冷冻食品中单核细胞增多性李斯特菌的分离鉴定

随机从锦州市的超市中采集速冻食品,通过细菌的增菌、分离纯化、生化鉴定确定是单核细胞增多性李斯特菌。通过细菌的血清学鉴定确定7株单增李斯特菌的血清型分别是3株为1/2b,2株为1/2 a,2株为1/2 c。通过本研究了解单增李斯特菌在速冻食品中的污染情况,为防治疾病提供依据。     

单核细胞增多性李斯特菌hlyA基因序列及溶血素活性测定

从3株不同来源的单核细胞增多性李斯特菌中PCR扩增溶血素编码基因hlyA,连接到T克隆载体转化到受体菌DH5α中,经菌落PCR和提取质粒酶切鉴定后测序,对测定的hlyA基因核苷酸序列及推导的氨基酸序列进行分析,结果表明,从3个菌株克隆的hlyA基因具有完整的开放阅读框,同源性在96.9%以上;其推导的LLO氨基酸序列同源性在97.9%以上,N端存在相同的PEST基序,C端存在相同的保守性11肽结构.建立和运用分光光度法对3个试验菌株的溶血素活性进行了检测,结果表明,在pH5.6时LLO溶血活性最高,在pH7.0时LLO溶血活性基本丧失.本试验单核细胞增多性李斯特菌的流行病学分析和基因工程疫苗载体研究提供了依据.     

单核细胞增多性李斯特菌的毒力因子及检测研究进展

单核细胞增多性李斯特菌是危害公共卫生和食品行业的一种重要人畜共患病致病菌,笔者就单核细胞增生李斯特氏菌的主要毒力因子、特异性鉴别基因和检测方法进行综述。     

单核细胞增多性李斯特菌LIPI-1全基因克隆与序列分析

参考GenBank中单核细胞增多性李斯特菌（LM）第I毒力岛（LIPI-1）有关基因序列设计引物,分段扩增、克隆了分离菌株XFL0605LIPI-1毒力岛全基因序列,序列全长8 558 bp,包括完整的prfA、plcA、hly、mpl、actA和plcB6个基因。对LIPI-1编码的6个毒力基因进行序列分析,各毒力基因反映的进化关系不尽一致,表明李斯特菌株在获得外源性毒力基因方面具有不同步性,各毒力基因来源也不尽相同。应用相应软件对各毒力基因编码蛋白的信号肽、跨膜区进行预测,确定了基因序列中调控蛋白PrfA结合区核苷酸序列。分析并确认了hlyA基因及推导氨基酸序列PEST基序和C端保守11肽序列。研究还发现LM菌株XFL0605actA基因编码604个氨基酸,缺失了105 bp富脯氨酸重复片段编码碱基,只有2个E/DFPPPPXD/E重复序列。     

单核细胞增多性李斯特菌LMO1847基因在不同应激条件下的表达

根据GenBank登陆的单增李斯特菌标准株EGD序列设计引物,PCR扩增LM01847基因片段后插入表达载体pET-30 a,构建重组原核表达载体,并在E. coliBL21中成功表达。纯化目的蛋白制备多抗,ELISA结果显示:该蛋白多抗与单增李斯特菌全菌、全菌多抗与该蛋白均可发生反应。证明该蛋白在天然状态下存在于单增李斯特菌表面,且具有良好的免疫反应性。该蛋白在45℃、高盐、酸性环境中表达水平基本稳定,但在低pH值条件下反应活性消失;高盐或低温条件下,表达量随盐浓度的升高或温度的下降而降低。除4℃以外,该蛋白均能保持良好的免疫反应活性。本研究为建立针对该蛋白的李斯特菌免疫微球凝集、免疫磁珠分离等检测方法奠定基础。     

单核细胞增生性李斯特氏菌hly基因的克隆表达

以单核细胞增多性李斯特氏菌（Listeria Monocytogengs,LMO）LMO-0586基因组DNA为模板,运用PCR扩增得到片段大小为1646bp的致病基因李氏溶血素（hly）基因,并克隆到pMD18-T载体上,构建克隆载体pMD18-T-hly。经测序正确后,将hly基因克隆至表达载体pGEX上构建表达质粒pGEX-6P-1-hly。在IPTG诱导下,携带pGEX-6P-1-hlyA的E．coli BL21（DE3）高效表达分子量约为82KDa的可溶性蛋白及包涵体形式的蛋白。为进一步研究溶血素蛋白的结构、功能,LMO的分子流行病学调查、诊断试剂盒的开发提供依据。     

