Differences in the genomic content of Bordetella pertussis isolates before and after introduction of pertussis vaccines in four European countries.

Resurgence of pertussis has been observed in many countries with high vaccination coverage and clonal expansion of certain Bordetella pertussis strains has been associated with recent epidemics in Europe. It is known that vaccinations have selected strains which are different from those used for vaccine production. However, little is known about the differences in genomic content of strains circulating before the vaccination was introduced. In this study, we compared the genomes of 39 vaccine strains and old clinical isolates (isolated 1941-1984) collected from Finland (n = 5), Poland (n = 14), Serbia (n = 10) and the UK (n = 10). The analysis included genotyping, pulsed-field gel electrophoresis (PFGE) and comparative genomic hybridisation (CGH). Compared to the strain Tohama I, the European isolates analyzed have lost three major regions of difference (RD3, 5 and 29). However, difference in frequency of the absent RDs 3 (BP0910A-BP0934), 5 (BP1135-BP1141) or 29 (BP1225) was observed among isolates from the four countries. Of the isolates with absent RD5, half had also a duplicated region in the genome. All four RDs (RD22 (BB0535-BB0541), 23 (BB0916-BB0921), 24 (BB1140-BB1158) and 26 (BB4880-BB4888)) absent in Tohama I were present in majority of the tested isolates. Results obtained from PFGE analysis correlated well with those of CGH. Recently a novel pertussis toxin promoter allele (ptxP3) was described. Isolates with ptxP3 have replaced resident ptxP1 isolates in the countries where this was investigated. When the recent isolates, collected in 2000-2004, selected from the four countries were examined, the ptxP3 allele was found in all countries except Poland. Our result indicates that at least three clusters of B. pertussis circulated in Europe in pre- and early vaccine era and their genomes were distinct from that of the reference strain Tohama I. Although progressive gene loss occurs in B. pertussis population with time, difference in frequency of the lost genes were observed among isolates from the four countries. The observed differences in genomic content might be vaccine-driven.

Bordetella pertussis vaccine strains and circulating isolates in Serbia.

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.

Pertussis before and after the introduction of acellular pertussis vaccines in Finland.

In Finland, the whole-cell pertussis vaccine was replaced with acellular pertussis vaccine in the national immunisation schedule in 2005. Adolescent booster vaccinations were also included in the programme. The aim of this study was to evaluate the effects of these changes on the epidemiology and strain characteristics of Bordetella pertussis. From the national register, we first analysed all the laboratory diagnosed cases during the study years in 1999-2006. The major pool of the 6876 cases was among adolescents and adults. After the change of the programme and the introduction of the adolescent boosters, a general reduction of the incidence was noticed but this might be related to the natural epidemic cycles of pertussis. Secondly, a questionnaire was sent to the families of the 517 young children (<2 years of age) with registered, laboratory confirmed pertussis diagnosed during the study years. Of these, 319 (62%) participated the study. Forty-five percents of the cases in this cohort were younger than 3 months, the age of the first pertussis immunisation in schedule. Only 4% of the children in vaccination age were totally unimmunised. Thirdly, isolates of B. pertussis were analysed and found to differ from the used whole-cell pertussis vaccine strains by their prn, ptxA and PFGE profiles. However, no significant differences were found between the strains from patients with different immunisation status or age. Despite marked changes in the virulence genes and the genomes of the circulating B. pertussis strains have occurred, the epidemiological data from the national reporting system indicates that the whole-cell and acellular vaccines still protect against pertussis, but the results stress the importance of early primary immunisations and the need for booster immunisations.

Population dynamics of Bordetella pertussis in Finland and Sweden, neighbouring countries with different vaccination histories.

Pertussis is an infectious disease of the respiratory tract in humans caused by Bordetella pertussis. Despite extensive vaccinations, pertussis has remained endemic and re-emerged. In Finland, a whole-cell pertussis vaccine has been used since 1952 with high coverage. In Sweden, whole-cell vaccinations were introduced in 1953 but ceased in 1979, and pertussis vaccinations with acellular vaccines were introduced in 1996. Two epidemic peaks occurred in Sweden in 1999 and 2002 and in Finland in 1999 and 2003. We compared Finnish (N=193) and Swedish (N=455) B. pertussis isolates circulating in 1998-2003 together with vaccine strains used in these neighbouring countries with different vaccination histories. The isolates were analysed by serotyping, genotyping of pertussis toxin S1 subunit and pertactin, and pulsed-field gel electrophoresis. The results suggest that the sequential epidemics were caused by clonal expansion of a certain B. pertussis strain possibly transmitted from Sweden to Finland. The roles of antigenic variation in immunity-driven evolution of B. pertussis in both countries are discussed.

Bordetella pertussis, Finland and France.

We used pulsed-field gel electrophoresis analysis and genotyping to compare clinical isolates of Bordetella pertussis recovered since the early 1990s in Finland and France, 2 countries with similar histories of long-term mass vaccination with whole-cell pertussis vaccines. Isolates from both countries were similar genetically but varied temporally.

Strain variation among Bordetella pertussis isolates in finland, where the whole-cell pertussis vaccine has been used for 50 years.

Pertussis is an infectious disease of the respiratory tract caused by Bordetella pertussis. Despite the introduction of mass vaccination against pertussis in Finland in 1952, pertussis has remained an endemic disease with regular epidemics. To monitor changes in the Finnish B. pertussis population, 101 isolates selected from 1991 to 2003 and 21 isolates selected from 1953 to 1982 were studied together with two Finnish vaccine strains. The analyses included serotyping of fimbriae (Fim), genotyping of the pertussis toxin S1 subunit (ptxA) and pertactin (prn), and pulsed-field gel electrophoresis (PFGE) after digestion of B. pertussis genomic DNA with XbaI restriction enzyme. Strains isolated before 1977 were found to harbor the same ptxA as the strains used in the Finnish whole-cell pertussis vaccine, and strains isolated before 1982 harbored the same prn as the strains used in the Finnish whole-cell pertussis vaccine. All recent isolates, however, represented genotypes distinct from those of the two vaccine strains. A marked shift of predominant serotype from Fim serotype 2 (Fim2) to Fim3 has been observed since the late 1990s. Temporal changes were seen in the genome of B. pertussis by PFGE analysis. Three PFGE profiles (BpSR1, BpSR11, and BpSR147) were distinguished by their prevalence between 1991 and 2003. The yearly emergence of the three profiles was distributed periodically. Our study stresses the importance of the continuous monitoring of emerging strains of B. pertussis and the need to obtain a better understanding of the relationship of the evolution of B. pertussis in vaccinated populations.

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6.

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.