Analysis of Bordetella pertussis 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.

Pertussis resurgence: waning immunity and pathogen adaptation - two sides of the same coin.

Pertussis or whooping cough has persisted and resurged in the face of vaccination and has become one of the most prevalent vaccine-preventable diseases in Western countries. The high circulation rate of Bordetella pertussis poses a threat to infants that have not been (completely) vaccinated and for whom pertussis is a severe, life-threatening, disease. The increase in pertussis is mainly found in age groups in which immunity has waned and this has resulted in the perception that waning immunity is the main or exclusive cause for the resurgence of pertussis. However, significant changes in B. pertussis populations have been observed after the introduction of vaccinations, suggesting a role for pathogen adaptation in the persistence and resurgence of pertussis. These changes include antigenic divergence with vaccine strains and increased production of pertussis toxin. Antigenic divergence will affect both memory recall and the efficacy of antibodies, while higher levels of pertussis toxin may increase suppression of the innate and acquired immune system. We propose these adaptations of B. pertussis have decreased the period in which pertussis vaccines are effective and thus enhanced the waning of immunity. We plead for a more integrated approach to the pertussis problem which includes the characteristics of the vaccines, the B. pertussis populations and the interaction between the two.

Pertussis disease burden in the household: how to protect young infants.

BACKGROUND: We conducted a population-based, nation-wide, prospective study to identify who introduced pertussis into the household of infants aged 6 months admitted to the hospital for pertussis in the Netherlands. METHODS: During the period 2006-2008, a total of 560 household contacts of 164 hospitalized infants were tested by polymerase chain reaction, culture, and serological examination to establish Bordetella pertussis infection. Clinical symptoms and vaccination history were obtained by a questionnaire submitted during sample collection and 4-6 weeks afterwards. RESULTS: Overall, 299 household contacts (53%) had laboratory-confired pertussis; 159 (53%) had symptoms compatible with typical pertussis infection, and 42 (14%) had no symptoms. Among children vaccinated with a whole-cell vaccine, 17 (46%) of 37 had typical pertussis 1-3 years after completion of the primary series, compared with 9 (29%) of 31 children who had been completely vaccinated with an acellular vaccine. For 96 households (60%), the most likely source of infection of the infant was established, being a sibling (41%), mother (38%), or father (17%). CONCLUSIONS: If immunity to pertussis in parents is maintained or boosted, 35%-55% of infant cases could be prevented. Furthermore, we found that, 1-3 years after vaccination with whole-cell or acellular vaccine, a significant percentage of children are again susceptible for typical pertussis. In the long term, pertussis vaccines and vaccination strategies should be improved to provide longer protection and prevent transmission.

Seasonal patterns in time series of pertussis.

To gain insight into pertussis disease dynamics, we studied age-specific long-term periodicity and seasonality of pertussis in The Netherlands. Hierarchical time-series models were used to analyse the monthly reported pertussis incidence in January 1996-June 2006 by age group. The incidence of pertussis showed a slightly increasing long-term trend with highest incidence rates seen in 1996, 1999, 2001 and 2004. For all age groups the annual peak incidence was found in August, except for the 13-18 years age group where the peak occurred in November. Monthly trends in adults showed high correlation with trends in age groups 0-4 years (0.94) and 5-12 years (0.92). We found no evidence for a relationship between annual rises in pertussis and the opening of schools. Concurrent annual fluctuations of pertussis incidence in adults and infants suggest frequent transmission within and between these age groups. Studying trends offers insight into transmission dynamics and may facilitate decisions on future vaccination strategies.

The evolution of epidemic Vibrio cholerae strains.

The emergence of the novel Vibrio cholerae strain, O139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms. Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed.

[Effect of vaccination against pertussis on the incidence of pertussis in The Netherlands, 1996-2003]. / Effect van vaccinatie tegen kinkhoest op de incidentie van kinkhoest in Nederland, 1996-2003.

OBJECTIVE: To determine whether booster vaccination of 4-year-old children with an acellular pertussis vaccine, which has been included in the national vaccination programme since October 2001, has decreased the incidence of pertussis. DESIGN: Descriptive. METHODS: Surveillance data were studied: mandatory notifications to the Health Inspectorate and reports of hospital admissions from the National Medical Register. RESULTS: During the past 7 years, there has been an increase in the incidence of pertussis every 2-3 years (1996, 1999, 2001). Moreover, the annual incidence in 1996-2003 was higher than in 1989-1995. As in previous years, the yearly peak incidence for hospital admissions due to pertussis was observed among nurslings, especially those younger than 3 months of age. In 2002 compared to 2000, the incidence among 3-4-year-olds on the basis of notifications and hospitalisations was 45% and 62% lower, respectively, very likely due to the booster vaccination for 4-year-olds introduced in 2001. The greatest decrease in the incidence was also observed among the 4-year-olds in 2003. CONCLUSION: Pertussis is still endemic in The Netherlands with a higher incidence than before the epidemic of 1996-1997. Severe disease often occurs, especially among unvaccinated children < 1 year of age. From January 2005 onwards, the vaccinations in the first year of life have been given with an acellular pertussis vaccine. However, since such infants are too young to be protected by vaccination alone, more information is needed on the most important sources of infection of nurslings in The Netherlands.

