Toxin Production and Pertussis Resurgence | CDC EID

Volume 15, Number 8–August 2009
Research
Bordetella pertussis Strains with Increased Toxin Production Associated with Pertussis Resurgence
Frits R. Mooi, Inge H.M. van Loo, Marjolein van Gent, Qiushui He, Marieke J. Bart, Kees J. Heuvelman, Sabine C. de Greeff, Dimitri Diavatopoulos, Peter Teunis, Nico Nagelkerke, and Jussi Mertsola
Author affiliations: National Institute for Public Health and the Environment, Bilthoven, the Netherlands (F.R. Mooi, M. van Gent, M.J. Bart, K.J. Heuvelman, S.C. de Greeff, D. Diavatopoulos, P. Teunis); Maastricht University Hospital, Maastricht, the Netherlands (I.H.M. van Loo); National Public Health Institute, Turku, Finland (Q. He); United Arab Emirates University, Al Ain, United Arab Emirates (N. Nagelkerke); and University of Turku, Turku (J. Mertsola)

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Abstract
Before childhood vaccination was introduced in the 1940s, pertussis was a major cause of infant death worldwide. Widespread vaccination of children succeeded in reducing illness and death. In the 1990s, a resurgence of pertussis was observed in a number of countries with highly vaccinated populations, and pertussis has become the most prevalent vaccine-preventable disease in industrialized countries. We present evidence that in the Netherlands the dramatic increase in pertussis is temporally associated with the emergence of Bordetella pertussis strains carrying a novel allele for the pertussis toxin promoter, which confers increased pertussis toxin (Ptx) production. Epidemiologic data suggest that these strains are more virulent in humans. We discuss changes in the ecology of B. pertussis that may have driven this adaptation. Our results underline the importance of Ptx in transmission, suggest that vaccination may select for increased virulence, and indicate ways to control pertussis more effectively.

Bordetella pertussis causes whooping cough or pertussis, a respiratory disease that is most severe in infants. Before childhood vaccination was introduced in the 1950s, pertussis was a major cause of infant deaths worldwide. Widespread vaccination of children reduced the incidence of illness and deaths caused by pertussis (1). However, globally pertussis remains 1 of the top 10 causes of death in children (2). Further, in the 1990s a resurgence of pertussis was observed in several countries with highly vaccinated populations (3,4), and pertussis has become the most prevalent vaccine-preventable disease in industrialized countries. In the Netherlands, the estimated incidence of infection was 6.6% per year for the 3–79-year age group from 1995 through 1996 (5). Similar percentages have been found in the United States (6). One of the hallmarks of the pertussis resurgence is a shift in disease prevalence toward older persons who have waning vaccine-induced immunity (7).

The reemergence of pertussis has been attributed to various factors, including increased awareness, improved diagnostics, decreased vaccination coverage, suboptimal vaccines, waning vaccine-induced immunity, and pathogen adaptation. The relative contribution of these factors may differ between countries and is the subject of ongoing debate. Pathogen adaptation is supported by several observations. We and others have shown that antigenic divergence has occurred between vaccine strains and clinical isolates with respect to surface proteins, which confer protective immunity: pertussis toxin (Ptx), pertactin (Prn), and fimbriae (8,9). Strain variation was shown to affect vaccine efficacy in a mouse model (10–13). Because adaptation may involve the structure of virulence factors (by antigenic variation) and their regulation, we extended our studies on the evolution of B. pertussis by investigating polymorphism in the promoter of Ptx (ptxP), a major virulence factor and component of all pertussis vaccines (1). We provide evidence that expansion of strains with increased Ptx production has contributed to the resurgence of pertussis in the Netherlands.

Methods
Pertussis Notifications
Pertussis became a notifiable disease in the Netherlands in 1976. Notifications are submitted online by local health authorities. Other notifiable diseases are also monitored through this system, which falls under the responsibility of the Dutch National Institute of Health and Environment (3).

