奶牛源单核细胞增生性李斯特菌分离鉴定及流行调查

为了解本地区规模化奶牛场单核细胞增生性李斯特菌的流行情况，本研究采集了320份奶牛鼻拭子，进行单核细胞增生性李斯特菌的分离、培养和PCR鉴定。鉴定结果表明分离出6株单核细胞增生性李斯特菌，分离率1.88%，本研究为本地区奶牛单核细胞增生性李斯特菌的防控及保障动物性食品安全奠定了技术基础。     

应用环介导等温扩增技术快速检测单核细胞增生李斯特菌的研究

目的建立环介导等温扩增技术快速检测单核细胞增生李斯特菌。方法根据单核细胞增生李斯特菌(LM)hlyA基因序列中的保守区域,采用在线引物设计软件Primer Explorer4.0进行设计,获得一套特异性的环介导等温扩增(LAMP)引物,对单核细胞增生李斯特菌hlyA基因进行LAMP扩增,并与常规PCR方法进行比较。结果建立的LAMP方法能成功扩增出梯形条带,LAMP检测单核细胞增生李斯特菌纯培养物和人工染菌的灵敏度为5.44×102cfu/mL,而对照PCR检测的灵敏度为5.44×104cfu/mL。对10株细菌进行LAMP扩增,仅单核细胞增生李斯特菌得到阳性结果。从DNA提取到报告结果,耗时仅1h。结论 LAMP检测单核细胞增生李斯特菌灵敏度高,特异性强,耗时短,方法简便,有望发展成为快速检测食品中单核细胞增生李斯特菌的有效手段。     

高度特异的李斯特菌单抗试剂的研制及鉴定

以单核细胞增生李斯特菌制备免疫原，应用淋巴细胞杂交瘤技术，建立了３株能稳定分泌抗单核细胞２增生李斯特菌，无害李斯特菌和格氏李斯特菌的单克隆抗体的细胞系，分别是ＬＪ１０Ａ，ＬＭ１１和ＬＢ５，而与其余种的李斯特菌和属外细菌不发生交叉反应。     

新疆不同来源单核细胞增生李斯特氏菌血清型的多重PCR鉴定

为鉴定分离自新疆北疆绵羊单核细胞增生李斯特氏菌,本研究采用多重PCR方法,鉴定来自病发地区部分羊场的发病绵羊、健康绵羊、羊舍环境和乌鸦粪分离的30株李斯特氏菌分离株的8株单核细胞增生李斯特氏菌分离株血清型。结果为4株发病绵羊株有3株鉴定为单核细胞增生李斯特氏菌,血清型为1/2a或4b,1株为非单核细胞增生李斯特氏菌;5株健康绵羊株血清型为1/2a;其余来自羊舍水源的3株、乌鸦粪的2株及健康绵羊16株为非单核细胞增生李斯特氏菌,表明来自发病绵羊、健康绵羊及参考菌株LM血清型之间具有相关性。     

广州地区禽产品单核细胞增生性李斯特菌污染情况调查

单核细胞增生性李斯特菌是食品卫生上重要的病原菌.它广泛存在于自然界,可导致人和动物脑膜炎、流产、败血症等,病死率高达30％～70％,是目前人类最重要的食物源性病原菌之一[1,2].在欧美许多发达国家常发生因食品中污染该菌而导致的人员死亡事件,因此,很多国家都将单核细胞增生性李斯特菌列入食品安全重点监测对象[2,3].本调查采集广州不同地点和来源的各种禽类产品,进行了单核细胞增生性李斯特菌的分离和鉴定,分析了广州地区禽类产品中该菌的污染情况,为单核细胞增生性李斯特菌病的防制提供依据.     

食品中产单核细胞李斯特菌PCR检测方法的建立

根据产单核细胞李斯特菌hlyA基因设计引物,进行PCR扩增,检测该方法的特异性和灵敏度。人工污染样品经Half-fraser和Fraser增菌后进行PCR检测。结果表明,产单核细胞李斯特菌扩增出234bp的条带,对照菌未扩增出目的条带。该方法的灵敏度为104cfu/mL。人工污染样品的检出限为8cfu/25g,说明PCR方法检测食品中产单核细胞李斯特菌具有快速、特异、敏感等特点,具有较高的实用价值。     

