聚合酶链反应在牛血清支原体检测中的应用

为了探讨聚合酶链反应在牛血清支原体检测上的应用价值,以支原体高度保守的rRNA操纵子(支原体基因组中16SrRNA的编码区序列)设计引物,采用碱裂解法提取牛血清中支原体DNA作为模板进行聚合酶链反应。结果表明,阳性、阴性和内控对照都扩增出了预期的条带,聚合酶链反应与支原体培养法比较,有灵敏、快速、特异性高的特点,可用于牛血清中支原体的常规检测。     

Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion

Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct.     

直接培养法检测支原体方法的建立

采用牛心消化液,加入支原体营养成份及琼脂,制备成固体培养基。以此培养基检测支原体,并根据其灵敏度及适用性确定培养基的配方。结果表明,用此法可以检测牛血清、胰酶、细胞及疫苗中的支原体生长,并且检测时间短,重复性好,灵敏度高,可以用于生物制品的质量控制。     

Hemolysin and Peroxide Activity of Mycoplasma Species

Various methods for the detection of hemolysin production by Mycoplasma species were compared. Inoculation of blood-agar by the push-block method and by use of concentrated mycoplasma cell suspensions was compared with the agar-overlay technique. The preferred method was direct surface inoculation of concentrated suspensions onto the blood-agar. Among the conditions tested, refrigeration of 48-hr cultures gave the best results. A wide variety of mycoplasma species were tested for hemolytic activity towards rabbit, sheep, guinea pig, duck, and chicken bloods. Guinea pig erythrocytes were found to be the most susceptible to lysis by mycoplasma, and rabbit erythrocytes were found to be the least susceptible. A sensitive technique for the detection of peroxide production by mycoplasma strains, employing agar containing benzidine and sheep blood, was used. With this method, peroxide production could be correlated with hemolysis on blood-agar. Peroxidase and catalase inhibited both the benzidine reaction and hemolysis. It was concluded that the major hemolysin of the Mycoplasma species examined is a peroxide.     

Mycoplasma phocidae sp. nov., isolated from harbor seals (Phoca vitulina L.).

In 1979 and 1980, more than 400 harbor seals (Phoca vitulina) along the New England coast of the United States died of epizootic pneumonia that was attributed to an influenza virus. Six mycoplasma isolates that were recovered from the respiratory tracts of affected seals were investigated and were found to be serologically identical and distinct from previously described species. These isolates required serum for growth, did not possess a cell wall, and did not hydrolyze urea. Arginine was hydrolyzed, glucose was not fermented, film and spots were observed on horse serum agar, phosphatase was produced, tetrazolium was not reduced, and serum and casein were not digested. The guanine-plus-cytosine content of the DNA was 27.8 mol%. We propose the name Mycoplasma phocidae for these isolates. The type strain of M. phocidae is strain 105 (= ATCC 33657).     

Mycoplasma growth factors in bovine serum fraction.

Mycoplasma growth factors in bovine serum fraction were separated by Sephadex G150 column chromatography and density ultracentrifugation. The major growth factor of bovine serum fraction eluted from the Sephadex column in the void volume. Its growth-supporting activity was greatly enhanced by the presence of bovine serum albumin in the mycoplasma culture media. Other investigators had previously identified the major growth factor in serum as an alpha-lipoprotein. Although density ultracentrifugation revealed the presence of traces of a high-density lipoprotein in bovine serum fraction, another, less dense component, isolated by ultracentrifugation (component 3) and containing cholesterol, cholesteryl esters, free fatty acids, triglycerides, and protein, but no lipoprotein, exhibited considerably more growth-supporting activity than did the high-density lipoprotein, thus indicating that at least two mycoplasma species do not require intact serum lipoprotein for growth. Both the high-density lipoprotein and component 3 exhibited maximum activity only in the presence of bovine serum albumin. A chloroform extract containing component 3 lipids combined with bovine serum albumin to form an effective, partially defined, less complex substitute for serum in mycoplasma culture media.     

Effect of mycoplasma on interferon production and interferon assay in cell cultures

Armstrong, D. (The Children's Hospital of Philadelphia, Philadelphia, Pa.), and K. Paucker. Effect of mycoplasma on interferon production and interferon assay in cell cultures. J. Bacteriol. 92:97-101. 1966.-The influence of mycoplasma on the production and action of interferon was studied in cultures of both L and human embryonic kidney (HEK) cells. Mycoplasma hominis 1, the Negroni agent, and the F12 mycoplasma were used for infection of L cells, and M. hominis 1 and M. pneumoniae for inoculation of HEK cells. All strains were capable of multiplication in the culture systems employed. None produced detectable levels of interferon, and responsiveness of the cells to induction of interferon by virus remained unaltered. Infection with mycoplasma did not impair the sensitivity of the cells to the action of interferon, nor was the replication of vesicular stomatitis virus noticeably diminished.     

