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Bordetella pertussis vaccine strains and circulating isolates in Serbia
Vaccine 28 (2010) 1188–1192
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Bordetella pertussis vaccine strains and circulating isolates in Serbia Gordana Dakic a , Teemu Kallonen b,c , Annika Elomaa b , Tatjana Pljesa a , Mirjana Vignjevic-Krastavcevic a , Qiushui He b,d,∗ a
Institute of Virology, Vaccine and Sera Torlak, Belgrade, Serbia Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), Turku, Finland c Turku Graduate School of Biomedical Sciences, University of Turku, Turku, Finland d Department of Pediatrics, University of Turku, Turku, Finland b
a r t i c l e
i n f o
Article history: Received 1 September 2009 Received in revised form 11 November 2009 Accepted 11 November 2009 Available online 27 November 2009 Keywords: Bordetella pertussis Pertussis Vaccine
a b s t r a c t In Serbia, whole cell pertussis vaccine was introduced in 1957. Current composition of the vaccine has been used since 1985 and contains four autochthonous strains of Bordetella pertussis isolated from 1957 to 1984. To monitor changes in bacterial population, 70 isolates collected from 1953 to 2000 were studied together with the vaccine strains. The methods included serotyping of fimbriae (Fim), genotyping of pertactin (prn) and pertussis toxin S1 subunit (ptxA), and pulsed-field gel electrophoresis analysis. Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. All isolates from 1980 to 1984 harbored ptxA1. Re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. The allele prn1 was predominant among the Serbian isolates, though prn3 and prn11 have been detected since 1981 and 1984. The allele prn2 was found only in two strains isolated in 2000. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant since then. The Serbian vaccine strains showed differences in ptxA and prn. The results of this present study indicate that the B. pertussis population in Serbia is different from other vaccinated populations and that this difference may be related to the vaccine used. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Despite extensive vaccinations, pertussis is still one of the leading causes of vaccine preventable deaths in the world today [1]. Resurgence of pertussis has been observed in many countries with long vaccination history [2–5]. Moreover, in these countries antigenic divergence with respect to pertussis toxin (Ptx) and pertactin (Prn) has been found between Bordetella pertussis vaccine strains and clinical isolates. In Serbia vaccination against pertussis has been used for 50 years. The diphtheria–tetanus-whole cell pertussis (DTPw) vaccine has been manufactured in the Institute of Virology, Vaccine and Sera Torlak, Belgrade, Serbia since 1957. The DTPw vaccine is given at 2, 4, 6, and 12 months of age. A second booster dose with the mono pertussis vaccine was given at 4 years of age during the periods from 1970 to 1981 and from 1990 to 2000. Current composition of the vaccine has been used since 1985 and contains four B. pertussis strains. The vaccine strains were chosen in compli-
∗ Corresponding author at: Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Kiinamyllynkatu 13, 20520 Turku, Finland. Tel.: +358 20 610 6634; fax: +358 20 610 6699. E-mail address: qiushui.he@thl.fi (Q. He). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.11.036
ance with serotype, immunogenicity and specific toxicity. The four strains represent three serotypes: Fim2 (8/84), Fim2,3 (1772/57 and 2047/57) and Fim3 (23/81) which are in equal amount in the vaccine composition. The reported vaccination coverage ranged from 79% to 98% (median, 90%) in 1981–2006 [6]. However, little is known of epidemiology of circulating B. pertussis strains in this country. Insight into the polymorphism of B. pertussis and its capacity to adapt to population immunity is important for the understanding of pertussis epidemiology. Therefore, we studied the Serbian vaccine strains and clinical isolates recovered before and after the introduction of vaccination by standardized typing methods [7]. 2. Materials and methods 2.1. Vaccine strains, clinical isolates and patient information Four vaccine strains and 70 clinical isolates were tested. Detailed information on each of vaccine strains and clinical isolates was included in the Supplementary data. The vaccine strain 2047/57 was isolated in 1957 and used for vaccine production from 1968. The strain 1772/57 was isolated in 1957 and added in the vaccine in 1972. The strains 23/81 and 8/84 were isolated in 1981 and 1984, and both were added in the vaccine in 1985. Clinical isolates were
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selected from the B. pertussis strain collection of the Institute of Virology, Vaccine and Sera Torlak, Belgrade, Serbia. The selection criteria included strains from different cities and not from same local outbreaks. Because of the low incidence of pertussis from 1990s, isolates were not available and were thus not tested. Information on age, gender and vaccination was available in 47 patients (Supplementary data). Their age ranged from 3 months to 33 years (mean, 6.4 years; median, 6 years). All isolates prior to 1960 were recovered from unvaccinated patients. 2.2. Laboratory methods for the typing of B. pertussis isolates The standardized typing methods included serotyping of fimbriae (Fim), genotyping of Ptx S1 subunit (ptxA) and prn by sequencing and LightCycler PCR, and pulsed-field gel electrophoresis (PFGE) [8–10]. These methods have been recommended for the epidemiological typing of B. pertussis isolates [7] and have been used for typing of the Finnish B. pertussis isolates [9,10]. For the PFGE analysis, six international reference strains were included [7] and nomenclature was based on the defined profiles already observed in Finland (BpFINR) and Sweden (BpSR) [8,9]. Profiles assigned BpSBR have been found only among the Serbian isolates analyzed. 2.3. Statistical analysis The Fisher’s exact test (two-sided) was used for statistical comparison of the frequencies of strain genotypes and serotypes between three time-periods (1953–1956, 1957–1984 and 1985–2000). The three time-periods were chosen according to the times 1957 when the pertussis vaccination was introduced and 1985 when the two strains harboring ptxA1/prn2 and ptxA1/prn1 were added in the vaccine. P value <0.05 was considered significant. 3. Results 3.1. Pertussis in Serbia In Serbia, pertussis is a notifiable infectious disease, which is collected in the Infectious Disease Register of the National Public Health Institute. Diagnosis is supplemented with culture and serology, and the diagnostic criteria and methods have remained unchanged in this country. The incidence of pertussis has been decreasing (Fig. 1). 3.2. Fimbrial serotypes All three serotypes, Fim2, Fim2.3, and Fim3, were observed among the vaccine strains as well as clinical isolates (Supplementary data). However, the frequency of each serotype has changed over time. Before the introduction of vaccination, the prevalent serotypes were Fim2 and Fim2.3. After the vaccination,
Fig. 1. The incidence (cases per 100,000) of reported pertussis cases in Serbia between 1965 and 2005. Source: Infectious Disease Register, National Public Health Institute, Serbia. In Serbia, vaccination against pertussis was started in 1957. Current composition of the vaccine has been used since 1985 and contains four strains. The strain 2047/57 was isolated in 1957 and used for vaccine production from 1968. The strain 1772/57 was isolated in 1957 and added in the vaccine in 1972. The strains 23/81 and 8/84 were isolated in 1981 and 1984, and both were added in the vaccine in 1985.