用四重PCR检测方法检测产单核细胞李斯特菌

为了同时快速准确的检测产单核细胞李斯特菌以及是否为有毒力的产单核细胞李斯特菌,根据相关文献报道的四重PCR方法,针对该菌的种特异性基因inl A基因进行引物设计,扩增片段大小为800bp,结果应用该PCR法可特异性的扩增出目的基因片段,与GenBank上发表的序列同源性为97.3%。同时通过对毒力基因inlB,inlC和inlJ的检测用来判断菌株的相关毒力。而同一属的其他菌种无害李斯特菌(L.innocua),韦氏李斯特菌(L.welshimeri)均无特异条带,嗜水气单胞菌(J-1),产气荚膜梭菌(C57-13),大肠埃希菌(ATCC 25922),鼠伤寒沙门菌(C79-32),金黄色葡萄球菌(ATCC 25923)结果均为阴性。对临床上送检的23份样品进行四重PCR方法检测并同时与国家标准GB/T22429-2008食品中单核细胞增生李斯特氏菌的快速筛选检验中使用的检测方法进行比对,两种检测方法的结果完全符合,均检测出7份阳性样品。并且四重PCR法表明这7份阳性样品中的产单核细胞李斯特菌均携带有3种毒力因子(inlB,inlC和inlJ),因此该四重PCR法可用于快速鉴定是否是有毒力的产单核细胞李斯特菌,为有效预防控制产单核细胞李斯特菌污染提供技术支撑。     

兔李斯特氏菌病的检疫

李斯特氏菌病或称李氏杆菌病,是一种散发性传染病,以全身血液中毒-败血症和子宫炎及流产为特征.本病的病原为单核细胞增多性李氏杆菌(或单核细菌增多性李斯特氏菌、产单核细胞李氏杆菌).     

单核细胞增生性李斯特菌LAMP-浊度/荧光检测方法的建立

将环介导等温扩增技术(LAMP)分别与浊度信号检测系统(turbidimeter)和荧光信号检测系统(fluorescence)相结合,建立了LAMP-浊度/荧光单核细胞增生性李斯特菌检测方法。选择单核细胞增生性李斯特菌保守毒力基因iap序列,通过环介导等温扩增引物设计在线软件设计4条特异性引物,进行反应条件的优化,并对建立的LAMP的特异性和灵敏度进行评价分析。结果表明,构建的LAMP-浊度信号检测方法优化后的扩增温度为61℃,菌液最低检测浓度为22.1 CFU/mL,灵敏度是普通PCR扩增方法的10倍;LAMP-荧光信号检测方法优化后的扩增温度为64℃,菌液最低检测浓度为2.21 CFU/mL,灵敏度是普通PCR扩增方法的100倍;建立的两种LAMP方法的特异性良好,其中1株单核细胞增生性李斯特菌标准菌株和1株本试验分离保存的单核细胞增生性李斯特菌检测结果均为阳性,8株非单核细胞增生性李斯特菌检测结果均为阴性。利用两种LAMP方法对630份肉类及其制品、人工污染样品等进行检测,检出45个LAMP阳性,与国标法(GB)检测结果一致。结果表明,建立优化的单核细胞增生性李斯特菌LAMP-浊度/荧光检测方法具有快速、特异、灵敏等特点,特别适用于基层兽医、食品及口岸一线部门对单核细胞增生性李斯特菌快速筛查工作。     

环介导等温扩增联合横向流动试纸条快速检测单核细胞增生李斯特菌的研究

将环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)与横向流动试纸条检测技术(lateral flow dipstick,LFD)联合,建立了1种可应用于单核细胞增生李斯特菌快速检测的新方法。针对单核细胞增生李斯特菌的actA基因设计3对引物(其中,上游内引物由生物素标记)和1条异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针,进行由生物素标记的LAMP扩增反应,并利用LFD对经FITC标记探针杂交的扩增产物完成检测。优化后的LAMP反应条件为65℃反应40min,从细菌基因组DNA提取到LFD结果判断只需90min左右,比常规PCR技术缩短近2h。LAMP-LFD可特异性地检出单核细胞增生李斯特菌,对哈维弧菌等常见弧菌及嗜水气单胞菌等的检测结果为阴性。灵敏性试验表明,LAMP-LFD针对病原纯培养物的检测灵敏度为3.2×101 CFU/mL或0.64CFU/反应,是LAMP检测的10倍,常规PCR检测的100倍;针对人工污染单核细胞增生李斯特菌的原料奶样品的检测灵敏度为1.6×102 CFU/mL或3.2CFU/反应。利用本方法可从采集样品中检测到单核细胞增生李斯特菌,检测结果与传统的细菌分离培养方法结果一致。试验表明,本研究建立的LAMP-LFD技术可特异、准确地应用于单核细胞增生李斯特菌检测,而且灵敏度高、操作简单、检测成本低,有望发展成为单核细胞增生李斯特菌快速检测的有效手段。     

