Importance of immunoelectrophoresis in agar and disc electrophoresis in polyacrylamide gel for characterizing different strains of Bordetella pertussis

The antigenic composition of typical and atypical B. pertussis strains obtained in the foci of pertussis infection, as well as experimentally obtained antibiotic-resistant B. pertussis strains, has been studied by the methods of immunoelectrophoresis in agar and electrophoresis in polyacrylamide gel (PAAG). Immunoelectrophoresis in agar has been found capable of differentiating B. pertussis culture from a group of unidentified morphologically similar Gram-negative bacilli by their antigenic composition and thus suitable for use as an additional criterion in the identification of atypical B. pertussis strains. PAAG electrophoresis has permitted finding differences in the set of protein antigens in the control strain and in its clones obtained by multiple subculturing in media with antibiotics added.     

Ultrastructure of freshly isolated strains of Bordetella pertussis

The study of the electronograms of B. pertussis strains isolated in the foci of pertussis revealed the existence of the morphological variants of these cells, differing in the character of the cell wall, the state of the cytoplasm, the presence of amorphous inclusions of medium electron-optical density. The morphological variants did not correlate with the character of B. pertussis colonies isolated from blood-charcoal agar. The ultrastructure of the cells belonging to the second morphological variant was similar to that of the cells from the museum strain, altered by tetracycline treatment in the course of the experiment.     

Glutamine synthetase content and immunochemical analysis of different strains of Bordetella pertussis

In this investigation 3 groups of strains isolated from pertussis patients have been studied: typical (group 1), atypical in their cultural properties (group 2), unidentified Gram-negative bacilli agglutinated by pertussis and parapertussis antitoxins (group 3). Besides, B. pertussis cultures, obtained by subculturing 2 museum strains and 2 newly isolated strains on synthetic casein-charcoal agar with subinhibiting doses of antibiotics or specific immune sera added, have been studied. As indicated by the results of this study, strains belonging to groups 1 and 2 contain glutamine synthetase, while in strains of group 3 this enzyme is absent. In immunoelectrophoresis strains of group 3 have been found to contain not a single antigen similar to the antigens of strains belonging to groups 1 and 2. Electrophoresis in polyacrylamide gel has revealed to differences in the protein spectrum of the strains of these 3 groups. The investigation has shown that the determination of glutamine synthetase and immunoelectrophoresis can be used for the differentiation of B. pertussis from similar Gram-negative bacilli. B. pertussis strains, changed as the result of experiments with antibiotics and specific immune sera, have also been shown to retain their antigenic composition and protein spectrum and to have no essential difference in the content of glutamine synthetase.     

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.     

Isolation of hemolytic cultures of Bordetella pertussis intended for the production of acellular pertussis preparations and the characterization of their biological properties

The heterogeneity of the population of B. pertussis laboratory strains with respect to the capacity of individual clones to lyse erythrocytes has been established. The complete or partial reduction of the antigens under study in nonhemolytic bacteria has been shown. The use of hemolytic cultures for the preparation of acellular pertussis vaccine makes it possible to increase the content of B. pertussis toxin in these vaccines.     

Toxigenicity conversion by pertussis phages in Bordetella parapertussis

For the first time toxigenicity conversion in B. parapertussis induced by B. pertussis phages was discovered. The clones of B. parapertussis recipient strain No. 17903 used in this study were subjected to lysogenization with 4 B. pertussis phages; as a result, 95% of these clones became immune to the repeated phage infection, developed spontaneous phage production and showed toxic properties (lethal toxicity due to the action of thermolabile and thermostable toxins) characteristic of the donor strains from which B. pertussis phages had been obtained. Differences in the degree of toxicity shown by the converted strains were determined by means of the spleen index. The convertants thus obtained did not possess protective potency.     

Genetic diversity and relationships in populations of Bordetella spp

Genetic diversity in 60 strains of three nominal Bordetella species recovered from humans and other mammalian hosts was assessed by analyzing electrophoretically demonstrable allelic variation at structural genes encoding 15 enzymes. Eleven of the loci were polymorphic, and 14 distinctive electrophoretic types, representing multilocus genotypes, were identified. The population structure of Bordetella spp. is clonal, and genetic diversity is relatively limited compared with most other pathogenic bacteria and is insufficient to justify recognition of three species. All isolates of Bordetella parapertussis were of one electrophoretic type, which was closely similar to 9 of the 10 electrophoretic types represented by isolates of Bordetella bronchiseptica. Bordetella pertussis 18-323, which is used in mouse potency tests of vaccines, is more similar genetically to isolates of B. bronchiseptica and B. parapertussis than to other isolates currently assigned to the species B. pertussis. Apart from strain 18-323, the isolates of B. pertussis represented only two closely related clones, and all isolates of B. pertussis from North America (except strain 18-323) were genotypically identical. Strain Dejong, which has been classified as B. bronchiseptica, was strongly differentiated from all of the other Bordetella isolates examined.     