the frequency of serotype Fim2.3 decreased, being significantly lower in 1985–2000 compared to those observed in 1953–1956 and 1957–1984 (P = 0.013 and 0.003) (Table 1). Although the serotype Fim3 started to appear, Fim2 has been the most prevalent serotype during the study period. 3.3. Genotypes of ptxA and prn The vaccine strains 2047/57 and 1772/57 represented ptxA2/prn1 genotypes, whereas the vaccine strains 23/81 and 8/84 harbored ptxA1/prn1 and ptxA1/prn2 (Supplementary data). Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. During the period of 1980–1984, all isolates represented ptxA1 genotype. Re-appearance of the isolates containing ptxA2 was noticed after the two strains harboring ptxA1 were added into the vaccine in 1985. Although the allele prn1 was predominant among the Serbian isolates analyzed (Supplementary data), prn2, prn3 and prn11 occurred in some isolates since 1980s. The amino acid sequences in variable regions of Prn1, Prn2, Prn3 and Prn11 were shown in Fig. 2. The allele prn3 and prn11 were first detected in 1981 and 1984, respectively. All the four isolates harboring prn3 contained ptxA1, whereas five of the six isolates harboring prn11 represented ptxA2. All the six isolates harboring prn11 were serotype Fim2, whereas three of the four isolates harboring prn3 were serotype Fim3. The prn2 allele was only found in two of the four strains isolated in 2000. The two isolates represented ptxA1 and serotype Fim3. The frequency of prn1 in 1985–2000 was significantly lower
Table 1 Characteristics of B. pertussis isolates from the time-periods 1953–1956, 1957–1984 and 1985–2000 by genotype and serotypea . Time-period
Isolates typed
prn1 no (%)
prn2 no (%)
prn3 no (%)
prn11 no (%)
ptxA1 no (%)
ptxA2 no (%)
Fim2 no (%)
Fim2.3 no (%)
Fim3 no (%)
1953–1956 1957–1984 1985–2000
10 47 17
10 (100) 43 (91) 8 (47)c , d
0 1 (2) 2 (12)
0 2 (4) 2 (12)
0 1 (2) 5 (29)d
0 31 (66)b 11 (65)c
10 (100) 16 (34)b 6 (35)c
5 (50) 19 (40) 13 (76)e
5 (50) 14 (30) 0c , f
0 14 (30) 4 (24)
a The three time-periods were chosen according to the times 1957 when the pertussis vaccination was introduced and 1985 when the two strains harboring ptxA1/prn2 and ptxA1/prn1 were added in the vaccine. The four vaccine strains were included in the isolates from the time-period 1957 to 1984. b P = 0.0001, comparing frequencies of isolates with ptxA1 or ptxA2 in 1957–1984 with 1953–1956. c P < 0.01, comparing frequencies of isolates with prn1, ptxA1 or ptxA2, or Fim2.3 in 1985–2000 with 1953–1956. d P < 0.01, comparing frequencies of isolates with prn1 or prn11 in 1985–2000 with 1957–1984. e P = 0.022, comparing frequencies of isolates with Fim2 in 1985–2000 with 1957–1984. f P = 0.013, comparing frequencies of isolates with Fim2.3 in 1985–2000 with 1957–1984.
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Fig. 2. Amino acid sequence of Prn. Numbering is relative to the first methionine of Prn1. Dashes represent deletions. The RGD motif implicated in adherence to host receptors is indicated.
than those observed in 1953–1956 and 1957–1984 (P = 0.009 and <0.001) (Table 1). The frequency of prn11 in 1985–2000 was significantly higher than that found in 1957–1984 (P = 0.006). No significant differences in frequencies of prn2 or prn3 were observed between the time-periods 1985–2000 and 1957–1984 (P = 0.17 and 0.285). 3.4. PFGE profiles The four vaccine strains represented four PFGE profiles (Fig. 3). The 56 isolates tested by PFGE produced 22 distinct profiles (Fig. 3). The five common profiles represented about two thirds of isolates (17 isolates with BpSR23, eight with BpFINR1, five with BpFINR9, four with BpSBR6 and three with BpSBR5). Twenty-four (43%) isolates had unique Serbian profiles (BpSBR). Change in PFGE profiles was observed over time. All PFGE profiles, except BpSR23, observed in 1950s disappeared since then. The profile BpSR23 was found in all the study periods.
