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Toxin Genes and Other Characteristics ofStaphylococcus aureus Isolates from Milk of Cows with Mastitis 乳腺炎奶牛乳中金黄色葡萄球菌分离物的毒素基因及其他特征
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.959-964.2001
Ö. Akineden, C. Annemüller, A. Hassan, Christoph Lämmler, W. Wolter, M. Zschöck
ABSTRACT In the present study, 103 Staphylococcus aureusstrains isolated from milk samples from 60 cows with mastitis from eight different farms in seven different locations in one region of Germany were compared pheno- and genotypically and by identification of various toxins. On the basis of culture and hemolytic properties and by determination of the tube coagulase reaction, all of the isolates could be identified as S. aureus. This could be confirmed by PCR amplification of species-specific parts of the gene encoding the 23S rRNA. In addition, all of the S. aureus isolates harbored the genes encoding staphylococcal coagulase and clumping factor and the genes encoding the X region and the immunoglobulin G binding region of protein A. These four genes displayed size polymorphisms. By PCR amplification, the genes for the toxins staphylococcal enterotoxin A (SEA), SEC, SED, SEG, SEI, SEJ, and TSST-1 but not those for SEB, SEE, SEH, and the exfoliative toxins ETA and ETB could be detected. To analyze the epidemiological relationships, the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNAs. According to the observed gene polymorphisms, the toxin patterns, and the information given by macrorestriction analysis of the isolates by pulsed-field gel electrophoresis, a limited number of clones seemed to be responsible for the cases of bovine mastitis on the various farms.
在本研究中,从德国一个地区7个不同地点的8个不同农场的60头乳腺炎奶牛的牛奶样本中分离出103株金黄色葡萄球菌,对其进行了表型和基因型比较,并鉴定了各种毒素。根据培养和溶血特性以及试管凝固酶反应的测定,所有分离株均可鉴定为金黄色葡萄球菌。这可以通过编码23S rRNA的基因的物种特异性部分的PCR扩增来证实。此外,所有金黄色葡萄球菌分离株均含有编码葡萄球菌凝固酶和凝块因子的基因以及编码a蛋白X区和免疫球蛋白G结合区的基因,这4个基因均表现出大小多态性。PCR扩增可检出葡萄球菌毒素肠毒素A (SEA)、SEC、SED、SEG、SEI、SEJ和TSST-1基因,但未检出SEB、SEE、SEH和剥脱性毒素ETA和ETB基因。为分析流行病学关系,对分离株染色体DNA进行了宏观限制性内切分析,建立了DNA指纹图谱。根据观察到的基因多态性、毒素模式和脉冲场凝胶电泳对分离物进行的宏观限制性内切分析所提供的信息,似乎是有限数量的克隆导致了各个农场的牛乳腺炎病例。
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引用次数: 271
Excretory-Secretory Antigens of Trypanosoma cruzi Are Potentially Useful for Serodiagnosis of Chronic Chagas' Disease 克氏锥虫排泄-分泌抗原在慢性恰加斯病血清诊断中的潜在价值
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.1024-1027.2001
M. Nakazawa, D. S. Rosa, V. Pereira, Milena O. Moura, V. C. Furtado, W. Souza, M. D. N. D. S. Barros, F. Abath, Y. Gomes
ABSTRACT The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of Trypanosoma cruzi strains with different biodemes were analyzed by TESA-blot and TESA–enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.
