Morphological heterogeneity of Bordetella pertussis populations and clones (based on electron microscopy data)

Populations belonging to different serovars of B. pertussis museum strains and antibiotic-resistant clones obtained from them have been studied by electron microscopy. As a result, morphological heterogeneity and differences in the ultrastructure of the cells with respect to the cell-wall structure, the character of the cytoplasm, the size of the cells, cytoplasmic inclusions and intracellular links have been demonstrated and, proceeding from these data, two main morphological variants of the cells have been defined. The cells of the morphological variant characterized by the pliciform surface of the outer membrane and the pronounced periplasmic space prevail among the populations of the museum strains. The possibility of the isolation of antibiotic-resistant clones, differing in their morphological structure and functional properties from the initial population, has been shown. The morphological diversity of B. pertussis population is the necessary condition for the existence and development of microbial populations.     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Spontaneous variability in a population of Candida albicans strains]

The spontaneous variability of the populations of C. albicans strains of different genesis in the morphological properties of their colonies and in the potential of the activity of their extracellular proteolytic and phospholipid enzymes has been studied. The isolated types of colonies, differing in their morphology, have the phenotypic character of variability. Different populations of strains exhibited variability in the activity of enzymes, depending on morphological variants isolated from these populations. Selected morphological variants with high potential of their proteolytic enzymes retained stability in this property for 5 generations and can be used in medical practice for the isolation of C. albicans antigens.     

Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus

Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the locus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir-) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir- phase variants. The vir locus and the adjacent 5' portion of the fhaB structural gene were cloned from the parental Vir+ B. bronchiseptica strain on a 23.5 kb BamHI fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir- variants of Bordetella pertussis and B. bronchiseptica as well as vir::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.     

Transparent solid nutrient medium for studying the lytic spectrum of bacteriophages of microbes of the genus Bordetella

A solid, transparent culture medium for the study of the lytic spectrum of the phages, active against B. pertussis and B. bronchiseptica, in respect to homologous and heterologous bacteria of the genus Bordetella has been developed. The Cohen-Wheeler liquid medium with nicotinic acid and nicotinamide added, solidified with agar, is nicotinamide added, solidified with agar, is used as the base of the new medium. This base ensures the growth of B. parapertussis and B. bronchiseptica. To stimulate the growth of B. pertussis, the tissue stimulant of B. pertussis growth (a transparent substrate obtained from the tissue of the large intestine of a rabbit) has been used. With 10% of this stimulant added, B. pertussis cells have been found to preserve their typical morphological and immunobiological properties.     

Cholesterol-dependent attachment of human respiratory cells by Bordetella pertussis

Bordetella pertussis is a re-emerging human respiratory pathogen whose infectious process is not fully understood, hampering the design of effective vaccines. The nature of bacterial attachment to host cells is a key event in the outcome of the infection. However, host cell receptors involved in B. pertussis colonization of the respiratory tract are still under investigation. Here, we report that cholesterol-rich domains are involved in B. pertussis adhesion to epithelial cells. Treatment of A549 cells with cholesterol-sequestering drugs such as methyl-β-cyclodextrin, nystatin, or filipin resulted in a significant decrease of B. pertussis attachment. Confocal laser microscopy studies showed B. pertussis associated with cholesterol-rich domains. Accordingly, B. pertussis was found in detergent-resistant membrane domain fractions isolated from bacterial-infected A549 cells. Our results indicate a main role of filamentous hemagglutinin, an environmentally regulated virulence factor, in this interaction, and a specific affinity for cholesterol, one of the major components of traqueal secretions, which might additionally contribute to the effective colonization of the respiratory tract.     

Antigenic similarity of the pertussis component of adsorbed DTP vaccine to human erythrocytes

The similarity of the heterogeneous antigens, types A and B, of human red blood cells to the most of B. pertussis strains constituting the pertussis component of commercial batches of adsorbed DPT vaccine has been established. This property makes the vaccine strains different from B. pertussis isolated from pertussis patients. One of the reasons of the insufficient effectiveness of immunization against pertussis has been determined: the intensity of immune response depends on the antigenic heterogeneity of the pertussis component of the vaccine and the AB0 group factors in the blood of the vaccinees. For the first time the accumulation of immune alpha- and beta-isoagglutinins in the blood of persons immunized with absorbed DPT vaccine has been established. This accumulation shows the medium degree of direct correlation with the manifestations of the clinical reaction to the injection of the vaccine. The data obtained in this study indicate the necessity to revise the existing method of obtaining the pertussis component of adsorbed DPT vaccine on solid culture media with human red blood cells added and to develop the technique of the additional purification of this component from heterogeneous antigens.     

