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Sample records for bordetella

  1. Bordetella

    Science.gov (United States)

    The genus Bordetella includes 8 formally recognized species, of which Bordetella parapertussis, Bordetella bronchiseptica, Bordetella avium, and Bordetella hinzii are of veterinary interest. Bordetella pertussis, the type species, is an obligate human pathogen and the causative agent of whooping co...

  2. Bordetella pertussis.

    Science.gov (United States)

    Nieves, Delma J; Heininger, Ulrich

    2016-06-01

    Pertussis is a highly infectious vaccine-preventable cough illness that continues to be a significant source of morbidity and mortality around the world. The majority of human illness is caused by Bordetella pertussis, and some is caused by Bordetella parapertussis. Bordetella is a Gram-negative, pleomorphic, aerobic coccobacillus. In the past several years, even countries with high immunization rates in early childhood have experienced rises in pertussis cases. Reasons for the resurgence of reported pertussis may include molecular changes in the organism and increased awareness and diagnostic capabilities, as well as lessened vaccine efficacy and waning immunity. The most morbidity and mortality with pertussis infection is seen in infants too young to benefit from immunization. Severe infection requiring hospitalization, including in an intensive care setting, is mostly seen in those under 3 months of age. As a result, research and public health actions have been aimed at better understanding and reducing the spread of Bordetella pertussis. Studies comparing the cost benefit of cocooning strategies versus immunization of pregnant women have been favorable towards immunizing pregnant women. This strategy is expected to prevent a larger number of pertussis cases, hospitalizations, and deaths in infants <1 year old while also being cost-effective. Studies have demonstrated that the source of infection in infants usually is a family member. Efforts to immunize children and adults, in particular pregnant women, need to remain strong.

  3. Bordetella pertussis transmission.

    Science.gov (United States)

    Trainor, Elizabeth A; Nicholson, Tracy L; Merkel, Tod J

    2015-11-01

    Bordetella pertussis and B. bronchiseptica are Gram-negative bacterial respiratory pathogens. Bordetella pertussis is the causative agent of whooping cough and is considered a human-adapted variant of B. bronchiseptica. Bordetella pertussis and B. bronchiseptica share mechanisms of pathogenesis and are genetically closely related. However, despite the close genetic relatedness, these Bordetella species differ in several classic fundamental aspects of bacterial pathogens such as host range, pathologies and persistence. The development of the baboon model for the study of B. pertussis transmission, along with the development of the swine and mouse model for the study of B. bronchiseptica, has enabled the investigation of different aspects of transmission including the route, attack rate, role of bacterial and host factors, and the impact of vaccination on transmission. This review will focus on B. pertussis transmission and how animal models of B. pertussis transmission and transmission models using the closely related B. bronchiseptica have increased our understanding of B. pertussis transmission.

  4. Evaluation of the Specificity of BP3385 for Bordetella pertussis

    Science.gov (United States)

    BP3385 has been proposed as a diagnostic PCR target for discriminating between Bordetella pertussis and other Bordetella species that also infect humans. Our results demonstrate this gene is also present in some strains of Bordetella hinzii and Bordetella bronchiseptica....

  5. 21 CFR 866.3065 - Bordetella spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... identification aids in the diagnosis of diseases caused by bacteria belonging to the genus Bordetella and provides epidemiological information on these diseases. Bordetella spp. cause whooping cough...

  6. Environmental Origin of the Genus Bordetella

    Science.gov (United States)

    Hamidou Soumana, Illiassou; Linz, Bodo; Harvill, Eric T.

    2017-01-01

    Members of the genus Bordetella include human and animal pathogens that cause a variety of respiratory infections, including whooping cough in humans. Despite the long known ability to switch between a within-animal and an extra-host lifestyle under laboratory growth conditions, no extra-host niches of pathogenic Bordetella species have been defined. To better understand the distribution of Bordetella species in the environment, we probed the NCBI nucleotide database with the 16S ribosomal RNA (16S rRNA) gene sequences from pathogenic Bordetella species. Bacteria of the genus Bordetella were frequently found in soil, water, sediment, and plants. Phylogenetic analyses of their 16S rRNA gene sequences showed that Bordetella recovered from environmental samples are evolutionarily ancestral to animal-associated species. Sequences from environmental samples had a significantly higher genetic diversity, were located closer to the root of the phylogenetic tree and were present in all 10 identified sequence clades, while only four sequence clades possessed animal-associated species. The pathogenic bordetellae appear to have evolved from ancestors in soil and/or water. We show that, despite being animal-adapted pathogens, Bordetella bronchiseptica, and Bordetella hinzii have preserved the ability to grow and proliferate in soil. Our data implicate soil as a probable environmental origin of Bordetella species, including the animal-pathogenic lineages. Soil may further constitute an environmental niche, allowing for persistence and dissemination of the bacterial pathogens. Spread of pathogenic bordetellae from an environmental reservoir such as soil may potentially explain their wide distribution as well as frequent disease outbreaks that start without an obvious infectious source. PMID:28174558

  7. Evolution of French Bordetella pertussis and Bordetella parapertussis isolates: increase of Bordetellae not expressing pertactin.

    Science.gov (United States)

    Hegerle, N; Paris, A-S; Brun, D; Dore, G; Njamkepo, E; Guillot, S; Guiso, N

    2012-09-01

    Bordetella pertussis and Bordetella parapertussis are closely related bacterial agents of whooping cough. Whole-cell pertussis (wP) vaccine was introduced in France in 1959. Acellular pertussis (aP) vaccine was introduced in 1998 as an adolescent booster and was rapidly generalized to the whole population, changing herd immunity by specifically targeting the virulence of the bacteria. We performed a temporal analysis of all French B. pertussis and B. parapertussis isolates collected since 2000 under aP vaccine pressure, using pulsed-field gel electrophoresis (PFGE), genotyping and detection of expression of virulence factors. Particular isolates were selected according to their different phenotype and PFGE type and their characteristics were analysed using the murine model of respiratory infection and in vitro cell cytotoxic assay. Since the introduction of the aP vaccines there has been a steady increase in the number of B. pertussis and B. parapertussis isolates collected that are lacking expression of pertactin. These isolates seem to be as virulent as those expressing all virulence factors according to animal and cellular models of infection. Whereas wP vaccine-induced immunity led to a monomorphic population of B. pertussis, aP vaccine-induced immunity enabled the number of circulating B. pertussis and B. parapertussis isolates not expressing virulence factors to increase, sustaining our previous hypothesis.

  8. Growth Phase dependent gene regulation in Bordetella bronchiseptica

    Science.gov (United States)

    Bordetellae are Gram negative bacterial respiratory pathogens. Bordetella pertussis, the causative agent of whooping cough, is a human-restricted variant of Bordetella bronchiseptica, which infects a broad range of mammals causing chronic and often asymptomatic infections. Growth phase dependent gen...

  9. Whooping cough in Pakistan: Bordetella pertussis vs Bordetella parapertussis in 2005-2009.

    Science.gov (United States)

    Bokhari, Habib; Said, Fahad; Syed, Muhammad A; Mughal, Amjad; Kazi, Yasmeen F; Heuvelman, Kees; Mooi, Frits R

    2011-10-01

    Pertussis, or whooping cough, is an acute respiratory disease mainly affecting infants and children and is caused by Bordetella pertussis and Bordetella parapertussis. The aim of this study was to investigate the share of Bordetella species from potential whooping cough cases during 2005-2009. Eight hundred and two samples from suspected pertussis cases were collected, mainly from 2 provinces of Pakistan. Bacterial culture, identification, DNA extraction and routinely used polymerase chain reaction (PCR) methods using IS1001, IS1002 and IS481 were used to identify the Bordetella species. The results were unexpected, because all of the isolates collected from the different cities were identified as B. parapertussis (7.4%); B. pertussis was not isolated from any sample. However, PCR results indicated the presence of a small percentage (0.6%) of B. pertussis among the total cases studied. This study suggests that vaccines to protect against both B. pertussis and B. parapertussis should be considered.

  10. Structure of Bordetella pertussis peptidoglycan

    Energy Technology Data Exchange (ETDEWEB)

    Folkening, W.J.; Nogami, W.; Martin, S.A.; Rosenthal, R.S.

    1987-09-01

    Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing (/sup 3/H)diaminopimelic acid and treated by a hot (96/sup 0/C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60/sup 6/). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and <2% protein. Radiochemical analyses indicated that /sup 3/H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelic acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived pepidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of >95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl-alanine.

  11. Bordetella pertussis diagnosed by polymerase chain reaction

    DEFF Research Database (Denmark)

    Birkebaek, N H; Heron, I; Skjødt, K

    1994-01-01

    The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity of...

  12. Genetic Variation of Bordetella pertussis in Austria

    NARCIS (Netherlands)

    Wagner, B.; Melzer, H.; Freymuller, G.; Stumvoll, S.; Rendi-Wagner, P.; Paulke-Korinek, M.; Repa, A.; Mooi, F.R.; Kollaritsch, H.; Mittermayer, H.; Kessler, H.H.; Stanek, G.; Steinborn, R.; Duchene, M.; Wiedermann, U.

    2015-01-01

    In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunit

  13. Diagnosis of whooping cough in Switzerland: differentiating Bordetella pertussis from Bordetella holmesii by polymerase chain reaction.

    Science.gov (United States)

    Pittet, Laure F; Emonet, Stéphane; François, Patrice; Bonetti, Eve-Julie; Schrenzel, Jacques; Hug, Melanie; Altwegg, Martin; Siegrist, Claire-Anne; Posfay-Barbe, Klara M

    2014-01-01

    Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR). In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old), formerly diagnosed as Bordetella pertussis (IS481+). No B. holmesii (IS481+, IS1001-, hIS1001+) was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested.

  14. Diagnosis of whooping cough in Switzerland: differentiating Bordetella pertussis from Bordetella holmesii by polymerase chain reaction.

    Directory of Open Access Journals (Sweden)

    Laure F Pittet

    Full Text Available Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR. In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old, formerly diagnosed as Bordetella pertussis (IS481+. No B. holmesii (IS481+, IS1001-, hIS1001+ was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested.

  15. Complete Genome Sequences of Four Different Bordetella sp. Isolates Causing Human Respiratory Infections

    Science.gov (United States)

    Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Bowden, Katherine E.; Cassiday, Pamela K.; Davis, Jamie K.; Johnson, Taccara; Juieng, Phalasy; Miner, Christine E.; Rowe, Lori; Sheth, Mili; Tondella, M. Lucia; Williams, Margaret M.

    2016-01-01

    Species of the genus Bordetella associate with various animal hosts, frequently causing respiratory disease. Bordetella pertussis is the primary agent of whooping cough and other Bordetella species can cause similar cough illness. Here, we report four complete genome sequences from isolates of different Bordetella species recovered from human respiratory infections.

  16. [Counterimmunoelectrophoresis in the diagnosis of Bordetella pertussis].

    Science.gov (United States)

    Grinstein, S; López, E L; Muchinik, G; Duhart, E; Bonesana, N

    1979-01-01

    The serum of 50 patients between 25 days to 15 years old who were admitted with the diagnosis of pertussis syndrome were investigated for precipitating antibodies by means of discontinuous counterimmunolectrophoresis (CIED). The presence of antibodies for Bordetella pertussis antigen was shown in 28 cases. All samples were taken within the first 24/48 hours of admission and at convalescence. The technique is easy to carry out, quick and of low cost. This test offers an adequate and fast means to differentiate a Bordetella pertussis syndrome form others produced by different viral etiologies. Besides, this serologic technique shows earlier results and of lower cost than classic techniques such as agglutination and complement fixation which are more difficult to apply.

  17. Bordetella bronchiseptica and fatal pneumonia of dogs and cats

    Science.gov (United States)

    Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less well-studied. The objectives of this study were to identify the frequency of Bordetella bronchiseptica infection in fatal cases of bronchopneumonia in dogs and cats and to compare the diag...

  18. Genomic island excisions in Bordetella petrii

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    Levillain Erwan

    2009-07-01

    Full Text Available Abstract Background Among the members of the genus Bordetella B. petrii is unique, since it is the only species isolated from the environment, while the pathogenic Bordetellae are obligately associated with host organisms. Another feature distinguishing B. petrii from the other sequenced Bordetellae is the presence of a large number of mobile genetic elements including several large genomic regions with typical characteristics of genomic islands collectively known as integrative and conjugative elements (ICEs. These elements mainly encode accessory metabolic factors enabling this bacterium to grow on a large repertoire of aromatic compounds. Results During in vitro culture of Bordetella petrii colony variants appear frequently. We show that this variability can be attributed to the presence of a large number of metastable mobile genetic elements on its chromosome. In fact, the genome sequence of B. petrii revealed the presence of at least seven large genomic islands mostly encoding accessory metabolic functions involved in the degradation of aromatic compounds and detoxification of heavy metals. Four of these islands (termed GI1 to GI3 and GI6 are highly related to ICEclc of Pseudomonas knackmussii sp. strain B13. Here we present first data about the molecular characterization of these islands. We defined the exact borders of each island and we show that during standard culture of the bacteria these islands get excised from the chromosome. For all but one of these islands (GI5 we could detect circular intermediates. For the clc-like elements GI1 to GI3 of B. petrii we provide evidence that tandem insertion of these islands which all encode highly related integrases and attachment sites may also lead to incorporation of genomic DNA which originally was not part of the island and to the formation of huge composite islands. By integration of a tetracycline resistance cassette into GI3 we found this island to be rather unstable and to be lost from

  19. Bordetella pertussis: why is it still circulating?

    Science.gov (United States)

    Guiso, Nicole

    2014-01-01

    Bordetella pertussis is the causal agent of whooping cough, a highly contagious respiratory disease that is life-threatening in infants under the age of three months and may also be very severe in pregnant women and seniors. This disease can be prevented by vaccination but it remains a public health problem in many developed and developing countries.(1) So, why is B. pertussis still circulating? We need to consider several aspects of this vaccine-preventable disease when answering this question: (i) the history of the disease and the historical context in which the vaccine was developed; (ii) the type of vaccine used; (iii) the vaccination strategy and coverage; (iv) the disease surveillance after the introduction of generalized vaccination and (v) the surveillance for the causal agent of the disease.

  20. Prevalence of Bordetella pertussis and Bordetella parapertussis in Samples Submitted for RSV Screening

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    Walsh, Paul

    2008-08-01

    Full Text Available BACKGROUND: The clinical presentation of Bordetella pertussis can overlap with that of respiratory syncytial virus (RSV; however, management differs.HYPOTHESIS: First, the prevalence of B. pertussis is less than 2% among patients screened for RSV, and second the prevalence of B. parapertussis is also less than 2% among these patients.METHODS: Nasal washings submitted to a clinical laboratory for RSV screening were tested for B. pertussis and B. parapertussis, using species-specific real-time polymerase chain reaction (PCR assays. These were optimized to target conserved regions within a complement gene and the CarB gene, respectively. A Bordetella spp. genus-specific real-time PCR assay was designed to detect the Bhur gene of B. pertussis, B. parapertussis, and B. bronchiseptica. RSV A and B subtypes were tested by reverse transcription-PCR.RESULTS: Four hundred and eighty-nine clinical samples were tested. There was insufficient material to complete testing for one B. pertussis, 10 RSV subtype A, and four RSV subtype B assays. Bordetella pertussis was detected in 3/488 (0.6% (95% CI 0.1% to 1.8%, while B. parapertussis was detected in 5/489 (1.0% (95% CI 0.3% to 2.4%. Dual infection of B. pertussis with RSV and of B. parapertussis with RSV occurred in two and in three cases respectively. RSV was detected by PCR in 127 (26.5%.CONCLUSION: The prevalence of B. pertussis in nasal washings submitted for RSV screening was less than 2%. The prevalence of parapertussis may be higher than 2%. RSV with B. pertussis and RSV with B. parapertussis coinfection do occur.

  1. Mouse lung adhesion assay for Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Burns, K.A.; Freer, J.H. (Department of Microbiology, Alexander Stone Building, Bearsden, Glasgow, Scotland)

    1982-03-01

    The ability of Bordetella pertussis to adhere to cell surfaces has been demonstrated by adhesion to tissue culture cells and adhesion to chicken, hamster or rabbit trachea in organ culture. In this report a mouse lung assay for adhesion is described and the results obtained using two virulent strains of B. pertussis and their avirulent counterparts. These were a C modulation of one of the original virulent strains and a phase IV variant of the other virulent strain. Organisms were radiolabelled by adding 1 ..mu..Ci (37 K Bq) of (/sup 14/C)glutamic acid per 10 ml of culture medium before inoculation and incubation for 5 days. The lungs were washed by perfusion in situ with at least two volumes (1 ml) of sterile 1% (w/v) casamino acids. The percentage of the inoculated organisms retained in the lungs was determined, after removal of the lungs, by one of the following two methods: viable count or radioactive count. Results for both methods were expressed as the percentage of the inoculum retained in the lungs plus or minus one standard deviation.

  2. Bordetella pertussis fimbriae (Fim): relevance for vaccines.

    Science.gov (United States)

    Gorringe, Andrew R; Vaughan, Thomas E

    2014-10-01

    Bordetella pertussis produces two serologically distinct fimbriae, Fim2 and Fim3. Expression of these antigens is governed by the BvgA/S system and by the length of a poly(C) tract in the promoter of each gene. Fim2 and Fim3 are important antigens for whole cell pertussis vaccines as clinical trials have shown an association of anti-fimbriae antibody-mediated agglutination and protection. The current five component acellular pertussis vaccine contains co-purified Fim2/3 and provided good efficacy in clinical trials with the anti-Fim antibody response correlating with protection when pre and post exposure antibody levels were analysed. The predominant serotype of B. pertussis isolates has changed over time in most countries but it is not understood whether this is vaccine-driven or whether serotype is linked to the prevailing predominant genotype. Recent studies have shown that both Fim2 and Fim3 are expressed during infection and that Fim2 is more immunogenic than Fim3 in the acellular vaccine.

  3. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

    Science.gov (United States)

    Williams, Margaret M; Sen, Kathryn; Weigand, Michael R; Skoff, Tami H; Cunningham, Victoria A; Halse, Tanya A; Tondella, M Lucia

    2016-02-01

    A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin-deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.

  4. Induction and maintenance of Bordetella pertussis specific immune responses

    NARCIS (Netherlands)

    Stenger, R.M.

    2010-01-01

    Pertussis, also referred to as whooping cough, is a serious respiratory disease mainly caused by the gram-negative bacterium Bordetella pertussis. The disease is most severe in neonates and children under the age of 1. Before childhood vaccination was introduced in the 1950s, pertussis was an import

  5. Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a Newly Described RT-PCR Targeting the Pertactin Gene

    Science.gov (United States)

    Recently a real-time PCR (RT-PCT) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here we report that the B. pertussis pertactin gene sequence for the region encompassing the RT-PCR probe and primers is nearly identical to that of many B....

  6. Genetic Variation of Bordetella pertussis in Austria.

    Directory of Open Access Journals (Sweden)

    Birgit Wagner

    Full Text Available In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32, Linz (n = 63 and Graz (n = 15 by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis (n = 77, by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin gene (n = 110, and by amplification refractory mutation system quantitative PCR (ARMS-qPCR (n = 110 to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB, a fimbrial adhesin (fimD, tracheal colonisation factor (tcfA, and the virulence sensor protein (bvgS. Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517. The major part of the samples (93% displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

  7. Genetic Variation of Bordetella pertussis in Austria.

    Science.gov (United States)

    Wagner, Birgit; Melzer, Helen; Freymüller, Georg; Stumvoll, Sabine; Rendi-Wagner, Pamela; Paulke-Korinek, Maria; Repa, Andreas; Mooi, Frits R; Kollaritsch, Herwig; Mittermayer, Helmut; Kessler, Harald H; Stanek, Gerold; Steinborn, Ralf; Duchêne, Michael; Wiedermann, Ursula

    2015-01-01

    In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

  8. A Multiplex PCR for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in Clinical Specimens

    Science.gov (United States)

    2007-11-02

    NAVAL HEALTH RESEARCH CENTER A MULTIPLEX PCR FOR DETECTION OF Mycoplasma pneumoniae,Chlamydophila pneumoniae, Legionella pneumophila, AND Bordetella...5300 2 A Multiplex PCR for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in Clinical...Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in uncultured patient specimens. These organisms cause similar symptomologies

  9. Epidemiology of whooping cough & typing of Bordetella pertussis.

    Science.gov (United States)

    Hegerle, Nicolas; Guiso, Nicole

    2013-11-01

    Bordetella pertussis is a Gram-negative human-restricted bacterium that evolved from the broad-range mammalian pathogen, Bordetella bronchiseptica. It causes whooping cough or pertussis in humans, which is the most prevalent vaccine-preventable disease worldwide. The introduction of the pertussis whole-cell vaccination for young children, followed by the introduction of the pertussis acellular vaccination (along with booster vaccination) for older age groups, has affected the bacterial population and epidemiology of the disease. B. pertussis is relatively monomorphic worldwide, but nevertheless, different countries are facing different epidemiological evolutions of the disease. Although it is tempting to link vaccine-driven phenotypic and genotypic evolution of the bacterium to epidemiology, many other factors should be considered and surveillance needs to continue, in addition to studies investigating the impact of current clinical isolates on vaccine efficacy.

  10. Comparative study of hemolytic activity of Bordetella species

    Directory of Open Access Journals (Sweden)

    C N Khobragade

    2009-11-01

    Full Text Available Background and objectives: Bordetella species colonize the respiratory tract of mammals and thereby cause the whooping cough. Most of the species produce adenylate cyclase - a toxin ( hemolysin responsible for increasing intracellular cyclic AMP (cAMP levels in mammalian neutrophils and macrophages and as a consequence their phagocytic function get impaired . This study was carried out to isolate species of Bordetella and to study the hemolytic activity of each species on RBCs of sheep, human and poultry at varied culture conditions by altering the temperature, pH and cell age."nMaterials and Methods: Three pathogenic Bordetella species were isolated from fifty suspected whooping cough patients on Bordet-Gengou agar and identified by their biochemical profiles. The hemolytic activity of B. pertussis, B. parapertussis and B. bronchiseptica was investigated in terms of cell bound and cell free hemolysin on human, poultry and sheep RBCs at variable pH, temperature and cell age in Stainer Scholt broth. The hemolysin activity was also determined qualitatively on blood agar containing different blood samples."nResults: All the species revealed optimum hemolytic activity in pH range 7.5-8.0 (in slight alkaline condition, temperature 37°C and cell age up to 20-24 hrs. The cell bound hemolytic activity was found to be maximum than cell free activity and varied with blood samples of different species. B. pertussis showed maximum hemolytic activity on human red blood cells followed by poultry and sheep RBCs. B. parapertussis and B. bronchiseptica showed maximum hemolytic activity on sheep and poultry RBCs respectively."nConclusion: The findings of our study revealed that different determinants are involved in host interactions and virulence of Bordetella species.

  11. Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats

    Science.gov (United States)

    Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less well-studied. The objectives of this study were to identify the frequency of Bordetella bronchiseptica infection in fatal cases of bronchopneumonia in dogs and cats and to compare the diag...

  12. Evaluation of 3 analyte-specific reagents for detection of Bordetella pertussis and Bordetella parapertussis in clinical specimens.

    Science.gov (United States)

    Hassan, Ferdaus; Hays, Lindsay; Bell, Jeremiah; Selvarangan, Rangaraj

    2014-11-01

    The performance of 3 analyte-specific reagents (ASRs), Elitech Biosciences, EraGen Biosciences, and Focus Diagnostic, was evaluated for detection of Bordetella pertussis (BP) and Bordetella parapertussis (BPP) in nasopharyngeal swab specimens. A total of 104 frozen, leftover clinical specimens obtained from pediatric patients during 2011-2012 were included in this study. Performance was compared to the Bordetella real-time polymerase chain reaction (PCR) laboratory-developed test (LDT). The positive percent agreement for detection of BP by Elitech was 96% (95% confidence interval [CI]: 85.14-99.30); EraGen and Focus was 98% (95% CI: 87.99-99.89) in comparison to LDT PCR assay. The negative percent agreement of Elitech, EraGen, and Focus in comparison to LDT was 96% (95% CI: 85.14-99.30), 92% (95% CI: 79.89-97.41), and 96% (95% CI: 85.14-99.30), respectively. Limit of detection (LOD) for BP was 0.1 CFU/reaction by both Focus and EraGen and 1.0 CFU/reaction by Elitech. However, LOD for BPP was lower by EraGen (0.1 CFU/reaction) compared to Focus (1.0 CFU/reaction) and Elitech (1.0 CFU/reaction). These results demonstrate that all 3 ASRs tested are comparable and reliable for routine clinical diagnosis of pertussis and parapertussis.

  13. The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae

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    Schneiker-Bekel Susanne

    2008-09-01

    Full Text Available Abstract Background Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. Results In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. Conclusion The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.

  14. Seroepidemiology of Bordetella pertussis infections in the twin cities of Pakistan

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    Fahad Said

    2009-12-01

    Full Text Available Background: Bordetella pertussis is the cause of whooping cough occurring mainly in children. The prevalence of this disease has been reduced largely due to worldwide mass vaccination with DTP vaccine. However, the immunity produced by the vaccination wanes by the passage of time. Still this disease kills around 2-4 million children annually. Adults may be a source of infection for infants and children. Furthermore, Bordetella pertussis has also been found to be associated with cases of persistent cough in adults in many countries. Aim: The aim of this study was to study the exposure of the adult population to the Bordetella pertussis by detecting IgG antibodies. Materials and Methods: We performed Seroepidemiology of Bordetella pertussis infections in multiethnic twin cities of Pakistan (Rawalpindi and Islamabad using a commercially available ELISA kit to have a picture of epidemiology of Bordetella pertussis in Pakistan. We targeted adults of age between 18-45 years (mean age 29.64 years. Results: The results of our study show a high percentage of seropositivity to Bordetella pertussis (89 percent, which indicates higher exposure to this organism and risk of infection to infants, children, adolescents and adults. Conclusion: A high percentage of seropositive individuals are alarming to health care professionals as well as policy makers. Bordetella pertussis infections may be associated with their atypical manifestation in Pakistan. Adult vaccination with DTP is recommended to reduce the risk of infection in infants and children through adult reservoirs.

  15. Seroepidemiology of Bordetella pertussis infections in the twin cities of Pakistan

    Directory of Open Access Journals (Sweden)

    Muhammad Ali Syed

    2009-01-01

    Full Text Available Background: Bordetella pertussis is the cause of whooping cough occurring mainly in children. The prevalence of this disease has been reduced largely due to worldwide mass vaccination with DTP vaccine. However, the immunity produced by the vaccination wanes by the passage of time. Still this disease kills around 2-4 million children annually. Adults may be a source of infection for infants and children. Furthermore, Bordetella pertussis has also been found to be associated with cases of persistent cough in adults in many countries. Aim: The aim of this study was to study the exposure of the adult population to the Bordetella pertussis by detecting IgG antibodies. Materials and Methods: We performed Seroepidemiology of Bordetella pertussis infections in multiethnic twin cities of Pakistan (Rawalpindi and Islamabad using a commercially available ELISA kit to have a picture of epidemiology of Bordetella pertussis in Pakistan. We targeted adults of age between 18-45 years (mean age 29.64 years. Results: The results of our study show a high percentage of seropositivity to Bordetella pertussis (89 percent, which indicates higher exposure to this organism and risk of infection to infants, children, adolescents and adults. Conclusion: A high percentage of seropositive individuals are alarming to health care professionals as well as policy makers. Bordetella pertussis infections may be associated with their atypical manifestation in Pakistan. Adult vaccination with DTP is recommended to reduce the risk of infection in infants and children through adult reservoirs.