Of the 56 isolates tested by PFGE, 53 (95%) belonged to two clusters, having a high similarity with a minimum of 78% overall relatedness (Fig. 3). Of the six isolates harboring prn11, four had the profile BpSBR6, one had BpSBR7, and one had BpSBR14. They all fell into the same cluster. Of the four isolates harboring prn3, two had the profile BpSBR8, one had BpSBR12, and one had BpSR127. They all fell into another cluster. Two isolates with prn2 had the profiles BpSBR9 and BpSR11. They fell into the same cluster as the isolates with prn3 (Fig. 3).
4. Discussion Vaccinations with Pw vaccines were introduced in the 1940s to 1960s and have successfully reduced morbidity and mortality of pertussis throughout the world [1]. Pw vaccines have been produced by different manufacturers, and the B. pertussis strains used for the production vary. The strains used for production of whole cell and acellular vaccines were usually isolated in the 1940s to 1960s, and in many countries the vaccinations have selected circulating isolates dissimilar to the vaccine strains [9,11,12]. Further, resurgence of pertussis has been observed in countries with longterm pertussis vaccination [2–5]. In Serbia Pw vaccine has been in use from 1957 and is manufactured in the Torlak Institute, Belgrade. In contrast to the many other countries, the Serbian vaccine contains four strains: two isolated in 1950s and two in 1980s. Of the four strains, one contained the prn2 allele and the other three
Fig. 3. Dendrogram analysis of 22 PFGE profiles of B. pertussis isolates circulating in Serbia during 1953–2000. The unweighted pair group method using arithmetic averages (UPGMA) with 1% band tolerance and 1% optimisation settings was used as the clustering method. The symbol (•) indicates international reference strains [7] and () Serbian vaccine strains. Of the isolates with identical profiles, only first one is shown.
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contained the prn1 allele. The vaccine has remained unchanged in this country since 1985. This unique formulation of the Serbian Pw vaccine provided us an opportunity to study possible effect of the inclusion of “contemporary” strains in the vaccine on temporal trends in B. pertussis population. The finding that frequency of the Serbian isolates representing prn2 was low and its appearance was late is striking. Prn, a 69 kDa outer membrane protein of B. pertussis contains two immunodominant regions, 1 and 2, composed of repeating units of five (GGXXP) or three (PQP) amino acids, respectively. It induces protective immunity and is included in some of the acellular vaccines. Polymorphism in Prn is essentially limited to region 1 and is located adjacent to an RDG motif implicated in adhesion [13]. So far, 12 prn alleles have been identified [1]. In many countries the allele prn1 or prn7 is present in most vaccine strains and predominated in prevaccine era [1]. However, the “vaccine type” strains were gradually replaced by “non-vaccine type” strains mainly prn2 after the introduction of vaccination. The prn2 is by far the most prevalent type in modern isolates [8,9,11,12,14]. Depending on the time when the pertussis vaccination was started and the potency of vaccine used, in most countries isolates containing prn2 were first observed in the 1970s to 1980s [8,9,11,12,14]. In line with these observations, in Serbia one isolate (8/84) harboring prn2 was detected in 1984. The isolate was added for vaccine production in 1985. From 1985 to 1991, almost all isolates were prn1 or prn11, and none of the isolates were found to be prn2. In 2000, two out of four isolates contained the prn2 allele. The low frequency of prn2 strains and their relatively late emergence in Serbia may be due to the fact that the vaccine contains an isolate having prn2 allele. In this present study, four different prn alleles (prn1, prn2, prn3 and prn11) were detected among the Serbian isolates. The alleles prn1 to prn3 have been observed in many countries [9,11,12,14,15]. The allele prn11 was only reported in a recent study carried out in Australia, where all 5 strains containing prn11 were isolated in the same year (1982) and the same region [15]. In our study, however, the 6 prn11 isolates were detected in different years from 1984 to 2000. Of the six isolates, four had identical serotype, genotype ptxA and PFGE profile. The difference between Prn1 and Prn11 was only one repeat in region 1: Prn1 has five repeats, whereas Prn11 has six repeats. These observations that most of the isolates from 1985 to 2000 contained the prn1 or prn11 allele suggested that the strains prevalent in the pre-vaccine era are still circulating in Serbia. The Serbian vaccine strains do not contain the allele prn3. However, strains harboring the allele prn3 were not isolated in the study at a frequency comparable to that seen in other countries [9,12,15]. The exact reasons for the difference are not known. In addition to the vaccine composition, many factors such as immunity, density and dynamics of population can contribute to selection of the circulating strains. So far 4 ptxA alleles have been reported [1]. In most countries, the allele ptxA2 and/or ptxA3 are present in most vaccine strains and predominated in the isolates circulating in pre-vaccine era [9,11,14]. However, the “vaccine type” strains were gradually replaced by “non-vaccine” type ptxA1 after the vaccination was introduced. Our result was in agreement with the earlier studies. Shift from ptxA2 to ptxA1 was observed in isolates since the late of 1960s, and all isolates from 1980 to 1984 represented ptxA1. Interestingly, re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. From 1985 to 2000, 35% of isolates tested in the study were ptxA2. The high frequency of strains harboring ptxA2 was not comparable to that noticed in many other countries [9,11,14]. Several studies have shown that Fim2 isolates predominate in unvaccinated population, while they are largely displaced by Fim3 strains when vaccination is introduced with a Pw vaccine containing both Fim2 and Fim3 [16,17]. Although the vaccination has been