The formation of a test system for assessing the safety of different types of pertussis preparations: their cytotoxic action on mouse thymocytes in vitro

The test for the evaluation of the toxicity of different types of pertussis preparations as manifested by their in vitro influence on mouse thymic cells (T test) has been finally worked out. The use of the T test has made it possible to reveal the nonstandard character of the production lots of adsorbed diphtheria-pertussis-tetanus vaccines, both whole-cell vaccine and Japanese acellular vaccine. The degree of the in vitro damaging action of pertussis preparations on mouse thymic cells greatly depends on the residual content of Bordetella pertussis nontoxoidized toxin which, in contrast to B. pertussis lipopolysaccharide and filamentous hemagglutinin, produces pronounced cytotoxic action on mouse thymic cells.     

A study of the ability of Bordetella pertussis toxin to induce the formation of B-suppressors

Mouse spleen cells not adhering to the plastic surface and B-cells isolated from them were treated with B. pertussis toxin in vitro, washed and injected into recipients (allogeneic, syngeneic, intact or lethally irradiated) whose immune response to sheep red blood cells was then evaluated by Jerne's method. Treatment with B. pertussis toxin was shown to induce the development of immunosuppressive activity in intact spleen cells and in B-cells, to abolish the activity of memory B-cells and to enhance the suppressor activity of autoimmune mice. Supernatants obtained after autoimmune mice. Supernatants obtained after the 18- to 24-hour cultivation of spleen cells, previously treated with B. pertussis toxin for 60 minutes, suppressed the reaction of blast transformation of spleen cells to Con A and lipopolysaccharide and induced the appearance of immunosuppressive activity in intact spleen cells. The suppressing effect of the cells studied in this investigation may be linked with the ability of B. pertussis cells to stimulate the synthesis of cAMP, prostaglandins E and/or suppressor factors.     

Evaluation of whole-cell and acellular pertussis vaccines effectiveness in clearance of experimental B. pertussis infection in mice

The study is based on assumption that B. pertussis strains harbouring different allele variants of genes encoding subunit S1 of pertussis toxin and pertactin might be eliminated with different efficiency from lung tissue of mice which were immunized with whole-cell and acellular pertussis vaccines. It has been assumed that strains containing combinations of genes alleles which were not prevalent since 1990-ties are consisting of mutated strains in respect to pertussis toxin subunit S1 and pertactin, and are capable to decrease efficiency of pertussis vaccines. Experiments performed in vivo dealt with activity of tested vaccines against B. pertussis strains of different combinations of ptxS1/prn. The study indicated for lowered efficiency of whole-cell and acellular pertussis vaccines in elimination of mutated strains of B. pertussis from animal lung tissue in comparison with strains currently used for vaccine production.     

Mitogenic action of the liquid phase of a microbial suspension of Bordetella pertussis

The suspension of B. pertussis cells in 0.15 M NaC1 solution, used for the preparation of corpuscular pertussis vaccine contains components loosely bound to microbial cells and producing pronounced mitogenic effect on mouse splenocytes at a concentration of 10 micrograms/ml. The mitogenic activity of B. pertussis is due to complex substances (lipopolysaccharide, protein, nucleic acids) with a wide range of molecular weights (70,000 to greater than 400,000). The mitogenic factor showing no leukocyte-stimulating and protective activity has been isolated by sedimentation with ammonium sulfate and gel filtration on Sephadex G-200. The mitogenic activity of B. pertussis lipopolysaccharide in the blast transformation test has been confirmed.     

Characterisation of major biological properties of Bordetella bacteriophages

The main biological properties (morphology of negative colonies, parameters of adsorption and single development cycle) of B. pertussis and B. bronchiseptica phages, isolated spontaneously and by induction with mitomycin C, were studied. To compare these characteristics, one B. parapertussis indicator strain was used, and the experiments were carried out under identical conditions. Highly active sera were obtained with the use of complete Freund's adjuvant. B. pertussis phages isolated from the strains of different serovars were serologically related, but not identical, and differed in their constant characterizing their rate of neutralization with homologous antisera. The adsorption of the phages on homologous strains was more intensive than on the cells of B. parapertussis indicator strain. However, the authors failed to observe the further development of the phages in the host cells.     

The phenotypic properties of freshly isolated strains of Bordetella]

As the result of our investigations, newly isolated B. pertussis and B. bronchiseptica strains were studied. The results of these investigations showed that B. pertussis strains isolated under the conditions of immunoprophylaxis were characterized by sufficient stability of the main phenotypical properties which determined their pathogenicity: B. pertussis toxin, fimbrial agglutinogens and filamentous hemagglutinin. At the same time B. bronchiseptica strains isolated from animals proved to be phenotypically variable both in vivo and in the process of in vitro passage.     