  16. Transcriptional analysis of the Bordetella alcaligin siderophore biosynthesis operon.

    Science.gov (United States)

    Kang, H Y; Armstrong, S K

    1998-02-01

    The alc gene cluster of Bordetella pertussis includes three genes, alcA, alcB, and alcC, which are involved in alcaligin siderophore biosynthesis in response to iron starvation. The production of AlcA, AlcB, and AlcC in Bordetella cells and the transcriptional organization of alcA, alcB, and alcC were investigated by using a set of three alc'-'lacZ gene fusion constructs that were contiguous with the known promoter upstream of alcA and extended to fusion junctions within each alc cistron. All three alc'-'lacZ fusions exhibited iron-repressible reporter gene expression which was abolished by deletion of the 105-bp alcA promoter-operator region. In an immunoblot analysis using a monoclonal antibody specific for beta-galactosidase, the AlcA-LacZ, AlcB-LacZ, and AlcC-LacZ hybrid proteins were detected in Bordetella cells grown under iron-depleted conditions. A B. pertussis mutant in which the 105-bp alcA promoter-operator region was deleted by allelic exchange was unable to produce detectable levels of siderophore. Hybridization analysis using gene-specific probes showed that alc-specific transcript levels in the mutant were negligible compared with those of the wild-type parent. These results confirm that alcA, alcB, and alcC are cotranscribed from an iron-regulated control region immediately upstream of alcA. Transcript analysis using hybridization probes representing regions downstream of alcC demonstrated that alc transcription extends approximately 3.6 kb further downstream from the alcC coding region, suggesting the cotranscription of additional, uncharacterized alcaligin system genes.

  17. Bordetella pertussis, B. parapertussis, vaccines and cycles of whooping cough.

    Science.gov (United States)

    Bouchez, Valérie; Guiso, Nicole

    2015-10-01

    Whooping cough is a vaccine-preventable disease due to Bordetella pertussis and B. parapertussis. This highly contagious respiratory disease occurs through epidemic cycles every 3-5 years and vaccination did not change this frequency. Models suggest that the cyclic increase of susceptibles is linked to demographic differences and different vaccine coverage. However, differences in surveillance of the disease as well as adaptation of the agents of the disease to their human hosts and to vaccine pressure might also play an important role. These parameters are discussed in this review.

  18. Pertussis in the Era of New Strains of Bordetella pertussis.

    Science.gov (United States)

    Souder, Emily; Long, Sarah S

    2015-12-01

    Despite implementation of a successful vaccination program, pertussis remains a significant health problem. Although the incidence of pertussis in the United States is reduced by approximately 80% compared with incidence before the introduction of vaccination in the 1940s, deaths still occur and the unrecognized disease burden remains high, with 1 million Bordetella pertussis infections annually in the United States estimated by serologic surveys. Reasons for the resurgence and current prevalence of pertussis may be multifactorial and include waning vaccine-induced protection as well as lower vaccine effectiveness, failure to vaccinate, and changes in the organism itself.

  19. Mejoras en el ensayo de potencia de Bordetella pertussis

    OpenAIRE

    Carmen Alina del Puerto; Aleida Mandiarote; Orialys Valle; Juan Francisco Núñez; Luis Izquierdo; Nadiezda Baños; Irma Labrador

    2013-01-01

    La potencia del componente pertussis, presente en la vacuna DPT-vax, se evalúa mediante el ensayo de Kendrick. La cepa recomendada por OMS para este ensayo es Bordetella pertussis 18 323, la cual debe ser conservada de manera que se asegure su viabilidad, pureza y virulencia. Como parte de dicho ensayo debe calcularse la concentración de una suspensión de la cepa, cuya opacidad se compara a simple vista con la 5ta Preparación Internacional de Referencia de Opacidad. Este método es muy inexact...

  20. Resident microbiota affect Bordetella pertussis infectious dose and host specificity.

    Science.gov (United States)

    Weyrich, Laura S; Feaga, Heather A; Park, Jihye; Muse, Sarah J; Safi, Chetan Y; Rolin, Olivier Y; Young, Sarah E; Harvill, Eric T

    2014-03-01

    Before contacting host tissues, invading pathogens directly or indirectly interact with host microbiota, but the effects of such interactions on the initial stages of infection are poorly understood. Bordetella pertussis is highly infectious among humans but requires large doses to colonize rodents, unlike a closely related zoonotic pathogen, Bordetella bronchiseptica, raising important questions about the contributions of bacterial competition to initial colonization and host selection. We observed that <100 colony-forming units (CFU) of B. bronchiseptica efficiently infected mice and displaced culturable host microbiota, whereas 10 000 CFU of B. pertussis were required to colonize murine nasal cavities and did not displace host microorganisms. Bacteria isolated from murine nasal cavities but not those from the human lower respiratory tract limited B. pertussis growth in vitro, indicating that interspecies competition may limit B. pertussis colonization of mice. Further, a broad-spectrum antibiotic treatment delivered before B. pertussis inoculation reduced the infectious dose to <100 CFU, and reintroduction of single Staphylococcus or Klebsiella species was sufficient to inhibit B. pertussis colonization of antibiotic-treated mice. Together, these results reveal that resident microorganisms can prevent B. pertussis colonization and influence host specificity, and they provide rationale for manipulating microbiomes to create more-accurate animal models of infectious diseases.

  1. Mejoras en el ensayo de potencia de Bordetella pertussis

    Directory of Open Access Journals (Sweden)

    Carmen Alina del Puerto

    2013-08-01

    Full Text Available La potencia del componente pertussis, presente en la vacuna DPT-vax, se evalúa mediante el ensayo de Kendrick. La cepa recomendada por OMS para este ensayo es Bordetella pertussis 18 323, la cual debe ser conservada de manera que se asegure su viabilidad, pureza y virulencia. Como parte de dicho ensayo debe calcularse la concentración de una suspensión de la cepa, cuya opacidad se compara a simple vista con la 5ta Preparación Internacional de Referencia de Opacidad. Este método es muy inexacto y con frecuencia conduce a errores. En este trabajo se elaboró y caracterizó un lote de siembra de Bordetella. pertussis 18 323; se calculó la concentración de dicha cepa empleando una curva de calibración. El lote se empleó en ocho ensayos de potencia y en los mismos se cumplieron los criterios de validez establecidos.

  2. Polymorphisms influencing expression of dermonecrotic toxin in Bordetella bronchiseptica.

    Directory of Open Access Journals (Sweden)

    Keisuke Okada

    Full Text Available Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT, when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases.

  3. Complete Genome Sequences of Bordetella pertussis Vaccine Reference Strains 134 and 10536

    Science.gov (United States)

    Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Burroughs, Mark; Johnson, Taccara; Juieng, Phalasy; Rowe, Lori; Tondella, M. Lucia; Williams, Margaret M.

    2016-01-01

    Vaccine formulations and vaccination programs against whooping cough (pertussis) vary worldwide. Here, we report the complete genome sequences of two divergent Bordetella pertussis reference strains used in the production of pertussis vaccines. PMID:27635001

  4. Patterns of Susceptibility in an Outbreak of Bordetella pertussis: Evidence from a Community-Based Study

    Directory of Open Access Journals (Sweden)

    David M Moore

    2002-01-01

    Full Text Available OBJECTIVE: To describe an outbreak of Bordetella pertussis and to assess which factors were associated with the development of clinical pertussis in children and adults during the outbreak.

  5. Complete Genome Sequences of Bordetella pertussis Vaccine Reference Strains 134 and 10536.

    Science.gov (United States)

    Weigand, Michael R; Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Burroughs, Mark; Johnson, Taccara; Juieng, Phalasy; Rowe, Lori; Tondella, M Lucia; Williams, Margaret M

    2016-09-15

    Vaccine formulations and vaccination programs against whooping cough (pertussis) vary worldwide. Here, we report the complete genome sequences of two divergent Bordetella pertussis reference strains used in the production of pertussis vaccines.

  6. Canine distemper virus infection with secondary Bordetella bronchiseptica pneumonia in dogs

    OpenAIRE

    HEADLEY, Selwyn Arlington; Graça,Dominguita Lühers; Costa,Mateus Matiuzzi da; Vargas,Agueda Castagna de

    1999-01-01

    Canine distemper virus infection and secondary Bordetella bronchiseptica pneumonia are described in mongrel dogs. Canine distemper was characterised by nonsuppurative demyelinating encephalitis with typical inclusion bodies in astrocytes. B. bronchiseptica was isolated from areas of purulent bronchopneumonia.

  7. Detection of small RNAs in Bordetella pertussis and identification of a novel repeated genetic element

    Directory of Open Access Journals (Sweden)

    Wulbrecht Bérénice

    2011-04-01

    Full Text Available Abstract Background Small bacterial RNAs (sRNAs have been shown to participate in the regulation of gene expression and have been identified in numerous prokaryotic species. Some of them are involved in the regulation of virulence in pathogenic bacteria. So far, little is known about sRNAs in Bordetella, and only very few sRNAs have been identified in the genome of Bordetella pertussis, the causative agent of whooping cough. Results An in silico approach was used to predict sRNAs genes in intergenic regions of the B. pertussis genome. The genome sequences of B. pertussis, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella avium were compared using a Blast, and significant hits were analyzed using RNAz. Twenty-three candidate regions were obtained, including regions encoding the already documented 6S RNA, and the GCVT and FMN riboswitches. The existence of sRNAs was verified by Northern blot analyses, and transcripts were detected for 13 out of the 20 additional candidates. These new sRNAs were named Bordetella pertussis RNAs, bpr. The expression of 4 of them differed between the early, exponential and late growth phases, and one of them, bprJ2, was found to be under the control of BvgA/BvgS two-component regulatory system of Bordetella virulence. A phylogenetic study of the bprJ sequence revealed a novel, so far undocumented repeat of ~90 bp, found in numerous copies in the Bordetella genomes and in that of other Betaproteobacteria. This repeat exhibits certain features of mobile elements. Conclusion We shown here that B. pertussis, like other pathogens, expresses sRNAs, and that the expression of one of them is controlled by the BvgA/BvgS system, similarly to most virulence genes, suggesting that it is involved in virulence of B. pertussis.

  8. Rapid increase in pertactin-deficient Bordetella pertussis isolates, Australia.

    Science.gov (United States)

    Lam, Connie; Octavia, Sophie; Ricafort, Lawrence; Sintchenko, Vitali; Gilbert, Gwendolyn L; Wood, Nicholas; McIntyre, Peter; Marshall, Helen; Guiso, Nicole; Keil, Anthony D; Lawrence, Andrew; Robson, Jenny; Hogg, Geoff; Lan, Ruiting

    2014-04-01

    Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008-2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (Prn). Multiple mechanisms of Prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of Prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing Prn arose independently multiple times since 2008, rather than by expansion of a single Prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.

  9. Bordetella pertussis evolution in the (functional) genomics era.

    Science.gov (United States)

    Belcher, Thomas; Preston, Andrew

    2015-11-01

    The incidence of whooping cough caused by Bordetella pertussis in many developed countries has risen dramatically in recent years. This has been linked to the use of an acellular pertussis vaccine. In addition, it is thought that B. pertussis is adapting under acellular vaccine mediated immune selection pressure, towards vaccine escape. Genomics-based approaches have revolutionized the ability to resolve the fine structure of the global B. pertussis population and its evolution during the era of vaccination. Here, we discuss the current picture of B. pertussis evolution and diversity in the light of the current resurgence, highlight import questions raised by recent studies in this area and discuss the role that functional genomics can play in addressing current knowledge gaps.

  10. Infectious Disease Report: Bordetella pertussis Infection in Patients With Cancer.

    Science.gov (United States)

    Yacoub, Abraham; Nanjappa, Sowmya; Janz, Tyler; Greene, John N

    2016-04-01

    We illustrate 2 cases of pneumonia associated with Bordetella pertussis infection in 72-year-old and 61-year-old patients with cancer receiving myelosuppressive therapy after hematopoietic stem cell transplantation. Bacterial infections are a significant cause of morbidity and mortality in patients with cancer, and those receiving hematopoietic stem cell transplant, solid organ transplant, or myelosuppressive therapy are at increased risk. The infection was detected and the 2 patients had good outcomes following azithromycin treatment. Pertussis, also known as whooping cough, is a contagious respiratory illness that has become a public health challenge due to decreased immunity of the pertussis vaccine. Therefore, it is critical to recognize pertussis early in the course of the disease.

  11. Harmonization of Bordetella pertussis real-time PCR diagnostics in the United States in 2012.

    Science.gov (United States)

    Williams, Margaret M; Taylor, Thomas H; Warshauer, David M; Martin, Monte D; Valley, Ann M; Tondella, M Lucia

    2015-01-01

    Real-time PCR (rt-PCR) is an important diagnostic tool for the identification of Bordetella pertussis, Bordetella holmesii, and Bordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in the B. pertussis genome and 32 to 65 copies in B. holmesii. The CDC developed a multitarget PCR assay to differentiate B. pertussis, B. holmesii, and B. parapertussis and provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these three Bordetella species in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viable Bordetella and non-Bordetella species in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481 in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiated B. pertussis and B. holmesii and 68% and 72% identified B. parapertussis. IS481 cycle threshold (CT) values for B. pertussis samples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiated B. pertussis and B. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCR Bordetella diagnostic protocols in USPHLs compared to that of the previous survey.

  12. Aislamiento y caracterización de cepas de Bordetella bronchiseptica de origen canino

    OpenAIRE

    Graciela Molina González; María Eugenia Rosales; Gabriela Bárcenas Morales; Juan Antonio Montaraz Crespo

    2006-01-01

    Se aislaron, identifi caron y caracterizaron cepas de Bordetella bronchiseptica de origen canino. Con ese propósito se tomaron exudados nasales de perros con semiótica de afecciones respiratorias y clínicamente sanos, con ayuda de hisopos impregnados con medio infusión cerebro-corazón. Éstos se cultivaron en agar Mac Conkey incubando a 37°C durante 48 h; las colonias presuntivas de Bordetella bronchiseptica se identifi caron por movilidad, tinción de Gram y pruebas bioquímicas. Las cepas se c...

  13. Real-time PCR-based detection of Bordetella pertussis and Bordetella parapertussis in an Irish paediatric population.

    LENUS (Irish Health Repository)

    Grogan, Juanita A

    2011-06-01

    Novel real-time PCR assays targeting the Bordetella pertussis insertion sequence IS481, the toxin promoter region and Bordetella parapertussis insertion sequence IS1001 were designed. PCR assays were capable of detecting ≤10 copies of target DNA per reaction, with an amplification efficiency of ≥90 %. From September 2003 to December 2009, per-nasal swabs and nasopharyngeal aspirates submitted for B. pertussis culture from patients ≤1 month to >15 years of age were examined by real-time PCR. Among 1324 patients, 76 (5.7 %) were B. pertussis culture positive and 145 (10.95 %) were B. pertussis PCR positive. Of the B. pertussis PCR-positive patients, 117 (81 %) were aged 6 months or less. A total of 1548 samples were examined, of which 87 (5.6 %) were culture positive for B. pertussis and 169 (10.92 %) were B. pertussis PCR positive. All culture-positive samples were PCR positive. Seven specimens (0.5 %) were B. parapertussis culture positive and 10 (0.8 %) were B. parapertussis PCR positive, with all culture-positive samples yielding PCR-positive results. A review of patient laboratory records showed that of the 1324 patients tested for pertussis 555 (42 %) had samples referred for respiratory syncytial virus (RSV) testing and 165 (30 %) were positive, as compared to 19.4 % of the total 5719 patients tested for RSV in this period. Analysis of the age distribution of RSV-positive patients identified that 129 (78 %) were aged 6 months or less, similar to the incidence observed for pertussis in that patient age group. In conclusion, the introduction of the real-time PCR assays for the routine detection of B. pertussis resulted in a 91 % increase in the detection of the organism as compared to microbiological culture. The incidence of infection with B. parapertussis is low while the incidence of RSV infection in infants suspected of having pertussis is high, with a similar age distribution to B. pertussis infection.

  14. New Data on Vaccine Antigen Deficient Bordetella pertussis Isolates

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    Valérie Bouchez

    2015-09-01

    Full Text Available Evolution of Bordetella pertussis is driven by natural and vaccine pressures. Isolates circulating in regions with high vaccination coverage present multiple allelic and antigenic variations as compared to isolates collected before introduction of vaccination. Furthermore, during the last epidemics reported in regions using pertussis acellular vaccines, isolates deficient for vaccine antigens, such as pertactin (PRN, were reported to reach high proportions of circulating isolates. More sporadic filamentous hemagglutinin (FHA or pertussis toxin (PT deficient isolates were also collected. The whole genome of some recent French isolates, deficient or non-deficient in vaccine antigens, were analyzed. Transcription profiles of the expression of the main virulence factors were also compared. The invasive phenotype in an in vitro human tracheal epithelial (HTE cell model of infection was evaluated. Our genomic analysis focused on SNPs related to virulence genes known to be more likely to present allelic polymorphism. Transcriptomic data indicated that isolates circulating since the introduction of pertussis vaccines present lower transcription levels of the main virulence genes than the isolates of the pre-vaccine era. Furthermore, isolates not producing FHA present significantly higher expression levels of the entire set of genes tested. Finally, we observed that recent isolates are more invasive in HTE cells when compared to the reference strain, but no multiplication occurs within cells.

  15. Antibodies to BrkA augment killing of Bordetella pertussis.

    Science.gov (United States)

    Oliver, D C; Fernandez, R C

    2001-10-12

    BrkA is a Bvg-regulated Bordetella pertussis protein that mediates serum resistance and adherence. It shares sequence identity with another B. pertussis virulence factor called pertactin, and it is a member of the diverse group of proteins found in Gram-negative bacteria that are secreted by an autotransporter mechanism. Sera, either from individuals who have been vaccinated with acellular pertussis vaccines, or from individuals who have no re-collection of recent infection with B. pertussis fail to kill wild-type B. pertussis, but kill brkA mutant strains very well. We examined whether BrkA could be neutralised in serum fitting this profile. BrkA is synthesised as a 103kDa precursor that is processed into a surface-associated N-terminal 73kDa passenger domain, and an outer-membrane embedded C-terminal 30kDa transporter moiety. Polyclonal antibodies were raised to a recombinant, re-folded histidine-tagged fusion protein representing the 73kDa passenger region. These anti-BrkA antibodies were shown to boost the existing bactericidal capacity of human serum against B. pertussis by neutralising BrkA.

  16. Clinical, laboratorial and radiographic predictors of Bordetella pertussis infection

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    Camila Vieira Bellettini

    2014-12-01

    Full Text Available OBJECTIVE: To identify clinical, laboratorial and radiographic predictors for Bordetella pertussis infection.METHODS: This was a retrospective study, which analyzed medical records of all patients submitted to a molecular dignosis (qPCR for B. pertussis from September 2011 to January 2013. Clinical and laboratorial data were reviewed, including information about age, sex, signs/symptoms, length of hospitalization, blood cell counts, imaging findings, coinfection with other respiratory pathogens and clinical outcome.RESULTS: 222 cases were revised. Of these, 72.5% had proven pertussis, and 60.9% were under 1 year old. In patients aging up to six months, independent predictors for B. pertussisinfection were (OR 8.0, CI 95% 1.8-36.3; p=0.007 and lymphocyte count >104/µL (OR 10.0, CI 95% 1.8-54.5; p=0.008. No independent predictors of B. pertussisinfection could be determined for patients older than six months. Co-infection was found in 21.4% of patients, of which 72.7% were up to six months of age. Adenovirus was the most common agent (40.9%. In these patients, we were not able to identify any clinical features to detect patients presenting with a respiratory co-infection, even though longer hospital stay was observed in patients with co-infections (12 vs. 6 days; p=0.009.CONCLUSIONS: Cyanosis and lymphocytosis are independent predictors for pertussis in children up to 6 months old.

  17. Monospecific antibody against Bordetella pertussis Adenylate Cyclase protects from Pertussis

    Directory of Open Access Journals (Sweden)

    Yasmeen Faiz Kazi

    2012-06-01

    Full Text Available Objectives: Acellular pertussis vaccines has been largely accepted world-wide however, there are reports about limitedantibody response against these vaccines suggesting that multiple antigens should be included in acellular vaccinesto attain full protection. The aim of present study was to evaluate the role of Bordetella pertussis adenylate cyclase as aprotective antigen.Materials and methods: Highly mono-specific antibody against adenylate cyclase (AC was raised in rabbits usingnitrocellulose bound adenylate cyclase and the specificity was assessed by immuoblotting. B.pertussis 18-323, wasincubated with the mono-specific serum and without serum as a control. Mice were challenged intra-nasally and pathophysiolgicalresponses were recorded.Results: The production of B.pertussis adenylate cyclase monospecific antibody that successfully recognized on immunoblotand gave protection against fatality (p< 0.01 and lung consolidation (p <0.01. Mouse weight gain showedsignificant difference (p< 0.05.Conclusion: These preliminary results highlight the role of the B.pertussis adenylate cyclase as a potential pertussisvaccine candidate. B.pertussis AC exhibited significant protection against pertussis in murine model. J Microbiol InfectDis 2012; 2(2: 36-43Key words: Pertussis; monospecific; antibody; passive-protection

  18. Waning and aging of cellular immunity to Bordetella pertussis.

    Science.gov (United States)

    van Twillert, Inonge; Han, Wanda G H; van Els, Cécile A C M

    2015-11-01

    While it is clear that the maintenance of Bordetella pertussis-specific immunity evoked both after vaccination and infection is insufficient, it is unknown at which pace waning occurs and which threshold levels of sustained functional memory B and T cells are required to provide long-term protection. Longevity of human cellular immunity to B. pertussis has been studied less extensively than serology, but is suggested to be key for the observed differences between the duration of protection induced by acellular vaccination and whole cell vaccination or infection. The induction and maintenance of levels of protective memory B and T cells may alter with age, associated with changes of the immune system throughout life and with accumulating exposures to circulating B. pertussis or vaccine doses. This is relevant since pertussis affects all age groups. This review summarizes current knowledge on the waning patterns of human cellular immune responses to B. pertussis as addressed in diverse vaccination and infection settings and in various age groups. Knowledge on the effectiveness and flaws in human B. pertussis-specific cellular immunity ultimately will advance the improvement of pertussis vaccination strategies.

  19. Bordetella pertussis iron regulated proteins as potential vaccine components.

    Science.gov (United States)

    Alvarez Hayes, Jimena; Erben, Esteban; Lamberti, Yanina; Principi, Guido; Maschi, Fabricio; Ayala, Miguel; Rodriguez, Maria Eugenia

    2013-08-01

    Bordetella pertussis is the etiologic agent of whooping cough, an illness whose incidence has been increasing over the last decades. Pertussis reemergence despite high vaccination coverage, together with the recent isolation of circulating strains deficient in some of the vaccine antigens, highlight the need for new vaccines. Proteins induced under physiological conditions, such as those required for nutrient acquisition during infection, might represent good targets for better preventive strategies. By mean of serological proteome analysis we identified two novel antigens of B. pertussis potentially involved in iron acquisition during host colonization. We had previously demonstrated that one of them, designated IRP1-3, is protective against pertussis infection in mice. In the present study, we show that the other antigen, named AfuA (BP1605), is a highly antigenic protein, exposed on the bacterial surface, conserved among clinical isolates and expressed during infection. Immunization of mice with the recombinant AfuA induced opsonophagocytic antibodies which could explain the protection against B. pertussis infection conferred by mice immunization with rAfuA. Importantly, we found that the addition of rAfuA and rIRP1-3 proteins to the commercial three pertussis components acellular vaccine significantly increased its protective activity. Taken together, our results point at these two antigens as potential components of a new generation of acellular vaccines.

  20. Review of the neutrophil response to Bordetella pertussis infection.

    Science.gov (United States)

    Eby, Joshua C; Hoffman, Casandra L; Gonyar, Laura A; Hewlett, Erik L

    2015-12-01

    The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10-14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28-35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors.

  1. The multifaceted RisA regulon of Bordetella pertussis

    Science.gov (United States)

    Coutte, Loïc; Huot, Ludovic; Antoine, Rudy; Slupek, Stephanie; Merkel, Tod J.; Chen, Qing; Stibitz, Scott; Hot, David; Locht, Camille

    2016-01-01

    The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA− background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD60N and the phosphomimetic RisAD60E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation. PMID:27620673

  2. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival.

    Science.gov (United States)

    Lamberti, Yanina; Gorgojo, Juan; Massillo, Cintia; Rodriguez, Maria E

    2013-12-01

    Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

  3. Competition, coinfection and strain replacement in models of Bordetella pertussis.

    Science.gov (United States)

    Nicoli, Emily J; Ayabina, Diepreye; Trotter, Caroline L; Turner, Katherine M E; Colijn, Caroline

    2015-08-01

    Pertussis, or whooping cough, is an important respiratory infection causing considerable infant mortality worldwide. Recently, incidence has risen in countries with strong vaccine programmes and there are concerns about antigenic shift resulting in vaccine evasion. Interactions between pertussis and non-vaccine-preventable strains will play an important role in the evolution and population dynamics of pertussis. In particular, if we are to understand the role strain replacement plays in vaccinated settings, it will be essential to understand how strains or variants of pertussis interact. Here we explore under what conditions we would expect strain replacement to be of concern in pertussis. We develop a dynamic transmission model that allows for coinfection between Bordetella pertussis (the main causative agent of pertussis) and a strain or variant unaffected by the vaccine. We incorporate both neutrality (in the sense of ecological/population genetic neutrality) and immunity into the model, leaving the specificity of the immune response flexible. We find that strain replacement may be considerable when immunity is non-specific. This is in contrast to previous findings where neutrality was not considered. We conclude that the extent to which models reflect ecological neutrality can have a large impact on conclusions regarding strain replacement. This will likely have onward consequences for estimates of vaccine efficacy and cost-effectiveness.

  4. The multifaceted RisA regulon of Bordetella pertussis.

    Science.gov (United States)

    Coutte, Loïc; Huot, Ludovic; Antoine, Rudy; Slupek, Stephanie; Merkel, Tod J; Chen, Qing; Stibitz, Scott; Hot, David; Locht, Camille

    2016-09-13

    The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA(-) background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD(60)N and the phosphomimetic RisAD(60)E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation.

  5. Bordetella pertussis modulates human macrophage defense gene expression.

    Science.gov (United States)

    Valdez, Hugo Alberto; Oviedo, Juan Marcos; Gorgojo, Juan Pablo; Lamberti, Yanina; Rodriguez, Maria Eugenia

    2016-08-01

    Bordetella pertussis, the etiological agent of whooping cough, still causes outbreaks. We recently found evidence that B. pertussis can survive and even replicate inside human macrophages, indicating that this host cell might serve as a niche for persistence. In this work, we examined the interaction of B. pertussis with a human monocyte cell line (THP-1) that differentiates into macrophages in culture in order to investigate the host cell response to the infection and the mechanisms that promote that intracellular survival. To that end, we investigated the expression profile of a selected number of genes involved in cellular bactericidal activity and the inflammatory response during the early and late phases of infection. The bactericidal and inflammatory response of infected macrophages was progressively downregulated, while the number of THP-1 cells heavily loaded with live bacteria increased over time postinfection. Two of the main toxins of B. pertussis, pertussis toxin (Ptx) and adenylate cyclase (CyaA), were found to be involved in manipulating the host cell response. Therefore, failure to express either toxin proved detrimental to the development of intracellular infections by those bacteria. Taken together, these results support the relevance of host defense gene manipulation to the outcome of the interaction between B. pertussis and macrophages.

  6. Differential regulation of type III secretion and virulence genes in Bordetella pertussis and Bordetella bronchiseptica by a secreted anti-σ factor.

    Science.gov (United States)

    Ahuja, Umesh; Shokeen, Bhumika; Cheng, Ning; Cho, Yeonjoo; Blum, Charles; Coppola, Giovanni; Miller, Jeff F

    2016-03-01

    The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues.

