Listeriosis in sheep. Isolation of Listeria monocytogenes from organs of slaughtered animals and dead animals submitted for post-mortem examination

The udders from 13 culled ewes and liver, spleen, kidney, lung and brain from 15 lambs, 11 months old, were examined for the presence of Listeria monocytogenes (Lm) at slaughter. Lm was isolated from 1 of 13 udders, from 6 of the 15 brains and from 0–4 of the other organs from each of the 15 lambs.Internal organs from 68 sheep submitted for post-mortem examination were examined in the same way. Lm was isolated from 25 of these animals. Lm was isolated from the brain of 7 of 9 animals with encephalitis, and from 0–3 of the other 4 organs examined. Lm was also isolated from 10–20 % of the organs from animals with other diagnoses. Altogether 9 of 10 animals with encephalitis and 16 of 58 with other diagnoses (28 %) were found to harbour this organism.     

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in healthy sheep.

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in healthy sheep. Acta vet. scand. 1979, 20, 168–179. — The excretion of Listeria monocytogenes (Lm) in the faeces and milk, and humoral and cell mediated immunity against Lm, were examined in a sheep flock where no cases of listeriosis had occurred during the last 3 years. The investigation was carried out during the indoor season. During the first part of the season 2 of the 10 pregnant, 8 months old lambs excreted Lm in the faeces, but none of the 106 ewes, 2–10 years old. At lambing the organism was isolated from the faeces of 6 of the 10 1 year old lambs and from 64% of the ewes, and from the milk of 1 of the lambs and 41% of the ewes. Nearly all the isolates (98.5%) belonged to serotype 1.Antibody titres against Lm were found in sera and whey by an indirect haemagglutination method. The titres were higher for the ewes than for the hoggs and seemed to be influenced by the number of foetuses the animals carried.Cell mediated immunity was determined by a skin test where delayed hypersensitivity against an antigen prepared from Lm, was measured. Animals fed grass silage had a stronger reaction than animals fed hay, and a stronger reaction was found in animals with ≥ 3 foetuses than in the remainder.The investigation indicates that even in a healthy sheep flock all the animals may be exposed to Lm, and the majority may be latent carriers and excrete this organism in the faeces and milk during periods of stress.     

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in sheep in flocks with clinical listeriosis.

The excretion of Listeria monocytogenes (Lm) in the faeces and milk, and humoral immunity against Lm, were examined in a sheep flock with outbreaks of listeric encephalitis and in a flock with outbreaks of listeric abortion. The encephalitis flock consisted of 86 ewes and 20 hoggs, the abortion flock of 45 ewes and 3 hoggs, all of them pregnant. Faecal excretion rate in the encephalitis flock varied from about 25 % in the first part of the indoor season to nearly zero 1 month later, to about 30 % 1 month before lambing and about 15 % at lambing. About 15 % of the animals also excreted Lm in the milk. Lm 4 was the dominating serotype.In the abortion flock about 2/3 of the animals excreted Lm in the faeces and 1/3 in the milk at lambing. All the isolates belonged to serotype 1, which also was isolated from grass silage and strawbedding samples.In the encephalitis flock ewes with ≥ 3 foetuses had a higher excretion rate than the remainder, while no such differences were found in the abortion flock.Antibody titres against Lm in sera and whey in the encephalitis flock were of the same order as in the healthy flock described in an earlier publication (Grønstøl 1979), except that the highest titres were found in the hoggs. Serum titres from the abortion flock after lambing were significantly higher than in the encephalitis flock, while whey titres were of the same order.Treatment with 2-mercapto-ethanol reduced the titres substantially in sera from the abortion flock, indicating that the antibodies belonged to the IgM-fraction, while only a slight reduction was seen after similar treatment of the whey.     

Listeriosis in Sheep: Listeria Monocytogenes in Sheep Fed Hay Or Grass Silage During Pregnancy. Immunological State,White Blood Cells,Total Serum Protein and Serum Iron

A comparison was made between a hay fed group, consisting of 23 ewes, and a grass silage fed group of 22 ewes, all pregnant. Excretion of Listeria monocytogenes (Lm) in the faeces and milk, antibody titres in sera and whey and delayed hypersensitivity against Lm, and several blood components were determined. The animals had previously been exposed to Lm, and Lm was isolated from the faeces from several animals when the experiment started.No significant difference in number of excretors between the 2 groups was found during the experimental period. The haemagglutination titres in both sera and whey were low and on the same level in both groups. The titres were higher in animals with 1 foetus than in animals with more than 1 foetus.In the first part of the experimental period the silage group had a reduced number of lymphocytes, lower total serum protein values and higher serum iron values, compared with the hay group.The silage group also had a stronger delayed hypersensitivity reaction against Lm than the hay group, and in the silage group the reaction was significantly stronger in ewes with 3 or more foetuses than in ewes with 1 foetus.In conclusion, the combined effect of some of the changes found in animals fed grass silage may leave them more susceptible to infections.     

