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Bordetella pertussis produced hemagglutinin in stationary cultures; in cultures kept under constant shaking, hemagglutinin was found only during the first 48 h of incubation but not after 3 to 5 days. The type of medium had a pronounced effect on production of hemagglutinin. Strain differences in ability to produce hemagglutinin were also detected.
Bordetella pertussis (B.p.) induces blast transformation of human lymphocytes; whole killed B.p. are more efficient than extracts obtained by sonication. Similar responses were obtained with each of the four strains used in the Danish pertussis vaccine. B.p. with low amounts of Protective Antigen and Histamine-Sensitizing Factor also induced lymphocyte transformation, but were less toxic to the lymphocytes at high concentrations. The supernatants of B.p. cultures were purified with respect to Lymphocytosis Promoting Factor; evidence is presented that these purified fractions possess T-lymphocyte mitogenic activity. Lymphocytes from all normal humans were stimulated by B.p., including cells from cord blood. Cells from childbearing women, obtained immediately after delivery, showed a general depression of lymphocyte transformation including the response to B.p. Children with whooping cough had a lower lymphocyte response to B.p. than healthy children. A highly significant correlation was observed between the responses to B.p. and to E. coli in the adults and newborn examined. It is concluded that the major part of the lymphocyte transformation induced by B.p. is non-specific.
The epidemiological and drug susceptibility data on whooping cough suggested a possibility that Bordetella pertussis converts in some way to Bordetella parapertussis. To prove this, B. pertussis strain 75 was treated with N-methyl-N'-nitro-N-nitrosoguanidine and a mutant resistant to staphcillin v and eight mutants resistant to trimethoprim were isolated. The staphcillin V-resistant mutant of B. pertussis agreed with all of the criteria of B. parapertussis and the trimethoprim-resistant mutants also agreed with many of these criteria. Thus, a hypothesis is presented that B. parapertussis is a mutant of B. pertussis which appeared in nature probably by a selective pressure of antibiotics.
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We have isolated from Bordetella pertussis an oligopeptide with characteristic amino acid composition. This peptide was applied to mice in standardized tests for pertussis immunization. In three tests with three independent isolates of peptide, a significant and dose dependent protection was observed. One microgram of peptide per mouse produces the same protective effect as 0.1 IU of pertussis vaccine. It is important to note that similar peptides can be isolated from other bacteria and other DNA containing cellular organisms which have specific amino acid compositions and which are antigens specific for the organism from which they were isolated. The antigens are very potent, e.g., one ng of Mycobacterium tuberculosis peptide is equivalent to one unit of tuberculin. It is conceivable that immunizing effects such as those observed for pertussis are common to the peptides of this group. Since all such peptides are isolated from a group of low molecular weight ribonucleoproteins, as first reported by WILHELM, we propose the term nucleopeptides for this group. Oligopeptides of the nucleopeptide group are now available for sequence analysis. We expect that synthetic peptides of this group will become available in time for diagnosis, prophylaxis and therapy of a number of diseases.
Five Bordetella pertussis strains of phase I were grown in conventional casamino-acid medium and in media modified by adding high concentrations of MgSO4 or nicotinic acid. Cells grown in high-magnesium media (in the C-mode) had only about 4% of the protective antigen (PA) and 6% of the histamine-sensitising factor (HSF) of cells from the normal medium. Envelopes from C-mode organisms when examined by SDS-PAGE showed a loss of 28K and 30K polypeptide bands. Similar parallel losses of PA, HSF and 28K and 30K bands were found with cells from the high-nicotinic-acid medium. A medium with a high concentration of nicotinamide gave cells with normal amounts of PA, HSF and 28K and 30K bands. Growth in high concentrations of Na2SO4 caused partial losses of PA, HSF and 28K and 30K bands, while a high-succinate medium gave cells with somewhat diminished PA and HSF but without appreciable attenuation of the 28K and 30K bands. Because of the close correlation between the presence or absence of PA, HSF and 28K and 30K envelope polypeptides, it is suggested that the latter may represent or be closely associated with the components responsible for PA and HSF activities.
Administration of Bordetella pertussis and some of their components to mice induced an increase of DNA-binding activity of the sera revealed under ionic strength conditions of physiological saline, mostly on the 14th day. It was shown by the inhibition method that interaction between mouse sera and native DNA was specific. Maximum increase in the quantity of mouse sera proteins reacting with DNA under low ionic strength condition of physiological saline (0.05 M NaCl) is revealed on the 7th day. However, in administration of Bordetella pertussis and their cytoplasmic membrane the elevated DNA-binding proteins content persisted up to 14 days.
Strains of Bordetella pertussis varied in their ability to elicit (in mice) an antibody bactericidal for an antiserum-sensitive strain of B. pertussis, although antibody was usually detectable after only one injection. High titres were produced by a course of seven injections with all strains of B. pertussis tested (six of phase I and three of phase IV) but not with three strains of other Bordetella species nor with two unrelated organisms, a finding of possible taxonomic value. Preliminary investigations have not revealed whether strain vaiations are due to quantitative or qualitative differences in either the bacterial lipopolysaccharide or the carrier protein necessary for antibody production, or whether they may be due to differences in heat lability of 'bactericidal antigen'.