单核细胞增生性李斯特菌检测研究进展

单核细胞增生性李斯特菌是近年来又重新抬头出现的病原微生物,已在世界许多港口被检测到,并造成了一定的经济损失被认为是影响畜禽和人类健康的重要病原微生物之一。文章就单核细胞增生性李斯特菌检测方法的研究进行了系统的综述,为控制该病原微生物的传播和蔓延提供参考。     

单核细胞增生性李斯特菌感染ICR小鼠模型的建立

单核细胞增生性李斯特菌(Listeria monocytogenes,LM)是一种人畜共患食源性致病菌,能引起人和动物较为严重的感染症状。现广泛使用具有免疫活性的小鼠模型测定半数致死量(LD₅₀)来评价不同LM菌株的致病性。为建立LM的ICR小鼠模型,本研究采用1/2a血清型的N21 LM菌株,分别以10⁶、10⁷、10⁸、10⁹和10¹⁰ CFU的剂量口腔灌注感染6周龄ICR小鼠,每组10只;另取10只接种PBS作为对照组,测定LM对ICR小鼠的LD₅₀;另取40只小鼠,平均分为2组,公母各半,以测定的LD₅₀剂量接种LM,分别对各组小鼠临床症状、组织病理变化、体重变化及组织中细菌载量进行评价。结果发现,N21 LM菌株对ICR小鼠的LD₅₀为10^9.25 CFU;感染周期为10 d左右,感染小鼠出现被毛粗糙、精神萎靡、阴茎垂出及体重下降等临床症状。组织病理学分析结果显示,肝脏先后出现实质内灶状细菌团块、形成血栓、细胞坏死等;脾脏主要表现为白髓内淋巴细胞减少;肺脏主要表现为纤维素性肺炎。菌落计数和Real-time PCR的检测结果发现肝脏中细菌载量最高,脾脏次之。结果表明,ICR小鼠能作为单核细胞增生性李斯特菌的感染模型。本试验结果为研究LM的致病机制、疫苗研究及抗菌肽转基因小鼠抗LM的评价奠定了基础。     

单核细胞增生性李斯特氏菌及其检验

单核细胞增生性李斯特氏菌是近年来又重新抬头出现的病原微生物,已在世界许多港口被检测到,并造成了一定的经济损失,被认为是影响畜禽和人类健康的重要病原微生物之一。本文就单核细胞增生性李斯特氏菌的生物学特性、流行病学、致病性及检验等方面进行了系统的综述,为控制此病原微生物的传播和蔓延提供了重要的理论和实践依据。     

Effects of dexamethasone on shedding of Listeria monocytogenes in dairy cattle

Ten lactating Holstein cows that had been given multiple injections of Listeria monocytogenes (serotype 4B, Scott A strain) via the intramammary route were allotted to 2 groups: group 1 (n = 5) was treated with the synthetic glucocorticoid, dexamethasone (0.04 mg/kg of body weight), for 3 consecutive days, and group 2 (n = 5) served as controls. Two days after the initial dexamethasone injection, the number of L monocytogenes in the milk had increased nearly 15-fold (1.16 log10) over pretreatment values. On day 3, Listeria numbers in the milk had increased by 1.83 log10, compared with pretreatment values. By day 4, Listeria numbers in the milk were approximately 100-fold (2.03 log10) greater than pretreatment numbers. Numbers remained high through day 7 and, by day 11, approached pretreatment numbers. Dexamethasone administration was accompanied by high total WBC and milk somatic cell counts and decreased eosinophil and lymphocyte numbers, and decreased milk production. The increase in shedding of L monocytogenes in the milk may reflect impairment of cell-mediated immune mechanisms and phagocytic cell functions that are critical for sustaining listerial immunity.     

猪血凝性脑脊髓炎病毒感染降低ZO-1表达致小鼠血脑屏障通透性升高

为明确猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus,PHEV)感染后对机体血脑屏障(blood-brain barrier,BBB)通透性的影响,本试验以PHEV易感的BALB/c小鼠为实验动物模型,将PHEV接种小鼠后静脉注射伊文氏蓝(Evans blue,EB),运用形态学和化学定量方法检测EB通过BBB进入脑组织的状况。结果显示,接种病毒后3d的小鼠脑组织即出现肉眼可见的蓝色,定量检测结果证实其含量随着感染时间的延长而增加。同时利用荧光定量PCR和Western blot方法分别对病毒感染后不同时间点紧密连接蛋白ZO-1、Occludin和Claudin-5进行核酸定量检测,证明感染组小鼠脑组织中ZO-1的基因转录水平随感染时间延长下调明显,而Occludin和Claudin-5的变化不明显。进而对脑组织中ZO-1蛋白表达水平进行检测,证实ZO-1的蛋白表达水平也发生下调,表明内皮细胞紧密连接完整性在病毒感染后期遭到了破坏。上述试验结果证实PHEV感染机体后可使BBB的通透性升高,而且这一变化与ZO-1的降低相关,为后期深入开展PHEV所致神经系统炎性损伤的机制奠定基础。     