Isolation of pathogenic Listeria monocytogenes in faeces of wild animals in captivity

The isolation of pathogenic Listeria spp. in faecal samples of captive wild animals was studied. Isolation of the pathogen was attempted from the samples by selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar, PALCAM agar and modified McBride Listeria agar. Pathogenicity of the isolates was tested by Christie, Atkins, Munch Petersen test, phosphotidylinositol-specific phospholipase C assay, mice inoculation test and chick embryo bioassay. Listeria monocytogenes was isolated from eight (16%) of 50 faecal samples from six different mammals and one bird. Out of eight isolates, one isolate from jackal proved to be pathogenic by all the pathogenicity testing assays. PCR amplification of virulence genes suggested that the isolate was potentially pathogenic.     

哈尔滨市鲜肉中单核细胞增生性李斯特菌的分离鉴定及耐药性分析

为了解哈尔滨市单核细胞增生性李斯特茵(Lm)的污染状况及耐药状况.在哈尔滨市市场随机采集158份鲜肉样品,采用显色培养基分离,API试剂条和PCR鉴定等方法对样品中的Lm进行分离鉴定,并通过Kirby-Barer法测定分离菌株对24种抗生素的耐药性.结果从鲜肉中共分离到Lm 23株,检出率为14.56%,其中鲜猪肉检出率最高,达20.00%(14/70);23株分离菌株中耐药菌株为22株,耐药率高达95.65%.这表明哈尔滨市鲜肉中存在一定程度的Lm污染,并且分离菌株存在较严重的耐药现象.应加强控制动物饲料亚治疗抗生素的使用并严格遵守休药期,防止耐药菌株产生进而控制食源性疾病的发生.     

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。     

产单核细胞李斯特菌毒力因子及免疫预防研究进展

产单核细胞李斯特茵在病原学上已被公认为一种人兽共患病和食源性疾病的致病菌,在各国已引起食品加工部门、公共卫生部门的高度重视和研究者的兴趣.文章就该菌的毒力因子及免疫预防研究做一简述.     

单核细胞增生症李斯特菌的分子亚分型法及其应用

细菌亚分型方法的建立不仅能检测人群李斯特菌病的暴发流行 ,还能追踪食物链中单核细胞增生症李斯特菌 (L M)的污染情况。亚分型法对更好的了解 LM的群体遗传学、流行病学及生态学有重要意义。过去的 5年内 ,在建立对 LM敏感、快速、自动化、简便易用的分子亚分型方面起得了重大的进展。文章对 L M不同的亚分型方法及其应用作了概述     

环境因子及接种量对单增李斯特菌生长的影响

为探究不同环境因子及接种量对单增李斯特菌（LM90）生长特性的影响,本试验通过测定LM90在各培养条件下的D_600nm值,分析了不同的温度、NaCl浓度、pH,以及温度（0~40℃,梯度为10℃）、NaCl浓度（3%~8%,梯度为1%）、pH （6~9,梯度为1）的交互作用下LM90的生长状况。结果显示,菌株的对数增长期为8~16 h,稳定期为16~20 h,20 h以后进入衰亡期;菌株在NaCl浓度为0.5%~4%时生长良好,其最适生长pH为7.5,最适生长温度为37℃。通过SPSS 20.0软件进行方差分析可知,各因素的交互作用均对LM90有极显著影响（P＜0.01）。本研究为进一步探讨温度、NaCl浓度、pH等因素对LM生长的影响及LM抗环境胁迫的作用机制奠定了基础。     

Effect of Environmental Factors and Different Inoculum Size on Growth State of Listeria monocytogenes

In order to explore the growth characteristics of the Listeria monocytogenes (LM90) strain under different environmental factors and inoculum size,the D_600nm value of bacteria culture broth were measured,the growth status of LM90 under different NaCl concentration,pH value,temperature and the interactions of NaCl concentration (3% to 8%,1% for per gradient),pH (6 to 9,one for per gradient),temperature (0 to 40℃,10℃ for per gradient) had been analyzed. The results showed that the strain was in the logarithmic phase when culturing for 8 to 16 h,then entered the stationary phase when culturing for 16 to 20 h,and finally went to decline phase when culturing after 20 h. It could grow well when the NaCl concentration was 0.5% to 4%. Its optimum growth pH and temperature were 7.5 and 37℃,respectively. The interaction effect of each two factor had been analyzed by variance analysis of SPSS software,and it found that the interaction effects was extremely significantly (P <0.01).The study lay a foundation for exploring the effects of environmental factors on LM90 growth and mechanisms of resistance to environmental stresses.     

