【作者】 梁启平；

【导师】 方维焕； 黄绍棠；

【作者基本信息】 浙江大学 ， 微生物与食品安全， 2009， 硕士

【摘要】 单增李斯特菌(Listeria monocytogenes)是重要的人兽共患食源性病原菌,可分为3个谱系(Lineage)与13个血清型,其中谱系Ⅰ包括1／2b、3b、4ab、4b、4d、4e和7型,谱系Ⅱ包括1／2a、1／2c、3a和3c型,谱系Ⅲ包括4a和4c型。单增李斯特菌具备抵抗各种应激(高盐、低pH、氧化应激等)的能力,主要通过污染食品或饲料感染人和动物。针对宁波口岸在2007年7月至2008年11月从北美洲、南美洲、欧洲以及亚洲共29个国家进口的1275批水产品,进行单增李斯特菌鉴定、谱系与血清型分析、小鼠毒力试验与多位点序列分析。单增李斯特菌在8个国家共计33个批次中检出,污染率为2.6%；其中智利的16个批次中,单增李斯特菌污染率高达31.3%；阿根廷／乌拉圭与美国次之,分别为11.5%与11.1%；越南、印度、秘鲁与墨西哥分别为5.9%、1.8%、1.8%与1.0%；而其余21个国家均未检出。在检出的33株单增李斯特菌中,选择23株代表不同国家与时间段(即批次不相连)的菌株进行分析。其中19个菌株(82.6%)属于谱系Ⅰ,4个菌株(17.4%)属于谱系Ⅱ,无谱系Ⅲ菌株。在谱系Ⅰ菌株中,4b型占78.9%[15／19,占总菌株的65.2%(15／23)]；而1／2b型占总菌株的17.4%(4／23)。在谱系Ⅱ菌株中,1／2a型与1／2c型各占总菌株的13.0%(3／23)与4.4%(1／23)。这些分离株对小鼠均具有与强毒参考株相当的毒力。选择毒力基因actA、看家基因ribC与应激调控基因sigB,对23个分离株及9个参考株进行了分析,其中hisJ含有比例最高的多态性位点,达15.2%; ribC次之,为15.0%；而actA与sigB分别为10.7%与5.6%。但actA却可分辨出最多的序列型(Sequence Type, ST),为19个；hisJ、ribC与sigB可分别分辨出16、11与7个序列型。actA的分辨力亦为最强,达0.95；hisJ为0.90；而sigB与ribC分辨力较弱,仅为0.80与0.77。将4个基因串联后,多态性位点比例为11.81%,可分辨出23个序列型,分辨力达0.97。在这些分离株中,来自阿根廷鱿鱼的N1、N14、N15与N16同为序列型8,来自智利鱿鱼的N6、N7与N8同为序列型10,而来自美国三文鱼的N4、来自智利鱿鱼的N13与来自秘鲁鱿鱼的N23同为序列型9。这3个主要序列型均分布于谱系Ⅰ的进化枝上。谱系Ⅰ共22个菌株(含参考株,下同),分为15个序列型；而谱系Ⅱ的7个菌株可分为5个序列型,谱系Ⅲ的3个菌株可分为3个序列型。其中17个4b型菌株形成10个序列型,而5个1／2b型菌株、5个1／2a型菌株、2个4a型菌株均各成一型,表明4b型菌株具有较高的同质性。研究结果表明,进口水产品中单增李斯特菌污染率与国内水产品相近,但部分南美国家的水产品中的检出率特别高,血清型分布以4b型为主,且有流行性克隆Ⅰ检出。因此,对进口水产品中李斯特菌污染的潜在危险性不容忽视,特别是要加强对单增李斯特菌检出率较高的部分南美国家水产品的监测。更多还原

【Abstract】 [Objective] We examined the prevalence, molecular characteristics and virulence potential of Listeria monocytogenes isolates from imported fishery products in an attempt to gain further insights on the public health risk caused by this important foodborne pathogen. [Methods] L. monocytogenes isolates, screened from 1275 batches of fishery products imported from July 2007 to November 2008, were studied by lineage classification, serotyping, in vivo virulence assessment in ICR mouse model and multilocus sequence typing (MLST). [Results] Thirty-three batches were contaminated by L. monocytogenes (33/1275,2.6%), in which serovar 4b dominated (65.2%),followed by serovars 1/2b (17.4%), 1/2a (13.0%) and 1/2c (4.4%). These isolates were all as virulent as L. monocytogenes reference strains. Of 23 selected L. monocytogenes isolates and 9 reference strains,23 sequence types (STs) were recognized with discrimination index (D.I.) of 0.97, based on the concatenated gene cluster covering one virulent gene actA, two housekeeping genes hisJ and ribC, and one stress-response gene sigB. Remarkably,3 isolates from American salmon, Argentina squid and Peru squid respectively belonged to ST9 which represented epidemic clone I (ECI). [Conclusion] The isolation rate of L. monocytogenes in imported fishery products (2.6%) was similar to that in domestic fishery products (2.7%). Serovar 4b, which was more virulent than other serovars in humans, was dominant in imported fishery products, and even ECI was recognized. All of these isolates were as virulent as L. monocytogenes reference strains, suggesting inspection and quarantine strategies of imported fishery products should be strengthened effectively.更多还原