Genotypic variation in the Bordetella pertussis virulence factors pertactin and pertussis toxin in historical and recent clinical isolates in the United Kingdom

The reemergence of pertussis has been reported in several countries despite high vaccination coverage. Studies in The Netherlands and Finland have investigated polymorphism in the genes coding for two important virulence factors of Bordetella pertussis, pertactin and pertussis toxin, and identified the emergence and subsequent dominance in circulating strains of pertactin and toxin variants not found in the whole-cell vaccine (WCV). The study described here investigated whether such variation had occurred in the United Kingdom, which presently has low levels of pertussis. Sequence analysis of the genes for pertactin (prnA) and the pertussis toxin S1 subunit (ptxA) among isolates of B. pertussis from 285 United Kingdom patients, from 1920 to 1999, revealed three prnA variants, prnA(1), prnA(2), and prnA(3), and two ptxA variants, ptxA(1) and ptxA(2), showing differences in nucleic acid sequence. The proportion of pertactin gene types not included in the United Kingdom WCV, i.e., prnA(2) and prnA(3), has increased in recent years and was found in 21 of 86 (24%) strains from the 1980s and 56 of 105 (53%) strains from the 1990s. To date, the presence of these nonvaccine prnA types has not been associated with a resurgence of pertussis in the United Kingdom. The distribution of prnA and ptxA types in The Netherlands, Finland, and the United Kingdom in the 1990s is distinct. The most striking difference in the United Kingdom isolates is that all 105 of the most recent circulating strains (from 1998 to 1999) are of a pertussis toxin type found in the United Kingdom WCV, i.e., ptxA(1).     

Polymorphism in the Bordetella pertussis Virulence Factors P.69/Pertactin and Pertussis Toxin in The Netherlands: Temporal Trends and Evidence for Vaccine-Driven Evolution

The Bordetella pertussis proteins P.69 (also designated pertactin) and pertussis toxin are important virulence factors and have been shown to confer protective immunity in animals and humans. Both proteins are used in the new generation of acellular pertussis vaccines (ACVs), and it is therefore important to study the degree of antigenic variation in these proteins. Sequence analysis of the genes for P.69 and the pertussis toxin S1 subunit, using strains collected from Dutch patients in the period 1949 to 1996, revealed three P.69 and three S1 variants which show differences in amino acid sequence. Polymorphism in P.69 was confined to a region comprised of repeats and located proximal to the RGD motif involved in adherence to host tissues. Variation in S1 was observed in two regions previously identified as T-cell epitopes. P.69 and S1 variants, identical to those included in the Dutch whole-cell pertussis vaccine (WCV), were found in 100% of the strains from the 1950s, the period when the WCV was introduced in The Netherlands. However, nonvaccine types of P.69 and S1 gradually replaced the vaccine types in later years and were found in ~90% strains from 1990 to 1996. These results suggest that vaccination has selected for strains which are antigenically distinct from vaccine strains. Analysis of strains from vaccinated and nonvaccinated individuals indicated that the WCV protects better against strains with the vaccine type P.69 than against strains with non-vaccine types (P = 0.024). ACVs contain P.69 and S1 types which are found in only 10% of recent Dutch B. pertussis isolates, implying that they do not have an optimal composition. Our findings cast a new light on the reemergence of pertussis in highly vaccinated populations and may have major implications for the long-term efficacy of both WCVs and ACVs.     

Strain variation among Bordetella pertussis isolates in finland, where the whole-cell pertussis vaccine has been used for 50 years

Pertussis is an infectious disease of the respiratory tract caused by Bordetella pertussis. Despite the introduction of mass vaccination against pertussis in Finland in 1952, pertussis has remained an endemic disease with regular epidemics. To monitor changes in the Finnish B. pertussis population, 101 isolates selected from 1991 to 2003 and 21 isolates selected from 1953 to 1982 were studied together with two Finnish vaccine strains. The analyses included serotyping of fimbriae (Fim), genotyping of the pertussis toxin S1 subunit (ptxA) and pertactin (prn), and pulsed-field gel electrophoresis (PFGE) after digestion of B. pertussis genomic DNA with XbaI restriction enzyme. Strains isolated before 1977 were found to harbor the same ptxA as the strains used in the Finnish whole-cell pertussis vaccine, and strains isolated before 1982 harbored the same prn as the strains used in the Finnish whole-cell pertussis vaccine. All recent isolates, however, represented genotypes distinct from those of the two vaccine strains. A marked shift of predominant serotype from Fim serotype 2 (Fim2) to Fim3 has been observed since the late 1990s. Temporal changes were seen in the genome of B. pertussis by PFGE analysis. Three PFGE profiles (BpSR1, BpSR11, and BpSR147) were distinguished by their prevalence between 1991 and 2003. The yearly emergence of the three profiles was distributed periodically. Our study stresses the importance of the continuous monitoring of emerging strains of B. pertussis and the need to obtain a better understanding of the relationship of the evolution of B. pertussis in vaccinated populations.     