Prolonged persistence of Listeria monocytogenes after intragastric infection in corticosteroid-treated mice

In an attempt to obtain a model more closely resembling natural listeriosis, we studied the course of infection in mice inoculated by the intragastric route with Listeria monocytogenes. Corticosteroid-treated, and untreated mice both developed subclinical infection without mortality, but faecal shedding and peristence of bacteria in the liver and spleen of corticosteroid-treated mice were significantly more protracted than in untreated mice. Untreated mice cleared the bacteria from their livers and spleens by day 5 postinfection (PI), whereas treated mice did not clear the organisms until 8–9 days PI. In untreated mice faecal shedding lasted 5 days PI, whereas in treated mice the organisms were recovered at significantly higher levels until day 9 PI. The only intestinal lesions observed were mild pyogranulomatous changes in the dome area of some Peyer's patches in treated mice.     

Listeria monocytogenes in food]

As in recent years laboratory diagnostics of listeria has become part of food microbiology, the frequency of occurrence of the bacteria Listeria monocytogenes has been followed in various kinds of foods for a year. A total of 51 strains of L. monocytogenes (7.2%) was isolated from 700 kinds of samples (raw milk, pasteurized milk, meat surface, poultry, cheeses, thermally not treated meat products, food--industry machinery). As can be seen in Tab. I, the highest number of strains was isolated from meat surfaces (13.5%), followed by meat--industry machinery (12.72%), poultry (10%) and cheeses (5%). The lower numbers of strains were found out in thermally not treated meat products (3.8%) and in raw milk (3.3%). Pasteurized milk did not contain any strains. Our findings in raw milk (3.3%) and in pasteurized milk (0) are in agreement with the data cited e. g. by authors from the USA (Lovett et al., 1987), who mention the value of 4.2% in raw milk and the zero value in pasteurized milk. The percentage of strains monitored in cheeses (5%) can be evaluated as low as the assortment of investigated cheeses was small (all strains were isolated from soft ripening cheeses). German authors (Tham et al., 1988) speak about the 2.5% percentage of L. monocytogenes strains; this is in keeping with our findings. The findings in thermally not treated meat products (3.8%) can be evaluated as low although the number of strains found in raw meat was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Listeria monocytogenes gastroenteritis in sheep

Extract

Listeria monocytogenes is widely distributed in the environment of farm animals. It is found in soil, faeces and nasal secretions of healthy animals, water troughs and animal feeds. Under certain conditions it becomes pathogenic, causing serious disease in cattle and sheep. Most commonly it causes meningoencephalitis but, on occasion, septicaemic listeriosis results in abortion, and more recently it has been associated with gastroenteritis.     

Induction of acquired cellular resistance in mice with viable and macrophage-processed Listeria monocytogenes

Intracutaneous immunization of mice with 10(5) or 10(6) viable listeria resulted in acquired cellular resistance (ACR) of short duration (7 days). The period during which viable Listeria monocytogenes had to be present in order to induce ACR was estimated by killing the listeria at different times after immunization by injecting the bactericidal antibiotic amoxycillin. The killing of listeria within 6 h after injection prevented the induction of A CR completely, between 6 and 12 h partially, while survival of listeria within animals for at least 18 h was required for the induction of complete protection. To determine whether multiplication of viable listeria was a prerequisite for the induction of ACR, the bacteriostatic antibiotic minocycline was injected for four days after immunization. Induction of ACR was only possible if the dose of viable listeria was large enough to permit a proportion of the listeria to escape bacteriostasis. Interaction of peritoneal macrophages of normal mice and viable listeria yielded a supernatant which induced specific ACR in normal recipient mice. No ACR could be induced with supernatant obtained from normal macrophages after digestion of killed listeria. A reduced level of ACR was obtained with supernatant collected after interaction of macrophages from immune mice and viable listeria. The immunogenic material present in the supernatant of normal macrophages after interaction with viable listeria is thermolabile, has a molecular weight of over 300,000, and is not affected by treatment with DNase, RNase, or trypsin.