Amplified fragment length polymorphism (AFLP) versus randomly amplified polymorphic DNA (RAPD) as new tools for inter- and intra-species differentiation within Bordetella.

Automated amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) techniques with fluorescently labelled primers were used to track differences among isolates of the eight known species of the Bordetella genus. Eighty-one representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247, and AFLP involving EcoRI/MseI or newly designed SpeI/ApaI restriction/ligation/amplification procedures. By comparing AFLP and RAPD data, it was concluded that the discriminatory power of AFLP is higher in comparison with RAPD for both intra- and inter-species differentiation of isolates of the Bordetella genus. The most precise level of inter-species discrimination and the highest level of intra-species discrimination of the Bordetella isolates of the eight species were observed in the AFLP EcoRI/MseI and SpeI/ApaI sets, respectively. Both techniques might provide alternative tools for the identification of Bordetella at the genomic species and strain levels, and thus may be valuable in human and veterinary diagnostics as well as in epidemiology. By applying the AFLP technique presented in this article, more precise data on the emergence of newly acquired and/or on expanded clones and transmission routes of isolates of the Bordetella genus in the human and animal environments might be obtained.

Potency of pertussis component in the DTP vaccine--an overview of three decade study in Poland.

In Poland, similar to many highly immunized Western countries, a recent increase in cases of pertussis has been observed. This study aims to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine due to the changes having occurred in production and potency testing procedures. We compared the potency of the pertussis component of DTP vaccine lots produced and evaluated in similar periods and with similar production and testing procedures. Records of Kendrick test results performed over a 30-year period were available for analysis. This study confirms the role of different manufacturers, changes in vaccine strain compositions, in-house reference preparations used as reference vaccines in the Kendrick tests, and in mice of single strain sources in the potency values obtained. In addition, the comparisons performed revealed a downtrend in potency levels since 1992. Potency decrease in vaccine lots produced during 1992-1997 has been positively correlated to the lowering of the number of IOU/dose. Strain compositions of the DTP vaccine pertussis component and in-house references have been found to be associated with the fluctuation in potency estimations, and confirmed their crucial role in ensuring vaccine efficacy. Our study reveals that relative efficacy of the DTP vaccine produced in 1992-1997 might be lower than that of vaccines produced in other periods. This might in turn explain the increase in pertussis cases among children aged 5-15 years which is presently being observed in Poland.

Sequence variation in pertussis S1 subunit toxin and pertussis genes in Bordetella pertussis strains used for the whole-cell pertussis vaccine produced in Poland since 1960: efficiency of the DTwP vaccine-induced immunity against currently circulating B. pertussis isolates.

The present study indicates that the appearance of the B. pertussis harbouring prn2 gene allele variant (not found among clinical isolates before 1990s) may have been induced by long-term vaccination in Poland with DTP-composed vaccine strains presenting exclusively prn1. However, ptxS1A allele of pertussis toxin subunit S1 encoding gene, predominant in the currently isolated B. pertussis strains, has been found in vaccine strains used for whole-cell pertussis component (wP) production of DTP vaccine in 1960-1978. This outrules the possibility that the appearance of ptxSIA allele might be related to vaccine pressure driven by non-ptxS1A vaccine strains used for long-term immunization with wP. Intranasal challenge animal model testing the efficiency of the clearance of B. pertussis strains harbouring different ptxS1/prn allele gene combinations revealed that currently produced DTwP vaccine may not contain adequate B. pertussis vaccine strains, since isolates with gene variants different from those observed in vaccine strains were eliminated from the lungs of the immunized animals with lower efficiency.

Pertussis in Poland.