  7. Manufacturing Vaccines: An Illustration of Using PAT Tools for Controlling the Cultivation of Bordetella pertussis

    NARCIS (Netherlands)

    Sprang, van E.N.M.; Streefland, M.; Ramaker, H.J.; Pol, van der L.A.; Beuvery, E.C.; Smilde, A.K.

    2007-01-01

    An illustration of the operational consistency of the upstream part of a biopharmaceutical process is given. For this purpose four batch cultivations of Bordetella pertussis have been executed under identical conditions. The batches have been monitored by means of two fundamentally different process

  8. Virulence of pertactin-negative Bordetella pertussis isolates from infants, France.

    Science.gov (United States)

    Bodilis, Hélène; Guiso, Nicole

    2013-03-01

    Bordetella pertussis isolates that do not express pertactin (PRN) are increasing in regions where acellular pertussis vaccines have been used for >7 years. We analyzed data from France and compared clinical symptoms among infants <6 months old infected by PRN-positive or PRN-negative isolates. No major clinical differences were found between the 2 groups.

  9. Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

    NARCIS (Netherlands)

    Lamberti, Yanina; Perez Vidakovics, Maria Laura; Van der Pol, Ludo-W.; Eugenia Rodriguez, Maria

    2008-01-01

    Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis However. the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis bindin

  10. Absence of Bordetella pertussis Among Infants Hospitalized for Bronchiolitis in Finland, 2008-2010.

    Science.gov (United States)

    Korppi, Matti; Kivistö, Juho; Koponen, Petri; Lehtinen, Pasi; Remes, Sami; Piippo-Savolainen, Eija; Piedra, Pedro A; Espinola, Janice A; Camargo, Carlos A; Jartti, Tuomas

    2016-02-01

    In 169 Finnish infants hospitalized for bronchiolitis at age Bordetella pertussis and 16 viruses. Respiratory viruses were detected in 89% (71% with respiratory syncytial virus), but no infant had B. pertussis. The latter finding may reflect a positive effect from the broadening of the Finnish pertussis vaccination program in 2005.

  11. Evidence of Bordetella pertussis infection in vaccinated 1-year-old Danish children

    DEFF Research Database (Denmark)

    von Linstow, Marie-Louise; Pontoppidan, Peter Lotko; von König, Carl-Heinz Wirsing;

    2010-01-01

    %. The apparent high Bordetella pertussis infection rate in Danish infants suggests that the monocomponent PT toxoid vaccine used in Denmark has limited efficacy against B. pertussis infection. A prospective immunization study comparing a multi-component vaccine with the present monocomponent PT toxoid vaccine...

  12. Bordetella pertussis attachment to respiratory epithelial cells can be impaired by fimbriae-specific antibodies

    NARCIS (Netherlands)

    Rodriguez, ME; Hellwig, SMM; Vidakovics, MLAP; Berbers, GAM; van de Winkel, JGJ

    2006-01-01

    Bordetella pertussis attachment to host cells is a crucial step in colonization. In this study, we investigated the specificity of antibodies, induced either by vaccination or infection, capable of reducing bacterial adherence to respiratory epithelial cells. Both sera and purified anti-B. pertussis

  13. Iron stress increases Bordetella pertussis mucin-binding capacity and attachment to respiratory epithelial cells

    NARCIS (Netherlands)

    Perez Vidakovics, Maria L. A.; Lamberti, Yanina; Serra, Diego; Berbers, Guy A. M.; van der Pol, W.-Ludo; Rodriguez, Maria Eugenia

    2007-01-01

    Whooping cough is a reemerging infectious disease of the respiratory tract caused by Bordetella pertussis. The incomplete understanding of the molecular mechanisms of host colonization hampers the efforts to control this disease. Among the environmental factors that commonly determine the bacterial

  14. Identification of Bordetella bronchseptica in fatal pneumonia of dogs and cats

    Science.gov (United States)

    Infection with Bordetella bronchiseptica is a common cause of tracheobronchitis and upper respiratory disease in dogs and cats, but it can also lead to fatal pneumonia. Identification of this pathogen is important due the risk of transmission to other animals, availability of vaccines and potential...

  15. Bordetella bronchiseptica in a paediatric cystic fibrosis patient: possible transmission from a household cat

    Science.gov (United States)

    Bordetella bronchiseptica was isolated from the sputum of a cystic fibrosis patient recently exposed to a kitten with an acute respiratory disease. Genetic characterization of the isolate and comparison with other isolates of human or feline origin strongly implicate the kitten as the source of infe...

  16. Evaluation of a commercial loop-mediated isothermal amplification assay for diagnosis of Bordetella pertussis infection.

    Science.gov (United States)

    Kamachi, Kazunari; Moriuchi, Takumi; Hiramatsu, Yukihiro; Otsuka, Nao; Shibayama, Keigo

    2017-02-01

    We evaluated a commercial loop-mediated isothermal amplification (LAMP) assay kit for Bordetella pertussis detection. The LAMP primers were designed to target the ptxP1 allele of the pertussis toxin promoter, but the assay could detect B. pertussis ptxP3 and ptxP8 strains in addition to ptxP1 strains, with high analytical sensitivity.

  17. Differentially expressed genes in Bordetella pertussis strains belonging to a lineage which recently spread globally

    NARCIS (Netherlands)

    de Gouw, Daan; Hermans, Peter W M; Bootsma, Hester J; Zomer, Aldert; Heuvelman, Kees; Diavatopoulos, Dimitri A; Mooi, Frits R

    2014-01-01

    Pertussis is a highly contagious, acute respiratory disease in humans caused by the Gram-negative pathogen Bordetella pertussis. Pertussis has resurged in the face of intensive vaccination and this has coincided with the emergence of strains carrying a particular allele for the pertussis toxin promo

  18. Global population structure and evolution of Bordetella pertussis and their relationship with vaccination

    NARCIS (Netherlands)

    Bart, M.J.; Harris, S.R.; Advani, A.; Arakawa, Y.; Bottero, D.; Bouchez, V.; Cassiday, P.K.; Chiang, C.S.; Dalby, T.; Fry, N.K.; Gaillard, M.E.; Gent, M. van; Guiso, N.; Hallander, H.O.; Harvill, E.T.; He, Q.; Heide, H.G. van der; Heuvelman, K.; Hozbor, D.F.; Kamachi, K.; Karataev, G.I.; Lan, R.; Lutylska, A.; Maharjan, R.P.; Mertsola, J.; Miyamura, T.; Octavia, S.; Preston, A.; Quail, M.A.; Sintchenko, V.; Stefanelli, P.; Tondella, M.L.; Tsang, R.S.; Xu, Y.; Yao, S.M.; Zhang, S.; Parkhill, J.; Mooi, F.R.

    2014-01-01

    Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-ce

  19. Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis

    Directory of Open Access Journals (Sweden)

    Cummings Craig A

    2007-05-01

    Full Text Available Abstract Background Bordetella pertussis, the causative agent of whooping cough, is a highly clonal pathogen of the respiratory tract. Its lack of genetic diversity, relative to many bacterial pathogens, could limit its ability to adapt to a hostile and changing host environment. This limitation might be overcome by phase variation, as observed for other mucosal pathogens. One of the most common mechanisms of phase variation is reversible expansion or contraction of homopolymeric tracts (HPTs. Results The genomes of B. pertussis and the two closely related species, B. bronchiseptica and B. parapertussis, were screened for homopolymeric tracts longer than expected on the basis of chance, given their nucleotide compositions. Sixty-nine such HPTs were found in total among the three genomes, 74% of which were polymorphic among the three species. Nine HPTs were genotyped in a collection of 90 geographically and temporally diverse B. pertussis strains using the polymerase chain reaction/ligase detection reaction (PCR/LDR assay. Six HPTs were polymorphic in this collection of B. pertussis strains. Of note, one of these polymorphic HPTs was found in the fimX promoter, where a single base insertion variant was present in seven strains, all of which were isolated prior to introduction of the pertussis vaccine. Transcript abundance of fimX was found to be 3.8-fold lower in strains carrying the longer allele. HPTs in three other genes, tcfA, bapC, and BP3651, varied widely in composition across the strain collection and displayed allelic polymorphism within single cultures. Conclusion Allelic polymorphism at homopolymeric tracts is common within the B. pertussis genome. Phase variability may be an important mechanism in B. pertussis for evasion of the immune system and adaptation to different niches in the human host. High sensitivity and specificity make the PCR/LDR assay a powerful tool for investigating allelic variation at HPTs. Using this method

  20. The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica.

    Directory of Open Access Journals (Sweden)

    Jun Kurushima

    Full Text Available Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator. The virulence of a BspR mutant (ΔbspR in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.

  1. Bordetella pertussis en estudiantes adolescentes de la Ciudad de México Bordetella pertussis em estudantes adolescentes da Cidade do México Bordetella pertussis in adolescents students in Mexico City

    Directory of Open Access Journals (Sweden)

    Patricia Tomé Sandoval

    2008-08-01

    Full Text Available OBJETIVO: Estimar la seroprevalencia a Bordetella pertussis en escolares y sus contactos escolares y familiares. MÉTODOS: Un total de 12.273 estudiantes de 12 a 15 años de edad, de 14 escuelas secundarias públicas de la Ciudad de México fueron estudiados durante los meses de Septiembre 2002 a Marzo 2003. Se tomó muestra de exudado nasofaríngeo en adolescentes con tos de más de 14 días de evolución. La infección fue confirmada por la técnica de reacción en cadena de polimerasa. Se realizó estudio de contactos escolares y familiares. RESULTADOS: La incidencia de tos fue de 5 para 1.000 estudiantes. De los 61 estudiantes con tos incluidos en la muestra, 20 (32,8% fueron positivos para Bordetella. De los 152 contactos escolares, 16 (10,6% resultaron positivos, y ocho tenían tos. Uno de esos contactos fue el director de una de las escuelas responsable de más del 60% de los casos positivos (12/20, quien también dio lecciones a diez de los estudiantes infectados. De los 29 familiares, ocho (27,6% fueron positivos, pertenecientes a tres familias. CONCLUSIONES: Los resultados muestran que la frecuencia de la enfermedad fue similar al comunicado en la población adolescente de otros países. Sin embargo, este trastorno no tiene necesariamente signos clínicos de la tos persistente y está sujeto a la existencia de infectados asintomáticos con Bordetella.OBJETIVO: Estimar a soroprevalência a Bordetella pertussis em escolares e seus contatos. MÉTODOS: Foram examinados 12.273 alunos entre 12 e 15 anos de idade, de 14 escolas secundárias públicas da Cidade do México, de setembro de 2002 a março de 2003. Amostras de exudado nasofaríngeo foram coletadas de adolescentes com tosse por mais de 14 dias. A infecção foi confirmada por reação em cadeia da polimerase. Todos os alunos e funcionários dos colégios dos casos confirmados por reação em cadeia da polimerase e seus familiares foram testados. RESULTADOS: A incidência de tosse

  2. Bordetella pertussis isolates in Finland: Serotype and fimbrial expression

    Directory of Open Access Journals (Sweden)

    Mertsola Jussi

    2008-09-01

    Full Text Available Abstract Background Bordetella pertussis causes whooping cough or pertussis in humans. It produces several virulence factors, of which the fimbriae are considered adhesins and elicit immune responses in the host. B. pertussis has three distinct serotypes Fim2, Fim3 or Fim2,3. Generally, B. pertussis Fim2 strains predominate in unvaccinated populations, whereas Fim3 strains are often isolated in vaccinated populations. In Finland, pertussis vaccination was introduced in 1952. The whole-cell vaccine contained two strains, 18530 (Fim3 since 1962 and strain 1772 (Fim2,3 added in 1976. After that the vaccine has remained the same until 2005 when the whole-cell vaccine was replaced by the acellular vaccine containing pertussis toxin and filamentous hemagglutinin. Our aims were to study serotypes of Finnish B. pertussis isolates from 1974 to 2006 in a population with > 90% vaccination coverage and fimbrial expression of the isolates during infection. Serotyping was done by agglutination and serotype-specific antibody responses were determined by blocking ELISA. Results Altogether, 1,109 isolates were serotyped. Before 1976, serotype distributions of Fim2, Fim3 and Fim2,3 were 67%, 19% and 10%, respectively. From 1976 to 1998, 94% of the isolates were Fim2 serotype. Since 1999, the frequency of Fim3 strains started to increase and reached 83% during a nationwide epidemic in 2003. A significant increase in level of serum IgG antibodies against purified fimbriae was observed between paired sera of 37 patients. The patients infected by Fim3 strains had antibodies which blocked the binding of monoclonal antibodies to Fim3 but not to Fim2. Moreover, about one third of the Fim2 strain infected patients developed antibodies capable of blocking of binding of both anti-Fim2 and Fim3 monoclonal antibodies. Conclusion Despite extensive vaccinations in Finland, B. pertussis Fim2 strains were the most common serotype. Emergence of Fim3 strains started in 1999 and

  3. Differential expression of alpha 4 integrins on effector memory T helper cells during Bordetella infections. Delayed responses in Bordetella pertussis.

    Directory of Open Access Journals (Sweden)

    Tuan M Nguyen

    Full Text Available Bordetella pertussis (B. pertussis is the causative agent of whooping cough, a respiratory disease that is reemerging worldwide. Mechanisms of selective lymphocyte trafficking to the airways are likely to be critical in the immune response to this pathogen. We compared murine infection by B. pertussis, B. parapertussis, and a pertussis toxin-deleted B. pertussis mutant (BpΔPTX to test the hypothesis that effector memory T-helper cells (emTh display an altered pattern of trafficking receptor expression in B. pertussis infection due to a defect in imprinting. Increased cell recruitment to the lungs at 5 days post infection (p.i. with B. parapertussis, and to a lesser extent with BpΔPTX, coincided with an increased frequency of circulating emTh cells expressing the mucosal-associated trafficking receptors α4β7 and α4β1 while a reduced population of these cells was observed in B. pertussis infection. These cells were highly evident in the blood and lungs in B. pertussis infection only at 25 days p.i. when B. parapertussis and BpΔPTX infections were resolved. Although at 5 days p.i., an equally high percentage of lung dendritic cells (DCs from all infections expressed maturation markers, this expression persisted only in B. pertussis infection at 25 days p.i. Furthermore, at 5 days p.i with B. pertussis, lung DCs migration to draining lymph nodes may be compromised as evidenced by decreased frequency of CCR7(+ DCs, inhibited CCR7-mediated in vitro migration, and fewer DCs in lung draining lymph nodes. Lastly, a reduced frequency of allogeneic CD4(+ cells expressing α4β1 was detected following co-culture with lung DCs from B. pertussis-infected mice, suggesting a defect in DC imprinting in comparison to the other infection groups. The findings in this study suggest that B. pertussis may interfere with imprinting of lung-associated trafficking receptors on T lymphocytes leading to extended survival in the host and a prolonged course of disease.

  4. Development of a multiplex real-time PCR assay for the detection of Bordetella pertussis and Bordetella parapertussis in a single tube reaction.

    Science.gov (United States)

    Arbefeville, Sophie; Levi, Michael H; Ferrieri, Patricia

    2014-02-01

    Pertussis is an infectious respiratory disease caused by the fastidious bacterium Bordetella pertussis, which may infect unvaccinated, previously vaccinated children, and adults in whom immunity has waned. Infants are at a particular risk for severe disease and complications. Bordetella parapertussis may cause a similar illness, however the symptoms are less severe and of shorter duration. Pertussis is a highly contagious disease and early diagnosis is essential. Studies have shown that PCR is 2-4 times more likely than culture to detect Bordetella pertussis. We developed a multiplex, real-time PCR assay using analyte-specific reagent (ASR) primers and probes dispensed in a convenient lyophilized bead format that targeted the multi-copy insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively. These specific ASRs were used in conjunction with Cepheid Smartmix. Included in the ASRs is a competitive internal control to evaluate the performance of the PCR reaction. After DNA extraction, amplification and detection were done on the Smart Cycler System, which performs integrated amplification and detection automatically in a single step. Specificity of the assay was confirmed using multiple distinct bacterial strains. Sensitivity of the assay and extraction efficiency were evaluated on DNA isolated from pure bacterial cultures and on spiked respiratory specimens. We also spiked different swab types and transport media to evaluate for interfering substances. To assess accuracy, we studied different patient specimen types received from two outside laboratories that used similar or different methods to detect B. pertussis and B. parapertussis. The sensitivity and the specificity of the assay for B. pertussis were 90% and 96%, respectively, and for B. parapertussis 71% (only 7 positive specimens were available for testing) and 100%, respectively. Our assay was found to be a valid method for the simultaneous detection of B. pertussis and B

  5. Bordetella pertussis and pertactin-deficient clinical isolates: lessons for pertussis vaccines.

    Science.gov (United States)

    Hegerle, Nicolas; Guiso, Nicole

    2014-09-01

    Bordetella pertussis causes whooping cough in humans, a highly transmissible respiratory disease life threatening for unvaccinated infants. Vaccination strategies were thus introduced worldwide with great success in developed countries reaching high vaccine coverage with efficacious vaccines. In the late 20th/early 21st century, acellular pertussis vaccines replaced whole cell pertussis vaccines but B. pertussis still circulates and evolves in humans, its only known reservoir. The latest transformation of this pathogen, and of its close relative Bordetella parapertussis, is the loss of pertactin production, a virulence factor included in different acellular pertussis vaccines. The real impact of this evolution on acellular pertussis vaccines efficacy and effectiveness should be assessed through standardized surveillance and isolation of B. pertussis and B. parapertussis worldwide.

  6. Bordetella avium Causes Induction of Apoptosis and Nitric Oxide Synthase in Turkey Tracheal Explant Cultures

    OpenAIRE

    Miyamoto, David M.; Ruff, Kristin; Beach, Nathan M.; Dorsey-Oresto, Angella; Masters, Isaac; Temple, Louise M.

    2011-01-01

    Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embr...

  7. Detection of IgG antibodies against Bordetella pertussis with /sup 125/I-protein A

    Energy Technology Data Exchange (ETDEWEB)

    Wirsing von Koenig, C.H.; Finger, H.

    1981-01-01

    A method for the detection of IgG antibodies against Bordetella pertussis is described, based on the principle of 'sandwich' radioimmunoassay. /sup 125/I protein A is used as radioactive tracer. The influence of amounts of antigen, antibody, radioactive tracer, incubation time and temperature were tested and the optimal conditions for the assay are described. The procedure offers a simple, quick, and sensitive method for detecting antibodies against B. pertussis. Application and limitation of the test are discussed.

  8. The potential role of subclinical Bordetella Pertussis colonization in the etiology of multiple sclerosis.

    Science.gov (United States)

    Rubin, Keith; Glazer, Steven

    2016-04-01

    It is established that (1) subclinical Bordetella pertussis colonization of the nasopharynx persists in highly vaccinated populations, and (2) B. pertussis toxin is a potent adjuvant that, when co-administered with neural antigens, induces neuropathology in experimental autoimmune encephalomyelitis, the principle animal model of multiple sclerosis. Building on these observations with supporting epidemiologic and biologic evidence, we propose that, contrary to conventional wisdom that subclinical pertussis infections are innocuous to hosts, B. pertussis colonization is an important cause of multiple sclerosis.

  9. Understanding receptor specificity through the massively variable major tropism determinant of Bordetella bacteriophage

    OpenAIRE

    Jason L Miller

    2006-01-01

    Only a few protein folds are known to tolerate massive sequence variation for the sake of binding diversity. The immunoglobulin (Ig)-fold found in the structural design of antibodies and T-cell receptors as well as the leucine- rich repeat-fold (LRR's) discovered in the variable lymphocyte receptors of the agnathous hagfish are capable of withstanding recombinant variability on the order of 1014-1016 unique protein sequences. Our studies have elucidated the structure of Bordetella bacteriopha...

  10. Piracy of adhesins: attachment of superinfecting pathogens to respiratory cilia by secreted adhesins of Bordetella pertussis.

    Science.gov (United States)

    Tuomanen, E

    1986-12-01

    Two proteins secreted by Bordetella pertussis are known to mediate adherence of these bacteria to mammalian respiratory cilia. When either ciliated cells or other pathogenic bacteria were pretreated with these adhesins, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus acquired the ability to adhere to cilia in vitro and in vivo. Such piracy of adhesins may contribute to superinfection in mucosal diseases such as whooping cough.

  11. Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice.

    Directory of Open Access Journals (Sweden)

    Matt S Conover

    Full Text Available Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA. In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.

  12. Biodegradation of endosulfan isomers and its metabolite endosulfate by two biosurfactant producing bacterial strains of Bordetella petrii.

    Science.gov (United States)

    Odukkathil, Greeshma; Vasudevan, Namasivayam

    2015-01-01

    The main objective of the investigation was to study the biodegradation of endosulfan isomers and its major metabolite endosulfate by two biosurfactant producing bacterial strains of Bordetella petrii. The significance of the study is to evaluate the capability of biosurfactant producing bacterial strains in enhancing the bioavailability of endosulfan. Sixty bacterial strains were isolated from the endosulfan degrading bacterial consortium and were screened for endosulfan degradation and biosurfactant production. Among those, two strains Bordetella petrii I GV 34 (Gene bank Accession No KJ02262) and Bordetella petrii II GV 36 (Gene bank Accession No KJ022625) were capable of degrading endosulfan with simultaneous biosurfactant production. Bordetella petrii I degraded 89% of α and 84% of β isomers of endosulfan whereas Bordetella petrii II degraded 82% of both the isomers. Both the strains were able to reduce the surface tension up to 19.6% and 21.4% with a minimum observed surface tension of 45 Dynes/cm and 44 Dynes/cm, respectively. The study revealed that the strains have the potential to enhance the degradation endosulfan residues in contaminated sites and water by biosurfactant production.

  13. Pasteurella multocida and Bordetella bronchiseptica in atrophic rhinitis and pneumonia in swine.

    OpenAIRE

    Cowart, R P; Bäckström, L; Brim, T A

    1989-01-01

    A total of 163 pigs from nine farrow-to-finish herds representing various levels of atrophic rhinitis (AR) were selected for postslaughter examination of AR and pneumonia. Nasal swabs and lungs were cultured for detection of Bordetella bronchiseptica and Pasteurella multocida. Seventy-three pigs were examined at eight weeks of age and 90 contemporaries at six months of age. Mean AR scores were 1.21 and 1.11 for the eight week and six month old pigs, respectively (0 = normal, 3 = severe). In i...

  14. Produccion de suspensiones de bordetella pertussis por fermentación

    OpenAIRE

    2011-01-01

    En este trabajo se estudió la producción de suspensión de Bordetella pertussis por fermentación para obtener el ingrediente activo de la vacuna contra tosferina. Se probaron diferentes medios de cultivo para el proceso, seleccionando el medio Stainer-Scholte adicionado con 3 g/L de casaminoacidos, el cual permite obtener altas concentraciones de células y suspensiones de buena calidad. Se estudió también la cinética de consumo de glutamato de sodio, producción de biomasa y evolución del pH. E...

  15. Respiratory disease associated with Bordetella bronchiseptica in a Hoffmann's two-toed sloth (Choloepus hoffmanni).

    Science.gov (United States)

    Hammond, Elizabeth E; Sosa, Daniel; Beckerman, Robert; Aguilar, Roberto F

    2009-06-01

    A 2-yr-old female captive-born Hoffmann's two-toed sloth (Choloepus hoffmanni) presented with respiratory disease. A severe inspiratory dyspnea with nasal congestion was observed with open-mouthed breathing and bilateral mucopurulent nasal exudate. Despite initial treatment with broad-spectrum antimicrobial therapy and anti-inflammatory and supportive care, the dyspnea persisted. The animal was anesthetized for bronchoscopy to obtain a deep tracheal sample. Based on culture of Bordetella bronchiseptica and sensitivity, a combination of systemic enrofloxacin, dexamethasone, and coupage with nebulization of saline, gentamicin, and albuterol as well as supportive care resulted in full recovery after 6 weeks of treatment.

  16. Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production.

    Science.gov (United States)

    Giardina, P C; Foster, L A; Toth, S I; Roe, B A; Dyer, D W

    1995-12-29

    The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced. The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846. Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron. The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin.

  17. Seroprevalence of pertussis in the Gambia : evidence for continued circulation of bordetella pertussis despite high vaccination rates

    NARCIS (Netherlands)

    Scott, Susana; van der Sande, Marianne; Faye-Joof, Tisbeh; Mendy, Maimuna; Sanneh, Bakary; Barry Jallow, Fatou; de Melker, Hester; van der Klis, Fiona; van Gageldonk, Pieter; Mooi, Frits; Kampmann, Beate

    2015-01-01

    BACKGROUND: Bordetella pertussis can cause severe respiratory disease and death in children. In recent years, large outbreaks have occurred in high-income countries; however, little is known about pertussis incidence in sub-Saharan Africa. METHODS: We evaluated antibody responses to pertussis toxin

  18. Pertactin-negative variants of Bordetella pertussis in New York State: a retrospective analysis, 2004-2013.

    Science.gov (United States)

    Quinlan, Tammy; Musser, Kimberlee A; Currenti, Salvatore A; Zansky, Shelley M; Halse, Tanya A

    2014-08-01

    The first report of pertactin-negative variants of Bordetella pertussis in the United States has raised questions about the role of acellular pertussis vaccines in the recent increase of pertussis cases. Our laboratory utilized a sequence-based method to identify mutations in the pertactin gene responsible for these variants and assessed vaccination status from the associated cases.

  19. Acquisition and loss of virulence-associated factors during genome evolution and speciation in three clades of Bordetella species

    Science.gov (United States)

    Bacteria in the genus Bordetella include nine species that are important pathogens. B. pertussis causes whooping cough, a serious and sometimes fatal disease in infants and in elderly people. Some strains of B. parapertussis also cause whooping cough-like disease in children while others cause pn...

  20. Evaluation of Amplification Targets for the Specific Detection of Bordetella pertussis Using Real-Time Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Mohammad Rubayet Hasan

    2014-01-01

    Full Text Available BACKGROUND: Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.

  1. Bordetella avium causes induction of apoptosis and nitric oxide synthase in turkey tracheal explant cultures.

    Science.gov (United States)

    Miyamoto, David M; Ruff, Kristin; Beach, Nathan M; Stockwell, Stephanie B; Dorsey-Oresto, Angella; Masters, Isaac; Temple, Louise M

    2011-09-01

    Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.

  2. Direct Detection of Erythromycin-Resistant Bordetella pertussis in Clinical Specimens by PCR.

    Science.gov (United States)

    Wang, Zengguo; Han, Ruijun; Liu, Ying; Du, Quanli; Liu, Jifeng; Ma, Chaofeng; Li, Hengxin; He, Qiushui; Yan, Yongping

    2015-11-01

    Resistance of Bordetella pertussis to erythromycin has been increasingly reported. We developed an allele-specific PCR method for rapid detection of erythromycin-resistant B. pertussis directly from nasopharyngeal (NP) swab samples submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA of B. pertussis, four primers, two of which were designed to be specific for either the wild-type or the mutant allele, were used in two different versions of the allele-specific PCR assay. The methods were verified with results obtained by PCR-based sequencing of 16 recent B. pertussis isolates and 100 NP swab samples submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin-susceptible and -resistant B. pertussis. Two amplified fragments of each PCR, of 286 and 112 bp, respectively, were obtained from a mutant allele of the isolates and/or NP swab samples containing B. pertussis DNAs. For the wild-type allele, only a 286-bp fragment was visible when the allele-specific PCR assay 1 was performed. No amplification was found when a number of non-Bordetella bacterial pathogens and NP swab samples that did not contain the DNAs of B. pertussis were examined. This assay can serve as an alternative for PCR-based sequencing, especially for local laboratories in resource-poor countries.