Comparative study of nutrient media for isolation of the agents of pertussis and parapertussis]

Taking into consideration literature data pointing to a possibility of a marked improvement of the quality of the medium for the isolation of the causative agents of pertussis and parapertussis, an experimental study was made of the sensitivity of the nutrient media produced by the Dagestan Institute of Nutrient Media, a medium made from dry and also of a number of media modified by the authors prepared on its basis. Pure freshly-isolated and museum B. pertussis cultures were used in the observations. Analysis of the results of a comparative study of the indices (detectability of the strains, the appearance of colonies and periods of their formation) allomed a recipee of the most sensitive medium to be elaborated. At the same time there were found considerable differences in the indices of the appearance of B. pertussis colonies and detectability of the museum cultures used for testing the industrial batches and of the freshly-isolated strains.     

Molecular evidence for the lysogenic state of microorganisms belonging to the genus Bordetella and characterization of Bordetella parapertussis temperate bacteriophage 66(2.2)

A new bacteriophage phiK of microorganisms belonging to the genus Bordetella was isolated from cells of the earlier characterized strains 66(2-2) (1 and 2) obtained upon phage conversion of B. parapertussis 17903 cells by B. pertussis bacteriophage phi134. Bacteriophage phiK is identical to previously described Bordetella bacteriophages phiT, phi134, and phi214 in morphology and some biological properties but has a permuted genome different from all other phages. DNA of bacteriophage phiK is not integrated in the chromosome of B. parapertussis 17903, similar to DNA of bacteriophages phiT, phi134, and phi214 that are not integrated into B. pertussis and B. bronchiseptica chromosomes, but may be present in a small part of the bacterial population as linear plasmids. Sequences homologous to DNA of bacteriophage phiK were detected in the chromosome of strain 66(2-2) (1 and 2) and in chromosomes of all tested strains B. pertussis and B. bronchiseptica. Prophage integration in chromosomes of microorganisms of the genus Bordetella may vary in different bacterial strains and species. An assumption about abortive lysogeny of B. parapertussis bacteria for phiK phage and of B. bronchiseptica for closely related phages phiT, phi134, and phi214 has been advanced. The possibility of involvement of B. pertussis insertion sequences in the formation of the chromosomal structure in 66(2-2) convertants and in phage genomes is considered.     

Isolation of a repeated DNA sequence from Bordetella pertussis

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.     

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.     

Adhesion of Bordetella pertussis to sulfatides and to the GalNAc beta 4Gal sequence found in glycosphingolipids

The adherence of the human respiratory pathogen, Bordetella pertussis, to purified glycosphingolipids was investigated using thin layer chromatography overlay assays. Both virulent and avirulent strains of B. pertussis bound to asialo GM1. The bacterium did not bind to the gangliosides GM1, GD1a, GD1b, and GT1b, nor to lactosylceramide, trihexosylceramide, globoside, or Forssman antigen. However, after treatment of the chromatography plates with sialidase, B. pertussis bound to the gangliosides GM1, GM2, GD1a, GD1b, and GT1b but not to GM3. Comparison of the oligosaccharide structures of these gangliosides suggests that the minimum sugar structure needed for avid bacterial binding is GalNAc beta 4Gal. This structure has been previously implicated as a receptor for other human respiratory pathogens (Krivan, H. C., Roberts, D. D., Ginsburg, V. (1988) Proc. Natl. Acad. Sci. U.S.A 85, 6157-6161). Virulent strains of B. pertussis also bound specifically to sulfatide. This response was dose-dependent and inhibited by the anionic polysaccharide dextran sulfate. The sulfated-sugars dextran sulfate, fucoidan, and heparin inhibited the attachment of virulent strains of B. pertussis to human WiDr cells and to hamster trachea cells indicating that sulfatides on the surface of mammalian cells may function as a receptor for B. pertussis. The occurrence of both sulfatides and asialo GM1 in human lung and trachea suggests that these glycolipids may serve as specific receptors for B. pertussis.     

