ABSTRACT
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.
Bordetella pertussis is the primary etiologic agent of the disease pertussis. Whole-cell and acellular pertussis vaccines have been very effective at inducing protection against B. pertussis infection (23, 24). In Japan, pertussis vaccination was started in 1950 using whole-cell vaccine. Then, in 1981, acellular pertussis vaccines containing detoxified pertussis toxin (PT) and filamentous hemagglutinin as major antigens were introduced and have successfully controlled the prevalence of pertussis ever since (24). Most people with cases of pertussis (98.7%) reported to the National Epidemiological Surveillance of Infectious Diseases from 1987 to 1996 had no history of pertussis vaccination in Japan (21). However, in recent years, a resurgence of pertussis has been found in several countries despite high vaccination coverage (1, 5, 8). Since Mooi et al. (20) found that the circulating clinical strains had antigens distinct from those of vaccine strains, they proposed that the circulating clinical strains might have escaped the immunity provided by pertussis vaccination. The antigenic divergence between recently circulating strains and vaccine strains has been reported in European and North American countries (3, 6, 9, 16, 19, 22, 29-31) but not in Asian countries.
In the United States and France, genetic diversity of circulating B. pertussis isolates has been observed using pulsed-field gel electrophoresis (PFGE) (10, 31). In addition, the genetic divergence between circulating strains and vaccine strains can also be detected by DNA typing analyses, by IS1002-based DNA fingerprinting, and by sequencing the structural genes encoding B. pertussis virulence factors. The antigenic variants have been observed in the genes encoding the S1 subunit of PT (ptxS1) and pertactin (prn), which are important virulence factors of B. pertussis. Recently, polymorphism was also found in the genes encoding the S3 subunit of PT (ptxS3) and tracheal colonization factor (tcf) in circulating clinical strains (28). However, it is not clear whether the antigenic variations and DNA types in B. pertussis are associated with each other. For example, van Loo et al. (29, 30) found congruence between clustering based on IS1002-based DNA fingerprint types of Dutch clinical strains and the ptxS1 allele but not the prn allele. In contrast, in those strains found recently in France, most of the circulating strains showed a correlation between the PFGE profile and prn allele but not ptxS1 (31). Moreover, there was no high correlation between the PFGE types and the combinations of ptxS1 and prn alleles in the strains found in Canada or the United States (3, 22).
In a previous study, most Japanese B. pertussis isolates (83%) collected in the period 1975 to 1996 had the same ptxS1 and prn alleles as the Japanese vaccine strain, whereas the two recently circulating isolates exhibiting a different PFGE profile had ptxS1 and prn alleles different from those of the vaccine strain (7). Since a limited number of isolates (n = 12) were used in the study, it was not clear whether the antigenic divergence in ptxS1 and prn alleles has progressed in circulating strains in Japan. In the present study, we collected 107 Japanese clinical isolates, including recently circulating strains, and the antigenic divergence in those strains was investigated using PFGE analysis and sequencing of ptxS1 and prn alleles. The possible association of the antigenic variations with the PFGE profiles in the isolates was also investigated.
MATERIALS AND METHODS
Isolates.One hundred seven B. pertussis clinical isolates, collected from 1988 to 2001 in Japan, were obtained from 22 medical institutes, hospital laboratories, private clinical laboratories, and prefectural public health institutes. The periods of isolation and numbers of strains isolated were as follows: 1988 to 1993, 35; 1994 to 1995, 15; 1996 to 1997, 18; 1998 to 1999, 12; 2000 to 2001, 27. All of the isolates were confirmed as B. pertussis in our laboratory using PCR identification (14). For comparison, the Japanese acellular vaccine strain B. pertussis Tohama was used as a reference strain. The strains were cultured on Bordet-Gengou agar (Difco) supplemented with 1% glycerol and 15% defibrinated horse blood and incubated at 36°C for 2 to 3 days.