采用TESA-blot和TESA酶联免疫吸附试验(ELISA)分析慢性血吸虫患者血清对不同生物组克氏锥虫菌株的胰蛋白酶体分泌抗原(TESA)的反应性。虽然两种检测方法都具有较高的灵敏度和特异性,但TESA-ELISA更适合于筛选大量的样品。
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引用次数: 39
Late Hematogenous Infection of Subcutaneous Implants in Rats 大鼠皮下植入物的晚期血液感染
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.980-983.2001
B. Gottenbos, F. Klatter, H. C. van der Mei, H. Busscher, P. Nieuwenhuis
ABSTRACT Late biomaterial-centered infection is a major complication associated with the use of biomaterial implants. In this study biomaterials that had been implanted subcutaneously in rats were hematogenously challenged with bacteria 4 weeks after implantation. Bacteria were spread either by intravenous injection or by stimulation of bacterial translocation. It was found that none of the biomaterials was infected by hematogenous spread, whereas 5% of the implants were infected by perioperative contamination. We conclude that late hematogenous infection of subcutaneous biomaterials does not occur in the rat. For humans as well, there are growing doubts whether implants actually become infected through hematogenous routes; it is thought that late infections may be caused by delayed appearance of perioperatively introduced bacteria.
晚期以生物材料为中心的感染是使用生物材料植入物的主要并发症。在本研究中,将植入大鼠皮下的生物材料在植入4周后进行细菌致血攻击。细菌通过静脉注射或刺激细菌易位传播。结果发现,所有生物材料均未发生血液传播感染,而5%的植入物因围手术期污染而感染。我们得出结论,皮下生物材料的晚期血行性感染不会发生在大鼠身上。对于人类也是如此,越来越多的人怀疑植入物是否真的会通过血液途径感染;据认为,晚期感染可能是由于围手术期引入细菌的延迟出现引起的。
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引用次数: 8
Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus 用于水痘-带状疱疹病毒细胞免疫应答定量的γ干扰素ELISPOT测定方法的建立和验证
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.871-879.2001
Jeffrey G. Smith, Xu Liu, R. Kaufhold, J. Clair, M. Caulfield
ABSTRACT Cell-mediated immunity appears to be critical for the prevention and control of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersome or lack sensitivity. We have developed a gamma interferon ELISPOT assay that provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (SFC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This assay has been validated by demonstrating the following: (i) the response detected is mediated almost entirely by CD4+ T cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those from freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a computer-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the use of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either during a herpes zoster disease outbreak or following vaccination.
细胞介导的免疫似乎对预防和控制水痘-带状疱疹病毒(VZV)感染和带状疱疹引起的并发症至关重要。目前的vzv特异性细胞介导免疫测定方法繁琐或缺乏敏感性。我们开发了一种γ干扰素ELISPOT检测方法,可直接测量抗原刺激后分泌细胞因子的T细胞数量。这种检测方法非常敏感和特异,能够检测到γ干扰素斑点形成细胞(SFC),范围为每百万外周血单个核细胞(PBMCs) 10至1,000 SFC。该分析已通过演示以下内容得到验证:(i)检测到的反应几乎完全由CD4+ T细胞介导,(ii)新鲜冷冻的PBMC的ELISPOT反应与新鲜分离的细胞相当,(iii)冷冻的PBMC可以在干冰上运输长达48小时而不失去活性,(iv)冷冻的PBMC样品可以在液氮中长期储存(>22个月)而没有任何明显的反应变化。(v)使用计算机成像系统计数的elispot数量与人类计数的数量相当,但变异性较低。使用重组核酸酶(苯并酶)可以在样品解冻时防止细胞结块,从而促进了使用冷冻细胞的能力。冷冻的PBMC样品可以在液氮和干冰之间多次循环储存,而不会检测到任何响应变化。这有利于在一个地点收集样品,并在远程地点进行测试。这种VZV ELISPOT检测提供了一种新的多功能工具,用于在带状疱疹疾病爆发期间或接种疫苗后监测细胞免疫反应。