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.     

Development of a method for the quantitative determination of Bordetella pertussis toxin and the prospects of its use for diagnosis

Two variants of the enzyme immunoassay (EIA) systems for the determination of B. pertussis toxin (BPT), the "double sandwich" system and the competitive assay system, have been developed. For the titration of BPT in B. pertussis antigens the use of fetuin as the affinity base is preferable, and not antibodies from different paired animals. Of the two variants, the competitive EIA is more promising for diagnostic purposes.     

Biochemical and immunological comparison of lipopolysaccharides from Bordetella species

Lipopolysaccharides (LPS) isolated from Bordetella pertussis, B. parapertussis and B. bronchiseptica were analysed for their chemical composition, molecular heterogeneity and immunological properties. All the LPS preparations contained heptose, 3-deoxy-D-manno-2-octulosonic acid, glucosamine, uronic acid, phosphate and fatty acids. The fatty acids C14:0, C16:0 and beta OHC14:0 were common to all the LPS preparations. LPS from B. pertussis strains additionally contained isoC16:0, those from B. parapertussis contained isoC14:0 and isoC16:0, and those from B. bronchiseptica contained C16:1. By SDS-PAGE, LPS from B. pertussis had two bands of low molecular mass, and the LPS from B. parapertussis and B. bronchiseptica showed low molecular mass bands together with a ladder arrangement of high molecular mass bands. Immunodiffusion, quantitative agglutination and ELISA demonstrated that the LPS from B. pertussis strains reacted with antisera prepared against whole cells of B. pertussis and B. bronchiseptica; LPS from B. parapertussis reacted with antisera to B. parapertussis and B. bronchiseptica, and LPS from B. bronchiseptica reacted with anti-whole cell serum raised against any of the three species. From these results, it is concluded that LPS from B. bronchiseptica has structures in common with LPS from B. pertussis and B. parapertussis, while the LPS from B. pertussis and B. parapertussis are serologically entirely different from each other.     

Restriction analysis of Bordetella pertussis DNA isolated from patients with whooping cough in 1968 and 1995-98 and B. pertussis used for production of national vaccine strains

The genotypic and serotypic analysis of B. pertussis strains isolated from the nasopharynx of children with whooping cough in the years 1968 and 1995-98 and B. pertussis vaccine strains was the aim of this study. The genotyping of the examined strains was done by electrophoretic division of DNA in pulsed field. The 3 types (A, B, C) and 2 subtypes (A1 and A2) of DNA restriction patterns were determined for the B. pertussis strains isolated in 1968. The 2 types (D and E) and 10 subtypes (D1-D10) of DNA restriction patterns were identified for B. pertussis strains from the years 1995-98. The DNA restriction patterns of B. pertussis strains isolated in the years 1968 and 1995-98 were not identical what was the evidence of the fact that in the sixties and nineties whooping cough was caused by different B. pertussis clones. The different DNA profiles were also observed for vaccine strains as well as for vaccine strains and current isolates. Differences in DNA patterns of vaccine strains and B. pertussis strains isolated in the years 1995-98 indicated a relationship possibility in some cases while lack of relationship between these strains in other cases. Serotyping of the examined B. pertussis strains was performed by the agglutination method with the sera against B. pertussis agglutinogens 1, 2 and 3. Most strains--15 (75%) isolated in 1968 possessed only agglutinogens 1 and 3. Serotype 1, 2, 3 was most frequently observed among isolates from the years 1995-98. This study indicates the expediency of periodical change of B. pertussis vaccine strains in the aspect of whooping cough resurgence in the years 1994-95 and 1997-98.     

Lectin-like binding of pertussis toxin to a 165-kilodalton Chinese hamster ovary cell glycoprotein