  3. In vivo phosphorylation dynamics of the Bordetella pertussis virulence-controlling response regulator BvgA.

    Science.gov (United States)

    Boulanger, Alice; Chen, Qing; Hinton, Deborah M; Stibitz, Scott

    2013-04-01

    We have used protein electrophoresis through polyacrylamide gels derivatized with the proprietary ligand Phos-tag™ to separate the response regulator BvgA from its phosphorylated counterpart BvgA∼P. This approach has allowed us to readily ascertain the degree of phosphorylation of BvgA in in vitro reactions, or in crude lysates of Bordetella pertussis grown under varying laboratory conditions. We have used this technique to examine the kinetics of BvgA phosphorylation after shift of B. pertussis cultures from non-permissive to permissive conditions, or of its dephosphorylation following a shift from permissive to non-permissive conditions. Our results provide the first direct evidence that levels of BvgA∼P in vivo correspond temporally to the expression of early and late BvgA-regulated virulence genes. We have also examined a number of other aspects of BvgA function predicted from previous studies and by analogy with other two-component response regulators. These include the site of BvgA phosphorylation, the exclusive role of the cognate BvgS sensor kinase in its phosphorylation in Bordetella pertussis, and the effect of the T194M mutation on phosphorylation. We also detected the phosphorylation of a small but consistent fraction of BvgA purified after expression in Escherichia coli.

  4. PROLIFERATION RESPONSES IN PREIMMUNIZED MICE LYMPHOCYTES BY BORDETELLA PERTUSSIS CELL WALL COMPONENTS

    Directory of Open Access Journals (Sweden)

    Ashraf Mohabbati Mobarez

    2003-03-01

    Full Text Available Bordetella pertussis infects the respiratory tract of the human host and causes whooping cough in children. The nature of immunity against Bordetella pertussis infection and disease is poorly understood. The aim of this study was to investigate cell mediated immunity in mice immunized with outer membrane component of cell wall, of B. Pertussis.A group of mice were immunized with outer membrane complex (OMC and killed whole cell (WCV of B. pertussis, with an interval of 2 weeks. During a period of 7 weeks following the immunization, lymphocytes were isolated from lymph nodes of immunized mice. The in vitro proliferative response of isolated lymphocyte to stimulation with 20 ^g of 30 and 69 kDa outer membrane protein (OMP were measured as parameters for cell mediated immunity (CMI. The data were expressed as mean count per minute (CPMxlO3 after subtraction of the CPM of unstimulated control cultures. Lymphoblastogenic response was observed in immunized mice with WCV and OMC. At 30 days of post immunization a significant increase in response to 30 and 69 kDa OMP was observed, a small decrease in the response was evident against P30 and P69 at 60 and 120 days of post immunization, but the response was still higher than what was observed in control mice.Current findings indicate strongly the potential of outer membrane protein component of B. pertussis in proliferating lymphocytes in the mice.

  5. Evidence of Bordetella pertussis infection in vaccinated 1-year-old Danish children

    DEFF Research Database (Denmark)

    von Linstow, Marie-Louise; Pontoppidan, Peter Lotko; von König, Carl-Heinz Wirsing

    2010-01-01

    We measured IgA and IgG antibodies to pertussis toxin (PT) and filamentous hemagglutinin (FHA) in sera from 203 1-year-old children who had received one to three doses of a monocomponent PT toxoid vaccine. Ten children (5%) had IgA antibody to PT indicating recent infection; seven of these children...... had received three doses of vaccine. PT IgA responders did not have significantly longer coughing episodes than PT IgA non-responders. Since an IgA antibody response occurs in only approximately 50% of infected children, the actual infection rate in our cohort is estimated to approximately 10......%. The apparent high Bordetella pertussis infection rate in Danish infants suggests that the monocomponent PT toxoid vaccine used in Denmark has limited efficacy against B. pertussis infection. A prospective immunization study comparing a multi-component vaccine with the present monocomponent PT toxoid vaccine...

  6. Mouse and pig models for studies of natural and vaccine-induced immunity to Bordetella pertussis.

    Science.gov (United States)

    Mills, Kingston H G; Gerdts, Volker

    2014-04-01

    The increasing incidence of whooping cough in many developed countries has been linked with waning immunity induced after immunization with acellular pertussis (aP) vaccines. The rational design of an improved aP vaccine requires a full understanding of the mechanism of protective immunity and preclinical studies in animal models. Infection of mice and pigs with Bordetella pertussis has many features of the infection seen in humans and has already provided valuable information on the roles of innate and adaptive immune responses in protection. Recent findings in these models have already indicated that it may be possible to develop an improved aP vaccine based on a formulation that includes a Toll-like receptor agonist as an adjuvant.

  7. Importance of (antibody-dependent) complement-mediated serum killing in protection against Bordetella pertussis.

    Science.gov (United States)

    Geurtsen, Jeroen; Fae, Kellen C; van den Dobbelsteen, Germie P J M

    2014-10-01

    Pertussis is a highly contagious respiratory disease that is caused by Bordetella pertussis. Despite being vaccine preventable, pertussis rates have been rising steadily over the last decades, even in areas with high vaccine uptake. Recently, experiments with infant baboons indicated that although vaccination with acellular pertussis vaccines prevented disease, no apparent effect was observed on infection and transmission. One explanation may be that current acellular pertussis vaccines do not induce high levels of opsonophagocytic and/or bactericidal activity, implying that engineering of vaccines that promote bacterial killing may improve efficacy. Here, we discuss the importance of complement-mediated killing in vaccine-induced protection against B. pertussis. We first examine how B. pertussis may have evolved different complement evasion strategies. Second, we explore the benefits of opsonophagocytic and/or bactericidal killing in vaccine-induced protection and discuss whether or not inclusion of new opsonophagocytic or bactericidal target antigens in pertussis vaccines may benefit efficacy.

  8. Pertactin deficient Bordetella pertussis present a better fitness in mice immunized with an acellular pertussis vaccine.

    Science.gov (United States)

    Hegerle, N; Dore, G; Guiso, N

    2014-11-20

    Bordetella pertussis is the etiologic agent of whooping cough and has been the target of vaccination for over fifty years. The latest strategies include the use of acellular pertussis vaccines that induce specific immunity against few virulence factors amongst which pertactin is included in three and five component acellular pertussis vaccines. Recently, it has been reported that B. pertussis clinical isolates loose the production of this adhesin in regions reaching high vaccine coverage with vaccines targeting this virulence factor. We here demonstrate that isolates not producing pertactin are capable of sustaining longer infection as compared to pertactin producing isolates in an in vivo model of acellular pertussis immunization. Loosing pertactin production might thus provide a selective advantage to these isolates in this background, which could account for the upraise in prevalence of these pertactin deficient isolates in the population.

  9. Complete genome sequence of a clinical Bordetella pertussis isolate from Brazil

    Directory of Open Access Journals (Sweden)

    Bruno Gabriel N Andrade

    2014-11-01

    Full Text Available There has been a resurgence in the number of pertussis cases in Brazil and around the world. Here, the genome of a clinical Bordetella pertussis strain (Bz181 that was recently isolated in Brazil is reported. Analysis of the virulence-associated genes defining the pre- and post-vaccination lineages revealed the presence of the prn2-ptxS1A-fim3B-ptxP3 allelic profile in Bz181, which is characteristic of the current pandemic lineage. A putative metallo-β-lactamase gene presenting all of the conserved zinc-binding motifs that characterise the catalytic site was identified, in addition to a multidrug efflux pump of the RND family that could confer resistance to erythromycin, which is the antibiotic of choice for treating pertussis disease.

  10. A dynamic metabolic flux balance based model of fed-batch fermentation of Bordetella pertussis.

    Science.gov (United States)

    Budman, Hector; Patel, Nilesh; Tamer, Melih; Al-Gherwi, Walid

    2013-01-01

    A mathematical model based on a dynamic metabolic flux balance (DMFB) is developed for a process of fed-batch fermentation of Bordetella pertussis. The model is based on the maximization of growth rate at each time interval subject to stoichiometric constraints. The model is calibrated and verified with experimental data obtained in two different bioreactor experimental systems. It was found that the model calibration was mostly sensitive to the consumption or production rates of tyrosine and, for high supplementation rates, to the consumption rate of glutamate. Following this calibration the model correctly predicts biomass and by-products concentrations for different supplementation rates. Comparisons of model predictions to oxygen uptake and carbon emission rates measurements indicate that the TCA cycle is fully functional.

  11. Comparison of lipopolysaccharide structures of Bordetella pertussis clinical isolates from pre- and post-vaccine era.

    Science.gov (United States)

    Albitar-Nehme, Sami; Basheer, Soorej M; Njamkepo, Elisabeth; Brisson, Jean-Robert; Guiso, Nicole; Caroff, Martine

    2013-08-30

    Endotoxins are lipopolysaccharides (LPS), and major constituents of the outer membrane of Gram-negative bacteria. Bordetella pertussis LPS were the only major antigens, of this agent of whooping-cough, that were not yet analyzed on isolates from the pre- and post-vaccination era. We compared here the LPS structures of four clinical isolates with that of the vaccine strain BP 1414. All physico-chemical analyses, including SDS-PAGE, TLC, and different MALDI mass spectrometry approaches were convergent. They helped demonstrating that, on the contrary to some other B. pertussis major antigens, no modification occurred in the dodecasaccharide core structure, as well as in the whole LPS molecules. These results are rendering these major antigens good potential vaccine components. Molecular modeling of this conserved LPS structure also confirmed the conclusions of previous experiments leading to the production of anti-LPS monoclonal antibodies and defining the main epitopes of these major antigens.

  12. Complete genome sequence of a clinical Bordetella pertussis isolate from Brazil.

    Science.gov (United States)

    Andrade, Bruno Gabriel N; Marin, Michel F Abanto; Cambuy, Diego Duque; Fonseca, Erica Lourenço; Souza, Nadjla Ferreira; Vicente, Ana Carolina P

    2014-11-01

    There has been a resurgence in the number of pertussis cases in Brazil and around the world. Here, the genome of a clinical Bordetella pertussis strain (Bz181) that was recently isolated in Brazil is reported. Analysis of the virulence-associated genes defining the pre- and post-vaccination lineages revealed the presence of the prn2-ptxS1A-fim3B-ptxP3 allelic profile in Bz181, which is characteristic of the current pandemic lineage. A putative metallo-β-lactamase gene presenting all of the conserved zinc-binding motifs that characterise the catalytic site was identified, in addition to a multidrug efflux pump of the RND family that could confer resistance to erythromycin, which is the antibiotic of choice for treating pertussis disease.

  13. Effects of polysaccharide on chicks co-infected with Bordetella avium and Avian leukosis virus.

    Science.gov (United States)

    Guo, Fanxia; Xue, Cong; Wu, Cun; Zhao, Xue; Qu, Tinghe; He, Xiaohua; Guo, Zhongkun; Zhu, Ruiliang

    2014-08-30

    Chicks' co-infection with immunosuppressive virus and bacteria seriously threaten the development of the poultry industry. In this study, a model was established in which chicks were injected with either subgroup B ALV (ALV-B)+Bordetella avium (B. avium), or ALV-B+B. avium+Taishan Pinus massoniana pollen polysaccharide (TPPPS), or B. avium only, or B. avium+TPPPS. The data showed that the group injected with ALV-B and B. avium exhibited significant inhibition of the immune function and therefore increased pathogenicity compared with the group injected with B. avium-only. Application of TPPPS effectively alleviated immunosuppression, and body weights increased sharply in the TPPPS groups compared with non-TPPPS groups. To some extent, TPPPS may reduce the proliferation of ALV-B. These results suggest that Pinus pollen polysaccharides are beneficial treating co-infections with immunosuppressive virus and bacteria and therefore have potential for development into safe and effective immunoregulator.

  14. Produccion de suspensiones de bordetella pertussis por fermentación

    Directory of Open Access Journals (Sweden)

    Algecira N.

    1998-12-01

    Full Text Available En este trabajo se estudió la producción de suspensión de Bordetella pertussis por fermentación para obtener el ingrediente activo de la vacuna contra tosferina. Se probaron diferentes medios de cultivo para el proceso, seleccionando el medio Stainer-Scholte adicionado con 3 g/L de casaminoacidos, el cual permite obtener altas concentraciones de células y suspensiones de buena calidad. Se estudió también la cinética de consumo de glutamato de sodio, producción de biomasa y evolución del pH. El crecimiento fue descrito por un modelo logístico. Se compara la tecnología de cultivo estacionario con el cultivo en fermentador presentándose esta última como la mejor alternativa de producción.

  15. Cough and fear of sleep: early clinical signs of Bordetella pertussis in an adult

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    Thomas C. Jones

    2004-08-01

    Full Text Available Pertussis is increasing in frequency among adults, but early diagnosis requires special attention to details in the medical history. We describe a 64 year-old male with classic signs and symptoms of pertussis and documented Bordetella pertussis infection that were overlooked because he presented with a chief complaint of cough and fear of falling asleep. Coughing paroxysms and a feeling of suffocation (30-60 seconds only occurred at night after short periods of deep sleep (30-45 minutes. The physicians did not observe these episodes during daytime examinations, and the basis of the patient's fear of sleep was not explored. We recommend reassessment of how adults describe symptoms of pertussis, including fear of sleep, and we suggest the use of PCR technology to allow early diagnosis and prompt treatment.

  16. Growth phase- and nutrient limitation-associated transcript abundance regulation in Bordetella pertussis.

    Science.gov (United States)

    Nakamura, Mari M; Liew, Sin-Yee; Cummings, Craig A; Brinig, Mary M; Dieterich, Christine; Relman, David A

    2006-10-01

    To survive in a host environment, microbial pathogens must sense local conditions, including nutrient availability, and adjust their growth state and virulence functions accordingly. No comprehensive investigation of growth phase-related gene regulation in Bordetella pertussis has been reported previously. We characterized changes in genome-wide transcript abundance of B. pertussis as a function of growth phase and availability of glutamate, a key nutrient for this organism. Using a Bordetella DNA microarray, we discovered significant changes in transcript abundance for 861 array elements during the transition from log phase to stationary phase, including declining transcript levels of many virulence factor genes. The responses to glutamate depletion exhibited similarities to the responses induced by exit from log phase, including decreased virulence factor transcript levels. However, only 23% of array elements that showed at least a fourfold growth phase-associated difference in transcript abundance also exhibited glutamate depletion-associated changes, suggesting that nutrient limitation may be one of several interacting factors affecting gene regulation during stationary phase. Transcript abundance patterns of a Bvg+ phase-locked mutant revealed that the BvgAS two-component regulatory system is a key determinant of growth phase- and nutrient limitation-related transcriptional control. Several adhesin genes exhibited lower transcript abundance during stationary phase and under glutamate restriction conditions. The predicted bacterial phenotype was confirmed: adherence to bronchoepithelial cells decreased 3.3- and 4.4-fold at stationary phase and with glutamate deprivation, respectively. Growth phase and nutrient availability may serve as cues by which B. pertussis regulates virulence according to the stage of infection or the location within the human airway.

  17. Survey and Rapid detection of Bordetella pertussis in clinical samples targeting the BP485 in China

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    Wei eLiu

    2015-03-01

    Full Text Available Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg/µl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 Bordetella pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing (MLST groups with different alleles of the virulence-related genes including 4 alleles of ptxA, 6 of prn, 4 of tcfA, 2 of fim2 and 3 of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.

  18. Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

    Science.gov (United States)

    Selwa, Edithe; Davi, Marilyne; Chenal, Alexandre; Sotomayor-Pérez, Ana-Cristina; Ladant, Daniel; Malliavin, Thérèse E

    2014-07-25

    Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

  19. A Comparative Study of Detection of Bordetella avium Antibodies in Turkeys by ELISA, SPAT, and AGID Test

    OpenAIRE

    TÜRKYILMAZ, Süheyla; TÜRKYILMAZ, Kenan; KAYA, Osman

    2006-01-01

    The aims of this study were to develop a serum plate agglutination test (SPAT) antigen and agar gel immunodiffusion (AGID) test antigen for the serological detection of turkeys that have been exposed to Bordetella avium; to compare the sensitivity of commercial enzyme-linked immunosorbent assay (ELISA) with SPAT, and AGID test, and to survey B. avium antibodies in turkey flocks in Aydın, Turkey. For these purposes, serum samples collected from 300 turkeys were examined by ELISA, SPAT, and AGI...

  20. Bordetella pertussis lipid A glucosamine modification confers resistance to cationic antimicrobial peptides and increases resistance to outer membrane perturbation.

    Science.gov (United States)

    Shah, Nita R; Hancock, Robert E W; Fernandez, Rachel C

    2014-08-01

    Bordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

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

    Science.gov (United States)

    Antila, Mia; He, Qiushui; de Jong, Caroline; Aarts, Ingrid; Verbakel, Harold; Bruisten, Sylvia; Keller, Suzanne; Haanperä, Marjo; Mäkinen, Johanna; Eerola, Erkki; Viljanen, Matti K; Mertsola, Jussi; van der Zee, Anneke

    2006-08-01

    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.

  2. A real-time PCR assay with improved specificity for detection and discrimination of all clinically relevant Bordetella species by the presence and distribution of three Insertion Sequence elements

    Directory of Open Access Journals (Sweden)

    Ossewaarde Jacobus M

    2011-01-01

    Full Text Available Abstract Background In Dutch laboratories molecular detection of B. pertussis and B. parapertussis is commonly based on insertion sequences IS481 and IS1001, respectively. Both IS elements are more widely spread among Bordetella species. Both Bordetella holmesii, and B. bronchiseptica can harbour IS481. Also, IS1001 is found among B. bronchiseptica. IS481, and IS1001 based PCR thus lacks specificity when used for detection of specific Bordetella spp. Findings We designed a PCR based on IS1002, another IS element that is present among Bordetella species, and exploited it as a template in combination with PCR for IS481, and IS1001. In combining the PCRs for IS481, IS1001, and IS1002, and including an inhibition control, we were able to detect and discriminate all clinically relevant Bordetella species. Conclusions We developed an improved PCR method for specific detection of B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica.

  3. Identification and regulation of expression of a gene encoding a filamentous hemagglutinin-related protein in Bordetella holmesii

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    Gross Roy

    2007-11-01

    Full Text Available Abstract Background Bordetella holmesii is a human pathogen closely related to B. pertussis, the etiological agent of whooping cough. It is able to cause disease in immunocompromised patients, but also whooping cough-like symptoms in otherwise healthy individuals. However, virtually nothing was known so far about the underlying virulence mechanisms and previous attempts to identify virulence factors related to those of B. pertussis were not successful. Results By use of a PCR approach we were able to identify a B. holmesii gene encoding a protein with significant sequence similarities to the filamentous hemagglutinin (FHA of B. avium and to a lesser extent to the FHA proteins of B. pertussis, B. parapertussis, and B. bronchiseptica. For these human and animal pathogens FHA is a crucial virulence factor required for successful colonization of the host. Interestingly, the B. holmesii protein shows a relatively high overall sequence similarity with the B. avium protein, while sequence conservation with the FHA proteins of the human and mammalian pathogens is quite limited and is most prominent in signal sequences required for their export to the cell surface. In the other Bordetellae expression of the fhaB gene encoding FHA was shown to be regulated by the master regulator of virulence, the BvgAS two-component system. Recently, we identified orthologs of BvgAS in B. holmesii, and here we show that this system also contributes to regulation of fhaB expression in B. holmesii. Accordingly, the purified BvgA response regulator of B. holmesii was shown to bind specifically in the upstream region of the fhaB promoter in vitro in a manner similar to that previously described for the BvgA protein of B. pertussis. Moreover, by deletion analysis of the fhaB promoter region we show that the BvgA binding sites are relevant for in vivo transcription from this promoter in B. holmesii. Conclusion The data reported here show that B. holmesii is endowed with a

  4. Global population structure and evolution of Bordetella pertussis and their relationship with vaccination.

    Science.gov (United States)

    Bart, Marieke J; Harris, Simon R; Advani, Abdolreza; Arakawa, Yoshichika; Bottero, Daniela; Bouchez, Valérie; Cassiday, Pamela K; Chiang, Chuen-Sheue; Dalby, Tine; Fry, Norman K; Gaillard, María Emilia; van Gent, Marjolein; Guiso, Nicole; Hallander, Hans O; Harvill, Eric T; He, Qiushui; van der Heide, Han G J; Heuvelman, Kees; Hozbor, Daniela F; Kamachi, Kazunari; Karataev, Gennady I; Lan, Ruiting; Lutyńska, Anna; Maharjan, Ram P; Mertsola, Jussi; Miyamura, Tatsuo; Octavia, Sophie; Preston, Andrew; Quail, Michael A; Sintchenko, Vitali; Stefanelli, Paola; Tondella, M Lucia; Tsang, Raymond S W; Xu, Yinghua; Yao, Shu-Man; Zhang, Shumin; Parkhill, Julian; Mooi, Frits R

    2014-04-22

    Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis

  5. Differentially expressed genes in Bordetella pertussis strains belonging to a lineage which recently spread globally.

    Science.gov (United States)

    de Gouw, Daan; Hermans, Peter W M; Bootsma, Hester J; Zomer, Aldert; Heuvelman, Kees; Diavatopoulos, Dimitri A; Mooi, Frits R

    2014-01-01

    Pertussis is a highly contagious, acute respiratory disease in humans caused by the Gram-negative pathogen Bordetella pertussis. Pertussis has resurged in the face of intensive vaccination and this has coincided with the emergence of strains carrying a particular allele for the pertussis toxin promoter, ptxP3, which is associated with higher levels of pertussis toxin (Ptx) production. Within 10 to 20 years, ptxP3 strains have nearly completely replaced the previously dominant ptxP1 strains resulting in a worldwide selective sweep. In order to identify B. pertussis genes associated with the selective sweep, we compared the expression of genes in ptxP1 and ptxP3 strains that are under control of the Bordetella master virulence regulatory locus (bvgASR). The BvgAS proteins comprise a two component sensory transduction system which is regulated by temperature, nicotinic acid and sulfate. By increasing the sulfate concentration, it is possible to change the phase of B. pertussis from virulent to avirulent. Until recently, the only distinctive phenotype of ptxP3 strains was a higher Ptx production. Here we identify additional phenotypic differences between ptxP1 and ptxP3 strains which may have contributed to its global spread by comparing global transcriptional responses under sulfate-modulating conditions. We show that ptxP3 strains are less sensitive to sulfate-mediated gene suppression, resulting in an increased production of the vaccine antigens pertactin (Prn) and Ptx and a number of other virulence genes, including a type III secretion toxin, Vag8, a protein involved in complement resistance, and lpxE involved in lipid A modification. Furthermore, enhanced expression of the vaccine antigens Ptx and Prn by ptxP3 strains was confirmed at the protein level. Identification of genes differentially expressed between ptxP1 and ptxP3 strains may elucidate how B. pertussis has adapted to vaccination and allow the improvement of pertussis vaccines by identifying novel

  6. Differentially expressed genes in Bordetella pertussis strains belonging to a lineage which recently spread globally.

    Directory of Open Access Journals (Sweden)

    Daan de Gouw

    Full Text Available Pertussis is a highly contagious, acute respiratory disease in humans caused by the Gram-negative pathogen Bordetella pertussis. Pertussis has resurged in the face of intensive vaccination and this has coincided with the emergence of strains carrying a particular allele for the pertussis toxin promoter, ptxP3, which is associated with higher levels of pertussis toxin (Ptx production. Within 10 to 20 years, ptxP3 strains have nearly completely replaced the previously dominant ptxP1 strains resulting in a worldwide selective sweep. In order to identify B. pertussis genes associated with the selective sweep, we compared the expression of genes in ptxP1 and ptxP3 strains that are under control of the Bordetella master virulence regulatory locus (bvgASR. The BvgAS proteins comprise a two component sensory transduction system which is regulated by temperature, nicotinic acid and sulfate. By increasing the sulfate concentration, it is possible to change the phase of B. pertussis from virulent to avirulent. Until recently, the only distinctive phenotype of ptxP3 strains was a higher Ptx production. Here we identify additional phenotypic differences between ptxP1 and ptxP3 strains which may have contributed to its global spread by comparing global transcriptional responses under sulfate-modulating conditions. We show that ptxP3 strains are less sensitive to sulfate-mediated gene suppression, resulting in an increased production of the vaccine antigens pertactin (Prn and Ptx and a number of other virulence genes, including a type III secretion toxin, Vag8, a protein involved in complement resistance, and lpxE involved in lipid A modification. Furthermore, enhanced expression of the vaccine antigens Ptx and Prn by ptxP3 strains was confirmed at the protein level. Identification of genes differentially expressed between ptxP1 and ptxP3 strains may elucidate how B. pertussis has adapted to vaccination and allow the improvement of pertussis vaccines by

  7. Evaluation of real-time PCR for diagnosis of Bordetella pertussis infection

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    Fox Julie D

    2006-03-01

    Full Text Available Abstract Background Nucleic acid amplification of the IS481 region by PCR is more sensitive than culture for detection and diagnosis of Bordetella pertussis but the assay has known cross-reactivity for Bordetella holmesii and its use as a routine diagnostic assay has not been widely evaluated. Methods The objectives of this study were: 1 to assess the diagnostic utility of real-time IS481 PCR by comparison of results with culture and direct fluorescent antigen (DFA testing for B. pertussis, 2 to employ a PCR assay designed against a different insertion sequence (IS1001 to assess the incidence of B. holmesii in symptomatic individuals and 3 to design and evaluate a new PCR-based assay which could be used for B. pertussis confirmation. A total of 808 nasopharyngeal specimens were included in the study the majority of which were submitted in charcoal transport medium (88% with the rest submitted in Regan-Lowe medium. Results Concordant results for PCR, DFA and culture were obtained for 21 B. pertussis positive and 729 B. pertussis negative specimens. DFA was prone to false positive and negative reactions when compared with both PCR and culture. The IS481 PCR identified 28 positive results for specimens that were DFA and culture negative. A novel real-time PCR targeting the B. pertussis toxin promoter was found to be specific and useful for confirming the majority of IS481 positive specimens as B. pertussis. B. holmesii was not detected in any of the submitted samples. Conclusion The potential pick up of B. holmesii by the IS481 PCR had minimal diagnostic relevance in the Alberta population during the time period of our study. The IS481 PCR assay is now used in our laboratory routinely for front-line screening of samples for B. pertussis with associated enhancement in diagnostic sensitivity compared with DFA and culture. Retrospectively, patients' samples are batched and tested by the IS1001 MB and TPR assays for research purposes and to ensure

  8. Membrane-Pore Forming Characteristics of the Bordetella pertussis CyaA-Hemolysin Domain

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    Chattip Kurehong

    2015-04-01

    Full Text Available Previously, the 126-kDa Bordetella pertussis CyaA pore-forming/hemolysin (CyaA-Hly domain was shown to retain its hemolytic activity causing lysis of susceptible erythrocytes. Here, we have succeeded in producing, at large quantity and high purity, the His-tagged CyaA-Hly domain over-expressed in Escherichia coli as a soluble hemolytically-active form. Quantitative assays of hemolysis against sheep erythrocytes revealed that the purified CyaA-Hly domain could function cooperatively by forming an oligomeric pore in the target cell membrane with a Hill coefficient of ~3. When the CyaA-Hly toxin was incorporated into planar lipid bilayers (PLBs under symmetrical conditions at 1.0 M KCl, 10 mM HEPES buffer (pH 7.4, it produced a clearly resolved single channel with a maximum conductance of ~35 pS. PLB results also revealed that the CyaA-Hly induced channel was unidirectional and opened more frequently at higher negative membrane potentials. Altogether, our results first provide more insights into pore-forming characteristics of the CyaA-Hly domain as being the major pore-forming determinant of which the ability to induce such ion channels in receptor-free membranes could account for its cooperative hemolytic action on the target erythrocytes.