Evaluation of farm management practices as risk factors for clinical listeriosis and fecal shedding of Listeria monocytogenes in ruminants

OBJECTIVE: To assess seasonal variation in prevalence of Listeria monocytogenes on ruminant farms and identify management practices associated with ruminant listeriosis and fecal shedding of L. monocytogenes. STUDY DESIGN: Case-control study. SAMPLE POPULATION: 2056 samples of feces, feed, soil, and water from 24 case farms with listeriosis and 28 control farms without listeriosis. PROCEDURE: Samples were collected and evaluated via bacterial culture for L. monocytogenes. Univariate associations between farm management practices and listeriosis and fecal shedding of L. monocytogenes were assessed. Multivariate models were developed to identify farm management practices associated with listeriosis and fecal shedding of L. monocytogenes. RESULTS: The prevalence of L. monocytogenes on cattle, goat, and sheep farms was seasonal, especially in fecal samples, with peak prevalence in winter. Although the prevalence of L. monocytogenes in feedstuffs from small-ruminant farms also peaked during winter, the bacterium was detected at a constant rate in cattle farm feedstuffs throughout the year. Farm management practices, animal health and hygiene, and feedstuff quality and storage were associated with ruminant listeriosis and fecal shedding of L. monocytogenes. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the prevalence of L. monocytogenes on ruminant farms is seasonal, management practices are associated with ruminant listeriosis and fecal shedding of L. monocytogenes, and the epidemiologic features of listeriosis differ in cattle versus small ruminants. Awareness of risk factors may be used to develop control measures to reduce animal disease and introduction of L. monocytogenes into the human food chain.     

Passive immunization with convalescent serum, or oral immunization with formalin-killed organisms, does not protect mice against gastrointestinal challenge with Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen in both humans and animals. In addition, murine listeriosis is a widely used model for studying the molecular pathogenesis of an intracellular pathogen, and the regulation of protective cellular immunity. Little attention has been paid to protective immunity against L. monocytogenes in the gastrointestinal tract, where a secretory immune response might prevent attachment of the bacteria to the intestinal epithelium. In this study we found that neither opsonization of L. monocytogenes with immune serum, nor repeated oral administration of killed L. monocytogenes, protected mice against gastrointestinal challenge with L. monocytogenes.     

A direct plating method for monitoring the contamination of Listeria monocytogenes in silage.

Twenty-two silage samples were analyzed for the presence of L. monocytogenes using five Listeria selective plating media, with and without previous selective enrichment step. L. monocytogenes was recovered from 3 samples by both procedures, but direct plating allowed the quantification of Listeria population. Two of these positive samples were implicated in outbreaks of listeriosis in sheep; the L. monocytogenes population in these samples was about 10(6) cells/g. The L. monocytogenes population in the other positive sample was 10(3) cells/g. Direct isolation of L. monocytogenes was only possible from LPM, PALCAM and LSAMm media. MOX and LSM media were not selective enough to allow direct Listeria isolation. In our hands, LSAMm was the most suitable plating medium for the direct isolation and specific quantification of L. monocytogenes from silage employing a red blood cells overlay technique.     

Survival of Listeria monocytogenes in cheese made of unpasteurized goat milk

Food-born infections with Listeria monocytogenes have been reported during recent years, cheese being mentioned as one of the foods responsible. A classical opinion is that cheese represents a very inhospitable environment for pathogens due to antagonism by the starter culture of lactic-acid-producing organisms. In order to study the survival of L. monocytogenes in goat cheese, cheeses were made with the addition of L. monocytogenes cultures. The maximum survival time for L. monocytogenes was 18 weeks in two of the cheeses. It is concluded that L. monocytogenes has the ability to survive in semi-soft cheese made of unpasteurized goat milk during normal curing (2–3 months).     

