Effect of the method of preparation of Bacille Calmette-Guérin (BCG) vaccine on the properties of four daughter strains

Samples of Bacille Calmette-Guerin (BCG) vaccines from four collaborating production laboratories, each of which had prepared vaccine from four different daughter strains of BCG, had previously been monitored for changes in colony morphology and the present study was undertaken to determine whether the changes observed were reflected in the patterns of protein secretion and lipid content. In the samples examined there was evidence for a correlation between colony morphology and the presence or absence of mycoside B. As the components of BCG that determine virulence and protective immunity are unknown, care must be taken to ensure constancy of the strains during the manufacture of vaccines.     

Effect of the method of preparation of bacille Calmette-Guérin (BCG) vaccine on the properties of four daughter strains

Samples of Bacille Calmette-Guérin (BCG) vaccines from four collaborating production laboratories, each of which had prepared vaccine from four different daughter strains of BCG, had previously been monitored for changes in colony morphology and the present study was undertaken to determine whether the changes observed were reflected in the patterns of protein secretion and lipid content. In the samples examined there was evidence for a correlation between colony morphology and the presence or absence of mycoside B. As the components of BCG that determine virulence and protective immunity are unknown, care must be taken to ensure constancy of the strains during the manufacture of vaccines.     

A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927

BCG vaccines are substrains of Mycobacterium bovis derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and mma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence of the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.     

Phage types of Mycobacterium bovis, substrains of BCG.

Nineteen substrains of Mycobacterium bovis, strain bacille Calmette-Guérin (BCG) used in laboratories throughout the world for the preparation of BCG vaccines were phage-typed with a battery of mycobacteriophages. The results revealed differences in their susceptibility to phage lysis that allow subdivision of these strains of BCG into two or more phage types.     

Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.

Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.     

BCG vaccine: an investigation of colony morphology from four different strains after their introduction as seed for vaccine preparation in four production laboratories

Four collaborating BCG production laboratories had each prepared vaccine from four different BCG strains. In this laboratory the morphology of colonies cultured from samples of the 16 vaccine lots thus available was examined, and it was found that changes monitored in earlier small-scale experimental reconstructions had also occurred during some of the full-scale production procedures, in accordance with prediction. In particular, a minority population carried by the Danish BCG strain had replaced the original majority when this strain had been employed as seed for the production of vaccine by the British procedure. Similarly, a minority carried by the Japanese strain had replaced the original majority when this strain was introduced as seed into the production procedures in the United Kingdom and France. Serial subcultures made in this laboratory showed, in accordance with previous experience, that the changes that had occurred in the Japanese strain could be completely reversed by serial subculture as a surface pellicle on Sauton medium.     

Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains

N.G. FOMUKONG, J.W. DALE, T.W. OSBORN AND J.M. GRANGE. 1992. Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.     

Cytochemical and biological properties ofMycobacterium bovis BCG

It was the aim of the present communication to find a simple test for a reliable discrimination ofMycobacterium bovis BCG fromMycobacterium tuberculosis. A total of 26 BCG strains, out of them 10 Czechoslovak strains (2 lyophilized cultures of BCG of different batch, 6 strains isolated from abscesses of children after BCG-vaccination and 2 strains from fatal cases after BCG-vaccination) and 16 strains obtained from foreign laboratories, were used. Of the tested characteristics a combination of 3 tests, sensitivity to 1 μg of 2-thiophene carbonylhydrazide (TCH), activity of 3 acylamidases (urease, nicotinamidase and pyrazinamidase) and a quantitative nitrate test, was found to be most advantageous. The Czechoslovak strains ofMycobacterium bovis BCG were fully sensitive to TCH, of the 3 acylamidases mentioned above only urease was positive and nitrate was reduced only little or not at all. On the other hand, strains ofMycobacterium tuberculosis were always resistant to TCH, had positive urease, nicotinamidase and pyrazinamidase and reduced nitrate very intensively.     

Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin

Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4+ and CD8+ T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses.     

DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG

DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.     

Subdivision of daughter strains of bacille Calmette-Guérin (BCG) according to secreted protein patterns

In order to identify proteins secreted by live organisms, daughter strains of the Bacillus Calmette-Guérin (BCG) were grown for 4-7 d in a defined medium containing [35S]methionine. Secreted components were then separated by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions, and analysed by autoradiography and in an Ambis beta-scanner. The results indicate that BCG daughter strains can be subdivided into two groups according to their secretion of a 46 kDa protein dimer consisting of two similar 23 kDa subunits. High-producer strains (Japanese, Brazilian and Russian) secrete very large quantities of this material, which constitutes approximately 23% of all secreted protein. These findings correlate with earlier studies in which degradation products of the protein dimer may have been identified, and with the data from patterns of cell wall lipids.     

多重PCR方法特异性鉴定卡介苗菌株多糖核酸的初探

与结核分枝杆菌H37Rv菌株进行比较,BCG菌株可找到一个特殊的缺失片段RD1,它存在于所有有毒分枝杆菌中,而在所有的卡介苗菌株中均缺失。应用多重PCR方法检测RD1区的存在与否,可以区别BCG和其它有毒的分枝杆菌。卡介菌多糖核酸来源于卡介菌,检测成品中DNA是否含有RD1区,能特异性地鉴别该制品。结果显示牛分枝杆菌标准株和结核分枝杆菌H37Rv存在RD1区;而卡介菌多糖核酸注射液和国内皮内注射用BCG疫苗生产用菌株扩增产物一致,提示均缺失RD1区。因此,这种多重PCR方法适用于卡介菌多糖核酸注射液的特异性鉴别试验。     

Variation in susceptibility to Bacillus thuringiensis toxins among unselected strains of Plutella xylostella

So far, the only insect that has evolved resistance in the field to Bacillus thuringiensis toxins is the diamondback moth (Plutella xylostella). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to B. thuringiensis toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to B. thuringiensis toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five B. thuringiensis toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.     