Bordetella pertussis causes whooping cough, a severe respiratory infectious disease. Studies have compared the currently dominant single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele, ptxP3) and previously dominant SNP cluster II (ptxP1) strains as planktonic cells. Since biofilm formation is linked with B. pertussis pathogenesis in vivo, this study compared the biofilm formation capabilities of representative strains of cluster I and cluster II. Confocal laser scanning microscopy found that the cluster I strain had a denser biofilm structure compared to the cluster II strain. Differences in protein abundance of the biofilm cells were then compared using tandem mass tagging and high-resolution multiple reaction monitoring. In total, 1,453 proteins were identified, of which 40 proteins had significant differential abundance between the two strains in biofilm conditions. Of particular interest was a large increase in the abundance of energy metabolism proteins (cytochrome proteins PetABC and BP3650) in the cluster I strain. When the abundance of these proteins was compared between six additional strains from each cluster, it was found that the protein abundance varied between all strains. These findings suggest that there are large levels of individual proteomic diversity between B. pertussis strains in biofilm conditions despite the highly conserved genome of the species. Overall, this study revealed visual differences in biofilm structure between B. pertussis strains and highlighted strain-specific variation in protein abundance that dominates potential cluster-specific changes that may be linked with the dominance of cluster I strains.IMPORTANCEBordetella pertussis causes whooping cough. The currently circulating cluster I strains have taken over previously dominant cluster II strains. It is important to understand the reasons behind this evolution to develop new strategies against the pathogen. Recent studies have shown that B. pertussis can form biofilms during infection. This study compared the biofilm formation capabilities of a cluster I and a cluster II strain and identified visual differences in the biofilms. The protein abundance between these strains grown in biofilms was compared, and proteins identified with varied abundance were measured with additional strains from each cluster. It was found that despite the highly conserved genetics of the species, there was varied protein abundance between the additional strains. This study highlights that strain-specific variation in protein abundance during biofilm conditions may dominate the cluster-specific changes that may be linked to the dominance of cluster I strains.
The activity of Bordetella pertussis extracytoplasmic adenylate cyclase is 100-fold higher in organisms grown on blood agar than in those grown in synthetic medium. This increase in activity is due to in vivo activation of the enzyme by a factor present in erythrocytes. Activation also occurs in killed or disrupted organisms. The activator can be separated from heme proteins and has been purified approximately 100-fold from erythrocytes, yielding material of approximately 105,000 daltons. It is sensitive to trypsin and alpha-chymotrypsin and exhibits considerable heat stability. Activation of cyclase in intact B. pertussis organisms exhibits a lag of 3 to 4 min and is not reversed by washing. Response to the activator decreases with increasing purification of the adenylate cyclase and is absent in the pure enzyme. The activation does not appear to be proteolytic and does not appear to change access to the substrate, ATP. The activator has no effect on a number of eukaryotic cyclases. We conclude that this is a new type of activation and that the activator differs from all those previously described.
Hydrolysis of the Bordetella pertussis endotoxin, extracted from both "phase I" and "phase IV" bacteria, with 4 M HCl for 1 h at 100 degrees C, released the disaccharide named in the title; it was isolated by paper electrophoresis or by ion-exchange chromatography in about 1% yield (w/w). The structure of the heptose could be rigorously established by chemical degradation; the facts that the glucosaminidic linkage was hydrolysed by an enzyme preparation containing both, alpha and beta-N-acetylglucosaminidase activities, whereas it was resistant to cleavage by pure beta-N-acetylglucosaminidase strongly support the assumption that the disaccharide contains an alpha-D-glucosaminide linkage.
A highly potent extract of the histamine sensitizing factor (HSF) of Bordetella pertussis was isolated by extraction of bacterial cells with urea buffer and subsequent gel filtration. This preparation of HSF also contained leukocytosis-promoting activity and adjuvant activity for reaginic and hemagglutinating antibodiesl Digestion of this extract with pronase or trypsin partially destroyed histamine-sensitizing activity, leukocytosis-promoting activity, and adjuvant activity for reaginic antibody, but did not affect adjuvant activity for hemagglutinating antibody. Antisera to HSF was prepared by immunizing rabbits with either whole bacteria or partially purfied extract. These antisera contained several precipitating antibodies to Bordetella pertussis extract demonstrated by immunodiffusion and immunoelectrophoresis. Antisera added in vitro to Bordetella pertussis extracts or passively administered in vivo to mice, reduced or abolished all biologic activities except adjuvant activity for hemagglutinating antibody. These results suggest that HSF might be an antigenic component of Bordetella pertussis which also possesses leukocytosis-promoting activity and adjuvant activity for reaginic antibody.