BACKGROUND: Since 1997, an unexpected 2-5-fold increase in the incidence of pertussis has been reported in Poland in comparison with the previous 10 years, although the introduction of the diphtheria-tetanus-pertussis (DTP) vaccination in 1960 reduced the incidence of pertussis approximately 100-fold in the 1980s. The aim of the study was to analyse all available data on pertussis in Poland to identify the risks associated with its re-emergence. METHODS: Available data on notification, incidence, mortality, hospitalization, geographical distribution, incidence according to age, and diagnosis of pertussis were collected from national surveillance monographs and statistically evaluated. RESULTS: Analyses performed in the study found two periods of rising and falling trends: in the incidence before and after 1989, respectively. Moreover, after 1989, the age-specific incidence among children aged 0-4 years decreased, and among 5-9, 10-14, and 15-19 year olds increased in comparison to the previous decade. The incidence rate of pertussis among infants was similar in both decades analysed. Clustering of pertussis incidence increase in provinces along a line from North East to South West was observed. CONCLUSIONS: As vaccination coverage did not decrease and diagnostics have not been improved since the 1980s, it is possible that waning immunity and the appearance of Bordetella pertussis vaccine escape mutants are involved in the changing pertussis epidemiological parameters. Further monitoring studies, together with improving diagnostics, might allow more precise epidemiological data to be obtained. An additional booster dose of acellular pertussis vaccine at age 6 years has been included in the current vaccination schedule.

[Increase in the incidence of pertussis and quality of whole-cell pertussis component of the DTP vaccine produced in Poland. Part I. Potency of DTP vaccine]. / Wzrost zachorowan na krztusiec a wyniki analizy jakosci komponentu krztuscowego polskiej szczepionki DTP. Czesc I: Moc szczepionki DTP.

This study aimed to identify and to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine lots produced within 1972-2001 due to the changes having occurred in production and potency testing procedures. The study confirms that higher potency values were obtained for vaccine lots produced since 80-ties, e.g. after changes of: references lots (1975), vaccine strains (1978) and source of animals used in Kendrick tests (1979). Additionally, the comparisons performed revealed a down trend in potency levels within 1992-1999 correlating to the lowering of the number of IOU/dose.

[Increase in the incidence of pertussis and quality of whole-cell pertussis component of the DTP vaccine produced in Poland. Part II. Consistency of control and production]. / Wzrost zachorowan na krztusiec a wyniki analizy jakosci komponentu krztuscowego polskiej szczepionki DTP. Czesc II: Jednorodnosc badan i produkcji.

This study aimed to identify and to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine lots produced within 1972-2001 due to the changes having occurred in production and potency testing procedures. Obtained results showed high consistency of the production and control tests confirming relevant quality of country-produced DTP vaccine.

Comparison of usefulness of randomly amplified polymorphic DNA and amplified-fragment length polymorphism techniques in epidemiological studies on nasopharyngeal carriage of non-typable Haemophilus influenzae.

Randomly amplified polymorphic DNA (RAPD) and automated amplified-fragment length polymorphism (AFLP) techniques with fluorescently labelled primers were used to type non-serotypable Haemophilus influenzae (NTHI) isolates. Eighty-seven isolates from healthy children attending day-care centres or living at orphanages in southern Poland were investigated. Through comparison of the AFLP data with RAPD analysis, it has been concluded that the discriminatory power of AFLP for NTHI typing is higher than RAPD. Generally, the NTHI isolates analysed were highly heterogeneous, as detected with a HindIII/TaqI AFLP genotyping scheme on intra/inter similarity levels of 94 and 96 % using Pearson's correlation coefficient. The range of similarity values found for isolates from children permanently residing at a particular day-care centre was much wider than that for isolates from orphanages. AFLP can efficiently access NTHI strain diversity and can monitor their turn-over for comparative typing in local and inter-local epidemiological investigations.

[Genetic variability of Bordetella pertussis strains. Part I. Current knowledge]. / Zmiennosc genetyczna szczepów Bordetella pertussis. Czesc I. Aktualny stan wiedzy.