实验用小型猪呼吸道支原体检测及方法比较

目的通过常用的三种不同方法对支原体的检测,了解实验用小型猪支原体感染情况,为今后实验用小型猪支原体检测方法国家标准的制定提供参考。方法采用培养法、PCR和ELISA方法分别对20头小型猪的气管、肺和血清进行检测。结果三种检测方法中,PCR方法支原体阳性检出率为15%,ELISA方法为20%,而培养法结果均为阴性。结论目前在普通级小型猪中存在支原体的感染。检测方法中PCR和ELISA方法较培养法更省时,敏感性更高。     

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii.

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

Rapid removal of mycoplasma from cell lines mediated by a direct effect of complement

Mycoplasma can be removed from the surface of contaminated human and murine cell lines by incubation for 4 h with human, rabbit, guinea pig, or mouse sera. Several lines of evidence suggest the involvement of complement in this process: (1) The activity can be abrogated by heat treatment (56 degrees C for 45 min). (2) Using monoclonal antibodies directed against C3a and C3b, the deposition of C3b fragments on the surface of mycoplasma-positive cells can be demonstrated after 1 h incubation with human serum. (3) Ca2+ depletion ablates the ability of serum to remove the activity. (4) C2def' sera are inactive while addition of purified C2 reconstitutes the activity. The latter two findings implicate that activation of the classical pathway of complement is responsible for the effect. Antibody, however, is not required as demonstrated by the uncompromised activity of Ig-deficient sera from bursectomized chicken. Treatment with human serum or rabbit serum was used successfully to permanently cleanse 10/10 tumor cell lines of human and of murine origin. The complete removal of mycoplasma was monitored over at least 8 weeks by direct DNA staining and confirmed by agar culture and transfer of supernatants to mycoplasma-free Vero cells followed by DNA staining. Thus the direct interaction of mycoplasma and complement appears to be an effective and rapid means of curing cell lines from mycoplasma.     

Cross comparison of rapid mycoplasma detection platforms

The use of animal and plant derived raw materials in mammalian cell culture processes may provide a possible route of entry for adventitious contaminants such as mycoplasma. Mycoplasma contaminations of cell culture represent a serious challenge to the production of biotechnology derived therapeutics. The slow growing nature of mycoplasma can disguise their infection of cultures since cells may continue to proliferate, though at reduced levels and with lesser output of engineered protein. Rapid identification of mycoplasma contaminated cell cultures and materials enables a faster response time to prevent the spread of the contamination. We describe here the comparison of different mycoplasma detection methods: two nucleic acid-based technologies, the standard mycoplasma culture procedure, and a hybrid culture-quantitative PCR assay. In this study, a cell line infected with two species of mycoplasma was used to compare the different detection methods. Our data demonstrates that the two nucleic acid-based techniques are robust methods for detection of mycoplasma and have similar detection capability. In contrast, no mycoplasma was detected in the standard culture assay or in a hybrid culture-quantitative PCR assay. This shows a potential limitation of the culture assay that relies on the ability of mycoplasma to grow in broth media.     

血清APOC1、LDH及炎性感染指标在难治性支原体肺炎患儿中的表达水平及其与肺通气功能、预后效果的相关性分析

摘要 目的：分析血清载脂蛋白C1（APOC1）、乳酸脱氢酶（LDH）及炎性感染指标在难治性支原体肺炎患儿中的表达水平及其与肺通气功能、预后效果的相关性。方法：选择我院自2019年1月至2022年12月接诊的130例难治性支原体肺炎患儿作为观察组，另选同期在我院体检且结果正常的儿童作为对照组。检测两组入选者血清APOC1、LDH及炎性感染指标[C反应蛋白（CRP）、白细胞介素-6（IL-6）]表达水平、肺通气功能指标[用力肺活量（FVC）、第1秒用力呼气容积（FEV₁）、呼气峰流速（PEF）]；观察组患儿均严格按照《儿童社区获得性肺炎诊疗规范（2019年版）》接受治疗，通过ROC曲线下面积（AUC）评价血清APOC1、LDH及炎性感染指标对难治性支原体肺炎患儿预后不良的预测效能。结果：观察组血清APOC1、LDH、CRP、IL-6表达水平均高于对照组（P＜0.05）；在观察组患儿中，重症组血清APOC1、LDH、CRP、IL-6表达水平均高于轻症组（P＜0.05）；经Pearson相关性分析，难治性支原体肺炎患儿血清APOC1、LDH、CRP、IL-6表达水平均与FVC、FEV1、PEF呈负相关（P＜0.05）；经多因素Logistic回归分析，难治性支原体肺炎患儿血清APOC1、LDH、CRP、IL-6均是其预后不良的独立预测因素（P＜0.05）；经ROC曲线分析，难治性支原体肺炎患儿血清APOC1、LDH、CRP联合IL-6预测其预后不良的AUC为0.930。结论：血清APOC1、LDH及炎性感染指标在难治性支原体肺炎患儿中的表达水平明显升高，与其肺通气功能密切相关，其中APOC1、LDH、CRP联合IL-6预测预后不良的效能较好，值得进一步研究应用。     

Growth of Mycoplasma hyorhinis cultivar alpha on semisynthetic medium.

