Analysis of Bordetella pertussis clinical isolates circulating in European countries during the period 1998-2012.

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998-2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.

Investigations into the emergence of pertactin-deficient Bordetella pertussis isolates in six European countries, 1996 to 2012.

Pathogen adaptation has been proposed to contribute to the resurgence of pertussis. A striking recent example is the emergence of isolates deficient in the vaccine component pertactin (Prn). This study explores the emergence of such Prn-deficient isolates in six European countries. During 2007 to 2009, 0/83 isolates from the Netherlands, 0/18 from the United Kingdom, 0/17 Finland, 0/23 Denmark, 4/99 Sweden and 5/20 from Norway of the isolates collected were Prn-deficient. In the Netherlands and Sweden, respectively 4/146 and 1/8 were observed in a later period (2010­12). The Prn-deficient isolates were genetically diverse and different mutations were found to inactivate the prn gene. These are indications that Prn-deficiency is subject to positive selective pressure. We hypothesise that the switch from whole cell to acellular pertussis vaccines has affected the balance between 'costs and benefits' of Prn production by Bordetella pertussis to the extent that isolates that do not produce Prn are able to expand. The absence of Prn-deficient isolates in some countries may point to ways to prevent or delay the spread of Prn-deficient strains. In order to substantiate this hypothesis, trends in the European B. pertussis population should be monitored continuously.

Pertussis disease burden in the household: how to protect young infants.

BACKGROUND: We conducted a population-based, nation-wide, prospective study to identify who introduced pertussis into the household of infants aged 6 months admitted to the hospital for pertussis in the Netherlands. METHODS: During the period 2006-2008, a total of 560 household contacts of 164 hospitalized infants were tested by polymerase chain reaction, culture, and serological examination to establish Bordetella pertussis infection. Clinical symptoms and vaccination history were obtained by a questionnaire submitted during sample collection and 4-6 weeks afterwards. RESULTS: Overall, 299 household contacts (53%) had laboratory-confired pertussis; 159 (53%) had symptoms compatible with typical pertussis infection, and 42 (14%) had no symptoms. Among children vaccinated with a whole-cell vaccine, 17 (46%) of 37 had typical pertussis 1-3 years after completion of the primary series, compared with 9 (29%) of 31 children who had been completely vaccinated with an acellular vaccine. For 96 households (60%), the most likely source of infection of the infant was established, being a sibling (41%), mother (38%), or father (17%). CONCLUSIONS: If immunity to pertussis in parents is maintained or boosted, 35%-55% of infant cases could be prevented. Furthermore, we found that, 1-3 years after vaccination with whole-cell or acellular vaccine, a significant percentage of children are again susceptible for typical pertussis. In the long term, pertussis vaccines and vaccination strategies should be improved to provide longer protection and prevent transmission.

Experimental studies on the infectivity of non-culturable forms of Campylobacter spp. in chicks and mice.

The significance of non-culturable forms of Campylobacter spp., especially with regard to the epidemiology of this organism in poultry flocks, was explored. Two different experiments were conducted to produce non-culturable Campylobacter spp. and test their ability to colonize the animal gut. In the first experiment a mixture of 28 different strains of Campylobacter spp. from various sources was inoculated in both sterilized surface water and potassium phosphate buffer and stored at 4 degrees C. After Campylobacter spp. were no longer detectable by culture in the microcosms, the mixtures of non-culturable cells were used to challenge both chicks and mice. Recovery of non-culturable Campylobacter spp. from the animals was not successful at 4 weeks after administration. In the second experiment the survival of six individual strains of Campylobacter spp. in sterilized surface water at 4 degrees C was studied and the resulting non-culturable cells were used to challenge chicks. None of the campylobacter strains could be recovered from the chicks at 2 weeks after administration. We conclude that occurrence of non-culturable forms of Campylobacter spp. capable of colonizing chicks is not a common phenomenon and that non-culturable forms of Campylobacter spp. are likely to be insignificant for importantly to the epidemiology of the organism in Dutch broiler flocks.

Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese.

The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth 18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10(3) cfu/0.5g could be visualized whereas in others the presence of 10(8) cfu/0.5 g did not yield a detectable quantity of amplified product.

The use of DNA probes for confirming enterotoxin production by Staphylococcus aureus and micrococci.

DNA-DNA colony hybridization was employed to evaluate the results obtained by different immunological methods for detection of staphylococcal enterotoxin. Staphylococcus aureus strains tested for staphylococcal enterotoxin production by immuno-assays and micrococci not previously tested for staphylococcal enterotoxin production were examined for presence of the genes encoding for staphylococcal enterotoxin A, B, C and E by using three corresponding DNA probes. The staphylococcal enterotoxin A probe also detected staphylococcal enterotoxin E gene because of 100% homology. The optimal sensitivity plate method showed the best accordance between the immuno-assay and the hybridization reactions. The enzyme-linked immunosorbent assay detected 12.5 to 17% staphylococcal enterotoxin producers without hybridization reactions. The microslide gel double diffusion test and the reversed passive latex agglutination test showed rather poor accordance with the hybridization reactions. All 17 strains of different micrococci investigated were negative in hybridization with all three DNA probes.

Detection of Mold in Food by Enzyme-Linked Immunosorbent Assay.

Evaluation of the enzyme-linked immunosorbent assay (ELISA) for detecting a mold-specific, heat-stable and water-soluble antigen demonstrated the potential of the method for detecting molds in food products. The mold antigen, as produced by Penicillium spp. and Aspergillus spp., was present in all food samples containing aflatoxin B1. The amount of mold antigen present in the test samples was related in each case to the aflatoxin B1 content. Experiments done with samples artificially inoculated with mycotoxin-producing molds revealed that mold contamination could be detected by ELISA at a very early stage. The minimum detectable amount of mold mycelium for three different species of Penicillium was 38 ng/g of sample.

The Salt Crystal Liquefaction Test - A Simple Method for Testing the Water Activity of Foods.

A simple method for testing the water activity (aw) of foods has been developed. The test can be used to check the aw of products which must comply with required aw standards. The test is based on the property of salt crystals to attract water vapor and to liquefy when they are placed in a jar containing a product with an aw above the specific aw of the salt. By using different salts with appropriate specific aw values, the aw of products at various aw levels can be examined. The specific aw values of salts which can be used for the test are: 0.68, 0.76, 0.79, 0.81, 0.86, 0.87, 0.91, 0.94 and 0.98. The sensitivity of the test is less than 0.02 aw with a reading time of 3-24 h, depending on the type of salt, type of product and temperature. The test using salt crystals with a high specific aw is sensitive to a change in temperature and must be performed in a thermostat-controlled cabinet. The test using CuCl2•2H2O crystals (0.68 aw) was evaluated under extreme environmental conditions and it appeared to be most promising for use in field conditions.