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used in Serbia since 1957, more than half of the isolates studied from 1957 to 2000 were serotype Fim2. The PFGE analyses of the Serbian isolates indicate that the prevalent PFGE profiles change temporally. However, the profile BpSR23, representing 30% of isolates studied persisted in the study periods. It has been reported that in Finland and Sweden the profile BpSR23 was prevalent in 1970s but not in 1990s [9,16]. Of the four strains isolated in 2000, one had the PFGE profile BpSR11. This PFGE profile was found to be predominant in six of the eight European countries [18]. Increases in the incidence of pertussis have been reported in many countries with long vaccination history. Moreover, in many of these countries divergence between vaccine strains and circulating isolates have been found. Interestingly, the incidence of pertussis has been decreasing in Serbia. It is known that in vaccinated populations, symptoms of pertussis can be mild and the patients do not usually seek for medical help. Therefore, the possibility that incidence of pertussis is underestimated in this country cannot be excluded. Whether the low incidence of pertussis in this country is related to the vaccine used remains to be illustrated. King et al. [19] were the first to show that variation in Prn affects vaccine efficacy in the mouse model. It has been recently shown that the adequate bacterial elimination rates were observed in mice immunized and challenged with the same vaccine type strain [20] and that the vaccine prepared from a recent isolate provided the highest mouse protection when compared to those prepared from the old isolates such as the strain Tohama I [21]. The limitations of this study include the lack of strains isolated later than 2000. Therefore, the question of whether prn2 strains eventually became predominant in this country remains to be shown. It should be kept in mind that bacterial amount of B. pertussis containing prn2 only accounts for one third in the vaccine. In conclusion, the Serbian vaccine strains showed differences in ptxA and prn as well as in the PFGE profiles. The B. pertussis population in Serbia is different from other vaccinated populations and this difference may be related to the vaccine used. Acknowledgments We thank Päivi Haaranen for excellent technical assistance and Prof. Dr. Milena Svabic Vlahovic, Medical Faculty, University of Belgrade for professional support. This work was supported in part by the Academy of Finland and Varsinais-Suomi Regional Fund of the Finnish Cultural Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2009.11.036. References [1] He Q, Mertsola J. Factors contributing to pertussis resurgence. Future Microbiol 2008;June (3):329–39. [2] Celentano LP, Massari M, Paramatti D, Salmaso S, Tozzi AE. Resurgence of pertussis in Europe. Pediatr Infect Dis J 2005;24(September (9)):761–5. [3] Halperin SA. The control of pertussis–2007 and beyond. N Engl J Med 2007;356(January (2)):110–3. [4] McIntyre P, Gidding H, Gilmour R, Lawrence G, Hull B, Horby P, et al. Vaccine preventable diseases and vaccination coverage in Australia, 1999 to 2000. Commun Dis Intell 2002;(May (Suppl.)), i-xi, 1–111. [5] Mooi FR, van Loo IH, King AJ. Adaptation of Bordetella pertussis to vaccination: a cause for its reemergence? Emerg Infect Dis 2001;Suppl. 7(3):526–8. [6] WHO. World Health Organisation: immunization, vaccination and biologicals. Vaccine preventable diseases Vaccines monitoring system 2009 Global Summary Reference Time Series: SERBIA http://www.who.int/immunization monitoring/en/globalsummary/timeseries/tscoveragebycountry.cfm?C=SRB. [7] Mooi FR, Hallander H, Wirsing von Konig CH, Hoet B, Guiso N. Epidemiological typing of Bordetella pertussis isolates: recommendations for a standard methodology. Eur J Clin Microbiol Infect Dis 2000;19(March (3)):174–81.
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[8] Advani A, Donnelly D, Hallander H. Reference system for characterization of Bordetella pertussis pulsed-field gel electrophoresis profiles. J Clin Microbiol 2004;42(July (7)):2890–7. [9] Elomaa A, Advani A, Donnelly D, Antila M, Mertsola J, Hallander H, et al. Strain variation among Bordetella pertussis isolates in Finland, where the whole-cell pertussis vaccine has been used for 50 years. J Clin Microbiol 2005;43(August (8)):3681–7. [10] Heikkinen E, Xing DK, Olander RM, Hytonen J, Viljanen MK, Mertsola J, et al. Bordetella pertussis isolates in Finland: serotype and fimbrial expression. BMC Microbiol 2008;8:162. [11] Cassiday P, Sanden G, Heuvelman K, Mooi F, Bisgard KM, Popovic T. Polymorphism in Bordetella pertussis pertactin and pertussis toxin virulence factors in the United States, 1935–1999. J Infect Dis 2000;182(November (5)):1402–8. [12] Mooi FR, van Oirschot H, Heuvelman K, van der Heide HG, Gaastra W, Willems RJ. Polymorphism in the Bordetella pertussis virulence factors P.69/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccinedriven evolution. Infect Immun 1998;66(February (2)):670–5. [13] Leininger E, Roberts M, Kenimer JG, Charles IG, Fairweather N, Novotny P, et al. Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes adherence of mammalian cells. Proc Natl Acad Sci USA 1991;88(January (2)):345–9. [14] Weber C, Boursaux-Eude C, Coralie G, Caro V, Guiso N. Polymorphism of Bordetella pertussis isolates circulating for the last 10 years in France, where a single effective whole-cell vaccine has been used for more than 30 years. J Clin Microbiol 2001;39(December (12)):4396–403.
