We tested 3 vaccine strains and 96 clinical isolates: 25 isolates from 1953–1958, 52 from 1963–1985, and 19 from 1997–2005. These 3 periods were chosen according to when Pw vaccines were introduced (1960) and when Pa vaccines were added to the vaccination program (1995). Information on vaccine strains and clinical isolates is shown in Figure A1 and in the Technical Appendix. Clinical information for patients from whom B. pertussis was isolated was not available. Vaccine strains P3s10 and CS were isolated in Beijing in 1951 (Figure A1). Vaccine strain 18530 originated in the United States. Strains P3s10 and 18530 have been used to produce Pw vaccines since the early 1960s, and strain CS has been used to produce Pa vaccines since 1995. All B. pertussis isolates were confirmed by slide agglutination with specific antiserum to B. pertussis and B. parapertussis (Murex Diagnostics, Dartford, UK) and by PCR according to insertion sequence IS481 (18). B. pertussis strains were grown on Bordet-Gengou agar supplemented with 15% defibrinated sheep blood, incubated at 37°C for 4–5 days.

Serotyping was performed by a microtiter plate–based monoclonal agglutination assay as described (17). Monoclonal antibodies against Fim2 (NIBSC 04/154) or Fim3 (NIBSC 04/156) were used.

PCR-based sequencing of 5 genes (prn, ptxA, tcfA, fim2, and fim3) was performed as described (17,19,20) with minor modifications. Nucleotide sequences were determined for the complete opening read frames of ptxA, fim2, fim3, and tcfA, and for part of prn. The same primers were used for amplification and sequencing (Table). The sequencing assays were performed with an ABI Prism 3100 DNA sequencing system (Applied Biosystems, Foster City, CA, USA), and data analysis was conducted with DNASTAR (Madison, WI, USA) software.

Pulsed-field gel electrophoresis (PFGE) was performed as recommended (17) with minor modifications. Briefly, after the DNA plugs were treated with 50 U of XbaI (New England Biolabs, Ipswich, MA, USA), the cleaved DNA fragments were separated by electrophoresis on a 1% agarose gel by using a Chef Mapper (Bio-Rad, Hercules, CA, USA) with pulse times of 2.16–44.69 s for 24 h. The band patterns were analyzed with BioNumerics program version 4.0 (Applied-Maths, Kortrijk, Belgium). The clustering method used was the unweighted pair group method with arithmetic mean. The Pertussis Reference Laboratory of the National Institute for Health and Welfare, Turku, Finland, used PFGE to retest 53 clinical isolates, 3 vaccine strains, and international reference strain 18323 (7).

The selection criteria for the 53 isolates included at least 1 strain for 1 unique profile. If 2 strains had identical profiles, both were retested. If multiple strains represented the same profile, the strains isolated in different years or different regions were included. For PFGE, 8 international reference strains were included. The nomenclature was based on the profiles already defined for Finland (BpFINR) and Sweden (BpSR) (7,21). Profiles assigned as BpCHR have been found only among the analyzed isolates from China. A 2-sided Fisher exact test was used to compare frequencies of strain serotypes and genotypes from the 3 periods.

Vaccine strains P3s10 and CS were serotype Fim2,3, and vaccine strain 18530 was serotype Fim3. Among the clinical isolates, all 3 serotypes were found (Figure 2, panel A). Significantly more isolates collected during 1963–1986 were serotype Fim2 than were isolates collected during 1953–1958 (p<0.001). Of the 19 isolates collected during 1997–2005, we found that 15 (79%) were Fim3, 3 (16%) were Fim2,3, and 1 (5%) was Fim2. Significantly more isolates collected during 1997–2005 were serotype Fim3 than were isolates collected during 1953–1958 and 1963–1986 (p<0.001 for each).

Vaccine strains P3s10 and CS contained ptxA2/prn1/tcfA2/fim2–1/fim3–1, whereas vaccine strain 18530 contained ptxA3/prn1/tcfA2/fim2–2/fim3–1. Among the clinical isolates, all 4 ptxA alleles (ptxA1, ptxA2, ptxA3, and ptxA4) were found (Figure 2, panel B). However, the frequency of each allele changed over time. In the prevaccine era, 64% (n = 16), 24% (n = 6), and 12% (n = 3) of isolates contained ptxA2, ptxA3, and ptxA4, respectively. The allele ptxA1 appeared in 1963 and has become predominant since then. After the 1980s, all isolates contained ptxA1.