Use of an immunoblotting method for identifying the individual proteins in the antigenic complexes of Bordetella pertussis

The composition of antigenic complexes isolated from the supernatant fluid of B. pertussis culture has been studied by means of immunoblotting techniques. In preparations obtained from B. pertussis strains 305 and 475 fragments of the molecule of fimbrial hemagglutinin, three subunits of B. pertussis toxin and agglutinogens 2 and 3 have been detected with the use of antisera to B. pertussis protective substances.     

Study of the DNA of the causative agent of whooping cough, Bordetella pertussis

A number of physico-chemical properties of Bordetella pertussis DNA has been studied. The values of its floating density and melting point have been established, which has allowed one to calculate the GC composition of B. pertussis DNA. The average molecular weights of the fragments of B. pertussis DNA, resulting from its hydrolysis with specific endonucleases EcoR, BamH 1, Sal 1, Pst 1, have been determined, and thus the basis has been provided for establishing the number of clones necessary for obtaining the complete lset of B. pertussis genes.     

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.     

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.     

Construction and characterization of Bordetella pertussis mutants lacking the vir-regulated P.69 outer membrane protein

The Bordetella pertussis P.69 protein is an immunogen with vaccine potential. The role of this protein in pathogenesis is unclear; it has been associated with the toxic adenylate cyclase and adhesion to eukaryotic cells. For further analysis of the role of P.69 in the biology of B. pertussis, we have constructed strains which specifically lack P.69. The cloned P.69 (prn) gene of B. pertussis was insertionally inactivated with a kanamycin-resistance cassette. This inactivated gene was used to construct P.69- mutants of B. pertussis by allelic exchange using plasmid pRTP1. B. pertussis P.69- strains produced normal levels of other vir-regulated factors, including adenylate cyclase. The serotype of B. pertussis, determined by Eldering and Preston typing sera and monoclonal antibodies, was also unaffected by the presence or absence of P.69. The ability of a prn mutant to adhere to and invade HEp2 cells was not significantly different from that of its parent strain. A strain containing a mutation in fhaB was significantly less adhesive and invasive than its parent, and a prn fhaB double mutant exhibited an even greater reduction in adhesiveness and invasiveness down to levels comparable with a Vir- strain. However, strains harbouring mutations in FHA and/or P.69 were able to colonize or multiply in the murine respiratory tract, although a Vir- strain was unable to survive and proliferate in the same infection model.     

Multiple-locus variable-number tandem repeat analysis of Dutch Bordetella pertussis strains reveals rapid genetic changes with clonal expansion during the late 1990s

Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953. In the 1990s, several epidemic periods have resulted in many cases of pertussis. We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands. Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology. For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B. pertussis. By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains. The relationships between the various genotypes were visualized by constructing a minimum spanning tree. MLVA of Dutch strains of B. pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s. Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods. Using this genotyping, we have been able to show that B. pertussis is much more dynamic than expected.     

Is Bordetella pertussis clonal?

OBJECTIVE--To establish whether Bordetella pertussis is essentially clonal. DESIGN--Analysis of restriction fragments of XbaI digests of DNA from clinical and control isolates of B pertussis by pulse field gel electrophoresis. MATERIALS--105 isolates of B pertussis: 67 clinical isolates from throughout the United Kingdom and 23 from Germany (collected during the previous 18 months); vaccine strains 2991 and 3700; and 13 control isolates from Manchester University''s culture collection. MAIN OUTCOME MEASURES--Frequency of DNA types according to country of origin and classical serotyping. RESULTS--17 DNA types were identified on the basis of the variation in 11 fragments, banding at 200-412 kilobases; 15 types were found in the clinical and control isolates from the United Kingdom and seven in those from Germany. There was no correlation with serotype. DNA type 1 was the commonest overall (22/105 strains, 22%), predominating in serotypes 1,2 and 1,2,3 and including the vaccine strains but not the isolates from Germany. CONCLUSIONS--Current infections due to B pertussis are not caused by a clonal pathogen as multiple strains are circulating in a given population at one time. There is also considerable epidemiological variation in the pathogen population between countries. These findings may have implications for the design of acellular vaccines.