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引用次数: 169
Clinical and Serological Follow-Up of Patients with Human Granulocytic Ehrlichiosis in Slovenia 斯洛文尼亚人粒细胞埃利希体病患者的临床和血清学随访
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.899-903.2001
S. Lotrič-Furlan, Tatjana Avšič-Županc, M. Petrovec, William L. Nicholson, J. W. Sumner, James E. Childs, F. Strle
ABSTRACT An evaluation of the clinical outcome and the duration of the antibody response of patients with human granulocytic ehrlichiosis (HGE) was undertaken in Slovenia. Adult patients with a febrile illness occurring within 6 weeks of a tick bite were classified as having probable or confirmed HGE based on the outcome of serological or PCR testing. Thirty patients (median age, 44 years) were enrolled, and clinical evaluations and serum collection were undertaken at initial presentation and at 14 days, 6 to 8 weeks, and 3 to 4, 6, 12, 18, and 24 months. An indirect immunofluorescence assay (IFA) was performed, and reciprocal titers of ≥128 were interpreted as positive. Patients presented a median of 4 days after the onset of fever and were febrile for a median of 7.5 days; four (13.3%) received doxycycline. Seroconversion was observed in 3 of 30 (10.0%) patients, and 25 (83.3%) showed >4-fold change in antibody titer. PCR results were positive in 2 of 3 (66.7%) seronegative patients but in none of 27 seropositive patients at the first presentation. IFA antibody titers of ≥128 were found in 14 of 29 (48.3%), 17 of 30 (56.7%), 13 of 30 (43.4%), and 12 of 30 (40.0%) patients 6, 12, 18, and 24 months after presentation, respectively. Patients reporting additional tick bites during the study had significantly higher antibody titers at most time points during follow-up. No long-term clinical consequences were found during follow-up.
斯洛文尼亚对人粒细胞埃利希体病(HGE)患者的临床结果和抗体反应持续时间进行了评估。根据血清学或PCR检测结果,在蜱虫叮咬后6周内出现发热性疾病的成年患者被归类为可能或确诊的HGE。30例患者(中位年龄44岁)入组,在首次就诊、14天、6至8周、3至4、6、12、18和24个月时进行临床评估和血清收集。间接免疫荧光测定(IFA),≥128的倒数滴度被解释为阳性。患者出现发热后中位时间为4天,发热时间中位时间为7.5天;4例(13.3%)接受强力霉素治疗。30例患者中有3例(10.0%)出现血清转化,25例(83.3%)抗体滴度变化大于4倍。3例血清阴性患者中2例(66.7%)的PCR结果为阳性,而27例首次就诊时血清阳性患者中无一阳性。29例患者中有14例(48.3%),30例中有17例(56.7%),30例中有13例(43.4%),30例患者中有12例(40.0%)在发病后6、12、18和24个月分别发现IFA抗体滴度≥128。在研究期间报告其他蜱虫叮咬的患者在随访期间的大多数时间点的抗体滴度显着较高。随访期间未发现长期临床后果。
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引用次数: 39
Chemokine Receptor CCR5 Δ32 Genetic Analysis Using Multiple Specimen Types and the NucliSens Basic Kit 趋化因子受体CCR5 Δ32使用多种标本类型和NucliSens基本试剂盒的遗传分析
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.965-971.2001
S. Tetali, Eun Mi Lee, M. Kaplan, Joseph P. Romano, C. Ginocchio
ABSTRACT Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Δ32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Δ32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Δ32 genotyping using multiple sample types.