[15] Poynten M, McIntyre PB, Mooi FR, Heuvelman KJ, Gilbert GL. Temporal trends in circulating Bordetella pertussis strains in Australia. Epidemiol Infect 2004;132(April (2)):185–93. [16] Hallander HO, Advani A, Donnelly D, Gustafsson L, Carlsson RM. Shifts of Bordetella pertussis variants in Sweden from 1970 to 2003, during three periods marked by different vaccination programs. J Clin Microbiol 2005;43(June (6)):2856–65. [17] Preston NW, Carter EJ. Serotype specificity of vaccine-induced immunity to pertussis. Commun Dis Rep CDR Rev 1992;2(December (13)):R155–6. [18] Hallander H, Advani A, Riffelmann M, von Konig CH, Caro V, Guiso N, et al. Bordetella pertussis strains circulating in Europe in 1999 to 2004 as determined by pulsed-field gel electrophoresis. J Clin Microbiol 2007;45(October (10)):3257–62. [19] King AJ, Berbers G, van Oirschot HF, Hoogerhout P, Knipping K, Mooi FR. Role of the polymorphic region 1 of the Bordetella pertussis protein pertactin in immunity. Microbiology 2001;147(November (Pt 11)): 2885–95. [20] Bottero D, Gaillard ME, Fingermann M, Weltman G, Fernandez J, Sisti F, et al. Pulsed-field gel electrophoresis, pertactin, pertussis toxin S1 subunit polymorphisms, and surfaceome analysis of vaccine and clinical Bordetella pertussis strains. Clin Vaccine Immunol 2007;14(November (11)): 1490–8. [21] Pereira A, Pereira AS, Moreira-Filho CA, Bando SY, Tambourgi DV. Comparative analysis of a Bordetella pertussis patient isolated strain and classical strains used in the pertussis vaccine. Vaccine 2005;23(July (34)):4353–8.
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Bordetella pertussis vaccine strains and circulating isolates in Serbia Gordana Dakic a , Teemu Kallonen b,c , Annika Elomaa b , Tatjana Pljesa a , Mirjana Vignjevic-Krastavcevic a , Qiushui He b,d,∗ a
Institute of Virology, Vaccine and Sera Torlak, Belgrade, Serbia Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), Turku, Finland c Turku Graduate School of Biomedical Sciences, University of Turku, Turku, Finland d Department of Pediatrics, University of Turku, Turku, Finland b
a r t i c l e
i n f o
Article history: Received 1 September 2009 Received in revised form 11 November 2009 Accepted 11 November 2009 Available online 27 November 2009 Keywords: Bordetella pertussis Pertussis Vaccine
a b s t r a c t In Serbia, whole cell pertussis vaccine was introduced in 1957. Current composition of the vaccine has been used since 1985 and contains four autochthonous strains of Bordetella pertussis isolated from 1957 to 1984. To monitor changes in bacterial population, 70 isolates collected from 1953 to 2000 were studied together with the vaccine strains. The methods included serotyping of fimbriae (Fim), genotyping of pertactin (prn) and pertussis toxin S1 subunit (ptxA), and pulsed-field gel electrophoresis analysis. Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. All isolates from 1980 to 1984 harbored ptxA1. Re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. The allele prn1 was predominant among the Serbian isolates, though prn3 and prn11 have been detected since 1981 and 1984. The allele prn2 was found only in two strains isolated in 2000. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant since then. The Serbian vaccine strains showed differences in ptxA and prn. The results of this present study indicate that the B. pertussis population in Serbia is different from other vaccinated populations and that this difference may be related to the vaccine used. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Despite extensive vaccinations, pertussis is still one of the leading causes of vaccine preventable deaths in the world today [1]. Resurgence of pertussis has been observed in many countries with long vaccination history [2–5]. Moreover, in these countries antigenic divergence with respect to pertussis toxin (Ptx) and pertactin (Prn) has been found between Bordetella pertussis vaccine strains and clinical isolates. In Serbia vaccination against pertussis has been used for 50 years. The diphtheria–tetanus-whole cell pertussis (DTPw) vaccine has been manufactured in the Institute of Virology, Vaccine and Sera Torlak, Belgrade, Serbia since 1957. The DTPw vaccine is given at 2, 4, 6, and 12 months of age. A second booster dose with the mono pertussis vaccine was given at 4 years of age during the periods from 1970 to 1981 and from 1990 to 2000. Current composition of the vaccine has been used since 1985 and contains four B. pertussis strains. The vaccine strains were chosen in compli-
∗ Corresponding author at: Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Kiinamyllynkatu 13, 20520 Turku, Finland. Tel.: +358 20 610 6634; fax: +358 20 610 6699. E-mail address: qiushui.he@thl.fi (Q. He). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.11.036
ance with serotype, immunogenicity and specific toxicity. The four strains represent three serotypes: Fim2 (8/84), Fim2,3 (1772/57 and 2047/57) and Fim3 (23/81) which are in equal amount in the vaccine composition. The reported vaccination coverage ranged from 79% to 98% (median, 90%) in 1981–2006 [6]. However, little is known of epidemiology of circulating B. pertussis strains in this country. Insight into the polymorphism of B. pertussis and its capacity to adapt to population immunity is important for the understanding of pertussis epidemiology. Therefore, we studied the Serbian vaccine strains and clinical isolates recovered before and after the introduction of vaccination by standardized typing methods [7]. 2. Materials and methods 2.1. Vaccine strains, clinical isolates and patient information Four vaccine strains and 70 clinical isolates were tested. Detailed information on each of vaccine strains and clinical isolates was included in the Supplementary data. The vaccine strain 2047/57 was isolated in 1957 and used for vaccine production from 1968. The strain 1772/57 was isolated in 1957 and added in the vaccine in 1972. The strains 23/81 and 8/84 were isolated in 1981 and 1984, and both were added in the vaccine in 1985. Clinical isolates were