PFGE analysis.PFGE was performed according to standardized recommendations for typing of B. pertussis (18), with minor modifications. Chromosomal DNA was digested with the restriction enzyme XbaI, and the digested fragments were separated using a CHEF DR II apparatus (Bio-Rad). Electrophoresis was performed at 6 V/cm at 16°C with the following ramped switch times: block 1, 4 to 8 s for 12 h; block 2, 8 to 50 s for 10 h. The PFGE patterns were analyzed by the unweighted pair-group method with arithmetic averages (UPGMA) using Diversity Database version 1.1 software (PDI, Inc.).
DNA sequencing.DNA sequencing of relevant regions of the pertactin (prn) and S1 subunit of pertussis toxin (ptxS1) genes was performed on PCR fragments as described previously (18). Chromosomal DNA of B. pertussis was isolated using a QIAGEN genomic tip (20G) and genomic DNA buffer set. Sequence reactions were carried out with a BigDye terminator version 3.1 cycle-sequencing kit (Applied Biosystems), and the products were sequenced on a PRISM 3100 genetic analyzer (Applied Biosystems). Regions 1 of prn and ptxS1 were sequenced for all 107 clinical isolates. On the other hand, region 2 of prn was sequenced for 40 of the 107 isolates, since polymorphism has been reported infrequently in the region (19, 20). The carbohydrate recognition domain (fragment A) of the fhaB gene was also sequenced between positions 3421 and 3837 (17). The sense primer fraAF (5′-CGACATCATCATGGATGCGA-3′) and the antisense primer fraAR (5′-TCTGGAAGGTGCCCCTGTTC-3′) were used for the sequencing.
Purification of PT variants and analysis of biological activity.Two PT variants, PT-194M and PT-194I, encoded by ptxS1B and ptxS1A, respectively, were purified from each culture supernatant using an Affi-gel blue column and a fetuin-Sepharose column as described previously (25). The protein concentrations of the purified PT variants were measured by Lowry assay with bovine serum albumin as a standard. The purity of PT-194M and PT-194I was >95%, as estimated by sodium dodecyl sulfate-14% polyacrylamide gel electrophoresis, followed by Coomassie blue R-250 staining. The biological activities of the purified PT variants were determined by the Chinese hamster ovary (CHO) cell-clustering test (13).
RESULTS
PFGE typing and polymorphism in PT-S1 and pertactin.Among 107 B. pertussis isolates collected in Japan during the period 1988 to 2001, 48 PFGE profiles were identified (Fig. 1). The 48 PFGE profiles were classified into three major groups using UPGMA; type A comprised 37 PFGE profiles, type B comprised 10 profiles, and type C had one profile. The type A strain was a major group of isolates, since 87 (81%) of the 107 isolates were type A. On the other hand, types B and C were minor groups: 19 isolates (18%) were type B and 1 isolate (1%) was type C. Surprisingly, the type C strain had the same profile as the Japanese vaccine strain, B. pertussis Tohama.
Dendrogram of PFGE profiles of 107 Japanese B. pertussis isolates from 1988 to 2001. The dendrogram was calculated by UPGMA. The regions including the ptxS1 and prn repeat were sequenced, and the combination of ptxS1 and prn alleles is shown as ptxS1/prn, e.g., B/1 indicates ptxS1B/prn1. *, one isolate had different ptxS1/prn alleles in the PFGE profile.
The ptxS1 and prn genes of all 107 isolates were sequenced. Two PT-S1 subunit alleles (ptxS1A and ptxS1B) and three pertactin alleles (prn1, prn2 and prn3) were identified among the isolates (Fig. 2). Four ptxS1 and eight prn variants have been described (6); however, only two ptxS1 and three prn variants were found in the Japanese isolates. The Japanese vaccine strain had the ptxS1B and prn1 alleles described previously (2, 7). The fhaB genes from the 40 selected isolates (type A, 20 isolates; type B, 19 isolates; type C, 1 isolate) were sequenced between bases 3421 and 3837. No polymorphism was observed in the sequence.
