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1.  Antigenic Divergence between Bordetella pertussis Clinical Isolates from Moscow, Russia, and Vaccine Strains▿  
Clinical and Vaccine Immunology  2007;14(3):234-238.
We analyzed temporal changes in the frequencies of the ptxA, prn, fim2, and fim3 alleles in Bordetella pertussis strains isolated from pertussis patients in Moscow, Russia, from 1948 to 2004. The three strains used for the whole-cell vaccine harbored the alleles ptxA2, ptxA4, prn1, fim2-1, and fim3A. Vaccine-type alleles of ptxA (ptxA2 and ptxA4) were characteristic for all prevaccination strains and for 96% of the strains isolated in the 1960s and 1970s. At the beginning of the 1970s, ptxA2 and ptxA4 were replaced by the ptxA1 allele. In the 1980s and to the present, strains with ptxA1 were predominant in the B. pertussis population. All prevaccination strains harbored the prn1 allele, which corresponds to the vaccine-type allele. In subsequent years, the proportion of strains with the prn1 allele decreased and the proportion of prn3 and prn2 strains increased. From 2002 to 2004 strains with prn2 or prn3 were predominant in the B. pertussis population. The vaccine-type alleles fim2-1 and fim3A were found in all prevaccination strains and in 92% of the strains isolated from 1960 to 1989. The fim2-2 and fim3B alleles were first observed at the beginning of the 1980s. In subsequent years, these strains became predominant. Together with waning immunity, the antigenic divergence between vaccine strains and clinical isolates observed in the Moscow area may explain the persistence of pertussis, despite the high rates of vaccine coverage. The results demonstrate that the selection of B. pertussis strains for vaccine manufacturing must be based on a thorough study of the B. pertussis population.
doi:10.1128/CVI.00294-06
PMCID: PMC1828859  PMID: 17202309
2.  Genetic Variation of Bordetella pertussis in Austria 
PLoS ONE  2015;10(7):e0132623.
In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.
doi:10.1371/journal.pone.0132623
PMCID: PMC4504479  PMID: 26182210
3.  Genetic Analysis of Bordetella pertussis Isolates from the 2008–2010 Pertussis Epidemic in Japan 
PLoS ONE  2013;8(10):e77165.
A large pertussis epidemic occurred between 2008 and 2010 in Japan. To investigate epidemic strains, we analyzed 33 Bordetella pertussis isolates from the epidemic period by sequencing virulence-associated genes (fim3, ptxP, ptxA, and prn) and performing multilocus variable-number tandem repeat analysis (MLVA), and compared these results with those of 101 isolates from non-epidemic, earlier and later time periods. DNA sequencing of the fim3 allele revealed that the frequency of fim3B was 4.3%, 12.8%, 30.3%, and 5.1% within isolates in 2002–2004, 2005–2007, 2008–2010, and 2011–2012, respectively. The isolation rate of the fim3B strain therefore temporarily increased during the epidemic period 2008–2010. In contrast, the frequencies of the virulence-associated allelic variants, ptxP3, ptxA1, and prn2, increased with time during overall study period, indicating that these variants were not directly involved in the occurrence of the 2008–2010 epidemic. MLVA genotyping in combination with analysis of allele types showed that the prevalence of an MT27d strain temporarily increased in the epidemic period, and that this strain carried virulence-associated allelic variants (fim3B, ptxP3, ptxA1, and prn2) also identified in recent epidemic strains of Australia, Europe, and the US. Phenotypic analyses revealed that the serotype Fim3 strain was predominant (≥87%) during all the periods studied, and that the frequency of adhesion pertactin (Prn) non-expressing B. pertussis decreased by half in the epidemic period. All MT27d strains expressed Prn and Fim3 proteins, suggesting that B. pertussis MT27d strains expressing Prn and Fim3B have the potential to cause large epidemics worldwide.
doi:10.1371/journal.pone.0077165
PMCID: PMC3790747  PMID: 24124606
4.  Strain Variation among Bordetella pertussis Isolates in Finland, Where the Whole-Cell Pertussis Vaccine Has Been Used for 50 Years 
Journal of Clinical Microbiology  2005;43(8):3681-3687.
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.
doi:10.1128/JCM.43.8.3681-3687.2005
PMCID: PMC1233997  PMID: 16081896
5.  Changes in Genetic Diversity of the Bordetella pertussis Population in the United Kingdom between 1920 and 2006 Reflect Vaccination Coverage and Emergence of a Single Dominant Clonal Type▿ † 
Journal of Clinical Microbiology  2009;47(3):680-688.