  9. Population diversity among Bordetella pertussis isolates, United States, 1935-2009.

    Science.gov (United States)

    Schmidtke, Amber J; Boney, Kathryn O; Martin, Stacey W; Skoff, Tami H; Tondella, M Lucia; Tatti, Kathleen M

    2012-08-01

    Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935-2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970-1990 (94%) but declined to ≈ 70% after 1991 and has remained constant. During 2006-2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population.

  10. The drug resistance of Bordetella pertussis%百日咳杆菌耐药

    Institute of Scientific and Technical Information of China (English)

    杨永弘; 杨颖

    2016-01-01

    大环内酯类抗生素是治疗百日咳的首选药物。然而,近年来出现了百日咳杆菌对红霉素等大环内酯类抗生素耐药的现象,局部监测耐药率甚至达到90%。现将从百日咳杆菌耐药情况、耐药机制、耐药后的抗生素选择、耐药的防治和控制等方面进行综述。%Macrolides have been the first line antibiotic choice for treatment and prophylaxis of pertussis.In re-cent years,several erythromycin -resistant Bordetella pertussis(B.pertussis)isolates have been reported worldwide.Fur-thermore,the resistant rate was high to 90% in some regions.This review aimes to summarize the current status of drug resistance,the resistance mechanism and the control and prevention of the resistance of B.pertussis.

  11. Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

    Science.gov (United States)

    Dirix, Violette; Mielcarek, Nathalie; Debrie, Anne-Sophie; Willery, Eve; Alonso, Sylvie; Versheure, Virginie; Mascart, Françoise; Locht, Camille

    2014-07-01

    In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

  12. Structural and functional studies of BapC protein of Bordetella pertussis.

    Science.gov (United States)

    Riaz, Muhammad Rizwan; Siddiqi, Abdul Rauf; Bokhari, Habib

    2015-05-01

    Bordetella pertussis, the causative agent of whooping cough, attaches to mucosal surface in upper respiratory tract, where it produces a variety of surface associated and secreted autotransporter molecules among others. In this study we have cloned newly identified member of autotransporter family BapC (B. pertussis autotransporter protein C); expressed it in Escherichia coli and characterized it for its different properties. We have also raised antisera to BapC protein; the antisera were used in immunofluorescence assay to determine the surface association of the protein. Results suggest that BapC in B. pertussis Taberman parent is surface exposed when compared with the respective BapC mutant. The neutralizing effect of anti-BapC serum was also evaluated in the presence of active complement proteins and results suggest that antiserum can potentiate the killing of B. pertussis cells in the presence of added source of complement. Structure of the protein was also studied, both α and β domains of the protein were modeled, β domain exhibits typical transmembrane β-barrel porin topology whereas α domain behaves as a characteristic bacterial autotransporter passenger domain.

  13. Genetic diversity and population dynamics of Bordetella pertussis in China between 1950-2007.

    Science.gov (United States)

    Xu, Yinghua; Zhang, Liu; Tan, Yajun; Wang, Lichan; Zhang, Shumin; Wang, Junzhi

    2015-11-17

    Pertussis is an acute respiratory infectious disease caused by the bacterium Bordetella pertussis. Although pertussis vaccination was introduced in the 1960s, pertussis is still an endemic disease in China. To better understand the genetic diversity of the Chinese B. pertussis population, we characterized 115 clinical isolates obtained in China during 1950-2007 using multilocus variable-number tandem repeat analysis (MLVA). Forty-six different B. pertussis MLVA profiles (MTs) were identified, of which 13 were new MTs. Analysis using a minimum-spanning tree showed that distinct MTs were prevalent during different periods, suggesting that a dynamic change in B. pertussis MTs occurred over time in China. The predominant MTs in recent isolates from China were different from those of many developed countries. A decreasing trend in genetic diversity of the B. pertussis population was observed following the introduction of pertussis vaccines. Similar to the pertactin 2 (prn2) allele, the novel pertussis toxin promoter (ptxP3) allele first emerged in 2000, but unlike trends elsewhere, ptxP1 remained predominant among the isolates, further reflecting the unique temporal trends in the B. pertussis population in China. Our results suggest that temporal changes in the B. pertussis population may be closely associated with vaccination coverage and the vaccine types used. These data may lead to an improved understanding of the virulence mechanism of B. pertussis and facilitate new strategies for controlling this infectious disease.

  14. Pertactin negative Bordetella pertussis demonstrates higher fitness under vaccine selection pressure in a mixed infection model.

    Science.gov (United States)

    Safarchi, Azadeh; Octavia, Sophie; Luu, Laurence Don Wai; Tay, Chin Yen; Sintchenko, Vitali; Wood, Nicholas; Marshall, Helen; McIntyre, Peter; Lan, Ruiting

    2015-11-17

    Whooping cough or pertussis is a highly infectious respiratory disease in humans caused by Bordetella pertussis. The use of acellular vaccines (ACV) has been associated with the recent resurgence of pertussis in developed countries including Australia despite high vaccination coverage where B. pertussis strains that do not express pertactin (Prn), a key antigenic component of the ACV, have emerged and become prevalent. In this study, we used an in vivo competition assay in mice immunised with ACV and in naïve (control) mice to compare the proportion of colonisation with recent clinical Prn positive and Prn negative B. pertussis strains from Australia. The Prn negative strain colonised the respiratory tract more effectively than the Prn positive strain in immunised mice, out-competing the Prn positive strain by day 3 of infection. However, in control mice, the Prn positive strain out-competed the Prn negative strain. Our findings of greater ability of Prn negative strains to colonise ACV-immunised mice are consistent with reports of selective advantage for these strains in ACV-immunised humans.

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

    Science.gov (United States)

    van Gent, M; Heuvelman, C J; van der Heide, H G; Hallander, H O; Advani, A; Guiso, N; Wirsing von Kőnig, C H; Vestrheim, D F; Dalby, T; Fry, N K; Pierard, D; Detemmerman, L; Zavadilova, J; Fabianova, K; Logan, C; Habington, A; Byrne, M; Lutyńska, A; Mosiej, E; Pelaz, C; Gröndahl-Yli-Hannuksela, K; Barkoff, A M; Mertsola, J; Economopoulou, A; He, Q; Mooi, F R

    2015-04-01

    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.

  16. Whole-genome sequencing reveals the effect of vaccination on the evolution of Bordetella pertussis.

    Science.gov (United States)

    Xu, Yinghua; Liu, Bin; Gröndahl-Yli-Hannuksila, Kirsi; Tan, Yajun; Feng, Lu; Kallonen, Teemu; Wang, Lichan; Peng, Ding; He, Qiushui; Wang, Lei; Zhang, Shumin

    2015-08-18

    Herd immunity can potentially induce a change of circulating viruses. However, it remains largely unknown that how bacterial pathogens adapt to vaccination. In this study, Bordetella pertussis, the causative agent of whooping cough, was selected as an example to explore possible effect of vaccination on the bacterial pathogen. We sequenced and analysed the complete genomes of 40 B. pertussis strains from Finland and China, as well as 11 previously sequenced strains from the Netherlands, where different vaccination strategies have been used over the past 50 years. The results showed that the molecular clock moved at different rates in these countries and in distinct periods, which suggested that evolution of the B. pertussis population was closely associated with the country vaccination coverage. Comparative whole-genome analyses indicated that evolution in this human-restricted pathogen was mainly characterised by ongoing genetic shift and gene loss. Furthermore, 116 SNPs were specifically detected in currently circulating ptxP3-containing strains. The finding might explain the successful emergence of this lineage and its spread worldwide. Collectively, our results suggest that the immune pressure of vaccination is one major driving force for the evolution of B. pertussis, which facilitates further exploration of the pathogenicity of B. pertussis.

  17. Better colonisation of newly emerged Bordetella pertussis in the co-infection mouse model study.

    Science.gov (United States)

    Safarchi, Azadeh; Octavia, Sophie; Luu, Laurence Don Wai; Tay, Chin Yen; Sintchenko, Vitali; Wood, Nicholas; Marshall, Helen; McIntyre, Peter; Lan, Ruiting

    2016-07-25

    Molecular epidemiological data indicates that the resurgence of pertussis (whooping cough) in populations with high vaccine coverage is associated with genomic adaptation of Bordetella pertussis, the causative agent of the disease, to vaccine selection pressure. We have previously shown that in the period after the introduction of acellular pertussis vaccine (ACV), the majority of circulating strains in Australia switched to single nucleotide polymorphism (SNP) cluster I (carrying ptxP3/prn2), replacing SNP cluster II (carrying ptxP1/prn3). In this study, we carried out an in vivo competition assay using a mouse model infected with SNP cluster I and II B. pertussis strains from Australia. We found that the SNP cluster I strain colonised better than the SNP cluster II strain, in both naïve and immunised mice, suggesting that SNP cluster I strains had better fitness regardless of immunisation status of the host, consistent with SNP cluster I strains replacing SNP cluster II. Nevertheless, we found that ACV enhanced clearance of both SNP cluster I and II strains from the mouse respiratory tract.

  18. Purification design and practice for pertactin, the third component of acellular pertussis vaccine, from Bordetella pertussis.

    Science.gov (United States)

    Li, Zenglan; Zhang, Yan; Wang, Qi; Li, Zhengjun; Liu, Yongdong; Zhang, Songping; Zhang, Guifeng; Ma, Guanghui; Luo, Jian; Su, Zhiguo

    2016-07-25

    Development of acellular pertussis vaccine (aPV) requires purification of several components from Bordetella pertussis. While the components pertussis toxin (PT) and filamentous hemagglutinin (FHA) have been successfully purified, the third component, pertactin, proves to be a difficult target due to its very low concentration. In order to solve its purification problem, we performed the surface potential analysis with GRASP2 program. The results demonstrated that there are two major charge patches, one negative and one positive, which are located separately on this linear protein. For this special feature, we designed a dual ion exchange chromatography strategy including an anionic exchange and a cationic exchange process for separation of pertactin from the heat extract of B. pertussis. The initial anionic exchange chromatography concentrated the product from 1.7% to 14.6%, with recovery of 80%. The second cationic exchange chromatography increased the purity to 33%, with recovery of 83%. The final purification was accomplished by hydrophobic interaction chromatography, yielding a purity of 96%. The total recovery of the three columns was 61%. Characterization of the purified antigen was performed with CD, intrinsic fluorescence, HP-SEC and western-blot, showing that the purified protein kept its natural conformation and immune-reactivity. The rationally designed process proved to be feasible, and it is suitable for large-scale preparation of the third aPV component pertactin.

  19. Proteomics-identified Bvg-activated autotransporters protect against bordetella pertussis in a mouse model.

    Science.gov (United States)

    de Gouw, Daan; Gouw, Daan de; de Jonge, Marien I; Jonge, Marien I de; Hermans, Peter W M; Wessels, Hans J C T; Zomer, Aldert; Berends, Alinda; Pratt, Catherine; Berbers, Guy A; Mooi, Frits R; Diavatopoulos, Dimitri A

    2014-01-01

    Pertussis is a highly infectious respiratory disease of humans caused by the bacterium Bordetella pertussis. Despite high vaccination coverage, pertussis has re-emerged globally. Causes for the re-emergence of pertussis include limited duration of protection conferred by acellular pertussis vaccines (aP) and pathogen adaptation. Pathogen adaptations involve antigenic divergence with vaccine strains, the emergence of strains which show enhanced in vitro expression of a number of virulence-associated genes and of strains that do not express pertactin, an important aP component. Clearly, the identification of more effective B. pertussis vaccine antigens is of utmost importance. To identify novel antigens, we used proteomics to identify B. pertussis proteins regulated by the master virulence regulatory system BvgAS in vitro. Five candidates proteins were selected and it was confirmed that they were also expressed in the lungs of naïve mice seven days after infection. The five proteins were expressed in recombinant form, adjuvanted with alum and used to immunize mice as stand-alone antigens. Subsequent respiratory challenge showed that immunization with the autotransporters Vag8 and SphB1 significantly reduced bacterial load in the lungs. Whilst these antigens induced strong opsonizing antibody responses, we found that none of the tested alum-adjuvanted vaccines - including a three-component aP - reduced bacterial load in the nasopharynx, suggesting that alternative immunological responses may be required for efficient bacterial clearance from the nasopharynx.

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

    Science.gov (United States)

    Elomaa, Annika; Advani, Abdolreza; Donnelly, Declan; Antila, Mia; Mertsola, Jussi; He, Qiushui; Hallander, Hans

    2007-01-15

    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.

  1. Drug Target Identification and Elucidation of Natural Inhibitors for Bordetella petrii: An In Silico Study

    Science.gov (United States)

    Ray, Manisha; Pattnaik, Animesh; Pradhan, Sukanta Kumar

    2016-01-01

    Environmental microbes like Bordetella petrii has been established as a causative agent for various infectious diseases in human. Again, development of drug resistance in B. petrii challenged to combat against the infection. Identification of potential drug target and proposing a novel lead compound against the pathogen has a great aid and value. In this study, bioinformatics tools and technology have been applied to suggest a potential drug target by screening the proteome information of B. petrii DSM 12804 (accession No. PRJNA28135) from genome database of National Centre for Biotechnology information. In this regards, the inhibitory effect of nine natural compounds like ajoene (Allium sativum), allicin (A. sativum), cinnamaldehyde (Cinnamomum cassia), curcumin (Curcuma longa), gallotannin (active component of green tea and red wine), isoorientin (Anthopterus wardii), isovitexin (A. wardii), neral (Melissa officinalis), and vitexin (A. wardii) have been acknowledged with anti-bacterial properties and hence tested against identified drug target of B. petrii by implicating computational approach. The in silico studies revealed the hypothesis that lpxD could be a potential drug target and with recommendation of a strong inhibitory effect of selected natural compounds against infection caused due to B. petrii, would be further validated through in vitro experiments. PMID:28154518

  2. Interaction of Bordetella bronchiseptica and Its Lipopolysaccharide with In Vitro Culture of Respiratory Nasal Epithelium

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    Carolina Gallego

    2013-01-01

    Full Text Available The nasal septa of fetal rabbits at 26 days of gestation were harvested by cesarean section of the does while under anesthesia and then exposed to Bordetella bronchiseptica or its lipopolysaccharide (LPS for periods of 2 and 4 hours. A total of 240 explants were used. The tissues were examined using the Hematoxylin & Eosin technique. Then, semithin sections (0.5 m were stained with toluidine blue and examined with indirect immunoperoxidase (IPI and lectin histochemistry. The most frequent and statistically significant findings were as follows: (1 cell death and increased goblet cell activity when exposed to bacteria and (2 cell death, cytoplasmic vacuolation and infiltration of polymorphonuclear leukocytes when exposed to LPS. The lesions induced by the bacterium were more severe than with LPS alone, except for the cytoplasmic vacuolation in epithelial cells. IPI stained the ciliated border of the epithelium with the bacterium more intensely, while LPS lectin histochemistry preferentially labeled the cytoplasm of goblet cell. These data indicate that B. bronchiseptica and its LPS may have an affinity for specific glycoproteins that would act as adhesion receptors in both locations.

  3. A Proteomic Characterization of Bordetella pertussis Clinical Isolates Associated with a California State Pertussis Outbreak

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    Yulanda M. Williamson

    2015-01-01

    Full Text Available Bordetella pertussis (Bp is the etiologic agent of pertussis (whooping cough, a highly communicable infection. Although pertussis is vaccine preventable, in recent years there has been increased incidence, despite high vaccine coverage. Possible reasons for the rise in cases include the following: Bp strain adaptation, waning vaccine immunity, increased surveillance, and improved clinical diagnostics. A pertussis outbreak impacted California (USA in 2010; children and preadolescents were the most affected but the burden of disease fell mainly on infants. To identify protein biomarkers associated with this pertussis outbreak, we report a whole cellular protein characterization of six Bp isolates plus the pertussis acellular vaccine strain Bp Tohama I (T, utilizing gel-free proteomics-based mass spectrometry (MS. MS/MS tryptic peptide detection and protein database searching combined with western blot analysis revealed three Bp isolates in this study had markedly reduced detection of pertactin (Prn, a subunit of pertussis acellular vaccines. Additionally, antibody affinity capture technologies were implemented using anti-Bp T rabbit polyclonal antisera and whole cellular proteins to identify putative immunogens. Proteome profiling could shed light on pathogenesis and potentially lay the foundation for reduced infection transmission strategies and improved clinical diagnostics.

  4. Modulation of the NF-kappaB pathway by Bordetella pertussis filamentous hemagglutinin.

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    Tzvia Abramson

    Full Text Available BACKGROUND: Filamentous hemagglutinin (FHA is a cell-associated and secreted adhesin produced by Bordetella pertussis with pro-apoptotic and pro-inflammatory activity in host cells. Given the importance of the NF-kappaB transcription factor family in these host cell responses, we examined the effect of FHA on NF-kappaB activation in macrophages and bronchial epithelial cells, both of which are relevant cell types during natural infection. METHODOLOGY/PRINCIPAL FINDINGS: Exposure to FHA of primary human monocytes and transformed U-937 macrophages, but not BEAS-2B epithelial cells, resulted in early activation of the NF-kappaB pathway, as manifested by the degradation of cytosolic IkappaB alpha, by NF-kappaB DNA binding, and by the subsequent secretion of NF-kappaB-regulated inflammatory cytokines. However, exposure of macrophages and human monocytes to FHA for two hours or more resulted in the accumulation of cytosolic IkappaB alpha, and the failure of TNF-alpha to activate NF-kappaB. Proteasome activity was attenuated following exposure of cells to FHA for 2 hours, as was the nuclear translocation of RelA in BEAS-2B cells. CONCLUSIONS: These results reveal a complex temporal dynamic, and suggest that despite short term effects to the contrary, longer exposures of host cells to this secreted adhesin may block NF-kappaB activation, and perhaps lead to a compromised immune response to this bacterial pathogen.

  5. Comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    Science.gov (United States)

    Kösters, K; Riffelmann, M; Dohrn, B; von König, C H

    2000-05-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for evaluation of the kits. The specimens contained 20 paired serum samples from patients with clinical pertussis, 15 samples were from children vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven specimens were taken from an interlaboratory comparison of ELISAs, and there were three reference preparations from the Food and Drug Administration's (FDA's) Laboratory of Pertussis and from our laboratory. Reference values were obtained from the FDA or from results obtained with an in-house ELISA. Commercial ELISAs were compared with respect to their reproducibility and variability, their ability to detect significant titer rises in paired serum samples, their ability to detect an immune response after vaccination, and the comparability of semiquantitative and quantitative results. Reproducibility was generally good (>89%), intra-assay variation ranged from 2.4 to 28.7%, and indeterminate results were recorded in up to 18.5% of all specimens. Most kits correctly identified the antibody response to an acellular pertussis vaccine. None of the commercial kits identified all cases of pertussis correctly, and the sensitivity ranged between 60 and 95%. All five commercial ELISAs showed great discrepancies when comparing semiquantitative results and contained obviously different antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.

  6. Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP

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    Hui Zhang

    2013-10-01

    Full Text Available Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

  7. Immunoproteomic Analysis ofBordetella bronchisepticaOuter Membrane Proteins and Identiifcation of New Immunogenic Proteins

    Institute of Scientific and Technical Information of China (English)

    JI Quan-an

    2014-01-01

    Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine ofB. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted fromB. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identiifed by matrix-assisted laser desorption/ionization time of lfight-mass spectrometry (MALDI-TOF-MS), a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins forB. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB (B. bronchiseptica strain isolated from a infected rabbit). The mortality of mice was 80% compared to 100% of positive controls. The identiifcation of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.

  8. Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetella pertussis

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    Hou Qiming

    2009-12-01

    Full Text Available Abstract Background Bordetella pertussis is a causative agent of pertussis or whooping cough in humans. Pertactin (Prn, fimbriae 2 (Fim2 and fimbriae 3 (Fim3 of B. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. In this present study, we cloned, expressed and purified Prn, Fim2 and Fim3, respectively. The immunogenicity and protective efficacy of the three recombinant proteins (rPrn, rFim2 and rFim3 were investigated in mouse model. Results Three recombinant proteins with amount of 12 to 25 mg/L were produced. Compared to the control mice only immunized with adjuvant, serum IgG antibody responses were significantly induced in the mice immunized with rPrn, rFim2 or rFim3 (P P B. pertussis (P Conclusions We have developed an efficient method to produce large amounts of rPrn, rFim2, and rFim3 from B. pertussis. The three recombinant proteins induced both humoral and cellular immune responses in mice. Immunization with rPrn also conferred protection against pertussis in mouse infection models. Our results indicated that the recombinant proteins still retain their immunological properties and highlighted the potential of the recombinant proteins for the future development of the B. pertussis vaccines.

  9. Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes.

    Science.gov (United States)

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-03-01

    Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.

  10. In Vitro and In Vivo Characterization of a Bordetella bronchiseptica Mutant Strain with a Deep Rough Lipopolysaccharide Structure

    Science.gov (United States)

    Sisti, Federico; Fernández, Julieta; Rodríguez, María Eugenia; Lagares, Antonio; Guiso, Nicole; Hozbor, Daniela Flavia

    2002-01-01

    Bordetella bronchiseptica is closely related to Bordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research on Bordetella has been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) during B. bronchiseptica infection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutant B. bronchiseptica LP39 on the waaC gene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although in B. bronchiseptica LP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS of B. bronchiseptica LP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, although B. bronchiseptica LP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form of B. bronchiseptica LPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from human B. bronchiseptica chronic

  11. Differential expression of type III effector BteA protein due to IS481 insertion in Bordetella pertussis.

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    Hyun-Ja Han

    Full Text Available BACKGROUND: Bordetella pertussis is the primary etiologic agent of the disease pertussis. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis; however, incidence of the disease, especially in adolescents and adults, has increased in several countries despite high vaccination coverage. During the last three decades, strains of Bordetella pertussis in circulation have shifted from the vaccine-type to the nonvaccine-type in many countries. A comparative proteomic analysis of the strains was performed to identify protein(s involved in the type shift. METHODOLOGY/PRINCIPAL FINDING: Proteomic analysis identified one differentially expressed protein in the B. pertussis strains: the type III cytotoxic effector protein BteA, which is responsible for host cell death in Bordetella bronchiseptica infections. Immunoblot analysis confirmed the prominent expression of BteA protein in the nonvaccine-type strains but not in the vaccine-type strains. Sequence analysis of the vaccine-type strains revealed an IS481 insertion in the 5' untranslated region of bteA, -136 bp upstream of the bteA start codon. A high level of bteA transcripts from the IS481 promoter was detected in the vaccine-type strains, indicating that the transcript might be an untranslatable form. Furthermore, BteA mutant studies demonstrated that BteA expression in the vaccine-type strains is down-regulated by the IS481 insertion. CONCLUSION/SIGNIFICANCE: The cytotoxic effector BteA protein is expressed at higher levels in B. pertussis nonvaccine-type strains than in vaccine-type strains. This type-dependent expression is due to an insertion of IS481 in B. pertussis clinical strains, suggesting that augmented expression of BteA protein might play a key role in the type shift of B. pertussis.

  12. sigE facilitates the adaptation of Bordetella bronchiseptica to stress conditions and lethal infection in immunocompromised mice

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    Barchinger Sarah E

    2012-08-01

    Full Text Available Abstract Background The cell envelope of a bacterial pathogen can be damaged by harsh conditions in the environment outside a host and by immune factors during infection. Cell envelope stress responses preserve the integrity of this essential compartment and are often required for virulence. Bordetella species are important respiratory pathogens that possess a large number of putative transcription factors. However, no cell envelope stress responses have been described in these species. Among the putative Bordetella transcription factors are a number of genes belonging to the extracytoplasmic function (ECF group of alternative sigma factors, some of which are known to mediate cell envelope stress responses in other bacteria. Here we investigate the role of one such gene, sigE, in stress survival and pathogenesis of Bordetella bronchiseptica. Results We demonstrate that sigE encodes a functional sigma factor that mediates a cell envelope stress response. Mutants of B. bronchiseptica strain RB50 lacking sigE are more sensitive to high temperature, ethanol, and perturbation of the envelope by SDS-EDTA and certain β-lactam antibiotics. Using a series of immunocompromised mice deficient in different components of the innate and adaptive immune responses, we show that SigE plays an important role in evading the innate immune response during lethal infections of mice lacking B cells and T cells. SigE is not required, however, for colonization of the respiratory tract of immunocompetent mice. The sigE mutant is more efficiently phagocytosed and killed by peripheral blood polymorphonuclear leukocytes (PMNs than RB50, and exhibits decreased cytotoxicity toward macrophages. These altered interactions with phagocytes could contribute to the defects observed during lethal infection. Conclusions Much of the work on transcriptional regulation during infection in B. bronchiseptica has focused on the BvgAS two-component system. This study reveals that the Sig

  13. Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange

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    Buasri Wasin

    2012-04-01

    Full Text Available Abstract Background Acellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT, must be chemically inactivated to obtain the corresponding toxoid (PTd. This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT with inactivated S1, Filamentous Hemagglutinin (FHA, and Pertactin (PRN has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN. Results Three recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 μg/mL compared to Bp-WWC (2.61 ± 0.16 μg/mL and wild type strain (2.2 μg/mL. In the third construct, a second copy of the prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ± 1.02 μg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 μg/mL and Bp-WWD (2.31 ± 0.17 μg/mL. Purified PTd from Bp-WWD at 0.8-1.6 μg/well did not show any toxicity against Chinese hamster ovary (CHO cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well. Conclusions We have constructed Bordetella

  14. Molecular evolution of the two-component system BvgAS involved in virulence regulation in Bordetella.

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    Julien Herrou

    Full Text Available The whooping cough agent Bordetella pertussis is closely related to Bordetella bronchiseptica, which is responsible for chronic respiratory infections in various mammals and is occasionally found in humans, and to Bordetella parapertussis, one lineage of which causes mild whooping cough in humans and the other ovine respiratory infections. All three species produce similar sets of virulence factors that are co-regulated by the two-component system BvgAS. We characterized the molecular diversity of BvgAS in Bordetella by sequencing the two genes from a large number of diverse isolates. The response regulator BvgA is virtually invariant, indicating strong functional constraints. In contrast, the multi-domain sensor kinase BvgS has evolved into two different types. The pertussis type is found in B. pertussis and in a lineage of essentially human-associated B. bronchiseptica, while the bronchiseptica type is associated with the majority of B. bronchiseptica and both ovine and human B. parapertussis. BvgS is monomorphic in B. pertussis, suggesting optimal adaptation or a recent population bottleneck. The degree of diversity of the bronchiseptica type BvgS is markedly different between domains, indicating distinct evolutionary pressures. Thus, absolute conservation of the putative solute-binding cavities of the two periplasmic Venus Fly Trap (VFT domains suggests that common signals are perceived in all three species, while the external surfaces of these domains vary more extensively. Co-evolution of the surfaces of the two VFT domains in each type and domain swapping experiments indicate that signal transduction in the periplasmic region may be type-specific. The two distinct evolutionary solutions for BvgS confirm that B. pertussis has emerged from a specific B. bronchiseptica lineage. The invariant regions of BvgS point to essential parts for its molecular mechanism, while the variable regions may indicate adaptations to different lifestyles. The

  15. Bordetella bronchiseptica como un riesgo importante de salud publica. Estudio clínico patológico en conejos

    OpenAIRE

    Valladares-Carranza B.; Ortega-Santana C; Velazquez-Ordoñez V; Zamora-Espinosa J.L.; Peñuelas-Rivas C.G.; Castro-Maruri J.; Talavera-Rojas M.; Alonso-Fresan M.U.; Zaragoza-Bastida A.

    2011-01-01

    B. bronchiseptica es reconocida como un patógeno primario inicial del tracto respiratorio en animales domésticos, puede provocar tos de las perreras (perro), respiración ruidosa (en conejos) y rinitis atrófica (en el cerdo). Conel objeto de enfatizar la importancia que representa en salud pública la infección ocasionada por Bordetella bronchiseptica se presenta un caso clínico ocurrido en una unidad de producción cúnicola en el Estado de México, México. Por la mortalidad observada en la gran...

