伊犁地区绵羊李氏杆菌药敏特性及平板凝集快速诊断方法的建立

[目的]建立一种快速检测李氏杆菌病的方法。[方法]通过对实验室保存的5株李氏杆菌分离株进行药敏试验,用甲醛灭活苗免疫试验动物,制备特异性抗体,将特异性抗体与抗原进行平板凝集试验。[结果]分离菌株对氨苄西林、庆大霉素、链霉素高度敏感,对头孢唑啉、左氧氟沙星和多粘菌素中度敏感,对头孢他啶、头孢呋辛钠耐药性较强。通过用甲醛灭活苗免疫试验动物来制备特异性抗体,将特异性抗体与抗原做平板凝集试验,建立一种快速准确检测李氏杆菌病的方法。[结论]成功建立了一种快速检测李氏杆菌病的方法。     

多花黑麦草幼穗分化进程对种子生产性状的影响

两年分期播种试验结果表明，多花黑麦草幼穗分化进程对穗部性状和种子产量有显著影响。幼穗分化的天数与每穗种子粒数呈极显著正相关。播种愈早，幼穗分化天数愈多，种子产量愈高。南京地区留种的多花草麦草的最佳播期为８月２０日至９月１０日。二棱期是多花黑麦草通过春化阶段的形态标志，也是幼穗能否分化完全的转折点。单棱至护颖分化期是影响种子粒数最关键的时期。     

猫尾草的栽培管理初探

本文对猫尾草种植过程中的整地、单播、混播、灌溉、病虫害、刈割和施肥、轮作、种子及其生产方面的栽培管理等情况进行初步论述,结合国内实际情况提出了建设性建议。     

单增李斯特菌lmo0331基因的克隆、序列分析及原核表达

试验旨在对单增李斯特菌临床分离株（LM90SB2）的LPXTG基序表面蛋白lmo0331基因进行克隆、序列分析和原核表达。根据GenBank中lmo0331基因序列设计特异性引物,利用PCR方法扩增LM90SB2 lmo0331基因,将扩增产物克隆到pMD19-T载体,测序后进行核苷酸序列和蛋白结构域分析;构建表达质粒pET32a-0331,转化大肠杆菌BL21（DE3）感受态细胞,经IPTG诱导后,利用SDS-PAGE和Western blotting检测重组蛋白。结果显示,LM90SB2 lmo0331基因核苷酸序列与NTSN株、F2365株、LL195株及WSLC 1033株的同源性均为100.0%,与N2306株、02-1792株、H34株的同源性均为99.9%。LM90SB2 lmo0331基因全长1 956 bp,ORF全长为1 857 bp,共编码618个氨基酸;蛋白结构域预测结果显示,lmo0331蛋白具有NEL、LRR、IR、PulA、LPXTG结构域。SDS-PAGE结果可见约88 ku重组蛋白带,Western blotting表明该蛋白可与小鼠抗His标签单克隆抗体发生特异性结合。本研究成功构建了原核表达载体pET32a-0331,实现了lmo0331融合蛋白的表达,为进一步研究lmo0331基因的功能及单克隆抗体的制备奠定了基础。     

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。     

Development of A Dual Real-time PCR for the Rapid Detection of Listeria monocytogenes

To establish a rapid assay for Listeria monocytogenes(LM) detection,a Real-time PCR method was developed targeting iap gene of LM.The results showed that the test for 15 bacteria strains,only LM was positive,indicated that the method had high specificity.In addition,the sensitivity of Real-time PCR was 6.5 CFU/mL.Stability and reproducibility of the test showed that the coefficient of variation for the same sample repeat the Ct values were less than 2%.Furthermore,a total of 3 positive samples for LM were detected from 139 clinical samples by the method,which was in accordance with the testing result by GB 478930-2010 standard detection protocol.Therefore,the Real-time PCR method provides a novel rapid,sensitive and good repeatability detection method for LM infection.     