Simple Algorithm for Identification of Bordetella pertussis Pertactin Gene Variants

Studies performed in several countries have demonstrated the recent emergence and subsequent dominance of circulating Bordetella pertussis strains harboring pertactin and pertussis toxin variants not included in pertussis vaccines. Determination of the pertactin gene variants is commonly performed using a time-consuming and expensive sequence analysis. We developed a simple and reliable pertactin typing algorithm suitable for large-scale screening. The assay correctly identified all pertactin alleles in representative strains. The typing of 231 clinical strains of B. pertussis routinely isolated in Belgium showed that this algorithm was adequate to identify less-frequent prn types like prn9 and prn11.     

Analysis of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> clinical isolates circulating in European countries during the period 1998–2012

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998–2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.     

Comparison of the Bordetella pertussis and Bordetella parapertussis isolates circulating in Saint Petersburg between 1998 and 2000 with Russian vaccine strains

We analyzed the Bordetella pertussis and Bordetella parapertussis isolates circulating in Saint Petersburg that were collected between 1998 and 2000 and compared them with isolates collected 40 years ago and Russian vaccine strains. The analysis involved serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion with XbaI and SpeI, and sequencing of the ptxS1 and prn genes, which encode the S1 subunit of the pertussis toxin and the major adhesin pertactin, respectively. The Russian isolates were classified in five of the six pulsed-field gel electrophoresis groups identified in other European countries. The B. pertussis isolates currently circulating in Saint Petersburg differed from the Russian whole-cell vaccine strains and the isolates collected in the prevaccine era. However, their repartition in the major pulsed-field gel electrophoresis groups was slightly different from that of isolates collected in countries that have had a high level of vaccine coverage for a long time, probably because the level of vaccine coverage in Saint Petersburg has increased only recently, after decreasing until the early 1990s. Most of the B. parapertussis isolates studied were similar to those circulating in France. However, some variants were observed, perhaps because B. parapertussis infections are more common in children in this area.     

Antigenic divergence suggested by correlation between antigenic variation and pulsed-field gel electrophoresis profiles of Bordetella pertussis isolates in Japan

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.     

Polymorphism of Bordetella pertussis isolates circulating for the last 10 years in France, where a single effective whole-cell vaccine has been used for more than 30 years.

We compared Bordetella pertussis isolates collected in France over the last 10 years, the vaccine strains used for more than 30 years, and isolates collected before the introduction of generalized vaccination. The analysis included serotyping, pulsed-field gel electrophoresis of chromosomal DNA after digestion with XbaI and SpeI, and sequencing of the pt S1 gene, encoding the S1 subunit of pertussis toxin, and the prn gene, encoding the adhesin pertactin. We found that the incidence of infection increases every 3 years. Ninety-five per cent of the isolates analyzed express type 3 fimbriae. Most of the isolates circulating since 1991, unlike the vaccinal strains, express a type A pertussis toxin and a type 2 pertactin. The isolates could be classified into five major groups by pulsed-field gel electrophoresis. Most of these groups correlated with the pertactin type expressed by the isolates. Pulsed-field gel electrophoresis is more discriminative than sequencing particular genes since it could differentiate isolates expressing type 2 pertactin into two subgroups: those circulating in 1993 to 1997 and those circulating in 1997 to 2001. This observation suggests that there has been continuous evolution of the B. pertussis population.     

Rapid typing of Bordetella pertussis pertussis toxin gene variants by LightCycler real-time PCR and fluorescence resonance energy transfer hybridization probe melting curve analysis

A LightCycler real-time PCR hybridization probe assay was developed for rapid typing of gene variants of the Bordetella pertussis virulence factor pertussis toxin. The assay correctly identified the ptxS1 alleles of all strains tested, comprising 57 Finnish clinical isolates and 2 vaccine strains. The method is simple, reliable, and suitable for large-scale screening of B. pertussis strains.     

Interleukin-12 is produced by macrophages in response to live or killed Bordetella pertussis and enhances the efficacy of an acellular pertussis vaccine by promoting induction of Th1 cells.