Pertussis (whooping cough) is a potentially fatal respiratory disease caused by the bacterium Bordetella pertussis. Despite effective vaccination programs, there has been concern in some developed countries that pertussis cases are on the increase. We characterized 703 clinical B. pertussis isolates collected in the United Kingdom between 1920 and 2006 using multilocus variable-number tandem repeat analysis (MLVA), pertactin (prnA) and pertussis toxin (ptxA) genotyping, and serotyping. The results showed that the genetic diversity of the bacterial population decreased during periods of high vaccine coverage. However, it was elevated between 1977 and 1986, when vaccine coverage in the United Kingdom was low and epidemics occurred. A high proportion of MLVA types during this epidemic period were novel, and the prnA(2) and prnA(3) alleles were seen for the first time in the United Kingdom. MLVA-27 appeared in 1982, was codominant during the 1998-to-2001 period, and comprised ∼70% of isolates during both the 2002-to-2004 and the 2005-to-2006 periods. The United Kingdom is dominated currently by an MLVA-27 prnA(2) ptxA(1) serotype Fim3 clonal type. Even during recent periods dominated by MLVA-27, many novel types were found at low frequencies, suggesting that either there are a large number of uncommon MLVA types circulating at low frequencies or new types are constantly arising. This supports a hypothesis that MLVA-27 is under some form of positive selection conferring increased survival in a highly vaccinated population. There has been no significant change to the bacterial population in the first 2 years since the United Kingdom switched from a whole-cell to an acellular vaccine.
doi:10.1128/JCM.01838-08
PMCID: PMC2650949  PMID: 19158267
6.  Genotypic Variation in the Bordetella pertussis Virulence Factors Pertactin and Pertussis Toxin in Historical and Recent Clinical Isolates in the United Kingdom 
Infection and Immunity  2001;69(9):5520-5528.
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).
doi:10.1128/IAI.69.9.5520-5528.2001
PMCID: PMC98665  PMID: 11500425
7.  Strain Variation among Bordetella pertussis Isolates Circulating in Poland after 50 Years of Whole-Cell Pertussis Vaccine Use▿† 
Journal of Clinical Microbiology  2011;49(4):1452-1457.
In the present study, clinical isolates of Bordetella pertussis collected in Poland from 1960 to 2005 were analyzed by pulsed-field gel electrophoresis (PFGE) according to protocols recommended in previous studies. Among the 110 isolates from 1995 to 2005, 59 PFGE patterns were found, most of which were different from those currently circulating in other European Union (EU) countries for which data are available. The PFGE patterns of currently disseminating B. pertussis clones were found within PFGE groups III and IV, as elsewhere in the EU, and in newly identified clusters A and C. Up to 70, 26, and 4%, respectively, of the currently isolated strains in Poland harbored ptxA1-prn1, ptxA1-prn2, and ptxA1-prn3 allele combinations, and most (82%) were found to be of the Fim2 phenotype. Differences in the extent of heterogeneity estimated by PFGE typing in B. pertussis populations circulating in Poland in comparison to other EU countries may be due to the different vaccine composition strategy, since routine pertussis vaccination was initiated in Poland in 1960.
doi:10.1128/JCM.01487-10
PMCID: PMC3122874  PMID: 21307213
8.  Molecular Epidemiology of the Pertussis Epidemic in Washington State in 2012 
Journal of Clinical Microbiology  2014;52(10):3549-3557.
Although pertussis disease is vaccine preventable, Washington State experienced a substantial rise in pertussis incidence beginning in 2011. By June 2012, the reported cases reached 2,520 (37.5 cases per 100,000 residents), a 1,300% increase compared with the same period in 2011. We assessed the molecular epidemiology of this statewide epidemic using 240 isolates collected from case patients reported from 19 of 39 Washington counties during 2012 to 2013. The typing methods included pulsed-field gel electrophoresis (PFGE), multilocus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), and pertactin gene (prn) mutational analysis. Using the scheme PFGE-MLVA-MLST-prn mutations-Prn deficiency, the 240 isolates comprised 65 distinct typing profiles. Thirty-one PFGE types were found, with the most common types, CDC013 (n = 51), CDC237 (n = 44), and CDC002 (n = 42), accounting for 57% of them. Eleven MLVA types were observed, mainly comprising type 27 (n = 183, 76%). Seven MLST types were identified, with the majority of the isolates typing as prn2-ptxP3-ptxA1-fim3-1 (n = 157, 65%). Four different prn mutations accounted for the 76% of isolates exhibiting pertactin deficiency. PFGE provided the highest discriminatory power (D = 0.87) and was found to be a more powerful typing method than MLVA and MLST combined (D = 0.67). This study provides evidence for the continued predominance of MLVA 27 and prn2-ptxP3-ptxA1 alleles, along with the reemergence of the fim3-1 allele. Our results indicate that the Bordetella pertussis population causing this epidemic was diverse, with a few molecular types predominating. The PFGE, MLVA, and MLST profiles were consistent with the predominate types circulating in the United States and other countries. For prn, several mutations were present in multiple molecular types.
