jejuni alone, Campylobacter coli alone and both C. jejuni and C. coli were detected in 20 (57%), 2 (6%) and 8 (23%) of the flocks respectively. SmaI/KpnI PFGE analysis identified 50 different genotypes across the 35 flocks. Genotype diversity richness was highest on the lifestyle block and farm properties with 43 different genotypes isolated, whilst urban properties displayed the least richness with 12 genotypes isolated. Rural flocks tended to have more different genotypes in a given flock than urban flocks. Comparison of the genotypes with the PulseNet Aotearoa Campylobacter database showed that 28 of the genotypes had previously been isolated from human cases of campylobacteriosis. Many of these were also indistinguishable from Campylobacter spp. previously isolated from retail chicken. Therefore, contact with backyard poultry or their faecal material is a potential additional infection pathway outside of exposure to the established pathways associated with the consumption of Campylobacter-contaminated commercial meat or foods cross-contaminated from contaminated poultry.