Using a murine respiratory infection model, we have demonstrated previously that infection with Bordetella pertussis or immunization with a whole-cell pertussis vaccine induced antigen-specific Th1 cells, which conferred a high level of protection against aerosol challenge. In contrast, immunization with an acellular vaccine, consisting of the B. pertussis components detoxified pertussis toxin, filamentous hemagglutinin, and pertactin adsorbed to alum, generated Th2 cells and was associated with delayed bacterial clearance following challenge. In this study, we demonstrated that addition of interleukin-12 (IL-12) either in vitro or in vivo enhanced type 1 T-cell cytokine responses induced with an acellular vaccine. Furthermore, the rate of bacterial clearance in mice coinjected with IL-12 and the acellular vaccine was similar to that observed following immunization with a potent whole-cell vaccine. Analysis of IL-12 secretion by murine macrophages suggested that this cytokine is produced in vivo following B. pertussis infection or immunization with the whole-cell vaccine. IL-12 was detected in the supernatants of lung, splenic, and peritoneal macrophages infected with live B. pertussis or stimulated with heat-killed whole B. pertussis or B. pertussis lipopolysaccharide. In contrast, IL-12 could not be detected following stimulation of macrophages with the bacterial antigens filamentous hemagglutinin, detoxified pertussis toxin, and pertactin, the components of acellular vaccines. Our findings suggest that induction of endogenous IL-12 may contribute to the high efficacy of pertussis whole-cell vaccines and also demonstrate that it is possible to attain these high levels of protection with a less reactogenic acellular vaccine incorporating IL-12 as an adjuvant.     

Analysis of Bordetella pertussis populations in European countries with different vaccination policies

Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.     

Shifts of Bordetella pertussis variants in Sweden from 1970 to 2003, during three periods marked by different vaccination programs

The Swedish population of Bordetella pertussis strains was characterized from 1,247 isolates covering a whole-cell vaccine program up to 1979, a 17-year period without vaccination (1979 to 1996), and a period after the introduction of general vaccination among newborns with acellular pertussis vaccines (1997 to 2003). Strains were characterized by serotyping and genotyping of pertactin and ptxA and by means of pulsed-field gel electrophoresis (PFGE). With emphasis on vaccine-related markers, the vast majority of circulating strains were of nonvaccine type. There were shifts of serotype connected with shifts of vaccination program. Serotype Fim3 was most frequent during the periods with general vaccination schedules, whereas serotype Fim2 was predominant during the 17-year vaccine-free period. Pertactin 1 was predominant during the pertussis whole-cell (Pw) vaccine period but was thereafter replaced by prn2 and has not reappeared after the introduction of acellular pertussis (Pa) vaccines. ptxA (1) was predominant over all three decades. There was a significant difference in the distribution of serotypes between vaccinated and unvaccinated individuals, but not for pertactin. A few PFGE profiles were predominant over the years: BpSR25 (serotype Fim3 prn1/7) and BpSR18 (serotype Fim3 prn2) during the Pw period, BpSR1 (serotype Fim2 prn2) during the 17 years without general vaccination, and BpSR11 (serotype Fim3 prn2) after the reintroduction of general vaccination in 1996. Despite differences between the pertactin and toxin types of Pa vaccines and circulating strains, there is no evidence that there is a threat, i.e., the vaccination program so far has been effective against whooping cough, and there seems to be no impact on the effectiveness of the vaccination program from the bacterial polymorphism.     

Multilocus sequence typing of Bordetella pertussis based on surface protein genes

Despite more than 50 years of vaccination, Bordetella pertussis has remained endemic in The Netherlands, causing epidemic outbreaks every 3 to 5 years. Strain variation may play a role in the persistence of B. pertussis and was studied by sequencing 15 genes coding for surface proteins, including genes for all five components of acellular pertussis vaccines: pertussis toxin (Ptx), pertactin (Prn), filamentous hemagglutinin, and fimbriae (Fim2 and Fim3). A low level of allelic variation was observed, confirming a recent evolutionary origin of B. pertussis. In modern isolates, polymorphism was observed only in prn, ptxS1, ptxS3, and tcfA. Polymorphism in ptxS1, ptxS3, and tcfA was used to categorize isolates in multilocus sequence types (MLSTs). Analysis of Dutch isolates from 1949 to 1999 revealed five MLSTs, which showed a highly dynamic temporal behavior. We observed significant changes in the MLSTs after the introduction of pertussis vaccination in The Netherlands. Epidemic years were found to be associated with the expansion of MLST-4 or MLST-5. MLST-5 showed a remarkable expansion from 10% in 1997 to 80% in 1999. The MLST analysis was extended to a number of widely separated geographic regions: Finland, Italy, Japan, and the United States. MLST-4 and MLST-5 were found to dominate in Italy and the United States. In Finland and Japan, MLST-3 and MLST-2, respectively, were predominant. Thus, although each region showed distinctive MLST frequencies, in three of the five regions MLST-4 and MLST-5 were predominant. These types may represent newly emerged, successful clones. The identification of highly successful clones may shed light on the question of how B. pertussis is able to maintain itself in vaccinated populations.     