Bordetella pertussis and Corynebacterium parvum are commonly used immunopotentiating agents. To explore the inflammatory environment induced by these agents, the peritoneal exudate response in mice following intraperitoneal injection of B. pertussis (PV) and C. parvum (CV) vaccines was investigated. The PV-induced exudate isolated by lavage was characterized by an early neutrophil influx followed by enhanced accumulation of mononuclear cells and fluid protein. The CV exudate was principally mononuclear in nature and displayed fewer numbers of cells and less fluid protein. Both vaccines also enhanced the leukocyte adherence inhibitory activity (LAIA) of peritoneal fluid as measured in vitro. The development of exudate LAIA was T lymphocyte independent. A similar LAIA was demonstrated in nonimmune mouse plasma and serum. Exudate fluid and serum LAIA were heat stable and trypsin sensitive. These studies suggest that significant differences exist in the composition of the local tissue environment following PV and CV injection and that exudate LAIA is serum derived. Further studies in this direction should result in a better understanding of the ways in which inflammatory cells and fluid substances affect lymphocyte-macrophage interaction subsequent to adjuvant administration.
The lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis was isolated from solutions obtained after cell disintegration by a novel affinity chromatographic method using an adsorbent composed of human haptoglobin covalently attached to a Sepharose 4B matrix. The haemagglutinin was bound to the adsorbent at pH 6.5 and eluted by a stepwise change to a pH 10 buffer. A 300--600-fold purification of the haemagglutinin was achieved by this one-step process. The chemical and biological properties of the haemagglutinin isolated by affinity chromatography were found to be similar to those of the protein isolated by other workers from culture supernatants. The affinity chromatographic method was found to be specific for the purification of the lymphocytosis promoting factor-haemagglutinin and no purification of the fimbrial-haemagglutinin of Bordetella pertussis was achieved by the method.
1. Rats and mice were vaccinated with Haemophilus influenzae in different vaccination schedules whereafter blood eosinophils were counted. In rats a single vaccination resulted in a dose-dependent effect on the blood eosinophil count in a pattern comparable with that after Bordetella pertussis vaccination. In a long-term vaccination schedule (five times a week for 5 weeks) rats developed a constant eosinophilia. In mice a single vaccination resulted in an eosinopenia of a consistent pattern which differed from the response after Bordetella pertussis vaccination; in a long-term vaccination schedule, eosinophilia was evoked for a period of about 13 days. 2. Thirty minutes after an adrenaline injection in vaccinated rats and mice with Haemophilus influenzae, hyperglycaemic and eosinophilic responses were measured. The eosinophilic response after adrenaline was inhibited in both species; the hyperglycaemic response in rats was unaltered, in mice the response was slightly but significantly (P less than 0.05) decreased. 3. The sensitivity to several drugs was tested in mice, 5 days after vaccination with Haemophilus influenzae or Bordetella pertussis. Haemophilus influenzae vaccination reduced the isoprenaline sensitivity and increased the noradrenaline sensitivity. Bordetella pertussis vaccination reduced the isoprenaline sensitivity while the sensitivity to histamine and adrenaline was raised. 4. The Haemophilus influenzae vaccinated experimental animal provides a model that is possibly more related to human atopy than the Bordetella pertussis vaccinated animal.
The effect of Bordetella pertussis vaccine on the cerebral vascular permeability in the mouse was studied by a radio-isotope method (131I-labelled HSA). Intravenous injection of 4 x 1010 heat-killed pertussis organisms caused a measurable increase in permeability in normal mice. Cryoinjury to the cerebral hemispheres resulted in a striking increase in vascular permeability at 24 h. This declined within 48 h and stabilized at a level fractionally higher than normal at 7 days ("healed lesion"). When pertussis organisms were injected into mice bearing ("healed lesion"). When pertussis organisms were injected into mice bearing "healed lesions" the increase in permeability was similar in magnitude to that in uninjured brain. The effect was increased by a second administration of pertussis 24 h after the first. The action of pertussis on a newly inflicted cryoinjury was protective. It is suggested that permeability changes in the cerebral vessels may be involved in the evolution of the encephalopathy attributed to the use of Bordetella pertussis vaccine in man.
The toxicity of 201 Bordetella pertussis strains isolated in Belgrade has been studied. The strains were grown on a modification of Cohen and Wheeler's medium. LD50 of examined strains ranging between 1.06 and 1.95 billion bacteria in 35 (17.41%), between 2.04 and 4.83 billion bacteria in 92 (45.77%) and between 5.31 and 7.79 billion bacteria in 46 (22.88%) were found. Low toxic cultures of B. pertussis strains with LD50 ranging between 9.0 and 14.1 billion bacteria in 28 (13.43%) were obtained. The culture of the strains with low toxicity was 7 to 14 times more toxic than those prepared from the strains with low toxicity. In the cultures of 51 B. pertussis strains, toxicity for mice was not correlated with the number of viable cells. The toxic substances which influenced the loss of mouse weight were found in the culture filtrate of B. pertussis. A considerable detoxifying effect of heating at 56 degrees C for 30 min on liquid cultures prepared from highly toxic strains of B. pertussis was observed. The susceptibility of two different strains of mice for pertussis vaccine has been examined. The Albany strain of mice was less susceptible to toxicity control of the vaccine by mouse-weight-gain test than the Torlak strain.