doi:10.1128/JCM.01189-14
PMCID: PMC4187741  PMID: 25031439
9.  Shifts of Bordetella pertussis Variants in Sweden from 1970 to 2003, during Three Periods Marked by Different Vaccination Programs 
Journal of Clinical Microbiology  2005;43(6):2856-2865.
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.
doi:10.1128/JCM.43.6.2856-2865.2005
PMCID: PMC1151881  PMID: 15956409
10.  Small Mutations in Bordetella pertussis Are Associated with Selective Sweeps 
PLoS ONE  2012;7(9):e46407.
Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite high vaccination coverage, pertussis has resurged and has become one of the most prevalent vaccine-preventable diseases in developed countries. We have proposed that both waning immunity and pathogen adaptation have contributed to the persistence and resurgence of pertussis. Allelic variation has been found in virulence-associated genes coding for the pertussis toxin A subunit (ptxA), pertactin (prn), serotype 2 fimbriae (fim2), serotype 3 fimbriae (fim3) and the promoter for pertussis toxin (ptxP). In this study, we investigated how more than 60 years of vaccination has affected the Dutch B. pertussis population by combining data from phylogeny, genomics and temporal trends in strain frequencies. Our main focus was on the ptxA, prn, fim3 and ptxP genes. However, we also compared the genomes of 11 Dutch strains belonging to successful lineages. Our results showed that, between 1949 and 2010, the Dutch B. pertussis population has undergone as least four selective sweeps that were associated with small mutations in ptxA, prn, fim3 and ptxP. Phylogenetic analysis revealed a stepwise adaptation in which mutations accumulated clonally. Genomic analysis revealed a number of additional mutations which may have a contributed to the selective sweeps. Five large deletions were identified which were fixed in the pathogen population. However, only one was linked to a selective sweep. No evidence was found for a role of gene acquisition in pathogen adaptation. Our results suggest that the B. pertussis gene repertoire is already well adapted to its current niche and required only fine tuning to persist in the face of vaccination. Further, this work shows that small mutations, even single SNPs, can drive large changes in the populations of bacterial pathogens within a time span of six to 19 years.
doi:10.1371/journal.pone.0046407
PMCID: PMC3460923  PMID: 23029513
11.  Variation in Bordetella pertussis Susceptibility to Erythromycin and Virulence-Related Genotype Changes in China (1970-2014) 
PLoS ONE  2015;10(9):e0138941.
Objectives
To investigate changes in virulence-related genotypes and in the antimicrobial susceptibility of Bordetella pertussis isolates collected from the 1970s to 2014 in the northern part of China.
Methods
A total of 124 B. pertussis isolates from three periods, the 1970s, 2000–2008, and May 2013–Sept 2014, were typed by multilocus sequence typing (MLST) and tested for antimicrobial susceptibility and virulence-related genes. A fragment of the 23S rRNA gene from each of the 99 isolates from 2013–2014 was amplified and sequenced.
Results
All isolates from 2000–2008 and 2013–2014 were identified as ST2, whereas isolates from the 1970s were ST1. PtxA2/ptxC1/ptxP1/prn1/fim2-1/fim3-1/tcfA2, which was the same as the vaccine strain, was the only type in the 1970s. During the 2000s and 2013–2014, the virulence type ptxA1/ptxC1/ptxP1/prn1/fim2-1/fim3-1/tcfA2 was dominant, with frequencies of 68.4% and 91.9%, respectively. Nine ptxP3 strains, which were more virulent, were detected after 2000. All 124 isolates were susceptible to levofloxacin, sulphamethoxazole/trimethoprim and tetracycline. The isolates from the 1970s and 2000–2008 were susceptible to all tested macrolides, whereas 91.9% of the 2013–2014 isolates were highly resistant (minimal inhibitory concentration, MIC >256 μg/ml). No ptxP3 strain was resistant to macrolides. All erythromycin-resistant strains except for one had the A2047G mutation in the 23S rRNA gene.
