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.     

Campylobacter spp. and their antimicrobial resistance patterns in poultry: an epidemiological survey study in Turkey

The current study aimed at determining the prevalence and the antimicrobial resistance profiles of thermophilic Campylobacter spp. infecting broiler chickens. A total of 240 caecal samples from six slaughterhouses were examined for the presence of Campylobacter spp. C. jejuni was detected in 40.4% (97/240) of the samples and C. coli in 12.1% (29/240). The agar disc diffusion method and the E-test were used for testing the antimicrobial susceptibility of C. jejuni and C. coli isolates. C. jejuni isolates were most resistant to nalidixic acid (79.4%) followed by tetracycline (76.3%), ciprofloxacin (74.2%) and enrofloxacin (15.5%). Among the C. coli isolates, the frequency of resistance to nalidixic acid and ciprofloxacin was the same at 65.5%. The predominant profiles of multidrug resistance to three or more antimicrobials in C. jejuni and C. coli were determined as tetracycline/nalidixic acid/ciprofloxacin resistance (48.5%) and tetracycline/nalidixic acid/ciprofloxacin/enrofloxacin resistance (51.7%), respectively. To prevent the transmission of antimicrobial-resistant bacteria of animal origin to humans, it should be noted that high proportions of multidrug resistance were found in both species.     

Concentrations of faecal coliforms,Escherichia coli,enterococci and Campylobacter spp. in equine faeces

Abstract

AIMS: To determine the concentration of Campylobacter spp. as well as faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci in the faeces of healthy adult horses in a sample of properties in the Canterbury region of New Zealand.

METHODS: The faeces of healthy adult horses (n=59), including ponies, pleasure horses and Thoroughbreds, were collected from eight properties around Christchurch, New Zealand. The faeces were analysed for concentrations of Campylobacter spp and faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci. The presence of other animals on the properties sampled as well as the age, feed and health of the horses at the time of sampling was recorded.

RESULTS: Enterococci and faecal coliforms were isolated from all samples, and E. coli was isolated from 58/59 samples. Mean concentrations of faecal coliforms and E. coli did not differ between properties, but there was a significant difference in mean concentration of enterococci between properties. Campylobacter spp. were detected in two faecal samples with one isolate being determined by PCR analysis to be a thermotolerant Campylobacter species, the other C. jejuni.

CONCLUSIONS: This is the first known report quantifying the concentration of Campylobacter spp. present in healthy adult horses in New Zealand. The presence of equine faecal material in water could elevate concentrations of faecal bacteria and therefore needs to be considered as a source of water contamination. The access of horses to waterways and coastal environments may also need to be restricted to prevent transmission of faecal indicator bacteria and potentially zoonotic agents.     

Sensitivity of Direct Culture,Enrichment and PCR for Detection of Campylobacter jejuni and C. coli in Broiler Flocks at Slaughter

Broiler chicken flocks are a significant source of Campylobacter jejuni and Campylobacter coli that result in the major public health problem of campylobacteriosis. Accurate estimates of the prevalence of both C. coli and C. jejuni in flocks would enhance epidemiological understanding, risk assessment and control options. This study combined results from a panel of 10 detection tests (direct culture, enrichment and PCR) on caecal samples from flocks at slaughter. A parallel interpretation approach was used to determine the presence of Campylobacter spp. and for C. jejuni and C. coli individually. The sample was considered positive if at least one method detected the target and this interpretation was taken to represent a ‘proxy gold standard’ for detection in the absence of a gold standard reference test. The sensitivity of each individual method to detect Campylobacter spp., C. jejuni and C. coli was then estimated relative to the proxy gold standard. Enrichment in adapted Exeter broth (deficient in polymyxin B) with a resuscitation step was 100% sensitive, whilst direct culture on modified charcoal cefoperazone deoxycholate agar (mCCDA) was highly sensitive (97.9%). Enrichment methods using Preston broth and Bolton broth were significantly less sensitive. Enrichment in Exeter broth promoted the recovery of C. jejuni, whilst enrichment in Bolton broth favoured C. coli. A RT‐PCR detection test could identify 80% of flocks that were co‐colonised with both species. This study found that 76.3% (n = 127) of flocks were colonised with Campylobacter spp. The majority (95.9%) of Campylobacter‐positive flocks were colonised with C. jejuni; however, approximately one‐third of positive flocks were simultaneously colonised with both C. jejuni and C. coli. The findings highlight the impact of different detection methodologies on the accuracy of the estimated incidence of both C. jejuni and C. coli entering the abattoir within broiler flocks and the associated public health risks.     

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...     

Occurrence and species level diagnostics of Campylobacter spp., enteric Helicobacter spp. and Anaerobiospirillum spp. in healthy and diarrheic dogs and cats

In order to study the occurrence and co-infection of different species of Campylobacter, enteric Helicobacter and Anaerobiospirillum in dogs and cats and define a possible association between these microrganisms and gastrointestinal disorders, 190 dogs and 84 cats, either healthy or with diarrhea, were sampled between 2002 and 2003. Thirty-three C. upsaliensis, 17 C. jejuni, 2 C. helveticus, 1 C. lari isolates from dogs and 14 C. helveticus, 7 C. jejuni, 6 C. upsaliensis isolates from cats were identified using species-specific PCR and phenotypic tests. Whole cell protein profile analysis, phenotypic tests, PCR-RFLP of gyrB and a phylogenetic study of partial groEL and 16S rRNA sequences were used to identify 37 H. bilis, 22 H. canis and 14 H. cinaedi in dogs and 12 H. canis, 5 H. bilis and 2 H. cinaedi in cats. Whole cell protein profile analysis, phenotypic tests and species-specific PCR of 16S rRNA were used to identify 14 A. succiniciproducens, 12 A. thomasii isolates and one unidentified Anaerobiospirillum sp. isolate in dogs and 3 A. thomasii isolates in cats. Fifty-two animals (19%) were positive for the isolation of more than one genus. No significant statistical correlation was found between any isolates of Campylobacter, Helicobacter or Anaerobiospirillum spp. or the various co-infection rates, and the presence of diarrhea in either dogs or cats. Campylobacter isolates were also tested for antibiotic resistance using the agar dilution method.     

Campylobacter spp. prevalence and fluoroquinolone resistance in chicken layer farms

Chicken is a major source of human campylobacteriosis. Chicken meat originates not only from broilers but also from spent layers; however, few reports have documented the prevalence and antimicrobial resistance of Campylobacter spp. in layers in Japan. Therefore, we investigated the prevalence and antimicrobial susceptibility of Campylobacter spp. in 47 layer farms in Japan. Fecal samples were collected from the youngest and oldest flocks on the farm, and Campylobacter spp. was isolated from 46/47 (97.9%) farms. Among the C. jejuni isolates, the resistance rates to ampicillin, tetracycline, and ciprofloxacin were 29.6%, 22.2%, and 19.8%, respectively. The ciprofloxacin resistance rate (7.3%) in C. jejuni isolated from old flocks was significantly (P<0.01) lower than that in young flocks (32.5%).     

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.     

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