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Antigenic divergence has been found between Bordetella pertussis vaccine strains and circulating strains in several countries. In the present study, we analyzed B. pertussis isolates collected in Japan from 1988 to 2001 using pulsed-field gel electrophoresis (PFGE) and sequencing of two virulence-associated proteins. The 107 isolates were classified into three major groups by PFGE analysis; 87 (81%) were type A, 19 (18%) were type B, and 1 (1%) was type C. Sequence analysis of the S1 subunit of pertussis toxin (ptxS1) and adhesion pertactin (prn) genes revealed the presence of two (ptxS1A and ptxS1B) and three (prn1, prn2, and prn3) variants, respectively, in the isolates. Among those isolates, 82 (95%) of the 87 type A strains and the type C strain had the same combination of ptxS1B and prn1 alleles (ptxS1B/prn1) as the Japanese vaccine strain. On the other hand, 17 (90%) of 19 type B strains had an allele (ptxS1A/prn2) distinct from that of the vaccine strain. A correlation was found between the antigenic variation and the PFGE profile in the isolates. In addition, the frequency of the type B strain was 0, 27, 0, 42, and 37% of the isolates in the periods 1988 to 1993, 1994 to 1995, 1996 to 1997, 1998 to 1999, and 2000 to 2001, respectively. In contrast, the number of reported pertussis-like and pertussis cases decreased gradually from 1991 on, suggesting that the antigenic divergence did not affect the efficacy of pertussis vaccination in Japan.

We examined the distribution of antibody levels against pertussis toxin (PT) and filamentous hemagglutinin (FHA) among a healthy Japanese population aged from 0 to 77 years. Levels of both antibodies in 1108 serum samples collected in 1994 from nine prefectures were assayed using polystyrene ball ELISA. The ratio of individuals positive (>or=10 ELISA U/ml) for anti-PT and anti-FHA antibodies at ages ranging from 0 to 3 years increased rapidly with the increase in the population vaccinated over three times with acellular pertussis (aP) vaccine. However, the ratio of those positive for anti-PT antibody tended to decrease until 6-8 years of age and to increase again from 9 to 19 years among the vaccinated population, although the ratio of individuals positive for anti-FHA antibody remained constant at 80-100% in children and adolescents over 3 years old. Moreover, positivity for anti-PT antibody was high (>or=50 ELISA U/ml) in some serum samples collected from adolescents and young adults, suggesting recent symptomatic or asymptomatic infection with circulating Bordetella pertussis. On the other hand, 50-60% of infants below 12 months of age was below the detection limit (1.0 ELISA U/ml) for anti-PT and anti-FHA antibodies, and most early infants were not vaccinated for pertussis. Since intermittent circulation of B. pertussis remains among the Japanese population, complete vaccination with aP vaccine for all infants should be highly recommended to prevent pertussis.

The internalization of the N-terminal catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) across the cytoplasmic membrane has been considered to occur independently from protein-protein interactions which can lead to oligomerization required for hemolytic activity by its C-terminal hemolysin domain. Here we report that when added in excess, this hemolysin domain stimulates the internalization, suggesting the involvement of protein-protein interactions in cell-invasive activity of ACT, as well as its hemolytic activity.

The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 microM)-induced aggregation of rabbit platelets at 3 micrograms/ml and strongly suppressed thrombin (0. 2 U/ml)-induced aggregation at 10 micrograms/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive point mutant of ACT did not show the suppressive effect. Since an increase of cAMP content is a known cause of platelet dysfunction, these results indicate that the observed platelet inactivation was due to the catalytic activity of ACT through increase of intracellular cAMP.

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.

The gelatin-particle-agglutination (PA) test for titrating antibodies against diphtheria, pertussis and tetanus toxins was developed and used for assaying 65 sera from healthy children to assess the antitoxin acquisition in relation to the administration of adsorbed diphtheria-purified pertussis-tetanus (DPT) combined vaccine. The antitoxin titers obtained by the PA test and the conventional methods were correlated well; the correlation coefficient of the diphtheria antitoxin titers between the PA test and the cell culture method was 0.908, that of the tetanus antitoxin titers between the PA test and the passive hemagglutination test 0.968, and that of anti-pertussis toxin titers between the PA test and polystyrene-ball ELISA 0.885. The PA test was shown to be useful in both developed and developing countries, since it is simple to perform, sensitive and specific, and the three antitoxins can be titrated by the same procedure.