Conclusions
Macrolide resistance of the B. pertussis population has been a serious problem in the northern part of China. Because most of the epidemic clone of the pathogen expresses the same antigen profiles as the vaccine strain, except ptxA, improvements in immunization strategies may prevent the spread of infection and drug resistance.
doi:10.1371/journal.pone.0138941
PMCID: PMC4583996  PMID: 26406905
12.  Synergic Effect of Genotype Changes in Pertussis Toxin and Pertactin on Adaptation to an Acellular Pertussis Vaccine in the Murine Intranasal Challenge Model▿  
The Bordetella pertussis pertussis toxin and pertactin (Prn) are protective antigens and are contained in acellular pertussis vaccines. Polymorphisms in the A subunit of pertussis toxin (PtxA) and pertactin have been proposed to mediate vaccine resistance and contribute to pertussis reemergence. To test this hypothesis, previous studies compared clinical isolates expressing different alleles for the proteins. However, other virulence factors or virulence factor expression levels also may vary, confounding the analysis. To overcome these limitations, we constructed isogenic mutants of B. pertussis Tohama expressing the alleles ptxA1 or ptxA2 and prn1 or prn2 and compared the efficacies of an acellular pertussis vaccine against the mutants in a mouse model. While the vaccine was effective against all of the B. pertussis strains regardless of the allele expression pattern, the strain expressing ptxA1 and prn2 displayed a survival advantage over the other strains. These results suggest that an allele shift to the ptxA1 prn2 genotype may play a role in the emergence of pertussis in vaccinated populations.
doi:10.1128/CVI.00449-09
PMCID: PMC2863393  PMID: 20357056
13.  Antigenic Divergence Suggested by Correlation between Antigenic Variation and Pulsed-Field Gel Electrophoresis Profiles of Bordetella pertussis Isolates in Japan 
Journal of Clinical Microbiology  2004;42(12):5453-5457.
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.
doi:10.1128/JCM.42.12.5453-5457.2004
PMCID: PMC535240  PMID: 15583264
14.  Rapid Increase in Pertactin-deficient Bordetella pertussis Isolates, Australia 
Emerging Infectious Diseases  2014;20(4):626-633.
Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008–2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (prn). Multiple mechanisms of prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing prn arose independently multiple times since 2008, rather than by expansion of a single prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.
doi:10.3201/eid2004.131478
PMCID: PMC3966384  PMID: 24655754
Bordetella pertussis; whooping cough; bacteria; outbreaks; pertactin; evolution; immunization; vaccination; vaccine; Australia
15.  Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function 
PLoS ONE  2015;10(9):e0137379.
Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one μg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice.
doi:10.1371/journal.pone.0137379
PMCID: PMC4573519  PMID: 26375454
16.  Analysis of Bordetella pertussis pertactin and pertussis toxin types from Queensland, Australia, 1999–2003 
Background
In Australia two acellular Bordetella pertussis vaccines have replaced the use of a whole cell vaccine. Both of the licensed acellular vaccines contain the following three components; pertussis toxoid, pertussis filamentous haemagglutinin and the 69 kDa pertactin adhesin. One vaccine also contains pertussis fimbriae 2 and 3. Various researchers have postulated that herd immunity due to high levels of pertussis vaccination might be influencing the makeup of endemic B. pertussis populations by selective pressure for strains possessing variants of these genes, in particular the pertactin gene type. Some publications have suggested that B. pertussis variants may be contributing to a reduced efficacy of the existing vaccines and a concomitant re-emergence of pertussis within vaccinated populations. This study was conducted to survey the pertactin and pertussis toxin subunit 1 types from B. pertussis isolates in Queensland, Australia following the introduction of acellular vaccines.
Methods
Forty-six B. pertussis isolates recovered from Queensland patients between 1999 and 2003 were examined by both DNA sequencing and LightCycler™ real time PCR to determine their pertactin and pertussis toxin subunit 1 genotypes.
Results
Pertactin typing showed that 38 isolates possessed the prn1 allele, 3 possessed the prn2 allele and 5 possessed the prn3 allele. All forty-six isolates possessed the pertussis toxin ptxS1A genotype. Amongst the circulating B. pertussis population in Queensland, 82.5% of the recovered clinical isolates therefore possessed the prn1/ptxS1A genotype.