Structure of the Bordetella pertussis gene coding for the serotype 3 fimbrial subunit.

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.

Evidence for an intracellular niche for Bordetella pertussis in broncho-alveolar lavage cells of mice.

Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B. pertussis. We found B. pertussis to be present in a viable state in BAL fluid cells until at least 19 days after infection, suggesting B. pertussis to be able to survive in those cells. This intracellular niche may play an important role in the pathogenesis of pertussis. Pertussis toxin and the RGD sequence of the virulence factor filamentous hemagglutinin (FHA) both play a role in the attachment of B. pertussis to human and mouse macrophages in vitro and we hypothesized these virulence factors to be required for invasion and subsequent intracellular survival of B. pertussis in macrophages in vivo. A B. pertussis double mutant, in which the FHA RGD motif was changed to RAD and the ptx genes were deleted, was also found in a viable state in BAL fluid cells, albeit at lower levels than the wild-type strain. In our model, uptake of B. pertussis by alveolar phagocytes in vivo is thus, at least in part, determined by the bacterial virulence factors FHA and pertussis toxin.

Rapid detection of the recently emerged Bordetella pertussis strains with the ptxP3 pertussis toxin promoter allele by real-time PCR.

Bordetella pertussis strains with the pertussis toxin promoter allele ptxP3 have expanded and replaced resident ptxP1 strains in several European countries. We developed an allele-specific real-time PCR method to identify strains with the allele ptxP3, and investigated the emergence of ptxP3 strains by genotyping Finnish clinical isolates (n = 524) from 1953 to 2010. The first ptxP3 strain was detected in 1994, and has become predominant since 2003. Our results demonstrate that the allele-specific real-time PCR is a suitable method for rapid detection of ptxP3 strains, and show the emergence and establishment of ptxP3 strains in Finland.

Analysis of Swedish Bordetella pertussis isolates with three typing methods: characterization of an epidemic lineage.

Three Bordetella pertussis typing methods, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable number tandem repeat analysis (MLVA) were compared using a collection of Swedish strains. Of the three typing methods used, PFGE was found to be the most discriminatory. MLVA and MLST were less discriminatory, but may be valuable for strain discrimination when culture is not possible as they are based on PCR. The combination of MLVA/MLST was found to be equally discriminatory as PFGE and should therefore also be considered. The relationship between predominant lineages in Sweden and The Netherlands, characterized by the PFGE type BpSR11 and the allele for the pertussis toxin promoter ptxP3, respectively, was investigated. Linkage was found between the PFGE type BpSR11 and ptxP3 in that all BpSR11 strains carried ptxP3. On the other hand ptxP3 was found in several other PFGE-types. The presence of the ptxP3 allele in different genetic backgrounds may indicate horizontal gene transfer within B. pertussis or homoplasy. Alternatively, this observation may be due to convergence of PFGE types.

Adaptive evolution of the Bordetella autotransporter pertactin.

The virulence factor pertactin is expressed by the closely related pathogens Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Pertactin is an autotransporter involved in adherence of Bordetella species to the lung epithelium of mammalian hosts, and it is an important component of most current acellular pertussis vaccines. These three species produce immunologically distinct pertactin molecules, resulting in a lack of cross-protection against B. parapertussis and probably also against B. bronchiseptica. Variation in pertactin is not only inter-specific, but also occurs between isolates from the same species. Knowledge about codons that are under positive selection could facilitate the development of more broadly protective vaccines. Using different nucleotide substitution models, pertactin genes from B. bronchiseptica, B. parapertussis and B. pertussis were compared, and positively selected codons were identified using an empirical Bayesian approach. This approach yielded 15 codons predicted to be under diversifying selection pressure. These results were interpreted in an immunological context and may help in improving future pertussis vaccines.

Differences of circulating Bordetella pertussis population in Argentina from the strain used in vaccine production.

In Argentina, as in other countries, the number of pertussis cases has been increasing, even in highly vaccinated zones. Many reports suggest that the decline of vaccine efficacy due to antigenic shifts in the circulating Bordetella pertussis might be among the factors that contribute to pertussis re-emergence in different parts of the world. To evaluate the incidence of this factor in Argentina, we decided to characterize the circulating bacteria of an important demographic area of this country in comparison with the strain used for vaccine production. From 1997 to 2003 we collected nasopharyngeal samples from pediatric patients with signs of Bordetella infection hospitalized in the metropolitan area of Buenos Aires and La Plata, Argentina. From these samples we identified 28 B. pertussis, which were characterized by biochemical techniques, PCR, DNA fingerprint, prn and ptx genes sequencing, and lipopolysaccharides (LPS) pattern. BOX-PCR from B. pertussis isolates yielded one cluster containing 13 isolates and some smaller ones, being all fingerprints different from the vaccine strain. Differences between Argentinean circulating bacteria and the vaccine strain were also observed for the Prn and Ptx variants as well as for the LPS pattern. Moreover, this last pattern seemed to change over the years. In addition, we identified two B. bronchiseptica. The presence of this Bordetella species together with the observed differences between circulating B. pertussis and the strain used in vaccine production should be considered for the development of an improved vaccine.