  16. Complete Genome Sequence of Bordetella pertussis Strain VA-190 Isolated from a Vaccinated 10-Year-Old Patient with Whooping Cough

    Science.gov (United States)

    Eby, Joshua C.; Turner, Lauren; Nguyen, Bryan; Kang, June; Neville, Carly

    2016-01-01

    The number of cases of pertussis has increased in the United States despite vaccination. We present the genome of an isolate of Bordetella pertussis from a vaccinated patient from Virginia. The genome was sequenced by long-read methodology and compared to that of a clinical isolate used for laboratory studies, D420. PMID:27634997

  17. Expression of bvg-repressed genes in Bordetella pertussis is controlled by RisA through a novel c-di-GMP signaling pathway

    Science.gov (United States)

    The BvgAS two component system of Bordetella pertussis controls virulence factor expression. In addition, BvgAS controls expression of the bvg-repressed genes through the action of the repressor, BvgR. The transcription factor RisA is inhibited by BvgR, and when BvgR is not expressed RisA induces th...

  18. Cyclic di-GMP regulation of the bvg-repressed genes and the orphan response regulator RisA in Bordetella pertussis

    Science.gov (United States)

    Expression of Bordetella pertussis virulence factors is activated by the BvgAS two-component system. Under modulating growth conditions BvgAS indirectly represses another set of genes through the action of BvgR, a bvg-activated protein. BvgR blocks activation of the response regulator RisA which is ...

  19. Complete Genome Sequence of Bordetella pertussis Strain VA-190 Isolated from a Vaccinated 10-Year-Old Patient with Whooping Cough.

    Science.gov (United States)

    Eby, Joshua C; Turner, Lauren; Nguyen, Bryan; Kang, June; Neville, Carly; Temple, Louise

    2016-09-15

    The number of cases of pertussis has increased in the United States despite vaccination. We present the genome of an isolate of Bordetella pertussis from a vaccinated patient from Virginia. The genome was sequenced by long-read methodology and compared to that of a clinical isolate used for laboratory studies, D420.

  20. Genomic content of Bordetella pertussis clinical isolates circulating in areas of intensive children vaccination.

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    Valérie Bouchez

    Full Text Available BACKGROUND: The objective of the study was to analyse the evolution of Bordetella pertussis population and the influence of herd immunity in different areas of the world where newborns and infants are highly vaccinated. METHODOLOGY: The analysis was performed using DNA microarray on 15 isolates, PCR on 111 isolates as well as GS-FLX sequencing technology on 3 isolates and the B. pertussis reference strain, Tohama I. PRINCIPAL FINDINGS: Our analyses demonstrate that the current circulating isolates are continuing to lose genetic material as compared to isolates circulating during the pre-vaccine era whatever the area of the world considered. The lost genetic material does not seem to be important for virulence. Our study confirms that the use of whole cell vaccines has led to the control of isolates that were similar to vaccine strains. GS-FLX sequencing technology shows that current isolates did not acquire any additional material when compared with vaccine strains or with isolates of the pre-vaccine era and that the sequenced strain Tohama I is not representative of the isolates. Furthermore, this technology allowed us to observe that the number of Insertion Sequence elements contained in the genome of the isolates is temporally increasing or varying between isolates. CONCLUSIONS: B. pertussis adaptation to humans is still in progress by losing genetic material via Insertion Sequence elements. Furthermore, recent isolates did not acquire any additional material when compared with vaccine strains or with isolates of the pre-vaccine era. Herd immunity, following intensive vaccination of infants and children with whole cell vaccines, has controlled isolates similar to the vaccine strains without modifying significantly the virulence of the isolates. With the replacement of whole cell vaccines by subunit vaccines, containing only few bacterial antigens targeting the virulence of the bacterium, one could hypothesize the circulation of isolates

  1. Prevalence and molecular characterization of pertactin-deficient Bordetella pertussis in the United States.

    Science.gov (United States)

    Pawloski, L C; Queenan, A M; Cassiday, P K; Lynch, A S; Harrison, M J; Shang, W; Williams, M M; Bowden, K E; Burgos-Rivera, B; Qin, X; Messonnier, N; Tondella, M L

    2014-02-01

    Pertussis has shown a striking resurgence in the United States, with a return to record numbers of reported cases as last observed in the 1950s. Bordetella pertussis isolates lacking pertactin, a key antigen component of the acellular pertussis vaccine, have been observed, suggesting that B. pertussis is losing pertactin in response to vaccine immunity. Screening of 1,300 isolates from outbreak and surveillance studies (historical isolates collected from 1935 up to 2009, isolates from the 2010 California pertussis outbreak, U.S. isolates from routine surveillance between 2010-2012, and isolates from the 2012 Washington pertussis outbreak) by conventional PCR and later by Western blotting and prn sequencing analyses ultimately identified 306 pertactin-deficient isolates. Of these pertactin-deficient strains, 276 were identified as having an IS481 in the prn gene (prnIS481 positive). The first prnIS481-positive isolate was found in 1994, and the next prnIS481-positive isolates were not detected until 2010. The prevalence of pertactin-deficient isolates increased substantially to more than 50% of collected isolates in 2012. Sequence analysis of pertactin-deficient isolates revealed various types of mutations in the prn gene, including two deletions, single nucleotide substitutions resulting in a stop codon, an inversion in the promoter, and a single nucleotide insertion resulting in a frameshift mutation. All but one mutation type were found in prn2 alleles. CDC 013 was a predominant pulsed-field gel electrophoresis (PFGE) profile in the pertactin-positive isolates (203/994) but was found in only 5% (16/306) of the pertactin-deficient isolates. Interestingly, PFGE profiles CDC 002 and CDC 237 represented 55% (167/306) of the identified pertactin-deficient isolates. These results indicate that there has been a recent dramatic increase in pertactin-deficient B. pertussis isolates throughout the United States.

  2. Immunological Signatures after Bordetella pertussis Infection Demonstrate Importance of Pulmonary Innate Immune Cells

    Science.gov (United States)

    Brummelman, Jolanda; van der Maas, Larissa; Tilstra, Wichard; Pennings, Jeroen L. A.; Han, Wanda G. H.; van Els, Cécile A. C. M.; van Riet, Elly; Kersten, Gideon F. A.; Metz, Bernard

    2016-01-01

    Effective immunity against Bordetella pertussis is currently under discussion following the stacking evidence of pertussis resurgence in the vaccinated population. Natural immunity is more effective than vaccine-induced immunity indicating that knowledge on infection-induced responses may contribute to improve vaccination strategies. We applied a systems biology approach comprising microarray, flow cytometry and multiplex immunoassays to unravel the molecular and cellular signatures in unprotected mice and protected mice with infection-induced immunity, around a B. pertussis challenge. Pre-existing systemic memory Th1/Th17 cells, memory B-cells, and mucosal IgA specific for Ptx, Vag8, Fim2/3 were detected in the protected mice 56 days after an experimental infection. In addition, pre-existing high activity and reactivation of pulmonary innate cells such as alveolar macrophages, M-cells and goblet cells was detected. The pro-inflammatory responses in the lungs and serum, and neutrophil recruitment in the spleen upon an infectious challenge of unprotected mice were absent in protected mice. Instead, fast pulmonary immune responses in protected mice led to efficient bacterial clearance and harbored potential new gene markers that contribute to immunity against B. pertussis. These responses comprised of innate makers, such as Clca3, Retlna, Glycam1, Gp2, and Umod, next to adaptive markers, such as CCR6+ B-cells, CCR6+ Th17 cells and CXCR6+ T-cells as demonstrated by transcriptome analysis. In conclusion, besides effective Th1/Th17 and mucosal IgA responses, the primary infection-induced immunity benefits from activation of pulmonary resident innate immune cells, achieved by local pathogen-recognition. These molecular signatures of primary infection-induced immunity provided potential markers to improve vaccine-induced immunity against B. pertussis. PMID:27711188

  3. The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production.

    Science.gov (United States)

    Zurita, E; Moreno, G; Errea, A; Ormazabal, M; Rumbo, M; Hozbor, D

    2013-07-01

    The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 μg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.

  4. Bordetella pertussis naturally occurring isolates with altered lipooligosaccharide structure fail to fully mature human dendritic cells.

    Science.gov (United States)

    Brummelman, Jolanda; Veerman, Rosanne E; Hamstra, Hendrik Jan; Deuss, Anna J M; Schuijt, Tim J; Sloots, Arjen; Kuipers, Betsy; van Els, Cécile A C M; van der Ley, Peter; Mooi, Frits R; Han, Wanda G H; Pinelli, Elena

    2015-01-01

    Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.

  5. Comparative genomics of Bordetella pertussis reveals progressive gene loss in Finnish strains.

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    Eriikka Heikkinen

    Full Text Available BACKGROUND: Bordetella pertussis is a gram-negative bacterium that infects the human respiratory tract and causes pertussis or whooping cough. The disease has resurged in many countries including Finland where the whole-cell pertussis vaccine has been used for more than 50 years. Antigenic divergence has been observed between vaccine strains and clinical isolates in Finland. To better understand genome evolution in B. pertussis circulating in the immunized population, we developed an oligonucleotide-based microarray for comparative genomic analysis of Finnish strains isolated during the period of 50 years. METHODOLOGY/PRINCIPAL FINDINGS: The microarray consisted of 3,582 oligonucleotides (70-mer and covered 94% of 3,816 ORFs of Tohama I, the strain of which the genome has been sequenced. Twenty isolates from 1953 to 2004 were studied together with two Finnish vaccine strains and two international reference strains. The isolates were selected according to their characteristics, e.g. the year and place of isolation and pulsed-field gel electrophoresis profiles. Genomic DNA of the tested strains, along with reference DNA of Tohama I strain, was labelled and hybridized. The absence of genes as established with microarrays, was confirmed by PCR. Compared with the Tohama I strain, Finnish isolates lost 7 (8.6 kb to 49 (55.3 kb genes, clustered in one to four distinct loci. The number of lost genes increased with time, and one third of lost genes had functions related to inorganic ion transport and metabolism, or energy production and conversion. All four loci of lost genes were flanked by the insertion sequence element IS481. CONCLUSION/SIGNIFICANCE: Our results showed that the progressive gene loss occurred in Finnish B. pertussis strains isolated during a period of 50 years and confirmed that B. pertussis is dynamic and is continuously evolving, suggesting that the bacterium may use gene loss as one strategy to adapt to highly immunized populations.

  6. Correlation of Real Time PCR Cycle Threshold Cut-Off with Bordetella pertussis Clinical Severity.

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    Shelly Bolotin

    Full Text Available Bordetella pertussis testing performed using real-time polymerase chain reaction (RT-PCR is interpreted based on a cycle threshold (Ct value. At Public Health Ontario Laboratories (PHOL, a Ct value <36 is reported as positive, and Ct values ≥36 and <40 are reported as indeterminate. PHOL reported indeterminate results to physicians and public health units until May 2012, after which these results were only reported to physicians. We investigated the association between Ct value and disease symptom and severity to examine the significance of indeterminate results clinically, epidemiologically and for public health reporting. B. pertussis positive and indeterminate RT-PCR results were linked to pertussis cases reported in the provincial Integrated Public Health Information System (iPHIS, using deterministic linkage. Patients with positive RT-PCR results had a lower median age of 10.8 years compared to 12.0 years for patients with indeterminate results (p = 0.24. Hospitalized patients had significantly lower Ct values than non-hospitalized patients (median Ct values of 20.7 vs. 31.6, p<0.001. The proportion of patients reporting the most indicative symptoms of pertussis did not differ between patients with positive vs. indeterminate RT-PCR results. Taking the most indicative symptoms of pertussis as the gold-standard, the positive predictive value of the RT-PCR test was 68.1%. RT-PCR test results should be interpreted in the context of the clinical symptoms, age, vaccination status, prevalence, and other factors. Further information on interpretation of indeterminate RT-PCR results may be needed, and the utility of reporting to public health practitioners should be re-evaluated.

  7. SNP-based typing: a useful tool to study Bordetella pertussis populations.

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    Marjolein van Gent

    Full Text Available To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA. In this study, a single nucleotide polymorphism (SNP typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in The Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis.

  8. High prevalence of erythromycin-resistant Bordetella pertussis in Xi'an, China.

    Science.gov (United States)

    Wang, Z; Cui, Z; Li, Y; Hou, T; Liu, X; Xi, Y; Liu, Y; Li, H; He, Q

    2014-11-01

    Resistance of Bordetella pertussis, the causative agent of pertussis, to erythromycin is rare. Recently, several Chinese isolates were found to be erythromycin resistant. This study aimed to investigate the occurrence of pertussis in children suffering persistent cough and the prevalence of B. pertussis resistance to erythromycin in Xi'an, China. Three hundred and thirteen patients with suspected pertussis admitted to Xi'an Children's Hospital from January 2012 through to December 2013 were included and their nasopharyngeal (NP) swabs were taken for culture and PCRs (targeting IS481 and ptx-Pr). PCR-based sequencing was used to identify the A2047G mutation of B. pertussis 23S rRNA directly from the NP samples. Sixteen (5.1%) and 168 (53.7%) patients were positive for culture and IS481 PCR. Of the 168 samples positive for IS481 PCR, 122 (72.6%) and 100 (59.5%) were positive for ptx-Pr and 23S rRNA PCRs, respectively. All culture-positive samples were also positive for the three PCRs. Fourteen (87.5%) of the 16 B. pertussis isolates were found to be resistant to erythromycin (MICs>256 mg/L). All the 14 isolates were confirmed to have a homogeneous A2047G mutation of 23S rRNA. Of the 100 samples positive for 23S rRNA PCR, 85 (85.0%) were found to have the A2047G mutation by sequencing. Our results indicate that in Xi'an, China, pertussis remains endemic in young children, and the circulating B. pertussis strains are mostly erythromycin resistant.

  9. Direct molecular typing of Bordetella pertussis from nasopharyngeal specimens in China in 2012-2013.

    Science.gov (United States)

    Du, Q; Wang, X; Liu, Y; Luan, Y; Zhang, J; Li, Y; Liu, X; Ma, C; Li, H; Wang, Z; He, Q

    2016-07-01

    Data on the molecular epidemiology of Bordetella pertussis are limited in developing countries where whole-cell pertussis vaccines (WCVs) have been used. The aim of this study was to determine the genotypes of circulating B. pertussis in China by direct molecular typing of clinical specimens. DNA extracts of 122 nasopharyngeal swabs (NPs) positive for B. pertussis by polymerase chain reaction (PCR) (targeting IS481 and ptx-Pr) from 2012 to 2013 were used for typing using the multiple-locus variable number tandem repeat analysis (MLVA) and also by PCR-based multilocus sequence typing (MLST) of B. pertussis virulence genes (ptxP, prn, and fim3). One hundred and eight DNA extracts (89 %) generated a complete MLVA type (MT). Among the 18 MTs obtained, MT55 (52 %) and MT104 (13 %) were the most common. MT27, which is linked to the ptxP3 allele and is prevalent in many developed countries using acellular pertussis vaccines (ACVs), was only found in 7 (6 %) DNA extracts. Eighty-seven DNA extracts (71 %) produced a complete multiantigen sequence typing (MAST) type. Of them, 77 (89 %) had the ptxP1/prn1/fim3-1 allele profile. Four DNA extracts (5 %) had the ptxP3/prn2/fim3-2 profile and 3 (4 %) had the ptxP3/prn1/fim3-2 allele profile. These seven DNA extracts also harbored MT27. Our result shows that B. pertussis circulating in China was different from those found in countries where ACVs have been in use, supporting the notion that selection pressure induced by WCVs and ACVs on the bacterial population differs.

  10. Antimicrobial Susceptibility of Bordetella bronchiseptica Isolates from Swine and Companion Animals and Detection of Resistance Genes.

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    Sandra Prüller

    Full Text Available Bordetella bronchiseptica causes infections of the respiratory tract in swine and other mammals and is a precursor for secondary infections with Pasteurella multocida. Treatment of B. bronchiseptica infections is conducted primarily with antimicrobial agents. Therefore it is essential to get an overview of the susceptibility status of these bacteria. The aim of this study was to comparatively analyse broth microdilution susceptibility testing according to CLSI recommendations with an incubation time of 16 to 20 hours and a longer incubation time of 24 hours, as recently proposed to obtain more homogenous MICs. Susceptibility testing against a panel of 22 antimicrobial agents and two fixed combinations was performed with 107 porcine isolates from different farms and regions in Germany and 43 isolates obtained from companion animals in Germany and other European countries. Isolates with increased MICs were investigated by PCR assays for the presence of resistance genes. For ampicillin, all 107 porcine isolates were classified as resistant, whereas only a single isolate was resistant to florfenicol. All isolates obtained from companion animals showed elevated MICs for β-lactam antibiotics and demonstrated an overall low susceptibility to cephalosporines. Extension of the incubation time resulted in 1-2 dilution steps higher MIC50 values of porcine isolates for seven antimicrobial agents tested, while isolates from companion animals exhibited twofold higher MIC50/90 values only for tetracycline and cefotaxime. For three antimicrobial agents, lower MIC50 and MIC90 values were detected for both, porcine and companion animal isolates. Among the 150 isolates tested, the resistance genes blaBOR-1 (n = 147, blaOXA-2, (n = 4, strA and strB (n = 17, sul1 (n = 10, sul2 (n = 73, dfrA7 (n = 3 and tet(A (n = 8 were detected and a plasmid localisation was identified for several of the resistance genes.

  11. Development of live attenuated Bordetella pertussis strains expressing the universal influenza vaccine candidate M2e.

    Science.gov (United States)

    Li, Rui; Lim, Annabelle; Ow, Stephanie T L; Phoon, Meng Chee; Locht, Camille; Chow, Vincent T; Alonso, Sylvie

    2011-07-26

    The attenuated Bordetella pertussis BPZE1 vaccine strain represents an attractive platform for the delivery of heterologous vaccine candidates via the nasal route. The filamentous hemagglutinin (FHA) has been used to secrete or expose the foreign antigens at the bacterial surface. In this study, one, two and three copies of the Cys-containing ectodomain of matrix protein 2 (M2e) from influenza A virus were genetically fused to full length FHA and expressed in BPZE1. The secretion efficacy of the FHA-(M2e)(1,2,3) chimera in the extracellular milieu and the ability of the recombinant bacteria to colonize the mouse lungs inversely correlated with the number of M2e copies fused to FHA. Nevertheless FHA-(M2e)(3)-producing bacteria (BPLR3) triggered the highest systemic anti-M2e antibody response upon nasal administration to BALB/c mice. Nasal immunization with BPLR3 bacteria resulted in a significant reduction in the viral loads upon challenge with H1N1/PR8 influenza A virus, but did not improve the survival rate compared to BPZE1-immunized mice. Furthermore, since previous work reported that disulfide bond formation in Cys-containing passenger antigens affects the secretion efficacy of the FHA chimera, the dsbA gene encoding a periplasmic disulfide isomerase was deleted in the FHA-(M2e)(3)-producing strain. Despite improving significantly the secretion efficacy of the FHA-(M2e)(3) chimera, the dsbA deletion did not result in higher anti-M2e antibody titers in mice, due to impaired bacterial fitness and colonization ability.

  12. Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    Science.gov (United States)

    Riffelmann, M; Thiel, K; Schmetz, J; Wirsing von Koenig, C H

    2010-12-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.

  13. Research on pharmacological mechanism of the treatment of Asthma by oral Bordetella pertussis

    Institute of Scientific and Technical Information of China (English)

    CHI Shen; SUN Yun; ZHANG Bao-yuan

    2008-01-01

    Objective To examine the effect of oral Bordetella pertussis on the asthma mice sensitized by ovalbumin (OVA), and explore the possible mechanism. Methods Culture the B. pertussis in Bordet-Gengou agar containing 25 % rabbit blood. Collect the bacteria and inactive them at 80 ℃ for 30 min to get whole killed B. pertussis. 32 BALB/C mice were randomly divided into control group, model-control group, model group and treatment group. The mice were sensitized and challenged with OVA to establish asthma model. Asthma mice in treatment group were orally administrated with B. pertussis 7 days before sensitization. The mice in control group and model-control group were challenged with saline. After 24 hours of last challenge, bronchoaveolar lavage fluid (BALF) and peripheral blood were collected. The total cells and eosinophils were counted in BALF. Results Compared with the control group (2.03±0.42, 0.33±0.82)× 105 mL-1 and model-control group (2.16±0.48,0.16±0.41)×105 mL-1, the total cells (10.13±1.33) ×105mL-1 and eosinophils (11.83±4.573)×105 mL-1 in BALF were more in asthma mice (P<0.01). The number of total cells (5.50±1.55)×105 mL-1 and eosinophils(0.66±0.82)×105 mL-1 in BALF were reduced in asthma mice treated with B. pertussis compared with asthma mice(P<0.01 ). Conclusions Oral B. pertussis can inhabit airway inflammation of asthma mice and has the potential of treating asthma.

  14. Prevalence and genetic characterization of pertactin-deficient Bordetella pertussis in Japan.

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    Nao Otsuka

    Full Text Available The adhesin pertactin (Prn is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn(- B. pertussis was observed in Japan. The Prn(- isolate was first discovered in 1997, and 33 (27% Prn(- isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn(- isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24 and an IS481 insertion in prn1 (prn1::IS481, n = 9. The frequency of Prn(- isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn(- isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA revealed that 24 (73% of 33 Prn(- isolates belong to MLVA-186, and 6 and 3 Prn(- isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn(- clinical strains (harboring prn1ΔSS and prn1::IS481 have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn(- isolates have a higher growth potential than the Prn(+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status that select for Prn(- strains have arisen and that Prn expression is not essential for fitness under these conditions.

  15. Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity.

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    Victor I Ayala

    Full Text Available Pertussis (whooping cough is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8 infection in mouse models and the role of pertussis toxin (PT in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT and subsequently (up to 14 days later infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.

  16. Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain.

    Science.gov (United States)

    Asgarian-Omran, Hossein; Amirzargar, Ali Akbar; Zeerleder, Sacha; Mahdavi, Marzieh; van Mierlo, Gerard; Solati, Shabnam; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Aarden, Leucien; Shokri, Fazel

    2015-02-01

    Filamentous hemagglutinin (FHA) is a major adhesion and virulence factor of Bordetella pertussis and also a main component of acellular pertussis vaccines. Interaction of FHA with different receptors on human epithelial and immune cells facilitates entrance and colonization of bacteria as well as immunomodulation of the host immune response. Three overlapping segments of the FHA gene were cloned in a prokaryotic expression vector and the recombinant proteins were purified. These recombinant fragments along with the native FHA protein were employed to assess their potential Toll-like receptor (TLR) stimulatory effects and to localize the TLR binding region. TLR stimulation was monitored by applying HEK293-Blue cell lines cotransfected with TLR2, 4, or 5 and a NF-κB reporter gene. Culture supernatants were checked for secretion of the reporter gene product and IL-8 as indicators of TLR stimulation. Native FHA was found to strongly stimulate TLR2, but not TLR4 or TLR5 transfected cells. Among recombinant FHA fragments only the fragment spanning amino acid residues 1544-1917 was able to exhibit the TLR2 stimulating property of FHA. Interaction of FHA with TLR2 suggests its involvement in induction of the innate immune system against Bordetella pertussis. The TLR2-binding domain of FHA may contribute to immunoprotection against pertussis infection.

  17. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and /sup 125/I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25/sup 0/C instead of 100/sup 0/C.

  18. Strong inhibition of fimbrial 3 subunit gene transcription by a novel downstream repressive element in Bordetella pertussis.

    Science.gov (United States)

    Chen, Qing; Boulanger, Alice; Hinton, Deborah M; Stibitz, Scott

    2014-08-01

    The Bvg-regulated promoters for the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis behave differently from each other both in vivo and in vitro. In vivo Pfim2 is significantly stronger than Pfim3 , even though predictions based on the DNA sequences of BvgA-binding motifs and core promoter elements would indicate the opposite. In vitro Pfim3 demonstrated robust BvgA∼P-dependent transcriptional activation, while none was seen with Pfim2 . This apparent contradiction was investigated further. By swapping sequence elements we created a number of hybrid promoters and assayed their strength in vivo. We found that, while Pfim3 promoter elements upstream of the +1 transcriptional start site do indeed direct Bvg-activated transcription more efficiently than those of Pfim2 , the overall promoter strength of Pfim3  in vivo is reduced due to sequences downstream of +1 that inhibit transcription more than 250-fold. This element, the DRE (downstream repressive element), was mapped to the 15 bp immediately downstream of the Pfim3 +1. Placing the DRE in different promoter contexts indicated that its activity was not specific to fim promoters, or even to Bvg-regulated promoters. However it does appear to be specific to Bordetella species in that it did not function in Escherichia coli.

  19. Expresión episomal de toxina de pertussis genéticamente inactivada en Bordetella pertussis

    Directory of Open Access Journals (Sweden)

    Ernesto Marcos

    2010-01-01

    Full Text Available Bordetella pertussis es una bacteria Gram negativa, la cual constituye el agente etiologico de la tos ferina. La enfermedad se desencadena por el efecto conjunto de una serie de factores de virulencia expresados por la bacteria, los cuales se encuentran regulados por el sistema bvg. Uno de los factores de virulencia mas importantes es la toxina de pertussis, razon por la cual, se emplea de forma inactivada como el componente principal de las vacunas acelulares contra la enfermedad. La toxina de pertussis posee una estructura del tipo A-B compuesta por seis polipeptidos codificados en un operon unico. El polipeptido S1 constituye la subunidad enzimaticamente activa, la cual cataliza la transferencia de ADP-ribosa del NAD a la subunidad ALPHA de las proteinas G en celulas eucariotas, lo cual genera una serie de efectos biologicos dentro de los que se incluye: sensibilizacion a histamina, incremento de la secrecion de insulina y efectos inmunoestimuladores e inmunosupresores. El presente trabajo describe los procedimientos realizados para la obtencion de cepas de Bordetella pertussis productoras de elevadas concentraciones de toxina pertusica atenuada geneticamente. Para esto, se realizaron las sustituciones aminoacidicas Arg9 por Lys y Glu129 por Gly de la subunidad S1. El operon de la toxina de pertussis mutada se clono en un vector de amplio rango de hospedero bajo la regulacion de un promotor de expresion temprana (fhaB. Los clones obtenidos pudieran ser empleados como sistemas de expresion para produccion de vacunas acelulares en Cuba.

  20. Comparison of the Genome Sequence of the Poultry Pathogen Bordetella avium with Those of B. bronchiseptica, B. pertussis, and B. parapertussis Reveals Extensive Diversity in Surface Structures Associated with Host Interaction

    Science.gov (United States)

    Sebaihia, Mohammed; Preston, Andrew; Maskell, Duncan J.; Kuzmiak, Holly; Connell, Terry D.; King, Natalie D.; Orndorff, Paul E.; Miyamoto, David M.; Thomson, Nicholas R.; Harris, David; Goble, Arlette; Lord, Angela; Murphy, Lee; Quail, Michael A.; Rutter, Simon; Squares, Robert; Squares, Steven; Woodward, John; Parkhill, Julian; Temple, Louise M.