Listeria Monocytogenes Excretion and Humoral Immunity in Goats in a Herd with Outbreaks of Listeriosis and in a Healthy Herd

In a herd of 65 goats with outbreaks of listeriosis (Herd A) blood, faeces and milk were collected just after the outbreaks, about 1 month later and at delivery about 4 months thereafter. Faeces and milk were examined bacteriologically and blood and milk serologically for Listeria monocytogenes (Lm), and the results were compared with those of 2 similar samplings in a healthy herd (Herd B).In Herd A Lm was isolated from faeces in 5 of 14 septicaemic does and in 6 of 48 other animals on the first sampling, and in 4 and 1 animals respectively, on the subsequent 2 samplings. In milk Lm was demonstrated just after the outbreaks only, viz. in 3 of 12 septicaemic does and in 16 of the other 32 examined. Four does excreted Lm in both faeces and milk on this date. In Herd B Lm was demonstrated only at delivery, i.e. from 10 of 43 animals. Most of the isolates belonged to serotype 1.Reciprocal geometrical mean titres (GMT) of antibodies in sera from the septicaemic group decreased from 236 to 140 and 136 respectively on the subsequent samplings, whereas GMT of the encephalitic animals and of the remainder of Herd A increased from about 20 to about 100 at delivery. GMT of Herd B increased toward delivery from 23 to 39, with largest increase for the does. GMT in whey were ≤ 18 for all groups.     

单增李斯特菌lmo2193基因克隆与原核表达

为了对单增李斯特菌新疆绵羊脑炎临床分离株LM90SB2的lmo2193基因进行克隆及其原核表达,采用PCR方法扩增lmo2193基因,连接pMD19-T载体进行克隆,筛选阳性菌进行测序比对。将目的基因克隆至原核表达质粒p ET32a中,构建重组质粒pET32a-2193,并转化大肠杆菌感受态细胞,经诱导表达后,利用SDS-PAGE和Western blot鉴定重组蛋白。结果显示:扩增得到的lmo2193基因序列长度为1 077 bp,与预期一致;该基因在大肠杆菌中大量表达,经SDS-PAGE检测和Western blot鉴定分析表明该产物为1个60 ku左右的融合重组蛋白。本研究成功克隆lmo2193基因,并获得大量表达,为进一步研究lmo2193基因功能奠定基础。     

单核细胞增生性李斯特菌的主要毒力因子及其致病机理

单核细胞增生性李斯特菌(Listeria monocytogenes,LM)是重要的食源性人兽共患病原菌,LM的致病性与毒力因子密切相关,研究其毒力因子对于充分了解李斯特菌病的致病机制及有效防制该病有重要意义。作者对LM的毒力因子(如李斯特溶血素、肌动蛋白聚合蛋白、C型磷脂酶、内化素、细胞壁水解酶、酰胺酶及毒力调节因子如转录活化因子和应答调控因子等)和致病机理进行了综述。     

产单核细胞李斯特菌的PFGE分子分型分析

对50株不同来源的产单核细胞李斯特菌（Lm）进行PFGE分型,比较食源、环境、人源菌株之间的相似性。结果显示,同一市场内,环境污水分离株和新鲜食品分离株表现出较高同源性,新鲜食品分离株与即食性（RTE）食品分离株相似性很高,临床脑膜炎病人的1株Lm与RTE食品分离株在进化上显示出了很近的亲缘关系。50株Lm的分子分型分析提示了菌株从市场环境污水-新鲜食品-RTE食品-人的传播模式,为食源性疾病溯源追踪提供了新的思路。     

李氏杆菌溶血素基因的表达及活性分析

根据LMO溶血素基因hlyA设计引物,PCR扩增hlyA,将扩增产物与pMD18-T连接,重组质粒经酶切鉴定、PCR分析以及确证性测序。将由重组质粒pMD18-hlyA上扩增的缺失信号肽序列的目的片段与表达载体DGEX-4T-1分别酶切连接后,转化BL21细胞。筛选阳性克隆,用IPTG诱导表达,SDS-PAGE和Western blot分析,纯化重组融合蛋白进行溶血试验、小鼠致病力试验和间接ELISA分析。结果表明hlyA基因在大肠杆菌中成功表达分子量82Ku的融合蛋白,能裂解红细胞,且能被LMO阳性血清所识别。一定剂量腹腔注射能将小鼠致死。以此为包被抗原的间接ELISA可以将LMO阴、阳性血清分开。这表明GST-LLO融合蛋白具有良好的生物活性,可用于李氏杆菌病的间接ELISA诊断。     

Survival of Listeria monocytogenes in wilted and additive-treated grass silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10(6)-10(7) cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in foodborne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8 x 10(5)/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25 degrees C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10(2) and 10(6) cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).     