Characterization of a highly conserved island in the otherwise divergent Bordetella holmesii and Bordetella pertussis genomes.

The recently discovered pathogen Bordetella holmesii has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of B. holmesii are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions, B. holmesii strains were analyzed by comparative genomic hybridization (CGH) to a Bordetella pertussis microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between B. pertussis and B. holmesii. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS481 elements that may have been laterally transferred from B. pertussis to B. holmesii. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by B. holmesii may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between B. pertussis and B. holmesii may also explain the unusually high sequence identity of their 16S rRNA genes.

Host genetics of Bordetella pertussis infection in mice: significance of Toll-like receptor 4 in genetic susceptibility and pathobiology.

The susceptibility to and the severity of Bordetella pertussis infections in infants and children varies widely, suggesting that genetic differences between individuals influence the course of infection. We have previously identified three novel loci that influence the severity of whooping cough by using recombinant congenic strains of mice: Bordetella pertussis susceptibility loci 1, 2, and 3 (Bps1, -2, and -3). Because these loci could not account for all genetic differences between mice, we extended our search for additional susceptibility loci. We therefore screened 11 inbred strains of mice for susceptibility to a pertussis infection after intranasal infection. Susceptibility was defined by the number of bacteria in the lungs, being indicative of the effect between the clearance and replication of bacteria. The most resistant (A/J) and the most susceptible (C3H/HeJ) strains were selected for further genetic and phenotypic characterization. The link between bacterial clearance and chromosomal location was investigated with 300 F2 mice, generated by crossing A/J and C3H/HeJ mice. We found a link between the delayed clearance of bacteria from the lung and a large part of chromosome 4 in F2 mice with a maximum log of the odds score of 33.6 at 65.4 Mb, which is the location of Tlr4. C3H/HeJ mice carry a functional mutation in the intracellular domain of Tlr4. This locus accounted for all detectable genetic differences between these strains. Compared to A/J mice, C3H/HeJ mice showed a delayed clearance of bacteria from the lung, a higher relative lung weight, and increased body weight loss. Splenocytes from infected C3H/HeJ mice produced almost no interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) upon ex vivo restimulation with B. pertussis compared to A/J mice and also showed a delayed gamma interferon (IFN-gamma) production. TNF-alpha expression in the lungs 3 days after infection was increased fivefold compared to uninfected controls in A/J mice and was not affected in C3H/HeJ mice. In conclusion, Tlr4 is a major host factor explaining the differences in the course of infection between these inbred strains of mice. Functional Tlr4 is essential for an efficient IL-1-beta, TNF-alpha, and IFN-gamma response; efficient clearance of bacteria from the lung; and reduced lung pathology.

Genetic control of Bordetella pertussis infection: identification of susceptibility loci using recombinant congenic strains of mice.

Susceptibility to and severity of Bordetella pertussis infection in infants and children vary widely. The spectrum of clinical symptoms ranges from subclinical infection to mild disease, severe whooping cough, and death. The aims of this study were to examine genetic susceptibilities of mice to B. pertussis and to identify genetic loci in the mouse genome that are involved in susceptibility to B. pertussis infection. For this purpose we screened two sets of recombinant congenic strains (RCS) of mice, HcB and CcS, for differences in the numbers of bacteria in the lung 7 days after inoculation. For both CcS and in HcB mice, a wide range in numbers of bacteria in the lung was found, suggesting that the course of infection is under multigenic control. From both RCS sets of mice, we selected one strain to identify possible susceptibility loci in F(2) hybrid mice. The degree of lung colonization 7 days postinoculation in these F(2) mice was evaluated in relation to genetic markers by linkage analysis. We found three novel loci that are involved in the control of B. pertussis infection. One locus, designated B. pertussis susceptibility locus 1 (Bps-1), was identified on chromosome 12. The presence of the C57BL/10 genome on this locus instead of the C3H genome significantly decreased the number of B. pertussis bacteria in the lung. Bps-1 has a dominant-positive effect on the clearance of B. pertussis from the lung. The function of most genes in this region is unknown. Two other loci, Bps-2 and Bps-3, showed genetic interaction and are located on chromosomes 5 and 11. We aim to identify the gene(s) in these regions which modify susceptibility to B. pertussis.

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.

Virulence factors of Bordetella pertussis.

Clearly, B. pertussis has evolved very elaborate mechanisms to maintain itself in the human host. Three different proteins (FHA, pertussis toxin and fimbriae) have been implicated in adherence. Furthermore, a number of toxins are produced (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or suppress the immune response. There is evidence that B. pertussis may survive intracellularly, and the possibility that it is a facultative intracellular parasite should certainly be explored. The availability of a large number of cloned virulence genes, and a system to construct well defined mutants by allelic exchange (Stibbitz et al. 1986) will greatly facilitate the study of Bordetella virulence factors at the molecular level. It opens the possibility to construct avirulent strains, which are still able to colonize and stimulate the local immune response. Such strains may be used as live, oral vaccines, to present (heterologous) antigens to the mucosal immune system of the respiratory tract.