Bordetella pertussis Infection: Pathogenesis, Diagnosis, Management, and the Role of Protective Immunity

 Whooping cough is presently one of the ten most common causes of death from infectious disease worldwide. Despite a high vaccine uptake, resurgences of this disease have been observed in several countries. Virulence factors of Bordetella pertussis include agglutinogens, fimbriae, P.69/pertactin, pertussis toxin, filamentous haemagglutinin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin, lipopolysaccharide, tracheal colonisation factor, serum resistance factor, and type III secretion. Virulence factor expression is regulated by the bvgAS locus, a two-component signal transduction system. The pathophysiologic sequence consists of attachment (fimbriae, P.69/pertactin, tracheal colonisation factor, pertussis toxin, filamentous haemagglutinin), evasion of host defence (adenylate cyclase, pertussis toxin, serum resistance factor), local effects (tracheal cytotoxin), and systemic effects (pertussis toxin). Bordetella pertussis is transmitted by respiratory droplets and causes disease only in humans. Various diagnostic methods are available, including culture, serological methods, and the polymerase chain reaction. Serotyping of isolates to detect agglutinogens 2 and 3 is useful because serotype 1,2 may be associated with higher mortality, and antibodies to these antigens (agglutinins) may be protective in both animals and humans. Immunisation using whole-cell vaccine is effective but is reactogenic. Acellular vaccines containing one to five components are being used increasingly in various countries. Protective immunity to pertussis correlates with high levels of antibody to each of pertactin, fimbriae, and pertussis toxin; however, doubt remains as to the relationship between agglutinogen 3 and fimbria 3, making results of trials investigating these virulence factors difficult to interpret.     

Determination of pertactin IgG antibodies for the diagnosis of pertussis

Objective  To compare increases in serum IgG antibody against pertactin with increases in IgG against pertussis toxin and filamentous hemagglutinin (FHA) in non-vaccinated children, children vaccinated with pertussis toxoid, and adults, all with culture-confirmed pertussis.
Methods  During a double-blind, placebo-controlled, efficacy trial of a monocomponent pertussis toxoid vaccine, acute and convalescent sera were obtained from study children and family members with suspected pertussis. In the present study, IgG antibodies against pertactin, pertussis toxin and FHA (determined by ELISA) were compared in 207 individuals with culture-verified pertussis and paroxysmal cough for ≥ 21 days.
Results  Significant increases in geometric mean serum IgG against all antigens occurred in non-vaccinated children, but more children responded against pertussis toxin and FHA than against pertactin (96%, 97%, and 62%, respectively). Of the children who had pertussis even though they were vaccinated with the pertussis toxoid vaccine, 97% responded to FHA, while responses to pertussis toxin and pertactin were less common (68% and 61%, respectively). In the 20 adults, the proportions of responders to FHA, pertussis toxin and pertactin were 90%, 80% and 55%, respectively.
Conclusion  Determination of IgG against pertussis toxin and FHA in paired sera in non-vaccinated children with pertussis is a more sensitive diagnostic tool than determination of IgG against pertactin. Pertactin IgG determinations might be of value as a complement to the other antibody assays in vaccinated children and in adults.     

Acellular pertussis vaccines and the role of pertactin and fimbriae

The introduction of acellular pertussis (Pa) vaccines in countries with a low uptake of whole-cell pertussis (Pw) vaccines has led to a dramatic reduction in pertussis disease. Diphtheria-tetanus-acellular pertussis (DTPa) vaccines have also ensured continued high level disease protection in these countries following the shift from Pw- to Pa-containing vaccines, and allowed pertussis booster programs to be implemented. Vaccines containing between one and five components have been licensed and implemented. Those with three or more components consisting of filamentous hemagglutinin (FHA), pertussis toxin (PT) and pertactin (PRN) are considered to be more effective than one/two-component Pa vaccines that contain only PT or both PT and FHA. Changes in circulating Bordetella pertussis strains may impact vaccine efficacy and, thus, incidence and transmission of pertussis and deserve to be followed carefully. To date, vaccine-induced shifts among fimbriae (FIM) are reported and this could impact the efficacy of FIM-containing vaccines. Currently, FIM3 appears to be dominant in most European countries, Canada and Australia. Data obtained from a DTPa5 vaccine containing FIM2 and FIM3 have indicated a shift towards an increase in FIM3-expressing B. pertussis clinical breakthrough cases when compared with control vaccine. By contrast, relatively minor PT and PRN sequence polymorphisms have been identified without demonstrable association with vaccination programs. Adsorption of PRN to aluminum salt appears critical for optimal protective capacity in murine pertussis lung challenge. In addition, clinical studies have shown anti-PRN antibody levels to be higher when PRN is adsorbed at a 8-microg dosage versus non-adsorbed PRN at a 3-microg dosage. The available data, therefore, demonstrate that appropriately formulated acellular vaccines containing PT and PRN are the preferred option for pertussis immunization.     