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selected from the B. pertussis strain collection of the Institute of Virology, Vaccine and Sera Torlak, Belgrade, Serbia. The selection criteria included strains from different cities and not from same local outbreaks. Because of the low incidence of pertussis from 1990s, isolates were not available and were thus not tested. Information on age, gender and vaccination was available in 47 patients (Supplementary data). Their age ranged from 3 months to 33 years (mean, 6.4 years; median, 6 years). All isolates prior to 1960 were recovered from unvaccinated patients. 2.2. Laboratory methods for the typing of B. pertussis isolates The standardized typing methods included serotyping of fimbriae (Fim), genotyping of Ptx S1 subunit (ptxA) and prn by sequencing and LightCycler PCR, and pulsed-field gel electrophoresis (PFGE) [8–10]. These methods have been recommended for the epidemiological typing of B. pertussis isolates [7] and have been used for typing of the Finnish B. pertussis isolates [9,10]. For the PFGE analysis, six international reference strains were included [7] and nomenclature was based on the defined profiles already observed in Finland (BpFINR) and Sweden (BpSR) [8,9]. Profiles assigned BpSBR have been found only among the Serbian isolates analyzed. 2.3. Statistical analysis The Fisher’s exact test (two-sided) was used for statistical comparison of the frequencies of strain genotypes and serotypes between three time-periods (1953–1956, 1957–1984 and 1985–2000). The three time-periods were chosen according to the times 1957 when the pertussis vaccination was introduced and 1985 when the two strains harboring ptxA1/prn2 and ptxA1/prn1 were added in the vaccine. P value <0.05 was considered significant. 3. Results 3.1. Pertussis in Serbia In Serbia, pertussis is a notifiable infectious disease, which is collected in the Infectious Disease Register of the National Public Health Institute. Diagnosis is supplemented with culture and serology, and the diagnostic criteria and methods have remained unchanged in this country. The incidence of pertussis has been decreasing (Fig. 1). 3.2. Fimbrial serotypes All three serotypes, Fim2, Fim2.3, and Fim3, were observed among the vaccine strains as well as clinical isolates (Supplementary data). However, the frequency of each serotype has changed over time. Before the introduction of vaccination, the prevalent serotypes were Fim2 and Fim2.3. After the vaccination,
Fig. 1. The incidence (cases per 100,000) of reported pertussis cases in Serbia between 1965 and 2005. Source: Infectious Disease Register, National Public Health Institute, Serbia. In Serbia, vaccination against pertussis was started in 1957. Current composition of the vaccine has been used since 1985 and contains four strains. The strain 2047/57 was isolated in 1957 and used for vaccine production from 1968. The strain 1772/57 was isolated in 1957 and added in the vaccine in 1972. The strains 23/81 and 8/84 were isolated in 1981 and 1984, and both were added in the vaccine in 1985.
the frequency of serotype Fim2.3 decreased, being significantly lower in 1985–2000 compared to those observed in 1953–1956 and 1957–1984 (P = 0.013 and 0.003) (Table 1). Although the serotype Fim3 started to appear, Fim2 has been the most prevalent serotype during the study period. 3.3. Genotypes of ptxA and prn The vaccine strains 2047/57 and 1772/57 represented ptxA2/prn1 genotypes, whereas the vaccine strains 23/81 and 8/84 harbored ptxA1/prn1 and ptxA1/prn2 (Supplementary data). Shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. During the period of 1980–1984, all isolates represented ptxA1 genotype. Re-appearance of the isolates containing ptxA2 was noticed after the two strains harboring ptxA1 were added into the vaccine in 1985. Although the allele prn1 was predominant among the Serbian isolates analyzed (Supplementary data), prn2, prn3 and prn11 occurred in some isolates since 1980s. The amino acid sequences in variable regions of Prn1, Prn2, Prn3 and Prn11 were shown in Fig. 2. The allele prn3 and prn11 were first detected in 1981 and 1984, respectively. All the four isolates harboring prn3 contained ptxA1, whereas five of the six isolates harboring prn11 represented ptxA2. All the six isolates harboring prn11 were serotype Fim2, whereas three of the four isolates harboring prn3 were serotype Fim3. The prn2 allele was only found in two of the four strains isolated in 2000. The two isolates represented ptxA1 and serotype Fim3. The frequency of prn1 in 1985–2000 was significantly lower
Table 1 Characteristics of B. pertussis isolates from the time-periods 1953–1956, 1957–1984 and 1985–2000 by genotype and serotypea . Time-period
Isolates typed
prn1 no (%)
prn2 no (%)
prn3 no (%)
prn11 no (%)
ptxA1 no (%)
ptxA2 no (%)
Fim2 no (%)
Fim2.3 no (%)
Fim3 no (%)
1953–1956 1957–1984 1985–2000
10 47 17
10 (100) 43 (91) 8 (47)c , d
0 1 (2) 2 (12)
0 2 (4) 2 (12)
0 1 (2) 5 (29)d
0 31 (66)b 11 (65)c
10 (100) 16 (34)b 6 (35)c
5 (50) 19 (40) 13 (76)e
5 (50) 14 (30) 0c , f
0 14 (30) 4 (24)
a The three time-periods were chosen according to the times 1957 when the pertussis vaccination was introduced and 1985 when the two strains harboring ptxA1/prn2 and ptxA1/prn1 were added in the vaccine. The four vaccine strains were included in the isolates from the time-period 1957 to 1984. b P = 0.0001, comparing frequencies of isolates with ptxA1 or ptxA2 in 1957–1984 with 1953–1956. c P < 0.01, comparing frequencies of isolates with prn1, ptxA1 or ptxA2, or Fim2.3 in 1985–2000 with 1953–1956. d P < 0.01, comparing frequencies of isolates with prn1 or prn11 in 1985–2000 with 1957–1984. e P = 0.022, comparing frequencies of isolates with Fim2 in 1985–2000 with 1957–1984. f P = 0.013, comparing frequencies of isolates with Fim2.3 in 1985–2000 with 1957–1984.