    2006-01-01

    Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these. PMID:16885469

  1. Analysis of Bordetella pertussis pertactin and pertussis toxin types from Queensland, Australia, 1999–2003

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    Slack Andrew T

    2006-03-01

    Full Text Available Abstract Background In Australia two acellular Bordetella pertussis vaccines have replaced the use of a whole cell vaccine. Both of the licensed acellular vaccines contain the following three components; pertussis toxoid, pertussis filamentous haemagglutinin and the 69 kDa pertactin adhesin. One vaccine also contains pertussis fimbriae 2 and 3. Various researchers have postulated that herd immunity due to high levels of pertussis vaccination might be influencing the makeup of endemic B. pertussis populations by selective pressure for strains possessing variants of these genes, in particular the pertactin gene type. Some publications have suggested that B. pertussis variants may be contributing to a reduced efficacy of the existing vaccines and a concomitant re-emergence of pertussis within vaccinated populations. This study was conducted to survey the pertactin and pertussis toxin subunit 1 types from B. pertussis isolates in Queensland, Australia following the introduction of acellular vaccines. Methods Forty-six B. pertussis isolates recovered from Queensland patients between 1999 and 2003 were examined by both DNA sequencing and LightCycler™ real time PCR to determine their pertactin and pertussis toxin subunit 1 genotypes. Results Pertactin typing showed that 38 isolates possessed the prn1 allele, 3 possessed the prn2 allele and 5 possessed the prn3 allele. All forty-six isolates possessed the pertussis toxin ptxS1A genotype. Amongst the circulating B. pertussis population in Queensland, 82.5% of the recovered clinical isolates therefore possessed the prn1/ptxS1A genotype. Conclusion The results of this study compared to historical research on Queensland isolates suggest that B. pertussis pertactin and pertussis toxin variants are not becoming more prevalent in Queensland since the introduction of the acellular vaccines. Current prevalences of pertactin variants are significantly different to that described in a number of other countries

  2. Comparative gene expression profiling in two congenic mouse strains following Bordetella pertussis infection

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    Demant Peter

    2007-10-01

    Full Text Available Abstract Background Susceptibility to Bordetella pertussis infection varies widely. These differences can partly be explained by genetic host factors. HcB-28 mice are more resistant to B. pertussis infection than C3H mice, which could partially be ascribed to the B. pertussis susceptibility locus-1 (Bps1 on chromosome 12. The presence of C57BL/10 genome on this locus instead of C3H genome resulted in a decreased number of bacteria in the lung. To further elucidate the role of host genetic factors, in particular in the Bps1 locus, in B. pertussis infection, and to identify candidate genes within in this region, we compared expression profiles in the lungs of the C3H and HcB-28 mouse strains following B. pertussis inoculation. Twelve and a half percent of the genomes of these mice are from a different genetic background. Results Upon B. pertussis inoculation 2,353 genes were differentially expressed in the lungs of both mouse strains. Two hundred and six genes were differentially expressed between the two mouse strains, but, remarkably, none of these were up- or down-regulated upon B. pertussis infection. Of these 206 genes, 17 were located in the Bps1 region. Eight of these genes, which showed a strong difference in gene expression between the two mouse strains, map to the immunoglobulin heavy chain complex (Igh. Conclusion Gene expression changes upon B. pertussis infection are highly identical between the two mouse strains despite the differences in the course of B. pertussis infection. Because the genes that were differentially regulated between the mouse strains only showed differences in expression before infection, it appears likely that such intrinsic differences in gene regulation are involved in determining differences in susceptibility to B. pertussis infection. Alternatively, such genetic differences in susceptibility may be explained by genes that are not differentially regulated between these two mouse strains. Genes in the Igh

  3. Infección por Bordetella pertussis: Una causa emergente de tos prolongada en adolescentes y adultos Bordetella pertussis infection: An emerging cause of prolonged cough illness in adolescents and adults

    Directory of Open Access Journals (Sweden)

    RODRIGO OSSES A

    2010-03-01

    Full Text Available La tos convulsiva o coqueluche está siendo reconocida cada vez con mayor frecuencia como causa de tos prolongada en adolescentes y adultos. La vacunación sistemática de la población pediátrica ha determinado un cambio en el perfl epidemiológico de la enfermedad, aumentando su prevalencia en la población adulta. Se presenta el caso clínico de una paciente de 45 años, fumadora, enfermera de unidad de hemodiálisis, que consulta por malestar general y tos seca de seis semanas de evolución. La radiografía de tórax era normal y la inmunofuorescencia directa de hisopado nasofaríngeo fue positiva para Bordetella pertussis. A propósito de este caso clínico, revisamos las principales causas de tos crónica: asma bronquial, enfermedad rinosinusal y refujo gastroesofágico; el cuadro clínico, evaluación diagnóstica y tratamiento de la infección por B. pertussis en población adulta.Whooping cough is increasingly recognized as a cause of prolonged cough illness in adolescents and adults. Systematic vaccination has changed its epidemiology, with the majority of cases now primarily affecting adolescents and adults. A 45-year-old female, active smoker, nurse, who works in a dialysis service, presented with a 6-week history of bothersome cough and malaise. Thorax x-ray was normal and direct immunofuorescence of nasopharyngeal swab was positive for Bordetella pertussis. This case illustrates pertussis infection in adulthood. We review the main causes of chronic cough in adults: asthma, chronic rhinosinusitis and gastroesophageal refux; the clinical features, prevalence, diagnostic tools, and management of adult patients with B. pertussis infection to increase awareness of this highly contagious disease.

  4. A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates.

    Science.gov (United States)

    Izac, Marie; Garnier, Dominique; Speck, Denis; Lindley, Nic D

    2015-01-01

    It has been claimed that citrate synthase, aconitase and isocitrate dehydrogenase activities are non-functional in Bordetella pertussis and that this might explain why this bacterium's growth is sometimes associated with accumulation of polyhydroxybutyrate (PHB) and/or free fatty acids. However, the sequenced genome includes the entire citric acid pathway genes. Furthermore, these genes were expressed and the corresponding enzyme activities detected at high levels for the pathway when grown on a defined medium imitating the amino acid content of complex media often used for growth of this pathogenic microorganism. In addition, no significant PHB or fatty acids could be detected. Analysis of the carbon balance and stoichiometric flux analysis based on specific rates of amino acid consumption, and estimated biomass requirements coherent with the observed growth rate, clearly indicate that a fully functional tricarboxylic acid cycle operates in contrast to previous reports.

  5. Changes in Predominance of Pulsed-Field Gel Electrophoresis Profiles of Bordetella pertussis Isolates, United States, 2000-2012.

    Science.gov (United States)

    Cassiday, Pamela K; Skoff, Tami H; Jawahir, Selina; Tondella, M Lucia

    2016-03-01

    To clarify the characteristics of circulating Bordetella pertussis isolates, we used pulsed-field gel electrophoresis (PFGE) to analyze 5,262 isolates collected in the United States during 2000-2012. We found 199 PFGE profiles; 5 profiles accounted for 72% of isolates. The most common profile, CDC013, accounted for 35%-46% of isolates tested from 2000-2009; however, the proportion of isolates of this profile rapidly decreased in 2010. Profile CDC237, first seen in 2009, increased rapidly and accounted for 29% of 2012 isolates. No location bias was observed among profiles during 2000-2010, but differences were observed among isolates from different states during 2012. Predominant profiles match those observed in recent European PFGE studies. PFGE profile changes are concurrent with other recent molecular changes in B. pertussis and may be contributing to the reemergence of pertussis in the United States. Continued PFGE monitoring is critical for understanding the changing epidemiology of pertussis.

  6. Detection of Bordetella pertussis from Clinical Samples by Culture and End-Point PCR in Malaysian Patients

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    Tan Xue Ting

    2013-01-01

    Full Text Available Pertussis or whooping cough is a highly infectious respiratory disease caused by Bordetella pertussis. In vaccinating countries, infants, adolescents, and adults are relevant patients groups. A total of 707 clinical specimens were received from major hospitals in Malaysia in year 2011. These specimens were cultured on Regan-Lowe charcoal agar and subjected to end-point PCR, which amplified the repetitive insertion sequence IS481 and pertussis toxin promoter gene. Out of these specimens, 275 were positive: 4 by culture only, 6 by both end-point PCR and culture, and 265 by end-point PCR only. The majority of the positive cases were from ≤3 months old patients (77.1% (. There was no significant association between type of samples collected and end-point PCR results (. Our study showed that the end-point PCR technique was able to pick up more positive cases compared to culture method.

  7. Attenuated Bordetella pertussis BPZE1 as a live vehicle for heterologous vaccine antigens delivery through the nasal route.

    Science.gov (United States)

    Li, Rui; Lim, Annabelle; Alonso, Sylvie

    2011-01-01

    Whereas the great majority of the current vaccines are delivered through the parenteral route, mucosal administration has been increasingly considered for controlling infection and preventing disease. Mucosal vaccination can trigger both humoral and cell-mediated protection, not only at the targeted mucosal surface, but also systemically. In this regard, nasal vaccination has shown great potential. The live attenuated strain of Bordetella pertussis, BPZE1, is particularly attractive and promising as a nasal vaccine delivery vector of heterologous antigen vaccine candidates. BPZE1 was originally developed as a live nasal pertussis vaccine candidate, and is currently undergoing phase I clinical trial in human (http://www.child-innovac.org). Highly adapted to the human respiratory tract and offering several potential protein carriers for presentation of the heterologous antigen vaccine candidates, BPZE1 represents an appealing platform for the development of live recombinant vaccines delivered via the nasal route that would confer simultaneous protection against pertussis and the targeted infectious disease(s).

  8. A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates.

    Directory of Open Access Journals (Sweden)

    Marie Izac

    Full Text Available It has been claimed that citrate synthase, aconitase and isocitrate dehydrogenase activities are non-functional in Bordetella pertussis and that this might explain why this bacterium's growth is sometimes associated with accumulation of polyhydroxybutyrate (PHB and/or free fatty acids. However, the sequenced genome includes the entire citric acid pathway genes. Furthermore, these genes were expressed and the corresponding enzyme activities detected at high levels for the pathway when grown on a defined medium imitating the amino acid content of complex media often used for growth of this pathogenic microorganism. In addition, no significant PHB or fatty acids could be detected. Analysis of the carbon balance and stoichiometric flux analysis based on specific rates of amino acid consumption, and estimated biomass requirements coherent with the observed growth rate, clearly indicate that a fully functional tricarboxylic acid cycle operates in contrast to previous reports.

  9. 百日咳流行病学研究进展%Update on bordetella pertussis epidemiology

    Institute of Scientific and Technical Information of China (English)

    张婉芳(综述); 陆敏(审校)

    2014-01-01

    百日咳主要是由百日咳杆菌、副百日咳杆菌引起的一种传染性强的急性呼吸系统疾病,人群普遍易患,小婴儿病情最重。尽管疫苗覆盖率较高,但是全球百日咳仍未完全根除。相反,过去20年,全球百日咳的发病率较之前升高,出现局部地区小流行,称百日咳再现,因此有必要对国内外百日咳发病现状做出评估,为更好地监测和控制百日咳提供依据。该文对百日咳的流行病学变化及其相关原因分析和免疫策略作一综述。%Pertussis or whooping cough is an acute infectious disease of the respiratory system,which is mainly caused by Bordetella pertussis and Bordetella parapertussis. It can occur at any age,but is most serious in young infants. Despite widespread use of vaccination, pertussis has not been eliminated. On the contrary, increased incidence rates have been reported worldwide during the last two decades,also called reemergence of pertussis. So it is necessary to evaluate current state on pertussis research,in order to better provide basis for mo-nitoring and control pertussis. This paper reviews the pertussis epidemiological changes and the related cause analysis and immunization strategy.

  10. Immunization with the Recombinant Cholera Toxin B Fused to Fimbria 2 Protein Protects against Bordetella pertussis Infection

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    Noelia Olivera

    2014-01-01

    Full Text Available This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2—cholera toxin B subunit (CTB in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2 were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside enzyme-linked immunosorbent assay (ELISA. To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2 or purified (BpFim2 Fim2, CTB, and phosphate-buffered saline (PBS were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P<0.01 or P<0.001, resp.. Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection.

  11. High-resolution melting analysis for the detection of two erythromycin-resistant Bordetella pertussis strains carried by healthy schoolchildren in China.

    Science.gov (United States)

    Zhang, Q; Li, M; Wang, L; Xin, T; He, Q

    2013-06-01

    Two erythromycin-resistant strains of Bordetella pertussis were isolated from nasopharyngeal specimens of two asymptomatic schoolchildren in China. High-resolution melting and sequencing analyses confirmed the homogeneous A2047G mutation in 23S rRNA genes of the two isolates. High-resolution melting (HRM) analysis is a useful assay for the rapid detection of erythromycin-resistant B. pertussis. The appearance of erythromycin-resistant B. pertussis strains in China is alarming.

  12. Factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, Chlamydophila felis and Bordetella bronchiseptica in cats: experience from 218 European catteries

    OpenAIRE

    Helps, C. R.; Lait, P.; Damhuis, A.; Björnehammar, U.; Bolta, D.; Brovida, C.; Chabanne, L.; Egberink, H; Ferrand, G.; Fontbonne, A.; Pennisi, M G; Gruffydd-Jones, T.; Gunn-Moore, D.; Hartmann, K.; Lutz, H

    2005-01-01

    A full history of the management practices and the prevalence of upper respiratory tract disease (URTD) at 218 rescue shelters, breeding establishments and private households with five or more cats was recorded. Oropharyngeal and conjunctival swabs and blood samples were taken from 1748 cats. The prevalences of feline herpesvirus (FHV), feline calicivirus (FCV), Chlamydophila felis and Bordetella bronchiseptica were determined by PCR on swab samples. An ELISA was applied to determine the prev...

  13. [Serological evaluation of Bordetella pertussis infection in adults with prolonged cough].

    Science.gov (United States)

    Sönmez, Cemile; Çöplü, Nilay; Gözalan, Ayşegül; Yılmaz, Ülkü; Bilekli, Selen; Demirci, Nilgün Yılmaz; Biber, Çiğdem; Erdoğan, Yurdanur; Esen, Berrin; Çöplü, Lütfi

    2016-07-01

    Pertussis is a vaccine-preventable disease that is transmitted from infected to susceptible individuals by respiratory route. Bordetella pertussis infection may occur at any age as neither vaccine nor natural infection induced immunity lasts life-long. This study was planned to demonstrate the serological evidence of infection among adults, to raise awareness among clinicians and to provide data for the development of strategies to protect vulnerable infants. A total of 538 patients (345 female, 193 male) ages between 18-87 years who had a complain of prolonged cough for more than two weeks were included in the study. Anti-pertussis toxin (PT) IgG and anti-filamentous hemagglutinin (FH) IgG levels from single serum samples were measured by an in-house ELISA test which was standardized and shown to be efficient previously. Anti-PT IgG antibody levels of ≥ 100 EU/ml were considered as acute/recent infection with B.pertussis. In our study, 9.7% (52/538) of the patients had high levels of anti-PT IgG (≥ 100 EU/ml) and among those patients 43 (43/52; 82.7%) also had high (≥ 100 EU/ml) anti-FHA IgG levels. There were no statistically significant differences in terms of age, gender, education level, DPT (diphtheria-pertussis-tetanus) vaccination history, smoking history or average daily cigarette consumption (p> 0.05) between the cases with high antibody levels (n= 52). When the symptoms and the presence of cases with high antibody levels were evaluated, it was detected that no one parameter was significantly different from others, except that 24.1% of the cases with inspiratory whooping had high anti-PT levels. There was also no statistically significant difference between high anti-PT levels ≥ 100 EU/ml and the patients with risk factors [smoking (21/200; 10.5%), presence of disease that cause chronic cough and/or drug usage (19/171; %11.1), and whole factors which cause chronic cough (32/306; %10.5)] and without risk factors (p= 0.581; p= 0.357; p= 0

  14. 婴幼儿百日咳的临床特征%Clinical manifestations of Bordetella pertussis infection in infants

    Institute of Scientific and Technical Information of China (English)

    黄建琼; 马卓娅; 郑跃杰; 刘恩梅

    2014-01-01

    Objective To explore the clinical manifestations and epidemiological profiles of hospitalized infants with Bordetella pertussis infection and to improve the knowledge for its early diagnosis and treatment.Methods Using a retrospective analysis method,the data of 142 hospitalized infants with Bordetella pertussis infection in Shenzhen Children's Hospital from 2011 to 2013 were collected,which was confirmed by a real-time polymerase chain reaction(PCR) of nasopharyngeal secretion.The SPSS 16.0 software was used for statistical analysis.Results Of the 142hospitalized infants with Bordetella pertussis infection,the ratio of male to female was 2.1∶ 1.0 (96/41 cases).Ninetythree (65.5%) cases were aged less than 3 months old,26 cases (18.3%) were 3-6 months old and 23 cases (16.2%) were 6-36 months old.One hundred and twenty-six (88.7%) patients were unvaccinated or incompletely vaccinated.The most common clinical manifestation was paroxysmal cough which accounts for 104/142 (73.2%) cases,and 6 cases of which had roaring(4.2%).Peripheral blood WBC level of 83 cases increased,and 75 (90.4%) cases were lymphocytosis.The positive rate of X-ray was 60.6%.Eighty-one cases were complicated with bacteria,respiratory virus or mycoplasma pneumonia infection.There were significant differences in symptom of fever and infection of respiratory tract pathogen among 3 groups.The symptom of fever in the group aged less than 3 months old group was significantly lower than the other 2 groups (x2 =11.45,P < 0.01 ;x2 =34.56,P < 0.01),and 6-36 months old group was significantly higher than the other 2 groups(x2 =7.590,P < 0.01 ;x2 =6.928,P < 0.01).Conclusions Bordetella pertussis is an important pathogen for the infants with persistent cough,especially in unvaccinated or incompletely vaccinated infants,but the clinical manifestation and signs of pertussis are atypical,which may lead to a misdiagnosis and delay proper treatment.As a rapid and sensitive method of

  15. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: finleynl@miamioh.edu [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  16. Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality.

    Science.gov (United States)

    Bibova, Ilona; Hot, David; Keidel, Kristina; Amman, Fabian; Slupek, Stephanie; Cerny, Ondrej; Gross, Roy; Vecerek, Branislav

    2015-01-01

    Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

  17. Amidate prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine as inhibitors of adenylate cyclase toxin from Bordetella pertussis.

    Science.gov (United States)

    Šmídková, Markéta; Dvoráková, Alexandra; Tloust'ová, Eva; Česnek, Michal; Janeba, Zlatko; Mertlíková-Kaiserová, Helena

    2014-01-01

    Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

  18. In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: a proof of principle.

    Science.gov (United States)

    Hoonakker, M E; Verhagen, L M; Hendriksen, C F M; van Els, C A C M; Vandebriel, R J; Sloots, A; Han, W G H

    2015-03-01

    Lot release testing of vaccines is primarily based on animal models that are costly, time-consuming and sometimes of questionable relevance. In order to reduce animal use, functional in vitro assays are being explored as an alternative approach for the current lot release testing paradigm. In this study, we present an evaluation of APC platforms assessing innate immune activation by whole cell Bordetella pertussis (wP) vaccines. Primary monocytes, monocyte-derived DC (moDC) and human monocyte/DC cell lines (MonoMac6 and MUTZ-3) were compared for their capacity to respond to wP vaccines of varying quality. To produce such vaccines, the production process of wP was manipulated, resulting in wP vaccines covering a range of in vivo potencies. The responses of MUTZ-3 cells and primary monocytes to these vaccines were marginal and these models were therefore considered inappropriate. Importantly, moDC and MonoMac6 cells responded to the wP vaccines and discriminated between vaccines of varying quality, although slight variations in the responses to wP vaccines of similar quality were also observed. This study provides a proof of principle for the use of in vitro APC platforms as part of a new strategy to assess wP vaccine lot consistency, though careful standardisation of assay conditions is necessary.

  19. Comparative genomic profiling of Dutch clinical Bordetella pertussis isolates using DNA microarrays: Identification of genes absent from epidemic strains

    Directory of Open Access Journals (Sweden)

    van Gent Marjolein

    2008-06-01

    Full Text Available Abstract Background Whooping cough caused by Bordetella pertussis in humans, is re-emerging in many countries despite vaccination. Several studies have shown that significant shifts have occurred in the B. pertussis population resulting in antigenic divergence between vaccine strains and circulating strains and suggesting pathogen adaptation. In the Netherlands, the resurgence of pertussis is associated with the rise of B. pertussis strains with an altered promoter region for pertussis toxin (ptxP3. Results We used Multi-Locus Sequence Typing (MLST, Multiple-Locus Variable Number of Tandem Repeat Analysis (MLVA and microarray-based comparative genomic hybridization (CGH to characterize the ptxP3 strains associated with the Dutch epidemic. For CGH analysis, we developed an oligonucleotide (70-mers microarray consisting of 3,581 oligonucleotides representing 94% of the gene repertoire of the B. pertussis strain Tohama I. Nine different MLST profiles and 38 different MLVA types were found in the period 1993 to 2004. Forty-three Dutch clinical isolates were analyzed with CGH, 98 genes were found to be absent in at least one of the B. pertussis strains tested, these genes were clustered in 8 distinct regions of difference. Conclusion The presented MLST, MLVA and CGH-analysis identified distinctive characteristics of ptxP3 B. pertussis strains -the most prominent of which was a genomic deletion removing about 23,000 bp. We propose a model for the emergence of ptxP3 strains.

  20. [Accumulation of the bvg- Bordetella pertussis a virulent mutants in the process of experimental whooping cough in mice].

    Science.gov (United States)

    Medkova, A Iu; Siniashina, L N; Rumiantseva, Iu P; Voronina, O L; Kunda, M S; Karataev, G I

    2013-01-01

    The duration of the persistence and dynamics of accumulation of insertion bvg- Bordetella pertussis mutants were studied in lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The capability of the virulent B. pertussis bacteria to long-term persistence in the body of mice was tested. Using the real-time PCR approximately hundred genome equivalents of the B. pertussis DNA were detected in lungs of mice in two months after infection regardless of the way of challenge. Using the bacterial test bacteria were identified during only four weeks after challenge. Bvg- B. pertussis avirulent mutants were accumulated for the infection time. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7-9 weeks after challenge. The obtained results show that the laboratory mice can be used for study of the B. pertussis insertion mutant formation dynamics in vivo and confirm the hypothesis about insertional bvg- B. pertussis virulent mutants accumulation during development of pertussis infection in human.

  1. Antigenic and genetic characterization of Bordetella pertussis recovered from Quebec, Canada, 2002-2014: detection of a genetic shift.

    Science.gov (United States)

    Shuel, Michelle; Lefebvre, Brigitte; Whyte, Kathleen; Hayden, Kristy; De Serres, Gaston; Brousseau, Nicholas; Tsang, Raymond S W

    2016-05-01

    Despite vaccination, cyclical peaks of Bordetella pertussis incidence rates are still observed in Canada and other developed countries, making pertussis one of the most prevalent vaccine preventable bacterial diseases. In the postacellular vaccine era, evolution of bacterial strains has resulted in strains with altered vaccine antigens. Previous Canadian studies have focused on isolates mainly from the provinces of Ontario and Alberta, with only small numbers of isolates from other provinces. Therefore, in this study, we examined a larger sample (n = 52) of isolates from Quebec, Canada, between 2002 and 2014. Isolates were characterized by serotype, sequence type, and prevalence of pertactin deficiency. The Quebec isolates shared characteristics similar to other Canadian isolates and to isolates circulating globally. Although pertactin-deficient isolates were not present, a significant shift in sequence type was observed in more recent years. This study highlights the importance of continually monitoring disease-causing isolates to track evolutionary trends and gain a better understanding of the molecular epidemiology of pertussis in Canada.

  2. Plasmacytoid dendritic cell-derived IFNα modulates Th17 differentiation during early Bordetella pertussis infection in mice.

    Science.gov (United States)

    Wu, V; Smith, A A; You, H; Nguyen, T A; Ferguson, R; Taylor, M; Park, J E; Llontop, P; Youngman, K R; Abramson, T

    2016-05-01

    Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis). T helper 17 (Th17) cells have a central role in the resolution of the infection. Emerging studies document that type I interferons (IFNs) suppress Th17 differentiation and interleukin (IL)-17 responses in models of infection and chronic inflammation. As plasmacytoid dendritic cells (pDCs) are a major source of type I IFNs, we hypothesize that during B. pertussis infection in mice, pDC-derived IFNα inhibits a rapid increase in Th17 cells. We found that IFNα-secreting pDCs appear in the lungs during the early stages of infection, while a robust rise of Th17 cells in the lungs is detected at 15 days post-infection or later. The presence of IFNα led to reduced Th17 differentiation and proliferation in vitro. Furthermore, in vivo blocking of IFNα produced by pDCs during infection with B. pertussis infection resulted in early increase of Th17 frequency, inflammation, and reduced bacterial loads in the airways of infected mice. Taken together, the experiments reported here describe an inhibitory role for pDCs and pDC-derived IFNα in modulating Th17 responses during the early stages of B. pertussis infection, which may explain the prolonged nature of whooping cough.

  3. Bordetella pertussis filamentous hemagglutinin itself does not trigger anti-inflammatory interleukin-10 production by human dendritic cells.

    Science.gov (United States)

    Villarino Romero, Rodrigo; Hasan, Shakir; Faé, Kellen; Holubova, Jana; Geurtsen, Jeroen; Schwarzer, Martin; Wiertsema, Selma; Osicka, Radim; Poolman, Jan; Sebo, Peter

    2016-01-01

    Filamentous hemagglutinin (FHA) is an important adhesin of the whooping cough agent Bordetella pertussis and is contained in most acellular pertussis vaccines. Recently, FHA was proposed to exert an immunomodulatory activity through induction of tolerogenic IL-10 secretion from dendritic cells. We have re-evaluated the cytokine-inducing activity of FHA, placing specific emphasis on the role of the residual endotoxin contamination of FHA preparations. We show that endotoxin depletion did not affect the capacity of FHA to bind primary human monocyte-derived dendritic cells, while it abrogated the capacity of FHA to elicit TNF-α and IL-10 secretion and strongly reduced its capacity to trigger IL-6 production. The levels of cytokines induced by the different FHA preparations correlated with their residual contents of B. pertussis endotoxin. Moreover, FHA failed to trigger cytokine secretion in the presence of antibodies that block TLR2 and/or TLR4 signaling. The TLR2 signaling capacity appeared to be linked to the presence of endotoxin-associated components in FHA preparations and not to the FHA protein itself. These results show that the endotoxin-depleted FHA protein does not induce cytokine release from human dendritic cells.

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

    Science.gov (United States)

    Zeddeman, A; van Gent, M; Heuvelman, C J; van der Heide, H G; Bart, M J; Advani, A; Hallander, H O; Wirsing von Konig, C H; Riffelman, M; Storsaeter, J; Vestrheim, D F; Dalby, T; Krogfelt, K A; Fry, N K; Barkoff, A M; Mertsola, J; He, Q; Mooi, F

    2014-08-21

    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 (2010–12). 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.

  5. Plasticity of fimbrial genotype and serotype within populations of Bordetella pertussis: analysis by paired flow cytometry and genome sequencing.

    Science.gov (United States)

    Vaughan, Thomas E; Pratt, Catherine B; Sealey, Katie; Preston, Andrew; Fry, Norman K; Gorringe, Andrew R

    2014-09-01

    The fimbriae of Bordetella pertussis are required for colonization of the human respiratory tract. Two serologically distinct fimbrial subunits, Fim2 and Fim3, considered important vaccine components for many years, are included in the Sanofi Pasteur 5-component acellular pertussis vaccine, and the World Health Organization recommends the inclusion of strains expressing both fimbrial serotypes in whole-cell pertussis vaccines. Each of the fimbrial major subunit genes, fim2, fim3, and fimX, has a promoter poly(C) tract upstream of its -10 box. Such monotonic DNA elements are susceptible to changes in length via slipped-strand mispairing in vitro and in vivo, which potentially causes on/off switching of genes at every cell division. Here, we have described intra-culture variability in poly(C) tract lengths and the resulting fimbrial phenotypes in 22 recent UK B. pertussis isolates. Owing to the highly plastic nature of fimbrial promoters, we used the same cultures for both genome sequencing and flow cytometry. Individual cultures of B. pertussis contained multiple fimbrial serotypes and multiple different fimbrial promoter poly(C) tract lengths, which supports earlier serological evidence that B. pertussis expresses both serotypes during infection.