Are vaccination programs and isolate polymorphism linked to pertussis re-emergence?

Whooping cough remains an endemic disease, and the re-emergence of pertussis in older children and adolescents has been reported in several countries, despite high vaccine coverage. Polymorphism of Bordetella pertussis has been observed over time, and some characteristics of pertussis isolates have gradually diverged from the vaccine strains. The present review summarizes the current knowledge on B. pertussis variability in countries with different vaccination programs and discusses its potential impact on the recently observed increased incidence of whooping cough. No direct association between B. pertussis isolate variability and vaccination programs has been observed to date, except for shifts from fimbriae Fim2 to Fim3. More likely explanations for the re-emergence of pertussis include the change in the epidemiology and transmission patterns of pertussis in highly vaccinated populations, and a shift of disease from young children to adolescents and adults due to waning protective immunity.     

T-cell immune response assessment as a complement to serology and intranasal protection assays in determining the protective immunity induced by acellular pertussis vaccines in mice

The relative value of antibodies and/or T-cell immune responses to Bordetella pertussis antigens in the immunity induced by acellular pertussis (aP) vaccines is still an open issue, probably due to the incomplete knowledge on the mechanisms of protective immunity to pertussis. The relevance of T-cell immune responses in protection from pertussis has been demonstrated in murine and human models of infection; thus, in this study, the ability of different vaccine preparations of three component (pertussis toxin, filamentous hemagglutinin, and pertactin) aP vaccines to induce T-cell responses was investigated in mice. All vaccine preparations examined passed the immunogenicity control test, based on antibody titer assessment, according to European Pharmacopoeia standards, and protected mice from B. pertussis intranasal challenge, but not all preparations were able to prime T cells to pertussis toxin, the specific B. pertussis antigen. In particular, one vaccine preparation was unable to induce proliferation and gamma interferon (IFN-gamma) production while the other two gave borderline results. The evaluation of T-cell responses to pertussis toxin antigen may provide information on the protective immunity induced by aP vaccines in animal models. Considering the critical role of the axis interleukin-12-IFN-gamma for protection from pertussis, our results suggest that testing the induction of a key protective cytokine such as IFN-gamma could be an additional tool for the evaluation of the immune response induced by aP vaccines.     

Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species.

Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically.     

Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins.

Bordetella pertussis expresses factors such as filamentous hemagglutinin, agglutinogens, pertactin, and pertussis toxin, which participate in bacterial adhesion; pertussis toxin, dermonecrotic toxin, lipopolysaccharide, and tracheal cytotoxin, which are responsible for toxic effects; and adenylate cyclase-hemolysin, which is required to initiate infection. By using a murine respiratory model, we showed that the RGD sequences of filamentous hemagglutinin and pertactin are important for bacterial persistence. However, mutants deficient in filamentous hemagglutinin and agglutinogens or in pertactin and the RGD sequence of filamentous hemagglutinin behaved as did wild-type B. pertussis, i.e., induced bronchopneumonia, alveolitis, and an influx of macrophages, lymphocytes, and polymorphonuclear leukocytes into bronchoalveolar lavage fluids. These results suggest that these adhesins are not involved in the induction of pulmonary lesions following infection. The intensity of inflammation was markedly reduced after infection with mutants deficient in either hemolytic activity or pertussis toxin expression, whereas a mutant devoid of adenylate cyclase activity behaved as did the avirulent mutant. Pertussis toxin and adenylate cyclase-hemolysin may act indirectly by altering immune cell functions and thus allowing other factors, such as filamentous hemagglutinin, agglutinogens, and pertactin, to trigger adhesion and lipopolysaccharide, dermonecrotic toxin, and tracheal cytotoxin to induce their toxic effects. However, it is possible that pertussis toxin is also responsible for the induction of some pulmonary alterations.