Immunoelectrophoretic analysis of the antigenic composition of Salmonella typhi in the process of L transformation and reversion

In the study of the antigenic composition of S. typhi L-forms and their revertants were determined. The stable L-forms were characterized by profound disturbances in the synthesis of Vi- and H-antigens. After reversion to bacterial forms all the revertants under study showed the complete restoration of their bacterial antigenic structure.     

细胞壁缺陷对白喉棒状杆菌致病性影响的研究

检测白喉棒状杆菌稳定L型对动物的致病性,探讨细胞壁缺陷对白喉棒状杆菌致病性的影响及其可能的分子机制。采用氨苄青霉素在非高渗培养基内诱导并获得产毒性白喉棒状杆菌稳定L型纯培养物。收集白喉棒状杆菌稳定L型纯培养物及其代谢产物,将收集的高于细菌型10 000倍浓度的白喉棒状杆菌稳定L型纯培养物及其代谢产物皮内注射家兔,观察局部注射部位皮肤或全身的病理改变。分别采用对流免疫电泳(CIEP)和SDS-不连续聚丙烯酰胺凝胶电泳(SDS-PAGE)检测白喉棒状杆菌稳定L型可溶性代谢产物中的白喉毒素蛋白质。结果显示,白喉棒状杆菌稳定L型不能引起动物局部或全身发生异常表现,在其可溶性代谢产物中并未检测到白喉毒素蛋白质。提示细胞壁缺陷变异可影响白喉棒状杆菌产生白喉毒素蛋白质,从而使其丧失了产生外毒素致病的作用。     

Biological properties and ultrastructure of brucellae during L-transformation and reversion]

There was shown a difference in the biological properties and the ultrastructure of two strains of brucellae, spheroplasts obtained from them under the action of penicillin, L-form and revertants obtained from the L-form. Spheroplasts formation was characterized by a change of brucellae into R-form and some virulence reduction. The cells had an outer and a cytoplasmic membranes, and usually lost their capacity to binary division. L-forms were obtained during the 9th and the 35th passage on a medium with penicillin; their formation was accompanied by the change in serological properties of the culture and significant reduction of the virulence; the cells were characterized by a marked polymorphism and the capacity to budding; they had 2 membranes on the cell surface and an intensively developed system of intracytoplasmic membranes. The revertants formed on the medium without penicillin during the 16th-30th passage or spontaneously on the medium with penicillin. They differed from the initial strains of brucella culture by a marked increase in penicillin-resistance, by the changes in serological properties, and also by polymorphism of cells, capable, however, of binary division.     

Electron microscopic findings on reversion of Listeria L-forms

Reversion in L-forms of Listeria monocytogenes was studied with the use of electron microscopy. In the culture undergoing the process of reversion cells differing in size, form and the electron density of the cytoplasm were present. The process of reversion was characterized by the increase of cytoplasmic density, the decrease of nucleoid, the appearance of fibrillary material of medium electron density on the membrane surface and the longitudinal elongation of the cell. The cell wall of the revertants was thinner and its outer layer with greater electron density was less pronounced than in the initial culture.     

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.     

Comparison of in vitro-cultured and wild-type Perkinsus marinus. I. Pathogen virulence

Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea virginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to using cultured parasites for infection studies, but results suggest that cultured parasites are less virulent than wild-type parasites In this paper, we report results of experiments designed to quantify differences between wild-type and cultured P. marinus virulence and to test the following hypotheses: (1) in vitro-cultured parasites are less virulent than wild-type parasites; (2) virulence decreases gradually during in vitro culture; (3) virulence of in vitro cultures can be restored by in vivo passage; (4) virulence changes with culture phase. Our results demonstrate that parasites freshly isolated from infected hosts are much more virulent than those propagated in culture, indicating a potential deficiency in the culture medium used. Virulence was lost immediately in culture and, for that reason, the practice of repassing cultured cells through the host to restore virulence does not work for P. marinus. Virulence was also associated with culture phase: log-phase parasites were significantly more virulent than those obtained from lag- or stationary-phase cultures.     