Variants of pertussis toxin S1 subunit and pertactin observed in Japanese B. pertussis isolates. (A) Primary structure of pertussis toxin S1 subunit gene showing regions of polymorphism. (B) Deduced amino acid sequence of region 1 of pertactin. The dots indicate sequence identity with the Japanese vaccine strain, B. pertussis Tohama. The dashed lines indicate gaps. The vaccine strain has a combination of ptxS1B/prn1 alleles.
Correlation between PFGE type and combination of ptxS1 and prn alleles.As shown in Fig. 1, there was a correlation between the PFGE profile and the combination of ptxS1 and prn alleles in the Japanese isolates, although the smallest genetic distance (10%) was observed between the type A and type B groups. Table 1 summarizes the correlation between the PFGE type and the combination of ptxS1 and prn alleles. For convenience, ptxS1 and prn alleles have been placed in one of three groups: ptxS1B/prn1, old; ptxS1A/prn1, transitional; and ptxS1A/prn2 and ptxS1A/prn3, new (3, 22). Of the 87 type A strains, 82 (95%) isolates had old ptxS1B/prn1 alleles and 3 and 2 isolates had transitional and new alleles, respectively. The Japanese vaccine strain also had the old ptxS1B/prn1. In contrast, 17 (90%) of 19 type B strains had new ptxS1A/prn2 alleles, and each isolate had old and transitional alleles.
Correlation between PFGE type and ptxS1/prn alleles in B. pertussis isolates collected from 1988 to 2001 in Japan
We also analyzed the PFGE data using the neighbor-joining clustering method instead of UPGMA. All of the type B strains were classified in the same group, and the same result was obtained (data not shown).
Trend in type B strain in Japan.As shown in Fig. 3, type B strains were collected in various areas of Japan, as were type A strains. Four type B strains were first collected in the Chugoku and Hokkaido districts in 1994 and 1995. Five were collected consecutively in the Kinki and Kanto districts in 1998 and 1999, and 10 were collected in the Tohoku, Kanto, and Kinki districts in 2000 and 2001 (data not shown). Thus, type B strains were widely distributed throughout Japan during these times.
Geographic distribution of type A (A) and type B (B) strains collected from 1988 to 2001 in Japan. The numbers of symbols indicate the numbers of isolates.
Figure 4B shows the temporal trend of the frequency of the type B strain according to the year of collection. Surprisingly, the percentage of the type B strain changed between 1994 and 1995 and between 2000 and 2001 (0% from 1988 to 1993, 27% from 1994 to 1995, 0% from 1996 to 1997, 42% from 1998 to 1999, and 37% from 2000 to 2001), although the numbers of reported pertussis-like and pertussis cases had decreased gradually from 1991 (Fig. 4A). The temporal trend of the type B strain harboring new ptxS1A/prn2 was the same as the trend of the type B strain. The frequency of the type B strain harboring ptxS1A/prn2 was 20, 0, 42, and 33% of the isolates in the periods 1994 to 1995, 1996 to 1997, 1998 to 1999, and 2000 to 2001, respectively.
Temporal trend in isolation of type B strains and reported pertussis cases in Japan. (A) Pertussis-like cases (○) and pertussis cases (•) reported by sentinel clinics and hospitals in Japan from 1988 to 2001. The data were obtained from Infectious Disease Surveillance data of the Ministry of Health, Labor and Welfare of Japan. The reporting of pertussis cases was discontinued in 1999. (B) Changes in the frequencies of type A, type B, and type C strains.
Biological activities of PT variants.The biological activities of vaccine-type PT (PT-194M) and nonvaccine-type PT (PT-194I) were assessed. Two PT variants, PT-194M and PT-194I, encoded by the old ptxS1B and the new ptxS1A, respectively, were purified from each culture supernatant, and the biological activities of the purified PT variants were determined by the CHO cell-clustering test. The minimum concentrations required for the clustering of PT-194M and PT-194I were assessed to be 68 (95% confidence interval, 52 to 89) and 44 pg/ml (95% confidence interval, 25 to 76 pg/ml), respectively. No significant difference was observed between the biological activities.