Chinese hamster ovary (CHO) cells cluster in the presence of pertussis toxin, a response that is correlated with the ADP-ribosylation of a Mr = 41,000 membrane protein by the toxin. A ricin-resistant line of CHO cells (CHO-15B) which specifically lacks the terminal NeuAc----Gal beta 4GlcNAc oligosaccharide sequence on glycoproteins did not cluster in response to pertussis toxin. These cells do contain the Mr = 41,000 protein substrate for the enzymatic activity of the toxin which suggests that pertussis toxin, like certain plant lectins, does not bind to or is not internalized by the CHO-15B cells. There was no evidence of pertussis toxin binding to gangliosides or neutral glycolipids isolated from CHO cells but the toxin bound to a Mr = 165,000 component in N-octyglucoside extracts of CHO cells that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose. Plant lectins from Ricinus communis and Erythina cristagalli detected a similar size band in CHO cells and also did not react with CHO-15B cells. Unlike pertussis toxin, these plant lectins recognized two other major bands in CHO cell extracts and reacted best after sialidase treatment of nitrocellulose transfers containing CHO cell extracts. Conversely, sialidase treatment abolished binding a pertussis toxin and wheat germ agglutinin, a plant lectin that reacts with multivalent sialic acid residues on glycoproteins, to the Mr = 165,000 band. Purified B oligomer of pertussis toxin also uniquely detected a Mr = 165,000 component in CHO cell extracts while the A subunit of pertussis toxin was unreactive. These results indicate that pertussis toxin binds to a CHO cell glycoprotein with N-linked oligosaccharides and that sialic acid contributes to the complementary receptor site for the toxin. In addition, they suggest that a glycoprotein may serve as a cell surface receptor for pertussis toxin and that this interaction is mediated by a lectin-like binding site located on the B oligomer.     

Efficacy of Whooping-cough Vaccines used in the United Kingdom before 1968: Final Report to the Director of the Public Health Laboratory Service by the Public Health Laboratory Service Whooping-Cough Committee and Working Party

The efficacy of pertussis vaccines was investigated in 33 areas in the United Kingdom from November 1966 until April 1968 inclusive. Bordetella pertussis was isolated from 1,293 persons, but there were only six isolations of B. papapertussis. Among vaccinated contacts under 5 years in homes in which B. pertussis was isolated 52% developed paroxysmal cough. The corresponding attack rate among unvaccinated contacts was 69%. These findings suggest that much of the pertussis vaccine in use for five or six years before 1968 was not very effective. However, vaccine from one producer was more effective than vaccine from another. Of the cultures of B. pertussis identified 89% were serotype 1, 3 and only about 9% were serotype 1, 2, 3. Serotype 1, 2, 3 was isolated much more frequently from unvaccinated than from vaccinated children, but some cultures identified as type 1, 2, 3 were found on re-examination to contain colonies of type 1, 3. Virological investigations were made in some areas during the first year of the study. Of the wide variety of viruses identified adenovirus and parainfluenza virus were the most common groups. Virus isolation rates were similar in patients and symptomless contacts, but B. pertussis was isolated far more often from patients than from symptomless contacts. The evidence suggests that B. pertussis remained the major cause of whooping cough in the U.K.     

Molecular genetic characteristics of the B. pertussis strains isolated in different periods of the pertussis epidemic process

Despite the fact that the mass immunization of the children population with the DPTs vaccine has been carried out in the Russian Federation since 1959, the pertussis infection persists to be one of the pressing problems for the children population. Although the vaccination coverage of the children population with pertussis vaccines is high in Russia, at present time the pertussis incidence rates are increasing among schoolchildren and remain high among infants younger than 12 months old. Many researchers believe that the variability of the genetic structure of the pertussis causative agent may be one of the causes of increasing pertussis incidence rates. This investigation provides the molecular genetic characteristics of 97 B. pertussis strains isolated in pertussis patients in Moscow in different periods of pertussis epidemic process since the 1950s up to present time. It shows the changes in the structures of genes, which are encoding the main protective antigens of the pertussis microbe that are the pertussis toxin (ptxS1) and the pertactin (pm). The structurre of the ptxS1 and pm gene of the B. pertussis vaccine strains was compared with the structures of these genes in the B. pertussis strains isolated from the pertussis patients at present time and also in past years. All B. pertussis strains isolated in the prevaccination period (1948-1959) and most strains (95%) isolated during the first twenty years of the mass immunization in Russia are characterized by the presence of the so called "vaccine" alleles of the pertussis toxin and pertactin genes that are ptxS1 B or ptxS1 D and pm 1 alleles that corresponds to the genetic structure of the vaccine producing strains. In the early 1970s the B. pertussis strains of another toxin and pertactin genetic structures with so-called "non-vaccinal" alleles ptxS1 A and pm 3 (pm 2 since 1980s) began to appear. The B. pertussis strains with "non-vaccinal" alleles have completely displaced the "old" strains. At present time in Moscow the pertussis disease is caused by the B. pertussis strains bearing ptxS1 A and pm 2 or pm 3 alleles of pertussis toxin and pertactin genes. There was no correlation between the genotype and serotype. Thus, the structure of the B. pertussis toxin and pertactin genes in strains which have been isolated since the 1980s up to now differs from the structure of these genes in strains which are used for producing DPTs vaccine. The data obtained in this investigation suggest that the genetic structure specificity of circulating B. pertussis strains that are producing the disease at present time should be used as one of the criteria for selecting vaccine producing strains.