Conclusion
The results of this study compared to historical research on Queensland isolates suggest that B. pertussis pertactin and pertussis toxin variants are not becoming more prevalent in Queensland since the introduction of the acellular vaccines. Current prevalences of pertactin variants are significantly different to that described in a number of other countries with high vaccine coverage. Relative paucity of recovered isolates compared to notified infections, due primarily to non culture based pertussis diagnostics is however a confounding factor in the assessment of variant prevalence.
doi:10.1186/1471-2334-6-53
PMCID: PMC1459169  PMID: 16542440
17.  Multilocus Sequence Typing of Bordetella pertussis Based on Surface Protein Genes 
Journal of Clinical Microbiology  2002;40(6):1994-2001.
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.
doi:10.1128/JCM.40.6.1994-2001.2002
PMCID: PMC130760  PMID: 12037054
18.  Pulsed-Field Gel Electrophoresis, Pertactin, Pertussis Toxin S1 Subunit Polymorphisms, and Surfaceome Analysis of Vaccine and Clinical Bordetella pertussis Strains▿  
Clinical and Vaccine Immunology : CVI  2007;14(11):1490-1498.
To add new insight to our previous work on the molecular epidemiology of Bordetella pertussis in Argentina, the prn and ptxS1 gene sequences and pulsed-field gel electrophoresis (PFGE) profiles of 57 clinical isolates obtained during two periods, 1969 to 1989 and 1997 to 2006, were analyzed. Non-vaccine-type ptxS1A was detected in isolates obtained since 1969. From 1989 on, a shift of predominance from the vaccine prn1 type to the nonvaccine prn2 type was observed. This was also reflected in a transition of PFGE group IV to group VI. These results show that nonvaccine B. pertussis strains are currently circulating. To analyze whether the observed genomic divergences between vaccine strains and clinical isolates have functional implications, protection assays using the intranasal mouse challenge model were performed. For such experiments, the clinical isolate B. pertussis 106 was selected as representative of circulating bacteria, since it came from the major group of the PFGE dendrogram (PFGE group VI). Groups of mice were immunized either with diphtheria-tetanus-whole-cell pertussis vaccine (ptxS1B prn1) or a vaccine prepared by us containing B. pertussis 106. Immunized mice were then challenged with a B. pertussis vaccine strain (Tohama, harboring ptxS1B and prn1) or the clinical isolate B. pertussis 106 (ptxS1A prn2). An adequate bacterial-elimination rate was observed only when mice were immunized and challenged with the same kind of strain. For further characterization, comparative proteomic profiling of enriched membrane proteins was done using three vaccine strains and the selected B. pertussis 106 clinical isolate. By matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, a total of 54 proteins were identified. This methodology allowed us to detect differing proteins among the four strains studied and, in particular, to distinguish the three vaccine strains from each other, as well as the vaccine strains from the clinical isolate. The differing proteins observed have cellular roles associated with amino acid and carbohydrate transport and metabolism. Some of them have been proposed as novel vaccine candidate proteins for other pathogens. Overall, the global strategy described here is presented as a good tool for the development of next-generation acellular vaccines.
doi:10.1128/CVI.00177-07
PMCID: PMC2168178  PMID: 17699837
19.  Analysis of Bordetella pertussis Populations in European Countries with Different Vaccination Policies 
Journal of Clinical Microbiology  2005;43(6):2837-2843.
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.
doi:10.1128/JCM.43.6.2837-2843.2005
PMCID: PMC1151907  PMID: 15956406
20.  Studies on Prn Variation in the Mouse Model and Comparison with Epidemiological Data 
PLoS ONE  2011;6(3):e18014.
The virulence factor pertactin (Prn) is a component of pertussis vaccines and one of the most polymorphic Bordetella pertussis antigens. After the introduction of vaccination shifts in predominant Prn types were observed and strains with the Prn vaccine type (Prn1) were replaced by strains carrying non-vaccine types (Prn2 and Prn3), suggesting vaccine-driven selection. The aim of this study was to elucidate the shifts observed in Prn variants. We show that, although Prn2 and Prn3 circulated in similar frequencies in the 1970s and 1980s, in the 1990s Prn2 strains expanded and Prn3 strains disappeared, suggesting that in vaccinated populations Prn2 strains are fitter than Prn3 strains. We established a role for Prn in the mouse model by showing that a Prn knock-out (Prn-ko) mutation reduced colonization in trachea and lungs. Restoration of the mutation resulted in a significant increase in colonization compared to the knock-out mutant. The ability of clinical isolates with different Prn variants to colonize the mouse lung was compared. Although these isolates were also polymorphic at other loci, only variation in the promoter for pertussis toxin (ptxP) and Prn were found to contribute significantly to differences in colonization. Analysis of a subset of strains with the same ptxP allele revealed that the ability to colonize mice decreased in the order Prn1>Prn2 and Prn3. Our results are consistent with the predominance of Prn1 strains in unvaccinated populations. Our results show that ability to colonize mice is practically the same for Prn2 and Prn3. Therefore other factors may have contributed to the predominance of Prn2 in vaccinated populations. The mouse model may be useful to assess and predict changes in the B. pertussis population due to vaccination.