The in vivo but not the in vitro am3 revertant frequencies increase linearly with increased ethylnitrosourea doses in spleen of mice transgenic for phiX174 am3, cs70 using the single burst assay

The am3 revertant frequencies (RF) in spleens from male mice transgenic for phiX174 am3, cs70 were analyzed 14 weeks after ethylnitrosourea (ENU) treatment, both by the single burst assay (SBA) and the mixed burst assay (MBA). The mean in vivo (burst size >30/assay plate) revertant frequency (MRF) for the vehicle control was 2.6x10(-7). The ENU induced in vivo RF were linear over the dose range 0-150mg/kg, (r(2)=0.999). The concomitant in (burst size G transitions. Sequence analysis of in vivo revertants from ENU treated animals revealed revertants that were 17% A-->G transitions and 83% A-->T transversions, the latter being consistent with the reported A:T base pair alterations induced by ENU. No A-->C transitions were seen. This suggests the occurrence of an ENU-induced O(2) ET-dT lesion leading to a dT base mismatch. The observations in this report both confirm and validate the use of the SBA for distinguishing between in vivo mutations that are fixed in the animal and in vitro mutations that arise from other sources. The ability of the SBA to distinguish the in vivo from the in vitro origin of mutations has increased the specificity, sensitivity and utility of the phiX transgenic system.     

Antibiotic sensitivity of S. typhi cultures that have reverted from L forms isolated from the blood of patients]

L-forms of bacteria were isolated in 18 out of 300 fever patients with diagnoses of typhoid-paratyphoid fever, grippe, virus respiration disease and others in the Diagnostic Department of an Infection Hospital during bacteriological tests of the blood. Among the cultures tested 13 were instable and reversed to the bacterial form. The type identification showed that only 9 revertants possessed properties characteristic of the typhoid fever microbes and belonged to S. typhi. Sensitivity of the typhoid fever revertants to levomycetin, sintomycin, streptomycin, pencillin and tetracycline was studied. The studies showed that the typhoid fever revertants from the L-forms isolated from the patients were sufficiently sensitive to levomycetin, sintomycin, penicillin and tetracycline. The minimum bactericidal concentrations of the above antibiotics ranged within 12.5--100 gamma/ml.     

Clonal structure of Yersinia pestis populations in experimental soil ecosystems

Two basic tendencies--formation of latent (uncultivable) form (LF) and hemin storage variability--has been revealed during study of clonal structure dynamics of Y. pestis populations in artificial soil ecosystems in long-term incubation conditions. Y. pestis populations disappeared within 3 - 6 months at 18 - 22 degrees C, whereas at 4 - 8 degrees C a subsequent replacement of vegetative cells on LF, which are capable to prolonged survival (up to 22 months) in soil with ability to reversion in the presence of abundance of nutrients, has been observed. Bacteria of virulent strain retained all determinants of pathogenicity when reverted to LF, whereas bacteria of avirulent strain (defective on plasmid of Ca-dependence), on the contrary, undergo further degradation that resulted in loss of a pgm locus and gradual disappearance of population. LF revertants of highly virulent strain restored properties of initial population and were highly virulent.     

无绿藻的研究进展:从基础到临床

无绿藻是一种直径约3 ～ 30 μm的单细胞生物,广泛存在于自然界和动物体表及体内,属于条件致病性真菌.目前主要通过直接镜检、真菌培养、组织病理学检查及分子生物学等手段对无绿藻进行鉴定.现已发现无绿藻属包括五个种,其中对人有致病性的仅为中型无绿藻基因型2、小型无绿藻和P.blaschkeae,其致病机制可能与外伤和免疫力低下有关.随着研究的深入,越来越多的无绿藻病被临床确诊.根据不同的类型及其临床表现,对无绿藻病的治疗也有所区别.为了提高对无绿藻这一条件真菌及其致病性的认识,该文对其生物学特性、鉴定方法、致病性、临床表现等研究进展做一简要综述.     

Characteristics of the virulence of a strain of S. typhimurium and its glycerin mutant in a HEp-2 cell line model]

Sensitivity of the HEp-2 cell culture to the infection with the virulent salmonella strains was shown; active penetration and multiplication of bacteria in the monolayer cells indicated this. Gly 90, a glycerine mutant defective by glycerokinase, obtained under the action of ethylmethanesulphonate, displayed a distinct difference from the initial virulent strains of salmonella by decreased invasive properties and the absence of any capacity to multiplication in the epithelial cells. The data on the avirulence of the glycerine mutant obtained on a model in vitro confirmed the observations carried out in vivo in intraperitoneal infection of albino mice (LD50 = 1-10(7) cells) and in keratoconjunctival infection of guinea pigs.     