DISCUSSION
In a previous study, most Japanese B. pertussis isolates collected from 1975 to 1996 had the same ptxS1B and prn1 alleles as the Japanese vaccine strain (7). However, since a limited number of isolates were used in the study, it was not clear whether the antigenic divergence between recently circulating strains and the vaccine strain has progressed. In the present study, 107 Japanese B. pertussis isolates, including recently circulating strains, were collected, and the antigenic divergence in those isolates was investigated. We found that the type B strain harboring nonvaccine ptxS1A and prn2 alleles appeared during the period 1994 to 1995. Thus, the antigenic divergence has progressed since the mid-1990s in Japan, similar to other countries, such as European and North American countries.
Despite high vaccination coverage, the resurgence of pertussis has been reported in several countries (1, 5, 8). In The Netherlands, the incidence of pertussis increased dramatically in 1996 and 1997 (5), and the antigenic divergence in the protective antigens encoded by ptxS1 and prn was observed in the circulating strains (20). In regard to the resurgence, Mooi et al. (20) proposed that circulating strains distinct from the vaccine strain might have escaped the immunity provided by vaccination. In contrast, in the United Kingdom, an antigenic divergence in the prn allele was observed in recently circulating strains (6), despite the relatively few pertussis cases there (27). These observations suggested that the presence of nonvaccine prn2 has not been associated with a resurgence of pertussis. In the present study, the type B strain harboring ptxS1A/prn2 distinct from those of the vaccine strain appeared in the mid-1990s, although the reported pertussis and pertussis-like cases had decreased since 1991 (Fig. 4). Most type B strains (90%) had not only prn2 but also nonvaccine ptxS1A. Therefore, our finding suggested that (i) the presence of nonvaccine ptxS1A, as with prn2, has not been associated with a resurgence of pertussis and (ii) the type B strain might not have escaped the immunity provided by vaccination in Japan.
In a previous study, Weber et al. (31) found no correlation between the PFGE type and the ptxS1 allele in French B. pertussis isolates but revealed a correlation with the prn allele. In the Canadian and U.S. isolates, there was no high correlation between the PFGE type and the combination of ptxS1 and prn alleles (3, 22). However, among the U.S. isolates, all isolates harboring ptxS1A/prn2 clustered in a relatedness group at the phylogenetic tree, whereas isolates harboring ptxS1B/prn1 and ptxS1A/prn1 were scattered throughout the tree (3). In contrast, among Japanese isolates, most (90%) type B strains had a combination of ptxS1A and prn2 (ptxS1A/prn2) and most (95%) type A strains and the type C strain had the same ptxS1B/prn1 as the Japanese vaccine strain (Table 1). Thus, there was a correlation between the PFGE types and ptxS1/prn alleles in the Japanese isolates. Strains harboring ptxS1B and prn1 had been collected prior to the 1970s in European countries and the United States, whereas strains harboring ptxS1A, prn2, and prn3 had been collected in those countries since the early 1980s (3, 19, 20). Therefore, ptxS1B and prn1 alleles have been called old alleles, while ptxS1A, prn2, and prn3 are called new alleles (3, 22). Moreover, strains harboring a combination of new and old alleles (ptxS1A/prn1) have been called transitional strains. Among strains collected in the United States from 1935 to 1999, 34 (22%) of 152 isolates were transitional strains (3). Similarly, among strains collected in Canada from 1985 to 1994, 17% of the isolates were transitional strains (22). These observations suggested that the B. pertussis strain had evolved from an old strain into a new strain by selective pressure from vaccination. However, only four transitional strains (4%) were found among the 107 Japanese isolates in this study. This finding strongly suggested that there was no genetic relationship between old strains (type A and type C) and the new strain (type B), i.e., the type B strain did not derive from the type A or type C strain genetically. One possible explanation for the appearance of the type B strain is that it was imported to Japan from other countries.
