【作者】 牛冬燕；

【导师】 徐永平； McAllister T.A.； Stanford K.；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 博士

【摘要】 食源性致病菌一大肠杆菌O157:H7(Escherichia coli O157:H7)已成为危害人类健康的严重世界性公共卫生问题。牛及其饲养环境是大肠杆菌O157:H7的主要寄居场所及重要的传播源,在饲养场建立切实可行的控制策略可以有效地从源头上降低甚至完全消除致病菌的流行,减少和切断致病菌通过食物链和环境污染等途径传播,从而保障人类健康。目前,控制动物个体及其饲养环境中的病原菌尚无有效方法。本论文主要通过动物实验及体外裂解实验考察噬菌体的体内控制效果、裂解能力和宿主范围,并通过探索牛及其饲养环境中天然噬菌体的分布特性、变化规律及在同一生态系统中噬菌体与大肠杆菌O157:H7之间的相互作用关系,探讨和评价在饲养场实施噬菌体控制策略的有效性和可行性,为保障食品源头安全和环境安全创建一个高效、绿色、安全、无公害的生物防治平台。以攻毒感染公牛为模型,选用直肠粪样法和直肠肛门连接处粘液拭子法监测大肠杆菌O157:H7的排泄量/检出率,考察四株噬菌体(rV5、wV7、wV8和wV11)口服给药的控制效果。综合对照组和给药组的检测结果,粪样的总阳性率(78.9%,202/256)显著高于拭子样本(62.9%,161/256;P＜0.01),二者之间的检测一致性为一般到中等(fairto moderate,κ=0.36～0.45);给药组口服噬菌体后,大肠杆菌O157:H7的总排泄量显著降低(P＜0.05)。粪样法比拭子法更能准确和有效地监测噬菌体的控制效果,口服四株噬菌体的混合制剂降低了牛大肠杆菌O157:H7的总排泄量。以加拿大阿尔伯塔(Alberta)地区商业化肉牛和奶牛饲养场及临床病例分离获得的422株大肠杆菌O157:H7菌株为研究对象,采用噬菌体分型技术和脉冲场电泳技术(pulsed-field gel electrophoresis,PFGE)鉴定牛源分离株的噬型特征和基因型,通过建立微孔板分析法计算噬菌体裂解分离株的感染复数,评价噬菌体的宿主谱和裂解能力。噬菌体wV7、wV8、rV5和wV11的宽噬率分别为100%、96.4%、81.0%和76.1%;wV7裂解分离株的感染复数最低(0.004～0.006,P＜0.0001),其次为rV5和wV11(0.002～0.04,P＜0.0001),wV8最高(25～29,P＜0.0001);牛源分离株对rV5、wV11和wV8的易感性与其噬型和/或PFGE基因型有关。研究表明噬菌体的宿主谱和裂解能力的评价指标感染复数应同时作为筛选治疗性噬菌体的标准;口服给药的四株噬菌体可以有效裂解大部分受检菌株,不同饲养场的菌株对噬菌体wV8表现出不同的易感性,发现噬菌体rV5、wV11和wV8的易感性受其噬菌体表型和/或PFGE基因型影响,而噬菌体wV7的宿主谱和裂解能力不受菌株的噬菌体表型和PFGE基因型的影响,能够高效裂解所有受检分离株。自2007年5～11月,采用初筛法和富集法从加拿大阿尔伯塔省南部两家商业化肉牛饲养场采集的个体直肠粪样、新鲜地面粪样、水槽中的饮用水和沉淀物样本(简称饮水样)以及地面粪尿泥浆样本中检测和分离天然大肠杆菌O157:H7噬菌体。噬菌体的总检出率为28.0%(239/855),其中粪尿泥浆样本检出率显著高于其它样本,达94.6%(35/37,P＜0.001);地面粪样中噬菌体的检出率呈周期性振荡变化,其中5月份最高(P＜0.05);另外,统计学分析显示若噬菌体在环境中的检出率较高时,大肠杆菌O157:H7在牛群中的检出率则较低(P＜0.01),Pearson相关性分析表明,同一个体粪样中噬菌体和大肠杆菌O157:H7的存在呈负向相关(r=-0.11,P＜0.05)。噬菌体广泛存在于牛场的动物个体及其周围环境中,粪尿泥浆中噬菌体的检出率高于地面粪样和饮水样,地面粪样中噬菌体检出率与大肠杆菌O157:H7呈现类似的周期性振荡变化规律;统计学分析显示存在于动物个体及其环境中的噬菌体均能显著降低大肠杆菌O157:H7在牛群中的检出率。综上,本论文证明了噬菌体控制大肠杆菌O157:H7的有效性和可行性,初步揭示了饲养牛的生态系统中天然噬菌体检出率的变化规律为周期性振荡,统计分析表明了天然噬菌体能够降低致病菌在牛群中的检出率,为噬菌体控制策略在商业化饲养场的真正实施提供了可靠的事实依据和理论支持,开启了应用绿色、安全、无公害的生物防治手段有效保障食品源头安全和环境安全的先河。更多还原