Bacterial Strains
B. pertussis strains examined were obtained from 1935 through 2004. A total of 1,566 isolates, 879 from the Netherlands and 687 from other countries, were analyzed for polymorphism in ptxP (Technical Appendix [Microsoft Excel, 191 KB]). Eight strains isolated from patients in the Netherlands from 1999 through 2001 were selected to study Ptx and Prn production: B1834 (ptxP1), B1868 (ptxP1), B1878 (ptxP1), B1920 (ptxP1), B1836 (ptxP3), B1865 (ptxP3), B1917 (ptxP3), and B2030 (ptxP3) (Table 1).

Sequencing
The primers 5´-AATCGTCCTGCTCAACCGCC-3´ and 5´-GGTATACGGTGGCGGGAGGA-3´ were used for amplification and sequencing of ptxP and correspond, respectively, to bases 60–79 and 633–614 of the ptx sequence with GenBank accession no. M14378. The ptx gene cluster from the strains B1834 (ptxP1), B1920 (ptxP1), B1917 (ptxP3), and B1831 (ptxP3) was sequenced completely. The sequences of the ptx gene clusters from strains B1834, B1920, B1917, and B1831 can be found under the following accession numbers, respectively: FN252334, FN252335, FN252336, and FN252333. The ptxP1-ptxP11 sequences have been assigned accession nos. FN252323, FN252322, FN252324, FN252325, FN252326, FN252327, FN252328, FN252329, FN252330, FN252331, and FN252332.

Pertussis Toxin and Pertactin Production
B. pertussis strains were grown on Bordet-Gengou agar plates supplemented with 15% sheep blood and incubated for 3 days at 35°C. Cells were harvested and suspended in 2 mL Verwey medium (14) per plate. Cells from 1 mL were collected by centrifugation and resuspended in Verwey medium to a concentration of 5 × 106 bacteria/mL. Subsequently, 100 μL of this suspension (5 × 105 CFU) was plated on Bordet-Gengou agar plates. After an incubation of 48 to 60 hours at 35°C, cells were harvested in 2.5 mL Verwey medium. The cell suspension was heat-inactivated for 30 min at 56°C and stored at 4°C. An ELISA was used to quantify Ptx and Prn. For Ptx, Maxisorp 96-well plates (Nunc International, Rochester, NY, USA) were coated with 100 μL of 0.04 mg/mL fetuin (Sigma-Aldrich, St. Louis, MO, USA) in 0.04 M carbonate buffer, pH 9.6, overnight at 4°C. For Prn, polystyrene 96-well plates (Immunolon II; Dynatech, Chantilly, VA, USA) were coated with 100 μL of a 2,000-fold dilution of polyclonal rabbit anti-Prn immunoglobulin (Ig)G (15) in 0.04 M carbonate buffer, pH 9.6, overnight at 20°C. Plates were blocked by incubation with 130 μL 1% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 1 hour at 37°C, after which plates were washed twice with PBS supplemented with 0.05% Tween. A 3-fold serial dilution of the heat-inactivated cell suspensions was made in 100 μL PBS supplemented with 0.1% Tween (PBST); 1 μg/mL of Prn and Ptx were used as reference. The suspensions were incubated for 1 hour at 37°C followed by 2 washings. The Prn monoclonal antibody (MAb) (PeM85) that was used binds to the linear epitope GGFGPGGFGP present in the repeat region 1 of all known Prn variants, except Prn13 (15). The Ptx MAb (3F10) binds to a conformational epitope in the PtxA subunit (16). All strains selected for the ELISA experiments produced Prn2 and PtxA1 (Table 1). The MAbs were diluted in PBST, added to the wells, and incubated for 1 hour at 37°C, followed by 2 washings. To detect bound MAbs, plates were incubated with horseradish peroxidase–conjugated polyclonal rabbit anti-mouse IgG (DakoCytmation, Glostrup, Denmark), diluted in PBST, for 1 hour at 37°C, and followed by 2 washings. The optical density at 450 nm was measured with a plate reader (PowerWave HT 340; Biotek, Winooski, VT, USA) and the amount of produced Ptx and Prn were calculated using the KC4 program (Biotek).The ratio of Ptx and Prn production by ptxP1 and ptxP3 strains was calculated as follows: Ptx (or Prn) production ptxP3 strains divided by Ptx (or Prn) production ptxP1 strains.

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