单增李斯特氏菌溶血素基因的克隆及原核表达

参考GenBank收录的单增李斯特菌Hly基因序列,设计1对引物,采用PCR技术扩增出单增李斯特氏菌的溶血素基因Hly(不含有信号肽部分),得到一条1590bp的条带。将其连入pMD18-T载体,经酶切、PCR鉴定和序列测定法进行鉴定。测序正确后,将该基因插入到pET-28a中构建原核表达载体pET-28a-sHly,将重组质粒转化到大肠杆菌BL21(DE3),经IPTG诱导,将诱导产物用SDS-PAGE和Western-blot鉴定。结果显示,Hly基因可以在大肠杆菌中获得表达,表达产物分子质量约为65kU,与预期蛋白质分子质量大小一致。经Western-blotting鉴定可知,诱导表达产物以可溶形式存在,可被兔抗LM阳性血清特异识别,具有较好的抗原活性,为进一步研制基于溶解素蛋白的诊断抗原和特异性单克隆抗体,开展LM的致病与免疫机理研究奠定基础。     

单增李斯特菌新疆分离株lmo2192基因克隆及生物信息学分析

试验旨在对单增李斯特菌新疆绵羊脑炎临床分离株LM90SB2的lmo2192基因进行克隆及生物信息学分析。根据GenBank中lmo2192基因序列（登录号：CAD00270）设计特异性引物,利用PCR方法对lmo2192基因进行扩增,回收目的基因,连接到pMD19-T载体上进行克隆,筛选阳性菌进行测序,测序后对lmo2192基因核苷酸序列进行分析,预测其编码蛋白质的二级结构、三级结构,对其进行同源性比对及遗传变异分析。结果显示,新疆分离株LM90SB2的lmo2192基因序列全长为1 277 bp,包含969 bp开放阅读框,共编码322个氨基酸;LM90SB2株lmo2192基因核苷酸序列与CⅡMS-PH-1同源性为100.0%,与81-0861、10-0809、81-0592、81-0558、NTSN、F2365和WSLC1033的同源性为99.8%~99.9%,与L2074和NH1同源性分别为97.0%和96.9%。推导的氨基酸序列同源性为91.6%~100.0%。系统进化树显示,LM90SB2菌株lmo2192基因与4b血清型菌株亲缘关系较近,聚为同一分支。蛋白质二级结构预测表明,LM90SB2 lmo2192蛋白为亲水性蛋白,无信号肽,不形成跨膜结构。蛋白结构域预测,lmo2192蛋白为ATP酶组分。本试验成功克隆LM90SB2分离株lmo2192基因,为进一步研究其基因功能提供理论依据。     

单增李斯特菌llsB基因克隆及序列分析

试验旨在对新疆绵羊脑炎临床分离株LM90SB2单增李斯特菌llsB基因进行克隆及生物信息学分析,以期进一步完善单增李斯特菌溶血素S的功能研究。根据GenBank中单增李斯特菌F2365基因全长序列（登录号为：AE017262）设计其特异性引物,利用PCR方法对新疆分离株LM90SB2的llsB基因进行扩增,回收目的基因与pMD19-T载体连接,采用PCR、双酶切鉴定筛选阳性菌并进行测序,对所获序列进行同源性比对及遗传变异分析。结果显示,新疆分离株LM90SB2的llsB基因序列全长为876 bp,共编码291个氨基酸;LM90SB2分离株llsB基因核苷酸序列与10-0809、81-0592、81-0558、02-1792、NTSN、02-1289不同分离株同源性均为100%,与CⅡMS-PH-1、NRRLB-57603株的同源性均为99.9%,与J1816、R2-502株的同源性为44.7%~45.0%。分子进化树显示,LM90SB2菌株llsB基因与血清型为4b的菌株亲缘关系较近,聚类为同一分支。蛋白质二级结构预测表明,LM90SB2 llsB蛋白为亲水性蛋白,无信号肽,不形成跨膜结构。本试验成功克隆了LM90SB2株llsB基因,为深入探讨该基因功能提供全面的理论依据。