Listeriosis in sheep. Isolation of Listeria monocytogenes from grass silage.

Two hundred and ninety-one grass silage samples from 113 farms with recent outbreaks of listeriosis were examined for the presence of Listeria monocytogenes (Lm). The frequency of Lm isolations increased with increasing pH. Lm was isolated from 22 % of the samples with pH < 4, from 37 % with pH 4–5 and from 56 % with pH > 5. Formic acid had been used as additive.A similar investigation was carried out on 32 samples from a farm with no outbreak of listeriosis during the investigation period. Lm was isolated from 9 samples.     

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.     

Evaluation of indirect and avidin-biotin enzyme linked immunosorbent assays for detection of anti-listeriolysin O antibodies in bovine milk samples

Listeria monocytogenes is a foodborne pathogen that causes a wide spectrum of diseases in humans and animals. Enzyme linked immunosorbent assays (ELISA) [indirect and avidin-biotin (A-B)] for detecting L. monocytogenes antibodies in bovine milk samples (n = 2060) were standardized and evaluated by comparison with bacteriological examination. The tests were standardized by checker board titration. Highly purified listeriolysin O (LLO) was used as an antigen. Receiver operating characteristic (ROC) analysis was performed to decide the cut-off values. The ROC analysis revealed the sensitivities of indirect and A-B ELISA as 100% and specificities as 97.1 and 99.9% respectively. Listeria monocytogenes was isolated from 105 (5.1%) milk samples collected from 52 farms. Anti-LLO IgG antibodies were detected from 137 and 112 milk samples when tested by indirect and A-B ELISA respectively. Of the 52 farms screened, 28 (53.8%) yielded one or more isolates of L. monocytogenes and 33 (63.5%) of the farms had one or more animals simultaneously positive by one or both the assays for anti-LLO antibodies.     

Survey on the Listeria contamination of ready-to-eat food products and household environments in Vienna, Austria

Qualitative and quantitative contamination of ready-to-eat food-stuffs with the pathogen Listeria monocytogenes was studied in 1586 samples collected from 103 supermarkets (n = 946) and 61 households (n = 640) in Vienna, Austria. Seventeen groups of ready-to-eat foods were classified into three risk categories for contamination (CP1-CP3). Three to four samples were randomly collected at the retail level from each CP. Regarding the households, the sampling procedure was started with food items of CP1, and if not available, was continued with sampling of food items of CP2 and finally of CP3. Additionally, 184 environmental samples (swabs from the kitchen area, dust samples from the vacuum cleaner) and faecal samples (household members and pet animals) were included. One-hundred and twenty-four (13.1%) and 45 (4.8%) samples out of 946 food samples collected from food retailers tested positive for Listeria spp. and L. monocytogenes, respectively, with five smoked fish samples exceeding the tolerated limit of 100 CFU/g food. Food-stuffs associated with the highest risk of contamination were twice as frequently contaminated with L. monocytogenes as food-stuffs associated with a medium risk of contamination. Products showing the highest contamination rate were fish and seafood (19.4%), followed by raw meat sausages (6.3%), soft cheese (5.5%) and cooked meat products/patés (4.5%). The overall contamination rate of foods collected at the household level was more than two times lower. Only 5.6% and 1.7% of 640 food-stuffs analysed tested positive for Listeria spp. and L. monocytogenes, respectively. However, CP1 foods were rarely collected. Pulsed-field gel electrophoresis (PFGE) typing of the collected L. monocytogenes isolates revealed a high degree of diversity between the isolates, with some exceptions. PFGE typing of isolates harvested from green-veined cheese revealed a match among strains, although the manufacturer seemed to be distinguishable. Typing of household strains revealed an epidemiological link within one family. In this case, food-stuffs and the kitchen environment were contaminated by an indistinguishable isolate. In addition, the same isolate was collected from a pooled faecal sample of the household members suggesting that consumption of even low contaminated food items (<100 CFU/g) results in Listeria shedding after the passage through the gut.