Bordetella holmesii DNA is not detected in nasopharyngeal swabs from Finnish and Dutch patients with suspected pertussis.

Bordetella holmesii is a Gram-negative bacterium first identified in 1995. It can cause pertussis-like symptoms in humans. B. holmesii contains insertion sequences IS481 and IS1001, two frequently used targets in the PCR diagnosis of Bordetella pertussis and Bordetella parapertussis infections. To investigate the prevalence of B. holmesii in Finnish and Dutch patients with pertussis-like symptoms and whether B. holmesii has caused any false-positive results in diagnostic PCRs, B. holmesii-specific real-time PCRs were developed. The Finnish methods were conventional IS481 PCR and B. holmesii-specific real-time PCR (LightCycler, Roche) targeting the B. holmesii recA gene. The Dutch methods were IS481 and IS1001 PCRs with conventional or real-time formats and B. holmesii-specific real-time PCR targeting the homologue of IS1001. Of 11,319 nasopharyngeal swabs, 2804 were collected from Finnish patients from 2000 to 2003, and 8515 from Dutch patients from 1992 to 2003. B. holmesii DNA was not found in the samples analysed. The results suggest that B. holmesii is not among the causative agents of pertussis-like symptoms in Finnish and Dutch patients and thus does not in practice confound IS481 and IS1001 PCRs.

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.

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.

Release of canine parvovirus from endocytic vesicles.

Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A(1), brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A(1), brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA(2) activity of the virus. These results suggest that parvoviral PLA(2) activity is essential for productive infection and presumably utilized in membrane penetration process of the virus, but CPV also needs other pH-dependent changes or factors to be released to the cytoplasm from endocytic vesicles.