【Abstract】 The food-borne pathogen Escherichia coli O157:H7(E.coli O157:H7) is a significant public health concern.Cattle are recognized as primary reservoirs of E.coli O157:H7 and contaminated foods of bovine origin or vegetable products contaminated with bovine feces are important vehicles of human infection;therefore,effective interventions targeted at reducing shedding of E.coli O157:H7 by live cattle(preharvest) and presence of their environment are desirable.Currently there is no one preferred on-farm method that has given satisfactory and consistent results in reducing E.coli O157:H7 populations in cattle and their environment.The objectives of this research were to assess the use of bacteriophage (phages) as an on-farm strategy in mitigating occurrence of E.coli O157:H7 in feedlot cattle and their environment and thereby construct an effective,green,safe and pollution-free biological control stage to ensure food safety from origin and environment safety.To evaluate oral administration of four bacteriophages(rV5,wV7,wV8,wV11) in mitigating the fecal shedding of E.coli O157:H7 by steers under production conditions,and compare fecal grab(FEC) and rectoanal mueosal swab(RAMS) techniques as sampling methods for surveillance of E.coli O157:H7 in conjunction with administration of a mitigation therapy.Overall,Oral treated steers presented fewer(P＜0.05) E.coli O157:H7 culture positive samples compared to control treatment.E.coli O157:H7 was detected more frequently(P＜0.01) by FEC than by RAMS.Kappa values(0.36 to 0.45) indicated only fair to moderate agreement between FEC and RAMS.Oral administration of bacteriophage could be an efficacious large-scale method for mitigating E.coli O157:H7 in cattle. Asessment of FEC samples by DP plus enrichment/IMS was more effective than RAMS/IMS for investigating E.coli O157:H7 mitigation strategies in E.coli O157:H7-challenged feedlot cattle.To evaluate host range and lyric capability of four bacteriophages(rV5,wV7,wV8, wV11) against E.coli O157:H7(n=422) obtained from cattle and humans in Alberta, CANADA.Multiplicity of infection(MOI) of the four phages were estimated via mircoplate virulence assay.All bovine E.coli O157:H7 isolates were subject to pulsed field gel electrophoresis(PFGE) and phage typing(PT).Phage wV7 lyzed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI(0.004 to 0.006,P＜0.0001) of all phages.Phages rV5 and wV11 exhibited a lower MOI(0.002 to 0.04,P＜0.0001) than phage wV8(25 to 29) and a narrower host range than phages wV7 or wV8.Phages rV5,wV11 and wV8 lyzed 342(81.0%),321(76.1%) and 407(96.4%), respectively,of the 422 isolates.Excluding phage wV7,susceptibility of bovine E.coli O157:H7 isolates to phages rV5,w11 and wV8 was influenced by both by the PFGE genotype and/or PT phenotype of the targeted host.Each of the four phages studied in the present experiment exhibited activity against the majority of bovine and human E.coli O157:H7 isolates.Susceptibility of bovine E.coli O157:H7 to phage may differ among farms.Both lytic capability based on MOI and host range should be considered in the selection of therapeutic phage.The present work indicates that a four-phage cocktail should be equally effective at mitigating E.coli O157:H7 isolates of both cattle and human origin.Distribution,fluctuation of endemic bacteriophages infecting E.coli O157:H7(PHAGE) and the relationship between PHAGE and shedding levels of E.coli O157:H7 by cattle were investigated in two commercial feedlots in southern Alberta.Between May and November 2007,10 pens of cattle were monitored by collection of pooled fecal pats(PAT),water with sediment from troughs(WAT),manure slurry from pen floor(when present,SLU) and rectal fecal samples from individual animals(FEC),collected at implantation and prior to slaughter. Bacteriophages infecting E.coli O157:H7 were recovered(by initial screening and/or enrichment) in 239 of 855 samples(109 PAT,76 FEC,19 WAT and 35 SLU).Overall, prevalence of PHAGE was highest(P＜0.001) in SLU.Recovery of PHAGE from PAT was highest(P＜0.05) in May.Overall,recovery did not differ(P＞0.10) between FEC and PAT.A higher prevalence of PHAGE in PAT or WAT was associated(P＜0.01) with reduced prevalence of E.coli O157:H7-positive FEC.There was a weak but significant negative correlation between isolation of PHAGE and E.coli O157:H7 in FEC(r＝-0.11; P＜0.05).These data demonstrate that the prevalence of PHAGE fluctuates periodically similar to that described for E.coli O157:H7.Endemic PHAGE were more prevalent in manure slurry than other environmental sources.The likelihood of fecal shedding of E.coli O157:H7 was reduced if cattle in the pen harbored PHAGE.In conclusion,this study demonstrated that phages.as biological control agents have great potential to effectively control E.coli O157:H7 on farm and thus pioneered a green,safe, pollution-free strategy to ensure food safety from origin and environment safety.更多还原