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Fig. 2. Amino acid sequence of Prn. Numbering is relative to the first methionine of Prn1. Dashes represent deletions. The RGD motif implicated in adherence to host receptors is indicated.
than those observed in 1953–1956 and 1957–1984 (P = 0.009 and <0.001) (Table 1). The frequency of prn11 in 1985–2000 was significantly higher than that found in 1957–1984 (P = 0.006). No significant differences in frequencies of prn2 or prn3 were observed between the time-periods 1985–2000 and 1957–1984 (P = 0.17 and 0.285). 3.4. PFGE profiles The four vaccine strains represented four PFGE profiles (Fig. 3). The 56 isolates tested by PFGE produced 22 distinct profiles (Fig. 3). The five common profiles represented about two thirds of isolates (17 isolates with BpSR23, eight with BpFINR1, five with BpFINR9, four with BpSBR6 and three with BpSBR5). Twenty-four (43%) isolates had unique Serbian profiles (BpSBR). Change in PFGE profiles was observed over time. All PFGE profiles, except BpSR23, observed in 1950s disappeared since then. The profile BpSR23 was found in all the study periods.
Of the 56 isolates tested by PFGE, 53 (95%) belonged to two clusters, having a high similarity with a minimum of 78% overall relatedness (Fig. 3). Of the six isolates harboring prn11, four had the profile BpSBR6, one had BpSBR7, and one had BpSBR14. They all fell into the same cluster. Of the four isolates harboring prn3, two had the profile BpSBR8, one had BpSBR12, and one had BpSR127. They all fell into another cluster. Two isolates with prn2 had the profiles BpSBR9 and BpSR11. They fell into the same cluster as the isolates with prn3 (Fig. 3).
4. Discussion Vaccinations with Pw vaccines were introduced in the 1940s to 1960s and have successfully reduced morbidity and mortality of pertussis throughout the world [1]. Pw vaccines have been produced by different manufacturers, and the B. pertussis strains used for the production vary. The strains used for production of whole cell and acellular vaccines were usually isolated in the 1940s to 1960s, and in many countries the vaccinations have selected circulating isolates dissimilar to the vaccine strains [9,11,12]. Further, resurgence of pertussis has been observed in countries with longterm pertussis vaccination [2–5]. In Serbia Pw vaccine has been in use from 1957 and is manufactured in the Torlak Institute, Belgrade. In contrast to the many other countries, the Serbian vaccine contains four strains: two isolated in 1950s and two in 1980s. Of the four strains, one contained the prn2 allele and the other three
Fig. 3. Dendrogram analysis of 22 PFGE profiles of B. pertussis isolates circulating in Serbia during 1953–2000. The unweighted pair group method using arithmetic averages (UPGMA) with 1% band tolerance and 1% optimisation settings was used as the clustering method. The symbol (•) indicates international reference strains [7] and () Serbian vaccine strains. Of the isolates with identical profiles, only first one is shown.
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contained the prn1 allele. The vaccine has remained unchanged in this country since 1985. This unique formulation of the Serbian Pw vaccine provided us an opportunity to study possible effect of the inclusion of “contemporary” strains in the vaccine on temporal trends in B. pertussis population. The finding that frequency of the Serbian isolates representing prn2 was low and its appearance was late is striking. Prn, a 69 kDa outer membrane protein of B. pertussis contains two immunodominant regions, 1 and 2, composed of repeating units of five (GGXXP) or three (PQP) amino acids, respectively. It induces protective immunity and is included in some of the acellular vaccines. Polymorphism in Prn is essentially limited to region 1 and is located adjacent to an RDG motif implicated in adhesion [13]. So far, 12 prn alleles have been identified [1]. In many countries the allele prn1 or prn7 is present in most vaccine strains and predominated in prevaccine era [1]. However, the “vaccine type” strains were gradually replaced by “non-vaccine type” strains mainly prn2 after the introduction of vaccination. The prn2 is by far the most prevalent type in modern isolates [8,9,11,12,14]. Depending on the time when the pertussis vaccination was started and the potency of vaccine used, in most countries isolates containing prn2 were first observed in the 1970s to 1980s [8,9,11,12,14]. In line with these observations, in Serbia one isolate (8/84) harboring prn2 was detected in 1984. The isolate was added for vaccine production in 1985. From 1985 to 1991, almost all isolates were prn1 or prn11, and none of the isolates were found to be prn2. In 2000, two out of four isolates contained the prn2 allele. The low frequency of prn2 strains and their relatively late emergence in Serbia may be due to the fact that the vaccine contains an isolate having prn2 allele. In this present study, four different prn alleles (prn1, prn2, prn3 and prn11) were detected among the Serbian isolates. The alleles prn1 to prn3 have been observed in many countries [9,11,12,14,15]. The allele prn11 was only reported in a recent study carried out in Australia, where all 5 strains containing prn11 were isolated in the same year (1982) and the same region [15]. In our study, however, the 6 prn11 isolates were detected in different years from 1984 to 2000. Of the six isolates, four had identical serotype, genotype ptxA and PFGE profile. The difference between Prn1 and Prn11 was only one repeat in region 1: Prn1 has five repeats, whereas Prn11 has six