3种革兰氏阴性细菌及其L型内毒素含量的测定

本文采用鲎试剂对大肠杆菌（ＡＴＣＣ２５９２２）、伤寒杆菌、绿脓杆菌（ＡＴＣＣ７８５３）及它们的Ｌ型内毒素的含量进行测定。结果显示细菌型与细菌Ｌ型均具有内毒素,但细菌Ｌ型内毒素含量较细菌型低（约为１／３￣１／２）。因此,认为细菌Ｌ型仍有一定的致病性。     

In vivo and in vitro assessment of the virulence of Listeria monocytogenes strains

To evaluate whether the in vitro model (invasion and intracellular growth in Caco-2 cells) for determining virulence is a suitable alternative to the in vivo model (50% lethal dose), we compared the levels of virulence obtained with the two models. We tested L. monocytogenes strains isolated from food and clinical samples during three episodes of listeriosis occurring in Italy in the period 1993-1995. We also tested L. monocytogenes strains isolated from food during official control activities. The results obtained from the tested strains varied according to the experimental method adopted: the L. monocytogenes strains featuring the same genetic pattern showed a greater uniformity of response in vivo than in vitro. We can conclude that the in vitro model may be used as an alternative to the animal model to determine Listeria spp pathogenicity, though it cannot distinguish levels of virulence within the L. monocytogenes species.     

Detection of protease specifically splitting actin in revertants of Shigella flexneri L-forms

Proteolytic activity of cell extracts from revertants of Shigella flexneri L-forms as well as biochemical properties of these strains and their sensitivity to antibiotics were studied. The protease found earlier in cells of strain E. coli A2 was shown to be synthesized by one of 8 revertants under study. This protease split actin and did not split some other proteins, its activity was inhibited by inhibitors of metalloproteases. Strain 5a2c which produced the protease was similar to the strain E. coli A2 and differed from other revertants in some biochemical properties, resistance to ampicillin and sensitivity to furazolidone. Thus the protease activity can be a marker of structural and functional transformation of Sh. flexneri under the influence of furazolidone.     

Theileria annulata: attenuation of a schizont-infected cell line by prolonged in vitro culture is not caused by the preferential growth of particular host cell types

Flow cytometry and monoclonal antibodies to bovine leucocyte surface antigens were used to identify the types of host cells that the sporozoites of Theileria annulata infect in cattle, to determine whether virulent schizont-infected cell lines (lines) differed phenotypically from avirulent lines, and to establish whether attenuation in vitro was accompanied by the preferential growth of particular host cell types. The surface antigens of four pairs of T. annulata (Ta) (Hisar) lines derived ex vivo and in vitro, including the virulent ex vivo-derived Ta Hisar S45 line, were consistent with a myeloid origin for all lines, irrespective of their derivation. The profiles of lines derived from cattle inoculated with a virulent line showed that the schizonts liberated from inoculated cells had transferred to myeloid cells. A number of other lines infected with different stocks of T. annulata expressed myeloid markers; a single line expressed CD21, a B cell marker. During prolonged in vitro culture, the parasites in the ex vivo (virulent)- and in vitro (avirulent)-derived Ta Hisar S45 myeloid lines became clonal, as defined by glucose phosphate isomerase (GPI) polymorphism, and the virulent line became attenuated. The two lines retained phenotypic profiles indicative of a myeloid origin but coexpressed some lymphoid antigens (CD2, CD4, CD8), although not CD3. Cloned schizont-infected lines, representing the three parasite GPI isotypes which constituted the virulent line, expressed similar patterns of myeloid and lymphoid markers to the virulent parent line. Some schizont-infected clones failed to establish as lines during the early weeks of culture because the cells died as the parasites differentiated into merozoites at 37 degrees C, the temperature at which schizont-infected cells normally grow exponentially. These results provided no evidence that prolonged culture induces preferential growth or loss of particular host cell types. However, a number of the alterations in host cell surface antigens induced by prolonged culture were shown to be linked to permanent changes in the parasite genome.     

