Effect of Vaccination on Bordetella pertussis Strains, China

Strains in China may differ from those in countries that have long histories of high vaccination coverage.

In the People's Republic of China, vaccination against pertussis was started in the early 1960s, when 3 doses of Pw vaccine combined with diphtheria and tetanus toxoids were given at 3, 4, and 5 months of age. In 1982, a booster dose at 18 months of age was added (10,11). Pw vaccines are free of charge in China. Since 1995, acellular pertussis (Pa) vaccines containing purifi ed pertussis toxin (Ptx) and fi lamentous hemagglutinin have been also introduced. However, because Pa vaccines are offered at the patient's expense, use of these vaccines has been minimal, especially in some resource-limited areas. Although since 2007 Pa vaccines have been included in the national expanded program on immunization, Pa and Pw vaccines are still used in most provinces because of limited availability and cost of Pa vaccines. Although the reported vaccination coverage for the primary 3 doses increased with time, before the 1980s it was low. From 1983 through 2008, coverage ranged from 58% to 99% (median 93%) ( Figure 1) (12).
In China, pertussis is a reportable infectious disease, and the number of reported cases has been decreasing (Figure 1). Since the 1990s, incidence has been <1 case/100,000 population (12). In China, pertussis is clinically diagnosed by physicians; laboratory methods such as culture, PCR, and serologic analysis are not used for diagnosis of pertussis. Therefore, the reported low incidence may be related to the diagnostic criteria used, suggesting substantial underreporting. Pertussis remains endemic to China (10)(11)(12), and a local outbreak was reported in April 1997.
To learn more about the B. pertussis strains circulating in China, we used standardized typing methods to analyze and compare B. pertussis isolates collected before and after the introduction of vaccination (17).

Bacterial Strains and Culture
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 online Appendix Figure 1, www.cdc.gov/EID/content/16/11/ 1695-appF1.htm) and in the online Technical Appendix (www.cdc.gov/EID/content/16/11/1695-Techapp.htm). Clinical information for patients from whom B. pertussis was isolated was not available. Vaccine strains P3s10 and CS were isolated in Beijing in 1951 (online Appendix Figure 1). 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 confi rmed by slide agglutination with specifi c 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% defi brinated sheep blood, incubated at 37°C for 4-5 days.

DNA Sequencing
PCR-based sequencing of 5 genes (prn, ptxA, tcfA, fi m2, and fi m3) was performed as described (17,19,20) with minor modifi cations. Nucleotide sequences were determined for the complete opening read frames of ptxA, fi m2, fi m3, and tcfA, and for part of prn. The same primers were used for amplifi cation 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
Pulsed-fi eld gel electrophoresis (PFGE) was performed as recommended (17) with minor modifi cations. Briefl y, 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 profi le. If 2 strains had identical profi les, both were retested. If multiple strains represented the same profi le, 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 profi les already defi ned for Finland (BpFINR) and Sweden (BpSR) (7,21). Profi les 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. 1696 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999
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 online Appendix Two isolates with prn11 contained ptxA2. All 5 isolates with prn2 or prn3 contained ptxA1.

PFGE Profi les
Vaccine strains P3s10 and CS had an identical profi le, BpCHR6, whereas vaccine strain 18530 represented Bp-FINR13 (Figure 3). The 96 isolates produced 27 distinct profi les, 4 of which (BpFINR9, BpSR127, BpSR23, and BpSR11) have been reported in Europe. The PFGE profi les obtained from vaccine strain 18530 (BpFINR13) and international reference strain 18323 were not detected among the isolates from China. The 6 common profi les represented 70% of isolates (41 isolates with BpCHR15, 7 with Bp-CHR6, 6 with BpCHR2, 5 with BpSR127, 5 with BpSR23, and 3 with BpCHR20). The PFGE profi les changed over time. Dendrogram analysis of the 27 profi les 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 identifi ed in our study, 1 consisted of 4 profi les (BpCHR1-3 and BpFIN13). Profi le 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 profi les BpCHR14 and BpCHR19, and the third contained profi les BpCHR20 and BpCHR22. In this study, 56 isolates tested belonged to cluster III ( Figure 3). Cluster III contained 6 profi les (BpCHR8, Bp-CHR15, 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 identifi ed; these 2 isolates had PFGE profi le BpSR11. Strains with BpSR11 were fi rst 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.