doi:10.1371/journal.pone.0018014
PMCID: PMC3064647  PMID: 21464955
21.  Prevalence and Genetic Characterization of Pertactin-Deficient Bordetella pertussis in Japan 
PLoS ONE  2012;7(2):e31985.
The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn−) B. pertussis was observed in Japan. The Prn− isolate was first discovered in 1997, and 33 (27%) Prn− isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn− isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn− isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn− isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn− isolates belong to MLVA-186, and 6 and 3 Prn− isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn− clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn− isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn− strains have arisen and that Prn expression is not essential for fitness under these conditions.
doi:10.1371/journal.pone.0031985
PMCID: PMC3279416  PMID: 22348138
22.  Population Diversity among Bordetella pertussis Isolates, United States, 1935–2009 
Emerging Infectious Diseases  2012;18(8):1248-1255.
Resurgence of pertussis was not directly correlated with changes in vaccine composition or schedule.
Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935–2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970–1990 (94%) but declined to ≈70% after 1991 and has remained constant. During 2006–2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population.
doi:10.3201/eid1808.120082
PMCID: PMC3414039  PMID: 22841154
whooping cough; Bordetella pertussis; pertussis; genetic diversity; molecular typing; wP; aP; acellular vaccines; bacteria; United States; vaccination; immunization; alleles; isolates
23.  Survey and Rapid Detection of Bordetella pertussis in Clinical Samples Targeting the BP485 in China 
Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP) method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg/μl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 B. pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing groups with different alleles of the virulence-related genes including four alleles of ptxA, six of prn, four of tcfA, two of fim2, and three of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.
doi:10.3389/fpubh.2015.00039
PMCID: PMC4350411  PMID: 25798436
BP485; B. pertussis; LAMP; sensitivity; specificity; rapid diagnosis; prevalence
24.  Comparative genomics of prevaccination and modern Bordetella pertussis strains 
BMC Genomics  2010;11:627.
Background
Despite vaccination since the 1950s, pertussis has persisted and resurged. It remains a major cause of infant death worldwide and is the most prevalent vaccine-preventable disease in developed countries. The resurgence of pertussis has been associated with the expansion of Bordetella pertussis strains with a novel allele for the pertussis toxin (Ptx) promoter, ptxP3, which have replaced resident ptxP1 strains. Compared to ptxP1 strains, ptxP3 produce more Ptx resulting in increased virulence and immune suppression. To elucidate how B. pertussis has adapted to vaccination, we compared genome sequences of two ptxP3 strains with four strains isolated before and after the introduction vaccination.
Results
The distribution of SNPs in regions involved in transcription and translation suggested that changes in gene regulation play an important role in adaptation. No evidence was found for acquisition of novel genes. Modern strains differed significantly from prevaccination strains, both phylogenetically and with respect to particular alleles. The ptxP3 strains were found to have diverged recently from modern ptxP1 strains. Differences between ptxP3 and modern ptxP1 strains included SNPs in a number of pathogenicity-associated genes. Further, both gene inactivation and reactivation was observed in ptxP3 strains relative to modern ptxP1 strains.
Conclusions
Our work suggests that B. pertussis adapted by successive accumulation of SNPs and by gene (in)activation. In particular changes in gene regulation may have played a role in adaptation.
doi:10.1186/1471-2164-11-627
PMCID: PMC3018138  PMID: 21070624
25.  Comparison of the Bordetella pertussis and Bordetella parapertussis Isolates Circulating in Saint Petersburg between 1998 and 2000 with Russian Vaccine Strains 
Journal of Clinical Microbiology  2003;41(8):3706-3711.
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.
doi:10.1128/JCM.41.8.3706-3711.2003
PMCID: PMC179792  PMID: 12904379

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