Among recently circulating strains in The Netherlands, polymorphism was observed only in prn, ptxS1, ptxS3, and tcfA by sequencing 15 genes coding for surface proteins (28). Polymorphism in prn is essentially limited to region 1, which has an important role in immunity (12, 15). However, Boursaux-Eude et al. (2) showed that acellular vaccine was highly effective against B. pertussis strains harboring nonvaccine ptxS1 and prn alleles by using a mouse intranasal challenge model. On the other hand, Hausman and Burns (11) suggested that significant amino acid changes could occur in PT sequence without affecting antibody neutralization. Thus, polymorphism in pertussis toxin may have no influence on the efficacy of antibody neutralization. In the present study, we also investigated the virulence of two PT variants, PT-194 M, encoded by vaccine ptxS1B, and PT194I, encoded by nonvaccine ptxS1A, but no significant difference between their biological activities was detected. These findings suggested that the virulence of PT-194I had not been associated with the wide spread of the type B strain harboring ptxS1A/prn2. The type B strain may have a more important virulent allele(s) than the ptxS1A and prn2 alleles associated with the wide spread. For analyzing differences in gene expression between bacterial strains, the proteomic approach is a powerful tool (4, 26). Thus, the comparative proteomic analysis of B. pertussis strains (type A and type B) was thought to be worth trying, and an attempt is now under way.
In conclusion, in the Japanese B. pertussis strains, the antigenic divergence between recently circulating strains and the vaccine strain has been observed since the mid-1990s, although reported pertussis-like and pertussis cases have decreased in number. In addition, the strains showed a correlation between the PFGE profile and the combination of ptxS1/prn alleles. Our findings strongly suggested that the antigenic divergence had no influence on the efficacy of pertussis vaccination in Japan. However, the reason for the appearance of the type B strain harboring nonvaccine ptxS1A/prn2 has remained unclear. Continuous surveillance and further analyses are needed to determine the virulence of the type B strain.
ACKNOWLEDGMENTS
We are grateful to the following for providing clinical isolates of B. pertussis: T. Fukui (Byotai-Seiri Laboratory, Tokyo, Japan), T. Hongou (Kurashiki Central Hospital, Okayama, Japan), M. Honma (Tokyo Metropolitan Kiyose Children's Hospital, Tokyo, Japan), T. Kato (St. Marianna University, Kanagawa, Japan), H. Kawai (Sano Kousei General Hospital, Tochigi, Japan), Y. Kouri (Chiba City Hospital, Chiba, Japan), K. Matsuda (Muroran City General Hospital, Hokkaido, Japan), T. Nakamura (Kansai Medical University, Osaka, Japan), M. Ohtsuka (Kotobiken Medical Laboratories Inc., Ibaraki, Japan), K. Okada (Kawasaki Municipal Hospital, Kanagawa, Japan), S. Saito (Akita Prefectural Institute of Public Health, Akita, Japan), K. Sugama (Fukushima Prefectural Institute of Public Health, Fukushima, Japan), S. Takahashi (Sapporo City General Hospital, Hokkaido, Japan), M. Yagoshi (Nihon University Itabashi Hospital, Tokyo, Japan), H. Yajima (BML, Inc., Tokyo, Japan), K. Yamanaka (Otemae Hospital, Osaka, Japan), and A. Yamauchi (Mie Prefectural Institute of Public Health and Environmental Science, Mie, Japan). We are also grateful to M. Kimura (Infectious Disease Surveillance Center of the National Institute of Infectious Diseases) and M. Fukui (Tokyo Metropolitan University) for their valuable comments and suggestions.
This work was supported by grants (H14-Shinkou-17 and H15-Shinkou-24) from the Ministry of Health, Labor and Welfare of Japan.
FOOTNOTES
- Received 12 April 2004.
- Returned for modification 28 May 2004.
- Accepted 9 August 2004.
- Copyright © 2004 American Society for Microbiology