HIV-1感染抵抗和疾病进展延迟与编码CCR5趋化因子受体的基因32 bp缺失(Δ32)有关。在本研究中,我们使用一种新的靶向特异性三明治寡核苷酸检测方法,对基于核酸序列扩增(NASBA)的CCR5基因分型检测方法进行了修改,用于NucliSens基本试剂盒(Organon Teknika, Durham, N.C.)格式。新方法允许使用NucliSens Basic Kit中提供的通用电化学发光探针,而原始的NASBA方法需要昂贵的靶向特异性钌检测探针。Basic Kit CCR5 Δ32基因型分析与原始NASBA测定和DNA PCR结果100%一致。本研究还评估了多种标本类型的使用,包括外周血单个核细胞(PBMC)、全血、干血斑、口腔刮痕和血浆,用于CCR5基因型分析。当PBMC或全血为标本来源时,这三种检测方法的敏感性可比较。相比之下,当使用干血斑、口腔刮痕或血浆作为样品源时,DNA PCR的灵敏度分别为80.95、42.8和0%,而原始NASBA和Basic Kit NASBA检测方法的灵敏度为100%。我们的研究表明,NucliSens Basic Kit NASBA检测对CCR5 Δ32多种样品类型的基因分型非常敏感和特异性。
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引用次数: 6
Effects of Calorie Restriction on Polymicrobial Peritonitis Induced by Cecum Ligation and Puncture in Young C57BL/6 Mice 热量限制对幼年C57BL/6小鼠盲肠结扎穿刺致多微生物性腹膜炎的影响
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.1003-1011.2001
Dongxu Sun, A. Muthukumar, R. Lawrence, G. Fernandes
ABSTRACT Calorie restriction (CR) is known to prolong the life span and maintain an active immune function in aged mice, but it is still not known if rodents under CR can respond optimally to bacterial infection. We report here on the influence of CR on the response of peritoneal macrophages to lipopolysaccharide, splenic NF-κB and NF–interleukin-6 (IL-6) activities, and mortality in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Macrophages from 6-month-old C57BL/6 mice on a calorie-restricted diet were less responsive to lipopolysaccharide, as evidenced by lower levels of IL-12 and IL-6 protein and mRNA expression. Furthermore, in vitro lipopolysaccharide-stimulated macrophages from mice under CR also expressed decreased lipopolysaccharide receptor CD14 levels as well as Toll-like receptor 2 (TLR2) and TLR4 mRNA levels. In addition, the phagocytic capacity and class II (I-Ab) expression of macrophages were also found to be significantly lower in mice under CR. Mice under CR died earlier (P < 0.005) after sepsis induced by CLP, which appeared to be a result of increased levels in serum of the proinflammatory cytokines tumor necrosis factor alpha and IL-6 and splenic NF-κB and NF–IL-6 activation 4 h after CLP. However, mice under CR survived significantly (P < 0.005) longer than mice fed ad libitum when injected with paraquat, a free radical-inducing agent. These data suggest that young mice under CR may be protected against oxidative stress but may have delayed maturation of macrophage function and increased susceptibility to bacterial infection.
已知热量限制(CR)可以延长老年小鼠的寿命并保持活跃的免疫功能,但目前尚不清楚CR下的啮齿动物是否能对细菌感染做出最佳反应。我们在此报道了CR对腹腔巨噬细胞对脂多糖的反应、脾脏NF-κB和NF-白细胞介素-6 (IL-6)活性以及盲肠结扎穿刺(CLP)引起的多微生物脓毒症死亡率的影响。限制热量饮食的6月龄C57BL/6小鼠巨噬细胞对脂多糖的反应较弱,IL-12和IL-6蛋白和mRNA表达水平较低。此外,体外脂多糖刺激下的CR小鼠巨噬细胞也表达了降低的脂多糖受体CD14水平以及toll样受体2 (TLR2)和TLR4 mRNA水平。此外,CR小鼠的吞噬能力和巨噬细胞II类(I-Ab)表达也明显降低,CLP诱导脓毒症后,CR小鼠死亡时间较早(P < 0.005),这可能是CLP后4小时血清促炎因子肿瘤坏死因子α和IL-6水平以及脾NF-κB和NF- IL-6活化水平升高所致。然而,CR组小鼠的存活时间明显长于注射百草枯(一种自由基诱导剂)的自由喂养小鼠(P < 0.005)。这些数据表明,CR下的幼鼠可能受到氧化应激的保护,但可能会延迟巨噬细胞功能的成熟,并增加对细菌感染的易感性。
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引用次数: 110
Plasmodium vivax Polymorphism in a Clinical Drug Trial 间日疟原虫在临床药物试验中的多态性
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.891-894.2001
T. Adak, N. Valecha, V. Sharma
ABSTRACT Data from a double-blind randomized clinical drug trial were analyzed to find the comparative responses of two antirelapse drugs, bulaquine and primaquine, against different relapsing forms ofPlasmodium vivax infection. A 1-year follow-up study strongly suggests that the duration of preerythrocytic development ofP. vivax is a polymorphic characteristic, exhibited by two strains of hypnozoites responsible for early and late manifestations after primary infection. Short-term relapses were significantly higher in the first half year than long-term relapses, and the reverse was true in the second half year. Clinical drug response data showed that the hypnozoites characterized for short-term relapse were not susceptible to either of the antirelapse drugs in the currently administered dose, whereas hypnozoites characterized for long incubation were significantly susceptible.