repeats. These observations that most of the isolates from 1985 to 2000 contained the prn1 or prn11 allele suggested that the strains prevalent in the pre-vaccine era are still circulating in Serbia. The Serbian vaccine strains do not contain the allele prn3. However, strains harboring the allele prn3 were not isolated in the study at a frequency comparable to that seen in other countries [9,12,15]. The exact reasons for the difference are not known. In addition to the vaccine composition, many factors such as immunity, density and dynamics of population can contribute to selection of the circulating strains. So far 4 ptxA alleles have been reported [1]. In most countries, the allele ptxA2 and/or ptxA3 are present in most vaccine strains and predominated in the isolates circulating in pre-vaccine era [9,11,14]. However, the “vaccine type” strains were gradually replaced by “non-vaccine” type ptxA1 after the vaccination was introduced. Our result was in agreement with the earlier studies. Shift from ptxA2 to ptxA1 was observed in isolates since the late of 1960s, and all isolates from 1980 to 1984 represented ptxA1. Interestingly, re-appearance of the ptxA2 allele followed an addition of the two strains harboring ptxA1 in the vaccine in 1985. From 1985 to 2000, 35% of isolates tested in the study were ptxA2. The high frequency of strains harboring ptxA2 was not comparable to that noticed in many other countries [9,11,14]. Several studies have shown that Fim2 isolates predominate in unvaccinated population, while they are largely displaced by Fim3 strains when vaccination is introduced with a Pw vaccine containing both Fim2 and Fim3 [16,17]. Although the vaccination has been
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used in Serbia since 1957, more than half of the isolates studied from 1957 to 2000 were serotype Fim2. The PFGE analyses of the Serbian isolates indicate that the prevalent PFGE profiles change temporally. However, the profile BpSR23, representing 30% of isolates studied persisted in the study periods. It has been reported that in Finland and Sweden the profile BpSR23 was prevalent in 1970s but not in 1990s [9,16]. Of the four strains isolated in 2000, one had the PFGE profile BpSR11. This PFGE profile was found to be predominant in six of the eight European countries [18]. Increases in the incidence of pertussis have been reported in many countries with long vaccination history. Moreover, in many of these countries divergence between vaccine strains and circulating isolates have been found. Interestingly, the incidence of pertussis has been decreasing in Serbia. It is known that in vaccinated populations, symptoms of pertussis can be mild and the patients do not usually seek for medical help. Therefore, the possibility that incidence of pertussis is underestimated in this country cannot be excluded. Whether the low incidence of pertussis in this country is related to the vaccine used remains to be illustrated. King et al. [19] were the first to show that variation in Prn affects vaccine efficacy in the mouse model. It has been recently shown that the adequate bacterial elimination rates were observed in mice immunized and challenged with the same vaccine type strain [20] and that the vaccine prepared from a recent isolate provided the highest mouse protection when compared to those prepared from the old isolates such as the strain Tohama I [21]. The limitations of this study include the lack of strains isolated later than 2000. Therefore, the question of whether prn2 strains eventually became predominant in this country remains to be shown. It should be kept in mind that bacterial amount of B. pertussis containing prn2 only accounts for one third in the vaccine. In conclusion, the Serbian vaccine strains showed differences in ptxA and prn as well as in the PFGE profiles. The B. pertussis population in Serbia is different from other vaccinated populations and this difference may be related to the vaccine used. Acknowledgments We thank Päivi Haaranen for excellent technical assistance and Prof. Dr. Milena Svabic Vlahovic, Medical Faculty, University of Belgrade for professional support. This work was supported in part by the Academy of Finland and Varsinais-Suomi Regional Fund of the Finnish Cultural Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2009.11.036. References [1] He Q, Mertsola J. Factors contributing to pertussis resurgence. Future Microbiol 2008;June (3):329–39. [2] Celentano LP, Massari M, Paramatti D, Salmaso S, Tozzi AE. Resurgence of pertussis in Europe. Pediatr Infect Dis J 2005;24(September (9)):761–5. [3] Halperin SA. The control of pertussis–2007 and beyond. N Engl J Med 2007;356(January (2)):110–3. [4] McIntyre P, Gidding H, Gilmour R, Lawrence G, Hull B, Horby P, et al. Vaccine preventable diseases and vaccination coverage in Australia, 1999 to 2000. Commun Dis Intell 2002;(May (Suppl.)), i-xi, 1–111. [5] Mooi FR, van Loo IH, King AJ. Adaptation of Bordetella pertussis to vaccination: a cause for its reemergence? Emerg Infect Dis 2001;Suppl. 7(3):526–8. [6] WHO. World Health Organisation: immunization, vaccination and biologicals. Vaccine preventable diseases Vaccines monitoring system 2009 Global Summary Reference Time Series: SERBIA http://www.who.int/immunization monitoring/en/globalsummary/timeseries/tscoveragebycountry.cfm?C=SRB. [7] Mooi FR, Hallander H, Wirsing von Konig CH, Hoet B, Guiso N. Epidemiological typing of Bordetella pertussis isolates: recommendations for a standard methodology. Eur J Clin Microbiol Infect Dis 2000;19(March (3)):174–81.
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