Serial passage of Mycoplasma hyorhinis cultivar alpha (formerly noncultivable strains) has been accomplished in modified CMRL-1066 medium with fetal bovine serum. In modified CMRL-1066 liquid medium, cultivar alpha strains grow at a similar rate and to equivalent titers when compared with BTS-7, the type strain of the species. Further experiments with BTS-7 demonstrate that the extent of growth obtained in the semidefined medium was comparable to growth in conventional mycoplasma medium. M. hyorhinis strains, including cultivar alpha strains, grow in serial passage when fetal bovine serum is replaced with bovine serum albumin, palmitic acid, and cholesterol. The results of these studies show that M. hyorhinis cultivar alpha strains are not nutritionally more fastidious than other mycoplasmas but that they are noncultivable on standard mycoplasma media because they are sensitive to high levels of inhibition activity by medium components.     

Rapid Detection of Bovine Mycoplasma Antigens by Counterimmunoelectrophoresis

Twelve reference strains of mycoplasma and acholeplasma previously reported to have been recovered from cattle were tested against hyperimmune rabbit serum by counterimmunoelectrophoresis. This technique detected antigen by the formation of precipitin lines with antibody within 1 h and promises to be a useful technique for detecting and identifying mycoplasma isolates in either pure or mixed cultures.     

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3μm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.     

清开灵注射液联合阿奇霉素对支原体肺炎患儿血清T-IgE和ECP水平的影响

目的:探讨清开灵注射液联合阿奇霉素对小儿支原体肺炎患者血清T-IgE和ECP影响。方法:选取我院收治的支原体肺炎患儿50例,随机分为实验组和对照组。两组均予阿奇霉素对症治疗,实验组在此基础上加用清开灵注射液治疗。观察并比较两组患儿治疗前后血常规各指标变化情况以及T-IgE、ECP水平的变化。结果:与治疗前相比,两组患者治疗后中性粒细胞比例降低,淋巴细胞比例升高,单核细胞比例降低(P0.05),且实验组治疗组中性粒细胞比例较对照组更低,淋巴细胞比例较高,单核细胞比例较低(P0.05)。治疗后3、7、14天,两组患儿的血清T-IgE及ECP水平呈下降趋势,与前一时间点比较,两组患者的T-IgE及ECP水平均较低(P0.05),与对照组比较,实验组同意时间点T-IgE及ECP水平较低(P0.05)。结论:清开灵注射液联合阿奇霉素能够明显降低小儿支原体肺炎患儿血清T-IgE和ECP水平。     

Elimination of Mycoplasma from various cell cultures

Elimination ofMycoplasma orale-I from chronically infected cell lines was achieved either by treatment with a mixture of antibiotics in a hypotonic solution, or with 10 vol % of anti -M.orale rabbit serum in tissue culture medium. The latter treatment was preferable in most cases, as it was practically harmless to the cells. Inactivation of this antiserum had no effect on its potency. The antibiotic-hypotonic treatment was rather destructive, but to a different degree for the various cell cultures. Both methods were equally useful for the treatment of a monkey kidney cell line contaminated with a mycoplasma strain related toM.hyorhinis. The available anti -M.hyorhinis rabbit serum was toxic for the monkey cells when not inactivated. The potency of the antiserum was rather low and even lower after inactivation. However, prolonged treatment successfully eliminated the mycoplasma. Pre-incubation of the inactivated anti -M.hyorhinis serum with tissue culture medium to which 10% non-inactivated calf serum had been added, favoured the elimination of the mycoplasma.During the treatment of contaminated cell cultures with single antibiotics a strain related toM.hyorhinis became resistant to chlortetracyclin.M.orale- I was found to be resistant to various single antibiotics.We are grateful to Professor Dr. A. Ch. Ruys (University of Amsterdam) and Dr. R. H. Leach (Mycoplasma Reference Laboratory, London) for helpful discussions and for identifying some of our mycoplasma strains; Dr. Leach also for kindly supplying us with his G. D. L.-strain. We thank Dr. H. Cohen and Dr. A. C. Hekker for their criticism and Mr. N. L. M. van Zwetselaar for his accurate technical assistance.