Comparison of bcg strains for delayed hypersensitivity induction tested in vivo in guinea pigs and in vitro

Previous studies performed on guinea pigs demonstrated a direct dependence of tuberculin reaction size (in vivo hypersensitivity) on immunogenicity in a number of BCG strains. The present work used an in vitro method, MIF detection, for assessing hypersensitivity and compared the results obtained with tuberculin hypersensitivity tests, correlating the data with the immunogenicity of the individual BCG strains employed. The following strains were used: the Czechoslovak BCG strain No. 725, Japanese BCG strain Tokyo, Danish BCG strain Copenhagen and Soviet BCG strain Moscow. The results obtained by the two hypersensitivity testing methods, in vivo and in vitro were in a direct correlation; a direct relationship was also demonstrated between hypersensitivity tested by the in vitro method and immunogenicity. The in vitro method of MIF detection is reproducible and comparable with the other two methods employed and may be used as an alternative approach to BCG vaccine efficacy testing. It might probably also be applicable to estimation of the status of cell-mediated immunity against intracellularly parasitizing bacteria in general.     

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.     

Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.     

Mapping quantitative trait loci for anxiety in chromosome substitution strains of mice

Anxious behavior in the mouse is a complex quantitative phenotype that varies widely among inbred mouse strains. We examined a panel of chromosome substitution strains bearing individual A/J chromosomes in an otherwise C57BL/6J background in open-field and light-dark transition tests. Our results confirmed previous reports of quantitative trait loci (QTL) on chromosomes 1, 4, and 15 and identified novel loci on chromosomes 6 and 17. The studies were replicated in two separate laboratories. Systematic differences in the overall activity level were found between the two facilities, but the presence of the QTL was confirmed in both laboratories. We also identified specific effects on open-field defecation and center avoidance and distinguished them from overall open-field activity.     

Effective nonliving vaccine against experimental tuberculosis in mice

Ribi, Edgar (Rocky Mountain Laboratory, Hamilton, Mont.), Carl Larson, William Wicht, Robert List, and Granville Goode. Effective nonliving vaccine against experimental tuberculosis in mice. J. Bacteriol. 91:975-983. 1966.-Antituberculosis vaccines were prepared in one of three manners: lyophilized BCG suspended in light mineral oil was disrupted in a Sorvall pressure cell and the "oil disruption product" was collected by centrifugation; BCG was disrupted in water, lyophilized, and worked into a paste with a small amount of oil (about 0.16 ml per 50 mg); BCG was disrupted in water, and the cell wall fraction was isolated, lyophilized, and prepared in an oil paste. These vaccines were suspended in Tween-saline to a concentration of 5 mg/ml and heated at 65 C for 30 min. In protection tests based on pulmonary infection with Mycobacterium tuberculosis H37Rv, the median number of virulent organisms in lung tissue of mice immunized with a few hundred micrograms of these three vaccines was 3 to 4 logs lower than in unvaccinated control mice. A similar dose of viable BCG standard vaccine reduced the lung count 1 to 2 logs below the controls. Protection afforded by nonviable, whole BCG, with or without oil, was of only borderline significance. Since oil-treated fractions containing cell walls produced effective immunity, while the oil-treated protoplasm or whole cells were not active, the protective antigen appeared to be an inner component of the cell wall, exposed when the cell was disrupted, and activated by oil. Extraction of oil from immunogenic disruption products resulted in loss of ability of the products to confer protection against the aerosol challenge, whereas high protection against the conventional challenge by intravenous infection with up to 1.4 x 10(8) cells of M. tuberculosis H37Rv was retained. Retreatment with oil of these nonimmunogenic products restored the immunogenicity if the oil was applied to dried products. The consistent finding that moisture interferes with the enhancement of the vaccine potency by oil suggested that such enhancement may not be the same as that ordinarily produced by water-in-oil emulsions.     

The potency testing of pseudorabies vaccines in pigs. A proposal for a quantitative criterion and a minimum requirement

A criterion for the potency testing of pseudorabies vaccine based on the difference between the mean weight gain during the seven days after pathogenic challenge of vaccinated or control piglets is proposed. This criterion, termed delta G7, has been studied as a function of initial weight variation of the animals, different challenge strains and the period between challenge and the end of the test. The statistical analysis of results optimizes the experimental parameters and gives rise to a quantitative test which discriminates between vaccinates and controls and substantiates a proposal for a minimum vaccine potency requirement. The implementation of the method proposed is compatible with current practice in control laboratories.     

Variation in Susceptibility to Bacillus thuringiensis Toxins among Unselected Strains of Plutella xylostella

So far, the only insect that has evolved resistance in the field to Bacillus thuringiensis toxins is the diamondback moth (Plutella xylostella). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to B. thuringiensis toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to B. thuringiensis toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five B. thuringiensis toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.