Discussion
Few B. pertussis isolates from China contained nonvaccine type alleles prn2 or prn3; those that did were found later. In many countries, the prn1 allele is found in most vaccine strains and predominated before introduction of vaccination. However, the vaccine type strains were gradually replaced by nonvaccine type strains, mainly with allele prn2, after the introduction of vaccination. The most prevalent type in modern isolates is prn2 (7,14,20,24). In Taiwan, Pw vaccines have been offered since 1954 (25). When 168 clinical isolates collected in Taiwan during 1993-2004 were analyzed, prn2 strains accounted for 39% in 1993-1996 and increased to 90% in 1998-2004. In contrast to fi ndings for many countries with long histories of high vaccination coverage, prn2 was fi rst found in China in 2000. The exact reasons for the low frequency of strains with prn2 and their relatively late emergence in China are not known. One explanation might be the low vaccination coverage in China before the 1980s and differences in vaccine coverage between urban and rural areas. Another reason might be the closed borders.
In Japan, divergence of Prn and Ptx between vaccine strains and circulating isolates (26-28) has been reported. Pw vaccines were introduced in Japan in 1958; the vaccine strain used was prn1/ptxA2. In 1971, reported vaccination coverage was 50% (8). In 1976, vaccination coverage dropped to only 9%, and pertussis returned. In 1981, a Pa vaccine containing Ptx and fi lamentous hemagglutinin was introduced in Japan (27). The strain used for production of the Pa vaccines was Tohama I (prn1/ptxA2), isolated in the 1950s. When 107 isolates collected from 1988 through 2001 were studied, the nonvaccine type prn2/ptxA1 appeared in 1994 and was found in 27%-42% of isolates from 1994 through 2001 (26). A recent study reported similar frequency (33%) of the nonvaccine type prn2/ptxA1 in Japan when 60 isolates collected during 1991-2007 were analyzed (28).
TcfA has been shown to be crucial for B. pertussis colonization (29). A total of 9 tcfA alleles have been reported 1698 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16 1953-1958, 1963-1985, and 1997-2005. (30,31), and the most common allele is tcfA2 (20,24). Our fi nding that 94% of isolates studied contained tcfA2 agreed with fi ndings from earlier studies (20,24). Allele tcfA1 has been described only for reference strain 18323. In our study, 3 clinical isolates recovered during 1953-1958 were found to contain tcfA1. All 3 isolates were recovered from the same geographic area. Allele tcfA1 contains a 75-bp segment not found in other tcfA variants. The strain with tcfA1 was postulated to be the progenitor of the strains with tcfA2, tcfA3, or tcfA5 (20). Several studies have demonstrated that serotype Fim3 is predominant in vaccinated populations, whereas serotypes Fim2 or Fim2,3 are predominant in unvaccinated populations (14,32,33). In Sweden, before 1979 when Pw vaccines were fi rst used, 70% of circulating strains were serotype Fim3 (32). During 1979-1995, when pertussis vaccination was stopped, Fim2 started to increase and reached 64% in the early 1990s. In 1996, when general vaccination with Pa vaccines was reintroduced, prevalence of Fim2 declined and Fim3 strains emerged rapidly. In 2002 and 2003, Fim3 was found in 96% of fully vaccinated persons. In China, before introduction of vaccination, the prevalent serotypes were Fim2,3 and Fim2. After vaccination, the frequency of serotype Fim2,3 decreased and Fim2 became predominant. The possible explanation for the predominance of Fim2 strains after vaccination is that the 2 vaccine strains used in China express Fim2,3 and Fim3. The shift from serotype Fim2 to Fim3 was observed in 1998 and coincided with the introduction of Pa vaccines in this country. Pa vaccines without fi mbrial antigens may have some effect on fi mbrial serotypes of circulating isolates, as was observed in Sweden (32); however, the exact reason remains to be shown.
In our study, most strains from China had different PFGE profi les than did those from Europe. However, many PFGE profi les detected among the strains from China fell into the same clusters as those reported in Europe (5,22). For example, the most common profi le, BpCHR15, fell into the same cluster (cluster III) as BpSR23 and BpSR127 (14). Cluster III includes most strains circulating in Europe before the 1990s (5,32). In China, strains with BpCHR15 had been prevalent during 1965-1983. Although the strains with BpCHR15 were recovered from several different regions and over 20 years, the possibility that some strains were isolated during outbreaks cannot be excluded.
The major PFGE cluster circulating in Europe since the 1990s is cluster IV (5,32). Cluster IV can be divided into 3 subgroups (α, β, and γ), the frequency of which differs among countries. However, since the late 1990s in many countries in Europe, subgroup IV-β became more prevalent than the other 2 subgroups (13). In our study, only 2 isolates with BpSR11 belonged to group IV-β, whereas no isolates fell into subgroups IV-α or IV-γ. This PFGE result correlates with genotyping results.
The emergence of B. pertussis strains carrying a novel allele (ptxP3) for the Ptx promoter has been recently observed in countries with long histories of high vaccination coverage, such as the Netherlands (34). Furthermore, all strains from the Netherlands with BpSR11 were found to carry the ptxP3 allele. In our study, only 2 isolates from China were found to have BpSR11, suggesting that ptxP3 strains are not common in China.
The limitations of this study include the small number of B. pertussis isolates collected during the study period and recent isolates collected mainly from Beijing and its surrounding area. Therefore, our data should be interpreted with caution, and more epidemiologic studies with a larger number of isolates should be conducted in this country.
In conclusion, B. pertussis strains in China may differ from those in countries with long histories of high vaccination coverage. Continuous monitoring of B. pertussis strains is needed.