In vitro study of Listeria monocytogenes infection to murine primary and human transformed B cells

Immunity to Listeria monocytogenes is largely mediated by T lymphocytes. Recently, B lymphocytes or their secreted products are implicated to provide immunity against L. monocytogenes infection. To understand whether L. monocytogenes can infect and kill B cells as a possible strategy to initiate an infection, we examined the effects of L. monocytogenes on a human B lymphoma (Ramos RA-1) and mouse primary B cells in vitro. L. monocytogenes infection resulted in significantly (p相似文献   

Influence of bacteriocin-like substance, generation times, and genetic profiles of Listeria innocua on the isolation of Listeria monocytogenes

Inhibition of isolation of Listeria monocytogenes by bacteriocin-like substance (BLS)-producing Listeria innocua after enrichment culture was investigated. When 26 L. monocytogenes strains were examined in combination with eight L. innocua strains using the spot on lawn method, 52/208 (25.0%) combinations showed the growth inhibition of L. monocytogenes. When two Listeria species were cultured simultaneously in selective enrichment broth, inhibition of isolation of L. monocytogenes was observed in 12/52 of the combinations at 24h (23.1%), in 24/52 at 48h (46.2%) and in 30/52 (57.7%) after 7 days of incubation. The randomly amplified polymorphic DNA profiles showed no interstrain similarities between either strains of the BLS-producing L. innocua or the BLS-sensitive L. monocytogenes strains. Therefore inhibition by BLS-producing L. innocua of isolation of L. monocytogenes after enrichment culture is unlikely to be dependent upon a particular genetic profile.     

Listeria monocytogenes contamination of finishing pigs: an exploratory epidemiological survey in France

Listeria monocytogenes is a foodborne pathogen of major concern for public health in industrialised countries. Since L. monocytogenes carriage by pigs at the herd level could be a primary source for carcass contamination, control measures should be designed to reduce the L. monocytogenes load at the pre-harvest stage. For this purpose, an exploratory analytical survey was carried out in 2000-2001 in 93 French farrow-to-finish pig farms concerning L. monocytogenes contamination in pigs before they left for the slaughterhouse. On each farm, the L. monocytogenes status of a batch of contemporary fattening pigs housed in the same room was assessed on faecal material samples taken by means of gauze swabs wiped on the perianal region of the pigs. Fourteen percent of the batches studied had at least one contaminated sample and were therefore classified as L. monocytogenes contaminated batches. Two logistic regression models were used to assess the association between managerial and hygiene practices and the risk of L. monocytogenes contamination of the batch at the end of the finishing period on the whole data set (n = 93) and in the wet feeding farms only (n = 57). Wet feeding during the fattening period was identified as a risk factor for L. monocytogenes contamination. Risk factors related to the introduction of L. monocytogenes in pig facilities were identified for both the general and wet feeding farm data sets. Poor care paid to hygiene on the farms was found to increase the risk of being infected (boots cleaning, change room presence). When the duration of the empty period prior to the introduction of growing pigs was less than one day in the fattening section, the risk of L. monocytogenes contamination was significantly increased. For wet feeding farms, a distribution pipeline cleaning procedure including disinfection was found to be associated with a higher risk of contamination than no cleaning or a procedure consisting of rinsing with water only.     

Respiratory infection of turkeys with Listeria monocytogenes Scott A

The pathogenesis of L. monocytogenes strain Scott A was studied by challenging day-old male turkey poults by air sac inoculation with tryptose phosphate broth containing 10(0) cfu (control), 10(4), 10(5), and 10(6) cfu (low challenge), or 10(7) and 10(8) cfu (high challenge) of the Scott A (serotype 4b) strain of L. monocytogenes. Mortality at 2 wk postinfection (PI) ranged from 25% for low challenge to 100% for high challenge (P= 0.0001). Gross and histopathological lesions were observed in heart, liver, spleen, lung, and bursa of Fabricius of mortalities at 4 days PI. Listeria monocytogenes challenge resulted in significantly decreased relative weight of the bursa of Fabricius and increased relative weight of the spleen, and L. monocytogenes was isolated by direct plating of liver, pericardium, brain, and both left and right stifle joint synovium (knee) cultures, as well as gall bladder, yolk sac, and cecal tonsil from transfer swabs onto Listeria-selective agar. Isolates were confirmed as positive using Gram stain, biochemical tests, and the Biolog system. High challenge resulted in confirmed L. monocytogenes isolation from 48% of left knee and 59% of right knee cultures. Low challenge resulted in isolation of L. monocytogenes from 11% of both left and right knee cultures. These results suggest that L. monocytogenes Scott A colonization of turkey knee synovial tissue can initiate in day-of-age poults and that L. monocytogenes Scott A can be invasive through air sac infection.