Loss of virulence of revertants from Staphylococcus aureus L-phase variants in comparison with the parent strain

The L-forms of the Klecha 155 strain were induced in vitro by methicillin. After reversion, the strains were tested for 19 biochemical features and for susceptibility to penicillin, ampicillin and methicillin. Two reverted strains were less virulent for mice than the parent strain. It is concluded that the decrease in virulence of reverted Staph. aureus strains is correlated with changes in the chemical structure and composition of the cell wall after reversion from the L-phase variants.     

Fidelity of replication of bacteriophage phi X174 DNA in vitro and in vivo

Seven different revertants of bacteriophage phi X174am16 (AB5276G leads to T) have been isolated and the nature of the reversions determined by sequencing their DNA. The revertants each differ from am16 by just a single base substitution. These may be distinguished with varying degrees of ease by characteristic temperature sensitivities of growth. This has facilitated the determination of the frequency at which DNA polymerase III catalyses different types of substitution mutations in copying phi X174 DNA in vitro and in vivo. During the replicative form (RF) leads to single-stranded (SS) stage of replication in vitro, four different revertants may be readily produced according to well-defined rate laws on biasing the concentrations of dNTPs. Transversion mutations are found to be formed predominantly by purine x purine mismatching, whilst transitions are formed predominantly by G x T mismatching. The substitutions via G x T and G x A mismatches are estimated to occur at similar frequencies in vivo. The two most common revertants isolated in vivo, however, are not those readily produced during the RF leads to SS stage in vitro but are those produced on purine x purine mismatching in the SS leads to RF stage. The accuracy of the DNA polymerase in vitro appears to be similar to that in this stage in vivo. However, the overall accuracy of the RF leads to SS replication in vivo is more accurate than predicted from the measurements of the accuracy in vitro.     

Models for the study of the toxic action of Legionella pneumophila

L. pneumophila virulent culture and the filtrate of this culture disintegrated with ultrasound were shown to be toxic for guinea-pig peritoneal macrophages in vitro and for AKR mice. The virulent culture of this infective agent and the filtrate of the supernatant fluid obtained from the washings of its virulent culture had no toxic effect on a macrophage monolayer culture and on mice. The use of these models for characterizing Legionella intracellular toxic activity, as well as for characterizing Legionella strains isolated from patients and the environment, is proposed.     

铜绿假单胞菌及L型诱导巨噬细胞凋亡的实验研究

目的研究铜绿假单胞菌（PA）及L型诱导巨噬细胞凋亡的能力,比较二者的差异。方法用生物素断端标记（TUNEL）法检测PA及L型感染巨噬细胞2、4、8、12、16和20h后各时间段的细胞凋亡率,Giemsa染色观察细胞凋亡情况,硝酸还原酶法检测培养液中一氧化氮（NO）的浓度变化。结果 PA及L型能诱导巨噬细胞发生凋亡,与对照组比较差异有统计学意义（P〈0.05）;L型诱导细胞的凋亡率弱于原菌（P〈0.05）;PA及L型感染组培养液NO浓度较对照组明显升高（P〈0.05）。结论 PA及L型可诱导巨噬细胞发生凋亡,L型较其原型诱导细胞凋亡的能力弱,NO可能在巨噬细胞凋亡中发挥一定作用。PA及L型可通过诱导巨噬细胞凋亡,发挥致病作用。     

Tumorigenicity of revertants from an SV40-transformed line

A syndrome of in vitro properties correlates with the tumorigenicity of SV40-transformed rodent cells. These properties are plasminogen activator production, loss of large actin cables, and anchorage-independent growth. An established rat fibroblast line, its SV40 transformant, several T-antigen negative revertants, and a spontaneous retransformant isolated form one of the revertants were analyzed in vivo for their tumorigenicity and in vitro for the syndrome. The two transformed lines were highly tumorigenic, and had clearly abnormal in vitro properties. The parental rat line was weakly tumorigenic in nude mice and demonstrated a slightly transformed response in the in vitro assays. The revertants were completely nontumorigenic. Expression of the in vitro syndrome was not uniform for all revertants; however, most cell lines maintained the correlation of the syndrome and tumorigenicity.