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本研究以纯化的多杀性巴氏杆菌毒素基因片段的原核表达产物作为抗原免疫小鼠制备单抗,并利用表达蛋白和多杀性巴氏杆菌毒素单抗酶结合物建立了竞争ELISA方法检测多杀性巴氏杆菌毒素抗体。经过研究确定抗原包被浓度为223ng/mL,待检血清最佳稀释度为1:2,酶标单抗工作浓度为1:3200,血清抑制率大于50%为阳性。应用单抗竞争ELISA和细胞毒性中和试验同时对82份血清进行猪多杀性巴氏杆菌毒素抗体检测,竞争ELISA的检出率为40.2%,细胞毒性中和试验检出率为36.6%,两者符合率达91.5%。试验结果表明,该ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。本方法的建立在实验室诊断的标准化、猪群萎缩性鼻炎疫苗免疫效果的评价及流行病学调查方面具有应用价值。 相似文献
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制备兔多杀性巴氏杆菌(Pasteurella multocida,Pm)外膜蛋白A(OmpA)重组蛋白单抗,为兔多杀性巴氏杆菌病的诊断提供特异性强,灵敏度高的单克隆抗体。本研究选取并扩增Pm外膜蛋白基因OmpA,原核表达获得了的大小为37.6kD的OmpA重组蛋白,以纯化复性的兔多杀性巴氏杆菌OmpA重组蛋白作为免疫原,按常规方法免疫BALB/c小鼠(Mus musculus),取其脾细胞与SP2/0细胞融合,ELISA筛选出了4株分泌Pm OmpA蛋白单克隆抗体的杂交瘤细胞,分别命名为5D2、BC11、2A2和6A4。其中2A2细胞培养液效价为1∶256,5D2、BC11和6A4效价为1∶128;2A2腹水效价为1∶12800,其余3株达1∶6400。杂交瘤细胞经反复冻存、复苏及多次传代,仍能稳定分泌高效价抗体。Western blot显示,4株单克隆抗体均能与Pm OmpA重组蛋白重组发生特异性反应。用ELISA方法鉴定2A2单克隆抗体亚型为IgG2b,Protein A亲和纯化2A2腹水抗体,获得的单抗浓度为130μg/mL。选取纯化的2A2单克隆抗体进行潜在应用研究,Western blot与间接免疫荧光结果显示,单克隆抗体能与兔多杀性巴氏杆菌分离菌株发生特异性反应。表明利用体外重组表达兔多杀性巴氏杆菌OmpA融合蛋白制成的杂交瘤能够稳定分泌效价高、特异性强的单克隆抗体,可用于兔多杀性巴氏杆菌诊断,本研究结果为兔多杀性巴氏杆菌诊断试剂盒的研制提供技术参数。 相似文献
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传染性萎缩性鼻炎是养猪业中的一种重要的呼吸道传染病,产毒素多杀性巴氏杆菌是其重要的病原之一,外膜蛋白H(OmpH)在该菌的致病和免疫中发挥重要作用。本研究克隆了产毒素多杀性巴氏杆菌HN-13株的ompH基因,进行了序列分析,并在大肠杆菌中高效表达,用表达产物建立了间接ELISA方法。同时用生物信息学方法对ompH基因及其编码蛋白的结构特性进行了分析和预测,表明OmpH是一种通道蛋白。 相似文献
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A.J.A. Vinten D.R. Lewis D.R. Fenlon K.A. Leach R. Howard I. Svoboda I. Ogden 《Soil Use and Management》2002,18(3):223-231
Abstract. Slurry from farm animals may contaminate water supplies, rivers and bathing waters with faecal coliforms, such as Escherichia coli . Where animals harbour the O157 strain the hazard to human health is particularly high, but both the hazard level, and the low incidence and sporadic nature of the excretion of E. coli O157 make it difficult to study this strain under field conditions. The survival of total E. coli and of E. coli O157 were compared in the laboratory for two soils under controlled temperature and moisture. E. coli O157 die-off rate was the same as or quicker than for total E. coli . This result meant that field experiments studying the fate of total E. coli should give a satisfactory evaluation of the risk of water contamination by the O157 strain. In four field experiments at three sites, slurry containing total E. coli numbers of 2.2 × 104 to 5.7 × 105 colony forming units per mL (c.f.u. mL–1 ) was applied to drained field plots. Field die-off was faster than expected from laboratory experiments, especially in one experiment where two weeks dry weather followed application. In all but this experiment, the first drain flow events after slurry application led to very high E. coli concentrations in the drains (103 to 104 c.f.u. mL–1 ). E. coli O157 was present in the slurry used for two of the experiments (33 c.f.u. per 100 mL in each case). However the proportion of E.coli O157 was very low (about 1 in 105 ) and it was not detected in the drainage water. After the first week E. coli drainage water numbers decreased rapidly but they were 1–10 c.f.u. mL–1 for much of the sampling period after slurry application (1–3 months). 相似文献
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《Soil Science and Plant Nutrition》2013,59(2):258-263
Abstract Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g?1 dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces. 相似文献
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Shengjin WU Motoki NISHIHARA Yoshie KAWASAKI Akitoshi YOKOYAMA Kei MATSUURA Tomohiro KOGA Daisuke UENO Koichi INOUE Takashi SOMEYA 《Soil Science and Plant Nutrition》2009,55(2):258-263
Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g−1 dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces. 相似文献
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CpG-DNA对大肠杆菌诱导的大鼠实验性乳腺炎的影响 总被引:3,自引:0,他引:3
72只雌性大鼠随机分成对照组和实验组(n =36)。对照组产后0 h于左后腿胫骨前肌肌肉注射0.01 mol/L,pH 7.2 磷酸盐缓冲液(PBS)100 μL,实验组肌注CpG-DNA 200 μg/只,72 h后经乳头管灌注2×1012 cfu (colony forming unit/mL)/mL,大肠杆菌(Escherichia coli )100 μL/侧到第4对(两侧)乳腺内,分别于灌注前0 h,灌注后8,16,24,48和72 h(n = 6)颈静脉放血处死。组织学观察显示大鼠感染后16 h为乳腺腺泡嗜中性粒细胞(polymorphonuclear leukocytes,PMN)浸润高峰,实验组PMN浸润迅速, 72 h已无浸润。乳腺组织细菌数在16 h上升至最高,实验组大鼠感染后16、24和48 h乳腺组织细菌数较对照组有显著下降。CpG-DNA能极显著地提高感染前乳腺组织中IL-2水平。乳腺组织IL-6在不同时间点均有显著上升,在16 h达最高。CpG-DNA能显著地提高16 h乳腺组织IL-6水平。实验组乳腺组织N-acetyl-β-D-glucosaminidase (NAGase)无显著变化。表明CpG-DNA可加速炎症初期PMN的快速浸润,促进IL-2和IL-6的产生,减少细菌数量,减轻炎症介质对细胞的损伤并缩短炎症过程,提示CpG-DNA对大肠杆菌诱发的大鼠实验性乳腺炎有一定的预防作用。 相似文献