摘要:通过对一项双盲随机临床药物试验的数据进行分析,比较两种抗复发药物布喹和伯氨喹对不同复发型间日疟原虫感染的疗效。一项为期1年的随访研究强烈表明,p的红细胞前发展持续时间。间日疟原虫是一种多态特征,在初次感染后的早期和晚期表现为两株催眠虫。上半年短期复发率明显高于长期复发率,下半年则相反。临床药物反应数据显示,短期复发的催眠虫对当前剂量的两种抗复发药物均不敏感,而长期潜伏的催眠虫则明显敏感。
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引用次数: 48
High Serum Interleukin-10 and Tumor Necrosis Factor Alpha Levels in Chronic Paracoccidioidomycosis 慢性副球孢子菌病患者血清白细胞介素-10和肿瘤坏死因子α水平升高
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.1036-1038.2001
M. Fornari, A. Bava, M. Guereño, V. Berardi, M. R. Silaf, R. Negroni, R. Diez
ABSTRACT In patients with chronic paracoccidioidomycosis (n= 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean ± standard error of the mean), were higher than in normal controls (n = 8): 186 ± 40 versus 40 ± 7 (P < 0.05), 203 ± 95 versus 20 ± 8 (P = 0.001), and 96.3 ± 78.57 versus 1.19 ± 1.19 (P = 0.045), respectively. Gamma interferon and interleukin-4 levels were similar in patients and controls.
文摘患者慢性paracoccidioidomycosis (n = 10),肿瘤坏死因子-α水平,白细胞介素- 10”,和血清中白介素2,以酶联免疫吸附试验(在每毫升皮克,平均值±标准平均误差),均高于正常对照组(n = 8): 186±40和40±7 (P < 0.05), 203±95和20±8 (P = 0.001),和96.3±78.57和1.19±1.19 (P = 0.045),分别。患者和对照组的γ干扰素和白细胞介素-4水平相似。
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引用次数: 33
Serum Immunoglobulin G Antibody Responses to Bordetella pertussis Lipooligosaccharide and B.parapertussis Lipopolysaccharide in Children with Pertussis and Parapertussis 百日咳和百日咳患儿血清免疫球蛋白G抗体对百日咳低脂多糖和百日咳双歧杆菌脂多糖的反应
Pub Date : 2001-09-01 DOI: 10.1128/CDLI.8.5.1015-1017.2001
B. Trollfors, T. Lagergård, J. Taranger, E. Bergfors, R. Schneerson, J. Robbins
ABSTRACT Serum immunoglobulin G (IgG) antibodies against the lipooligosaccharide (LOS) of Bordetella pertussis and the lipopolysaccharide (LPS) of Bordetella parapertussiswere measured by enzyme-linked immunosorbent assay in paired sera from 40 children with pertussis and 14 with parapertussis. Wide differences in the individual responses were noted. Both anti-LOS and -LPS IgG levels increased significantly in the children with pertussis, as did anti-LPS but not anti-LOS in those with parapertussis.
采用酶联免疫吸附法测定了40例百日咳患儿和14例百日咳患儿血清中抗百日咳杆菌脂寡糖(LOS)和副百日咳杆菌脂多糖(LPS)的免疫球蛋白G (IgG)抗体。人们注意到个体反应的巨大差异。百日咳患儿抗- los和-LPS IgG水平均显著升高,百日咳患儿抗-LPS水平升高,但抗- los水平未升高。
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引用次数: 21
期刊
Clinical Diagnostic Laboratory Immunology
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