Altogether 6 prn alleles (prn1, prn2, prn3, prn7, prn10, and prn11) were detected. The amino acid sequences in variable regions of the 6 alleles are shown in Figure A2. Allele prn1 remained predominant at 81% (78/96) during the study period (Figure 2, panel C). No significant difference was found in the frequency of finding prn2 and prn3 in isolates collected during 1997–2005 than in those collected during 1953–1958 (p = 0.749) and 1963–1986 (p = 0.0513). All 7 isolates with prn7 contained ptxA3, whereas all 3 isolates with prn10 contained ptxA4. Two isolates with prn11 contained ptxA2. All 5 isolates with prn2 or prn3 contained ptxA1.

Four tcfA alleles (tcfA1, tcfA2, tcfA5, and tcfA9) were identified. Allele tcfA2 was most common, representing 94% (n = 90) of the isolates (Technical Appendix). Also detected were 2 fim2 (fim2–1 and fim2–2) and 3 fim3 (fim3–1, fim3–2, and fim3–4) alleles. Of the 96 isolates, 90 (94%) contained fim2–1. All 6 isolates with fim2–2 were recovered during 1953–1958. Five of the 6 isolates with fim2–2 contained prn7. For the fim3 alleles, 96% of isolates were fim3–1. All 3 isolates with fim3–2 and 1 isolate with fim3–4 were recovered in 1997–2005. Identical amino acid sequences were found for fim3–1 and fim3–4, but a silent mutation at nt 87 was found for fim3–4. All 3 isolates harboring fim3–2 contained prn2.

Vaccine strains P3s10 and CS had an identical profile, BpCHR6, whereas vaccine strain 18530 represented BpFINR13 (Figure 3). The 96 isolates produced 27 distinct profiles, 4 of which (BpFINR9, BpSR127, BpSR23, and BpSR11) have been reported in Europe. The PFGE profiles obtained from vaccine strain 18530 (BpFINR13) and international reference strain 18323 were not detected among the isolates from China. The 6 common profiles represented 70% of isolates (41 isolates with BpCHR15, 7 with BpCHR6, 6 with BpCHR2, 5 with BpSR127, 5 with BpSR23, and 3 with BpCHR20). The PFGE profiles changed over time. Dendrogram analysis of the 27 profiles indicated that they belonged to 8 clusters. Among these clusters, 5 (clusters I–IV and VII) have been reported in Europe (5,22,23).

Previous studies have shown that cluster I includes international reference strain 18323 and 1 clinical isolate (21,22). Clusters II and III include the vaccine strains and most strains that were circulating before the 1990s. Cluster IV contains most strains currently circulating in Europe. Cluster VII, a new group, consists of some isolates collected from Finland in 2004 (23). Of the 3 clusters identified in our study, 1 consisted of 4 profiles (BpCHR1–3 and BpFIN13). Profile BpFINR13 was found only in pertussis vaccine strain 18530 (7). The strain was obtained from the United States and used as a vaccine strain in Finland and China. The second cluster contained profiles BpCHR14 and BpCHR19, and the third contained profiles BpCHR20 and BpCHR22.

In this study, 56 isolates tested belonged to cluster III (Figure 3). Cluster III contained 6 profiles (BpCHR8, BpCHR15, BpCHR17, BpFINR9, BpSR23, and BpSR127) with a minimum of 79% overall relatedness. All isolates belonging to the cluster were collected during 1963–1986 and 1997–2005. Of the 96 clinical isolates, only 2 isolates that belonged to cluster IV (IV-β) were identified; these 2 isolates had PFGE profile BpSR11. Strains with BpSR11 were first detected in France in 1996 (22) and have been prevalent in Europe since then (13). The 2 isolates with BpSR11 were recovered in 2000 and 2001 and contained prn2.