Variability of B. pertussis genome concerning S1 subunit of pertussis toxin (ptxS1) and pertactin (prn) genes has recently been recognised. Studies clearly showed differences in the DNA patterns and ptxS1 and prn alleles present in B. pertussis strains used for production of pertussis component in the DTP vaccine, in comparison to strains presently isolated from clinical materials. This trend has been observed in highly immunized populations with a long history of pertussis vaccination (The Netherlands, Finland, Poland, USA). Different ptxS1 and prn allele distribution for different countries needs further careful monitoring.

[Genetic variability of Bordetella pertussis strains. Part II. Perspectives]. / Zmiennosc genetyczna szczepów Bordetella pertussis. Czesc II. Perspektywy na przyszlosc.

In some highly immunized countries with long history of pertussis vaccination (The Netherlands, Finland, Poland, USA, France, Great Britain) differences in the DNA patterns and ptxS1 and prn alleles present in B. pertussis strains used for production of pertussis component in the DTP vaccine, in comparison to strains presently isolated from clinical materials have been found. Studies on efficacy of whole-cell and acellular pertussis vaccines in elimination of B. pertussis strains harbouring different gene variants in a particular country might indicate a need for a change of B. pertussis strains used for production of the vaccine against pertussis.

[Prevalence of antibodies and vaccination against measles, mumps and rubella in Polish population]. / Stan uodpornienia populacji polskiej przeciw odrze, swince i rózyczce, a szczepienia ochronne.

The data according number of cases and morbidity rates of measles, mumps and rubella as well as antibody prevalence against diseases above mentioned in Polish population were shown. The impact of mass vaccination on decreasing of measles incidence and number of reported rubella congenital syndrome was observed. The possibility of elimination these diseases in the near future was discussed.

[Phenotype and genotype of Clostridium sp. strains producing botulism neurotoxin]. / Fenotyp i genotyp szczepów z rodzaju Clostridium wytwarzajacych neurotoksyny botulinowe.

Neurotoxins produced by strains of Clostridium sp. are belonging to the most toxic biological substances. In the study phenotypes and genotypes of C. botulinum strains in animal studies in vivo and on the DNA level were evaluated, respectively. Additionally, the presence of genes encoding BoNT toxins of A, B, and E types among strains of Clostridium sp. were identified. In case of C. botulinum DNA was isolated from vegetative bacterial cells and from spores. Two different genes encoding two different neurotoxins harboured by three strains of Ae biotype/ae genotype, and by two strains of B biotype/be genotype were detected. Additionally, above E type C. botulinum strains, the presence of gene encoding E type neurotoxin, was found in genome of two C. baratii, two C. butyricum, and C. bifidobacterium, and C. oedematicum strains. C. bifidobacterium and C. oedematicum strains positive for presence of gene encoding E type neurotoxin, were found negative for E neurotoxin production in vivo in TN test. The study indicates that genes encoding BoNT/E neurotoxins are very common among Clostridium species. Phenotype and genotype analysis indicated co-presence of B phenotype together with be genotype and A phenotype together with ae genotype among C. botulinum strains.

[Immunity of children aged 6-8 against pertussis, tetanus and diphtheria]. / Stan uodpornienia dzieci w wieku 6-8 lat przeciw krztuscowi, tezcowi i blonicy.

Vaccination against pertussis has been performed since 1960. Whole cell pertussis vaccine produced by Plant of Sera and Vaccines Biomed S.A. in Kraków, has been used. After vaccination has been introduced, epidemiological situation of pertussis in Poland improved, but in 90's the decrease of immunity was observed. The aim of this work was to determine pertussis immunity of children after several years from the last dose of pertussis vaccine. For comparison purposes immunity against tetanus and diphtheria was tested. Protective antibody levels were detected in 70%, 58%, and 45% children aged 6, 7, and 8, respectively. It shows that decrease of immunity may cause increasing number of pertussis in children above 5. Taking into consideration our results, it seems necessary to introduce additional dose of pertussis vaccine among children aged 5 years. The level of tetanus and diphtheria antibodies was high in all tested groups.