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Kiyoshi Sato Yoshiyuki Taniyama Ayami Yoshida Kazuhiko Toyomasu Noriko Ryuda Daisuke Ueno 《Soil Science and Plant Nutrition》2013,59(3):234-242
Multiple outbreaks of food poisoning associated with fresh vegetable consumptions have occurred in many countries. Numerous reports have described human pathogenic bacteria, such as Escherichia coli O157:H7 and Salmonella spp., that can internalize into fresh vegetables via root or leaf surfaces. While attempting to obtain the threshold concentration of internalization of E. coli inoculated into hydroponic medium during vegetable cultivation, we observed a rapid decrease in E. coli numbers. In the present study, we determined that the rapid decline in E. coli was not due to a physiological change into a viable but non-culturable (VNC) state. The population crash was instead caused by true bacterial death, as the rapid descent was also confirmed by micro-colony fluorescence in situ hybridization, a culture-independent method that can detect VNC cells. We next monitored the number of E. coli inoculated into intact or filter-sterilized hydroponic medium after cultivation of various types of plants. We found that the number of E. coli in intact hydroponic medium decreased markedly, whereas the level in filter-sterilized hydroponic medium was completely unchanged. This result suggests that biotic factors were present that could be eliminated by filtering. Robust predation of E. coli by protozoa (ciliates and flagellates) was observed using fluorescently labeled bacteria incorporated into the hydroponic medium. Finally, morphological identification of flagellates by scanning electron microscopy revealed the presence of a species of Stramenopiles. These findings suggest the importance of protozoa as bacterial feeders in hydroponic systems and hence the use of these organisms as potential control agents of human pathogenic bacteria. 相似文献
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Chun-Ming GONG Junichi KOSHIDA Noriko MORIYAMA Xiaodan WANG Takezo UDOU Koichi INOUE Takashi SOMEYA 《Soil Science and Plant Nutrition》2006,52(1):135-136
pp. 865–874
Occurrence and survival of fecal-contamination indicator bacteria (coliform bacteria, Escherichia coli and Salmonella ) in various manure and compost samples collected from 23 composting facilities mostly in Kyushu were investigated by using selective media. Coliform bacteria were detected on desoxycholate agar from 11 (38%) of 29 product samples (15 cow dung manure, 4 poultry manure, 2 biosolid compost and 8 food waste compost) at a range of 102 to 106 cfu g1 dry matter. From positive samples, 21 isolates of possible coliform bacteria were purified. Among them, species of coliform bacteria ( E. coli , E. vulneria , Pantoea sp. and Buttiauxella agrestis ) were identified whereas isolates of Serratia marcescens , not coliform bacteria, were also obtained, suggesting that careful observation was necessary to avoid false positive counting due to the presence of a red colony of S. marcescens that resembled coliform bacteria. Isolates of E. coli were tested for slide aggregation with a set of antiserum against pathogenic E. coli serotypes and negative reaction was obtained for all the isolates tested. Direct detection of E. coli on Chromocult coliform agar and Salmonella on MLCB agar resulted in none and 2 (17%) of 12 samples tested, respectively. The fate of fecal-contamination indicator bacteria as above was followed during compost production on 7 cases at 6 compost facilities and 4 patterns were observed: fecal-contamination indicator bacteria 1) decreased and finally disappeared, 2) decreased once but re-growth was occurred on products, 3) decreased to some extent but remained in products, 4) was not detected throughout production. These results suggest that some fecal-contamination indicator bacteria may survive compost production and appropriate temperature control would be significant for hygiene control of manure and compost. 相似文献
Occurrence and survival of fecal-contamination indicator bacteria (coliform bacteria, Escherichia coli and Salmonella ) in various manure and compost samples collected from 23 composting facilities mostly in Kyushu were investigated by using selective media. Coliform bacteria were detected on desoxycholate agar from 11 (38%) of 29 product samples (15 cow dung manure, 4 poultry manure, 2 biosolid compost and 8 food waste compost) at a range of 10