  6. Homologs of the LapD-LapG c-di-GMP Effector System Control Biofilm Formation by Bordetella bronchiseptica

    Science.gov (United States)

    Ambrosis, Nicolás; Boyd, Chelsea D.; O´Toole, George A.; Fernández, Julieta; Sisti, Federico

    2016-01-01

    Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica PMID:27380521

  7. Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella

    Directory of Open Access Journals (Sweden)

    E. Lesne

    2016-03-01

    Full Text Available The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil’s dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases.

  8. Development and validation of a multiplex immunoassay for the simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus.

    Science.gov (United States)

    van Gageldonk, Pieter G M; van Schaijk, Frank G; van der Klis, Fiona R; Berbers, Guy A M

    2008-06-01

    To increase testing of vaccine induced humoral immunity in immune surveillance studies and vaccine trials, a rapid and simple microsphere-based multiplex assay (pentaplex) was developed for the quantitation of IgG serum antibodies directed against the Bordetella pertussis antigens: Pertussis Toxin (Ptx), Filamentous hemagglutinin (FHA), Pertactin (Prn) and to Diphtheria toxin and Tetanus toxin. All individual antigens were covalently linked to carboxylated microspheres. The method was validated with different serum panels (n=60-78 samples). With the Multiplex Immunoassay (MIA) no evidence for bead interference between monoplex and pentaplex was found. The specificity of the method was shown by a heterologous inhibition of 92%. The pentaplex MIA appeared sensitive with lower limits of quantitation (LLOQ) well below those for ELISA (enzyme-linked immuno-sorbant assay). Assay reproducibility was high with intra-assay variability less than 10% and inter-assay variability below 14%. The reproducibility of the bead conjugation was good and beads could be stored up to at least 6 months without quality reduction. Importantly, the correlation of the pentaplex MIA with the individual ELISAs was excellent, R>0.98 for the Pertussis antigens and R=0.95 for Diphtheria and R=0.98 for Tetanus. Serum IgG antibodies to B. pertussis, Diphtheria and Tetanus can be measured easily, specific and reproducible using the pentaplex MIA. The pentaplex MIA shares features of the ELISA with the additional advantages of high sample throughput and small sample volumes and antigen required.

  9. Performance of transport and selective media for swine Bordetella bronchiseptica recovery and it comparison to polymerase chain reaction detection

    Science.gov (United States)

    Coutinho, Tania Alen; Bernardi, Mari Lourdes; de Itapema Cardoso, Marisa Ribeiro; Borowski, Sandra Maria; Moreno, Andrea Micke; de Barcellos, David Emilio Santos Neves

    2009-01-01

    Three comparative assays were performed seeking to improve the sensitivity of the diagnosis of Bordetella bronchiseptica infection analyzing swine nasal swabs. An initial assay compared the recovery of B. bronchiseptica from swabs simultaneously inoculated with B. bronchiseptica and some interfering bacteria, immersed into three transport formulations (Amies with charcoal, trypticase soy broth and phosphate buffer according to Soerensen supplemented with 5% of bovine fetal serum) and submitted to different temperatures (10°C and 27°C) and periods of incubation (24, 72 and 120 hours). A subsequent assay compared three selective media (MacConkey agar, modified selective medium G20G and a ceftiofur medium) for their recovery capabilities from clinical specimens. One last assay compared the polymerase chain reaction to the three selective media. In the first assay, the recovery of B. bronchiseptica from transport systems was better at 27°C and the three formulations had good performances at this temperature, but the collection of qualitative and quantitative analysis indicated the advantage of Amies medium for nasal swabs transportation. The second assay indicated that MacConkey agar and modified G20G had similar results and were superior to the ceftiofur medium. In the final assay, polymerase chain reaction presented superior capability of B. bronchiseptica detection to culture procedures. PMID:24031390

  10. Pathogenical Studies And Preliminary Prevention on Bordetella Avium%禽波氏杆菌病原性研究及防治初探

    Institute of Scientific and Technical Information of China (English)

    王玉燕; 朱瑞良

    2001-01-01

    Straining characters, antibiotic sensibilities and biochemical characters of 8 strains of Bordetella avium have been experimented. Their pathogenicities to chicks were concentrated on , and proved they caused chicks acute death. The main pathogenic changes were haemorrhage on lung, liver, kidney and serous membrane of glandular stomach and muscular stomach. The resistance of the tested individuals was positively correlated with their age. The older, the resistance of chicks to bordetella avium is stronger. We also developed a preprolis vaccine with 8 strains of bordetella avium coming from different area. Used the vaccine to vaccinate 1-day-old and 11-day-old chick groups, the preventive rate is low.%本文介绍了禽波氏杆菌的培养特性、药物敏感性及生化特性,重点进行了禽波氏杆菌对雏鸡的致病性研究,结果证明本菌主要引起雏鸡的急性死亡,主要病变为肺脏出血,肝脏出血、边缘坏死,肾脏出血,腺胃、肌胃浆膜出血,肠道出血。不同日龄的雏鸡对禽波氏杆菌的抵抗力不同,与日龄成正相关。同时还用不同来源的8株禽波氏杆菌制成蜂胶灭活苗,并分别免疫1日龄和11日龄雏鸡,结果发现本疫苗对雏鸡的保护率低。

  11. Caracterización molecular y funcional de la respuesta de la acidez en Bordetella bronchiseptica : Posible rol en la infección persistente

    OpenAIRE

    Fingermann, Matías

    2011-01-01

    El presente trabajo de tesis se ha focalizado en el estudio del comportamiento de Bordetella bronchiseptica frente a una condición de estrés ambiental como lo es la acidez. La elección de esta temática responde a que este patógeno respiratorio, capaz de inducir un conjunto de enfermedades denominadas bordetellosis, enfrenta y sobrelleva esta condición de estrés durante su ciclo de vida. La capacidad de resistir a a condiciones de acidez podría permitir a B. bronchiseptica inducir estadíos cró...

  12. Expresión de las fimbrias Fim2 y Fim3 desde el plasmidio pMMB67 en cepas de Bordetella pertussis

    OpenAIRE

    Lourdes Proenza; Javier Campos; Beatriz Ferrán; Ernesto Marcos; Celso Pérez

    2010-01-01

    Bordetella pertussis, el agente etiológico de la tos ferina, produce dos tipos de fimbrias serológicamente diferentes, denominadas Fim2 y Fim3. Las fimbrias 2 y 3 están compuestas por una subunidad proteica mayoritaria Fim2, de 22,5 kDa y Fim3, de 22 kDa. Las fimbrias desempeñan un papel inmunomodulador y además, existe una correlación entre la presencia de anticuerpos aglutinantes antifimbrias y la protección contra B. pertussis en pacientes humanos, todo esto ha conducido a la inclusión de ...

  13. Antibody response patterns to Bordetella pertussis antigens in vaccinated (primed) and unvaccinated (unprimed) young children with pertussis.

    Science.gov (United States)

    Cherry, James D; Heininger, Ulrich; Richards, David M; Storsaeter, Jann; Gustafsson, Lennart; Ljungman, Margaretha; Hallander, Hans O

    2010-05-01

    In a previous study, it was found that the antibody response to a nonvaccine pertussis antigen in children who were vaccine failures was reduced compared with the response in nonvaccinated children who had pertussis. In two acellular pertussis vaccine efficacy trials in Sweden, we studied the convalescent-phase enzyme-linked immunosorbent assay (ELISA) geometric mean values (GMVs) in response to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM 2/3) in vaccine failures and controls with pertussis. In Germany, the antibody responses to Bordetella pertussis antigens PT, FHA, PRN, and FIM-2 were analyzed by ELISA according to time of serum collection after onset of illness in children with pertussis who were vaccine failures or who were previously unvaccinated. Antibody values were also compared by severity of clinical illness. In Sweden, infants who had received a PT toxoid vaccine and who were vaccine failures had a blunted response to the nonvaccine antigen FHA compared with the response in children who had received a PT/FHA vaccine. Similarly, infants who had pertussis and who had received a PT/FHA vaccine had a blunted response to the nonvaccine antigens PRN and FIM 2/3 compared with the response in children who were vaccine failures and who had received a PT, FHA, PRN, and FIM 2/3 vaccine. In Germany, in sera collected from 0 to 15 days after pertussis illness onset, the GMVs for all 4 antigens (PT, FHA, PRN, and FIM-2) were significantly lower in an unvaccinated group than in children who were diphtheria-tetanus-acellular pertussis (DTaP) vaccine failures. In the unvaccinated group, the GMV of the PT antibody rose rapidly over time so that it was similar to that of the DTaP vaccine recipients at the 16- to 30-day period. In contrast, the antibody responses to FHA, PRN, and FIM-2 at all time periods were lower in the diphtheria-tetanus vaccine (DT) recipients than in the DTaP vaccine failures. In both Sweden and Germany

  14. Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

    Science.gov (United States)

    Boulanger, Alice; Moon, Kyung; Decker, Kimberly B; Chen, Qing; Knipling, Leslie; Stibitz, Scott; Hinton, Deborah M

    2015-02-10

    Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.

  15. Isolated CyaA-RTX subdomain from Bordetella pertussis: Structural and functional implications for its interaction with target erythrocyte membranes.

    Science.gov (United States)

    Pandit, Riyaz Ahmad; Meetum, Kanungsuk; Suvarnapunya, Kittipong; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2015-10-01

    The 126-kDa Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was previously expressed in Escherichia coli as a soluble precursor that can be acylated to retain hemolytic activity. Here, we investigated structural and functional characteristics of a ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of CyaA-Hly. Initially, we succeeded in producing a large amount with high purity of the His-tagged CyaA-RTX fragment and in establishing the interaction of acylated CyaA-Hly with sheep red blood cell (sRBC) membranes by immuno-localization. Following pre-incubation of sRBCs with non-acylated CyaA-Hly or with the CyaA-RTX fragment that itself produces no hemolytic activity, there was a dramatic decrease in CyaA-Hly-induced hemolysis. When CyaA-RTX was pre-incubated with anti-CyaA-RTX antisera, the capability of CyaA-RTX to neutralize the hemolytic activity of CyaA-Hly was greatly decreased. A homology-based model of the 100-kDa CyaA-RTX subdomain revealed a loop structure in Linker II sharing sequence similarity to human WW domains. Sequence alignment of Linker II with the human WW-domain family revealed highly conserved aromatic residues important for protein-protein interactions. Altogether, our present study demonstrates that the recombinant CyaA-RTX subdomain retains its functionality with respect to binding to target erythrocyte membranes and the WW-homologous region in Linker II conceivably serves as a functional segment required for receptor-binding activity.

  16. Sequence variation in virulence-related genes of Bordetella pertussis isolates from Poland in the period 1959-2013.

    Science.gov (United States)

    Mosiej, E; Zawadka, M; Krysztopa-Grzybowska, K; Polak, M; Augustynowicz, E; Piekarska, K; Lutyńska, A

    2015-01-01

    This study aimed to characterise Bordetella pertussis isolates circulating in Poland since 1959. Sequence analysis of ptxA, ptxC, prn, tcfA, fim2, fim3 and ptxP for 175 clinical isolates and currently and previously used vaccine strains was performed. Clinical isolates from the period 1995-2013 were found to be different to three currently used vaccine strains harbouring the allelic combination ptxA2-ptxC1-ptxP1-prn1-tcfA2-fim2-1-fim3-1, seen frequently in Poland in the early pertussis vaccination period but not found after 1995. Generally, among B. pertussis isolates from the period 2000-2013, two genotypes predominated, ptxA1-ptxC1-ptxP1-prn1-tcfA2-fim2-2-fim3-1 and ptxA1-ptxC1-ptxP1-prn2-tcfA2-fim2-1-fim3-1, with frequencies of 45% and 32.5%, respectively. The isolates harbouring ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-2 and ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 profiles, currently highly prevalent within other European Union (EU) countries, were rarely found in Poland, as they circulated in the period 2000-2013 with frequencies of 10% and 5%, respectively. We hypothesise that several previous changes of strain composition in whole-cell pertussis vaccine produced locally and used since 1960 in Poland resulted in a more diverse immune pressure in the population, resulting in different prevalence of alleles compared to elsewhere.

  17. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Masure, H.R.; Donovan, M.G.; Storm, D.R.

    1991-01-01

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.

  18. Pulsed-field gel electrophoresis analysis of Bordetella pertussis isolates circulating in Europe from 1998 to 2009.

    Science.gov (United States)

    Advani, Abdolreza; Hallander, Hans O; Dalby, Tine; Krogfelt, Karen Angeliki; Guiso, Nicole; Njamkepo, Elisabeth; von Könnig, Carl Heinz Wirsing; Riffelmann, Marion; Mooi, Frits R; Sandven, Per; Lutynska, Anna; Fry, Norman K; Mertsola, Jussi; He, Qiushui

    2013-02-01

    Between 1998 and 2009, Bordetella pertussis clinical isolates were collected during three periods, i.e., 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), and 2007 to 2009 (n = 140), from nine countries with distinct vaccination programs, i.e., Denmark, Finland, France, Germany, The Netherlands, Norway, Poland, Sweden, and the United Kingdom. Pulsed-field gel electrophoresis (PFGE) analysis was performed according to standardized recommendations for epidemiological typing of B. pertussis. There were 81 different PFGE profiles, five of which (BpSR3, BpSR5, BpSR10, BpSR11, and BpSR12) were observed in 61% of the 396 isolates and shown to be predominant in almost all countries. The major profile, BpSR11, showed a decreasing trend from 25% to 30% in 1998 to 2005 to 13% in 2007 to 2009, and there were increases in BpSR3 and BpSR10 from 0% and 8% to 21% and 22%, respectively. One difference between these profiles is that BpSR11 contains isolates harboring the fim3-2 allele and BpSR3 and BpSR10 contain isolates harboring the fim3-1 allele. The total proportion of the five predominant profiles increased from 44% in 1998 to 2001 to 63% in 2004 to 2005 to 70% in 2007 to 2009. In conclusion, common PFGE profiles were identified in B. pertussis populations circulating in European countries with different vaccination programs and different vaccine coverages. These prevalent isolates contain the novel pertussis toxin promoter ptxP3 allele. However, there is evidence for diversifying selection between ptxP3 strains characterized by distinct PFGE profiles. This work shows that, even within a relatively short time span of 10 years, successful isolates which spread through Europe and cause large shifts in B. pertussis populations may emerge.

  19. Studying Bordetella pertussis populations by use of SNPeX, a simple high-throughput single nucleotide polymorphism typing method.

    Science.gov (United States)

    Zeddeman, Anne; Witteveen, Sandra; Bart, Marieke J; van Gent, Marjolein; van der Heide, Han G J; Heuvelman, Kees J; Schouls, Leo M; Mooi, Frits R

    2015-03-01

    Large outbreaks of pertussis occur despite vaccination. A first step in the analyses of outbreaks is strain typing. However, the typing of Bordetella pertussis, the causative agent of pertussis, is problematic because the available assays are insufficiently discriminatory, not unequivocal, time-consuming, and/or costly. Here, we describe a single nucleotide primer extension assay for the study of B. pertussis populations, SNPeX (single nucleotide primer extension), which addresses these problems. The assay is based on the incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) at the 3' end of allele-specific poly(A)-tailed primers and subsequent analysis with a capillary DNA analyzer. Each single nucleotide polymorphism (SNP) primer has a specific length, and as a result, up to 20 SNPs can be determined in one SNPeX reaction. Importantly, PCR amplification of target DNA is not required. We selected 38 SNPeX targets from the whole-genome sequencing data of 74 B. pertussis strains collected from across the world. The SNPeX-based phylogenetic trees preserved the general tree topology of B. pertussis populations based on whole-genome sequencing, with a minor loss of details. We envisage a strategy whereby SNP types (SnpTs) are quickly identified with the SNPeX assay during an outbreak, followed by whole-genome sequencing (WGS) of a limited number of isolates representing predominant SnpTs and the incorporation of novel SNPs in the SNPeX assay. The flexibility of the SNPeX assay allows the method to evolve along with the pathogen, making it a promising method for studying outbreaks of B. pertussis and other pathogens.

  20. Assessment of antibody level and avidity against Bordetella pertussis in a cohort of Egyptian individuals aged 1-18 years.

    Science.gov (United States)

    Ibrahim, Nihal M; El-Kady, Ebtsam M; Eissa, Somiaa A; Wahby, Ahmed F

    2016-01-01

    Pertussis specific antibodies were studied with respect to quality and quantity in a cohort of apparently healthy Egyptian children and adolescents, with their age range between 1 and 18 years, in an attempt to get a close and clear insight into the current humoral immunization status in this specified group and to try find a relation between the antibody levels and their avidities in eradication of this devastating infectious disease. Our results showed that avidity increase was most marked in young school children (6-8 years) where it seemed to reach a plateau in older children and adolescents. Antibody titer was highest in toddlers (1-2 years) and young school children (6-8 years) groups, most probably following vaccination and/or booster doses. Among children aged 1-5 years, 28% had highly avid and 50% had high titer antibodies, whereas in adolescents aged 13-18 years, 70% had highly avid antibodies and only 30% had high titer antibodies. The results clearly demonstrated that while levels of anti-Bordetella pertussis (B. pertussis) antibodies wane with growing age, the avidity seems to increase, to a plateau, irrespective of further antigen exposure in a pattern showing complete independence of avidity on concentration. The present study draws attention to the importance of avidity measurements, together with conventional ELISAs, for evaluating immunity against pertussis. Being based on a limited sample size, it could open doors for larger-scale surveys to be possible indicators for the need and timing of booster vaccination doses among Egyptians.

  1. The Bordetella pertussis Bps polysaccharide enhances lung colonization by conferring protection from complement-mediated killing.

    Science.gov (United States)

    Ganguly, Tridib; Johnson, John B; Kock, Nancy D; Parks, Griffith D; Deora, Rajendar

    2014-07-01

    Bordetella pertussis is a human-restricted Gram-negative bacterial pathogen that causes whooping cough or pertussis. Pertussis is the leading vaccine preventable disease that is resurging in the USA and other parts of the developed world. There is an incomplete understanding of the mechanisms by which B. pertussis evades killing and clearance by the complement system, a first line of host innate immune defence. The present study examined the role of the Bps polysaccharide to resist complement activity in vitro and in the mouse respiratory tract. The isogenic bps mutant strain containing a large non-polar in-frame deletion of the bpsA-D locus was more sensitive to serum and complement mediated killing than the WT strain. As determined by Western blotting, flow cytometry and electron microscopic studies, the heightened sensitivity of the mutant strain was due to enhanced deposition of complement proteins and the formation of membrane attack complex, the end-product of complement activation. Bps was sufficient to confer complement resistance as evidenced by a Bps-expressing Escherichia coli being protected by serum killing. Additionally, Western blotting and flow cytometry assays revealed that Bps inhibited the deposition of complement proteins independent of other B. pertussis factors. The bps mutant strain colonized the lungs of complement-deficient mice at higher levels than that observed in C57Bl/6 mice. These results reveal a previously unknown interaction between Bps and the complement system in controlling B. pertussis colonization of the respiratory tract. These findings also make Bps a potential target for the prevention and therapy of whooping cough.

  2. Bordetella pertussis isolates from Argentinean whooping cough patients display enhanced biofilm formation capacity compared to Tohama I reference strain

    Directory of Open Access Journals (Sweden)

    Laura eArnal

    2015-12-01

    Full Text Available Pertussis is a highly contagious disease mainly caused by Bordetella pertussis. Despite the massive use of vaccines since the 1950´s the disease has become re-emergent in 2000 with a shift in incidence from infants to adolescents and adults. Clearly, the efficacy of current cellular or acellular vaccines, formulated from bacteria grown in stirred bioreactors is limited, presenting a challenge for future vaccine development. For gaining insights into the role of B. pertussis biofilm development for host colonization and persistence within the host, we examined the biofilm forming capacity of eight argentinean clinical isolates recovered from 2001 to 2007. All clinical isolates showed an enhanced potential for biofilm formation compared to the reference strain Tohama I. We further selected the clinical isolate B. pertussis 2723, exhibiting the highest biofilm biomass production, for quantitative proteomic profiling by means of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE coupled with mass spectrometry (MS, which was accompanied by targeted transcriptional analysis. Results revealed an elevated expression of several virulence factors, including adhesins involved in biofilm development. In addition, we observed a higher expression of energy metabolism enzymes in the clinical isolate compared to the Tohama I strain. Furthermore, all clinical isolates carried a polymorphism in the bvgS gene. This mutation was associated to an increased sensitivity to modulation and a faster rate of adhesion to abiotic surfaces. Thus, the phenotypic biofilm characteristics shown by the clinical isolates might represent an important, hitherto underestimated, adaptive strategy for host colonization and long time persistence within the host.

  3. Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

    Science.gov (United States)

    Cannella, Sara E.; Ntsogo Enguéné, Véronique Yvette; Davi, Marilyne; Malosse, Christian; Sotomayor Pérez, Ana Cristina; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Ladant, Daniel; Chenal, Alexandre

    2017-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins. PMID:28186111

  4. Bordetella pertussis Isolates from Argentinean Whooping Cough Patients Display Enhanced Biofilm Formation Capacity Compared to Tohama I Reference Strain.

    Science.gov (United States)

    Arnal, Laura; Grunert, Tom; Cattelan, Natalia; de Gouw, Daan; Villalba, María I; Serra, Diego O; Mooi, Frits R; Ehling-Schulz, Monika; Yantorno, Osvaldo M

    2015-01-01

    Pertussis is a highly contagious disease mainly caused by Bordetella pertussis. Despite the massive use of vaccines, since the 1950s the disease has become re-emergent in 2000 with a shift in incidence from infants to adolescents and adults. Clearly, the efficacy of current cellular or acellular vaccines, formulated from bacteria grown in stirred bioreactors is limited, presenting a challenge for future vaccine development. For gaining insights into the role of B. pertussis biofilm development for host colonization and persistence within the host, we examined the biofilm forming capacity of eight argentinean clinical isolates recovered from 2001 to 2007. All clinical isolates showed an enhanced potential for biofilm formation compared to the reference strain Tohama I. We further selected the clinical isolate B. pertussis 2723, exhibiting the highest biofilm biomass production, for quantitative proteomic profiling by means of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, which was accompanied by targeted transcriptional analysis. Results revealed an elevated expression of several virulence factors, including adhesins involved in biofilm development. In addition, we observed a higher expression of energy metabolism enzymes in the clinical isolate compared to the Tohama I strain. Furthermore, all clinical isolates carried a polymorphism in the bvgS gene. This mutation was associated to an increased sensitivity to modulation and a faster rate of adhesion to abiotic surfaces. Thus, the phenotypic biofilm characteristics shown by the clinical isolates might represent an important, hitherto underestimated, adaptive strategy for host colonization and long time persistence within the host.

  5. Sequence Variation of the Pertussis Toxin S1 Subunit Encoding Gene in the Clinical Isolates of Bordetella pertussis in Iran

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    Hosseinpour

    2015-08-01

    Full Text Available Background Whooping cough (pertussis is an acute respiratory disease caused by Bordetella pertussis (B. pertussis. Pertussis toxin is an important virulence factor of B. pertussis and plays a major role in the immune and inflammatory responses. Likewise, allelic variations in the genes of virulence factors have led to the non-responsiveness of the new strains to both whole-cell and acellular vaccines. Given the importance of pertussis vaccine, we sought to address the lack of fundamental studies on the polymorphisms of the virulence genes of B. pertussis in Iran. Objectives The aim of this study was to identify the polymorphisms of the pertussis toxin S1 subunit (ptxS1 gene in the circulating strains and compare them to the vaccine strain. Patients and Methods In this study, 50 strains of B. pertussis isolated from patients with pertussis were investigated in the pertussis reference laboratory of Pasteur institute of Iran. Cultivation, biochemical tests, and the specific antisera were used to confirm B. pertussis. The sequencing of the polymerase chain reaction products was performed to determine the ptxS1 alleles, and B. pertussis 134 was studied as the vaccine strain. Results The results showed that all the strains had the dominant allele ptxS1A. There were differences between the alleles of the clinical strains and the vaccine strain. Conclusions In recent years, a significant increase in the incidence of pertussis has been reported worldwide. Our findings regarding the allelic shift of the ptxS1 gene are similar to those reported in many European and American countries showing the difference of the dominant allele of ptxS1 between the circulating isolates and the vaccine strains.

  6. Bordetella pertussis, the causative agent of whooping cough, evolved from a distinct, human-associated lineage of B. bronchiseptica.

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    Dimitri A Diavatopoulos

    2005-12-01

    Full Text Available Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu, and B. parapertussis(ov are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu are assumed to have evolved from a B. bronchiseptica-like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu, and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than

  7. Characterization of the N-terminal domain of BteA: a Bordetella type III secreted cytotoxic effector.

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    Chen Guttman

    Full Text Available BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287 and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain.

  8. Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis.

    Science.gov (United States)

    Sugisaki, Kentaro; Hanawa, Tomoko; Yonezawa, Hideo; Osaki, Takako; Fukutomi, Toshiyuki; Kawakami, Hayato; Yamamoto, Tomoko; Kamiya, Shigeru

    2013-07-01

    Bordetella pertussis, the causative agent of whooping cough, is highly adapted to cause human infection. The production of virulence factors, such as adhesins and toxins, is just part of an array of mechanisms by which B. pertussis causes infection. The stringent response is a global bacterial response to nutritional limitation that is mediated by the accumulation of cellular ppGpp and pppGpp [termed together as (p)ppGpp]. Here, we demonstrate that production of (p)ppGpp was controlled by RelA and SpoT proteins in B. pertussis, and that mutation-induced loss of both proteins together caused deficiencies in (p)ppGpp production. The (p)ppGpp-deficient mutants also exhibited defects in growth regulation, decreases in viability under nutritionally limited conditions, increases in susceptibility to oxidative stress and defects in biofilm formation. Analysis of the secreted proteins and the respective transcripts showed that lack of (p)ppGpp led to decreased expression of fim3 and bsp22, which encode a fimbrial subunit and the self-polymerizing type III secretion system tip protein, respectively. Moreover, electron microscopic analysis also indicated that (p)ppGpp regulated the formation of filamentous structures. Most virulence genes - including fim3 and bsp22 - were expressed in the Bvg(+) phase during which the BvgAS two-component system was activated. Although fim3 and bsp22 were downregulated in a (p)ppGpp-deficient mutant, normal expression of fhaB, cyaA and ptxA persisted. Lack of coherence between virulence gene expression and (p)ppGpp production indicated that (p)ppGpp did not modulate the Bvg phase. Taken together, our data indicate that (p)ppGpp may govern an as-yet-unrecognized system that influences B. pertussis pathogenicity.

  9. Isolamento de bactérias do gênero Bordetella e provas sorológicas, a partir de crianças com sintomas de coqueluche, atendidas no Hospital de Isolamento Emílio Ribas de São Paulo Isolation of bacteria of genus Bordetella and serological studies in Brazilian children with symptoms of whoping-cough

    Directory of Open Access Journals (Sweden)

    Sebastião Timo Iaria

    1973-12-01

    Full Text Available A partir de 255 crianças com sintomas de coqueluche atendidas no Hospital de Isolamento Emílio Ribas de São Paulo, procedeu-se a pesquisa de bactérias do gênero Bordetella. Em 46 delas, determinou-se, também, a elevação dos níveis de anticorpos circulantes aglutinantes e fixadores de complemento contra a B. pertussis, aglutinantes contra a B. parapertussis e fixadores de complemento contra adenovirus. Por outro lado, relacionaram-se os resultados do exame bacteriológico com o período da doença, em semanas, e com alguns sintomas principais apresentados. Foram também relacionados os resultados das provas bacteriológica e sorológica com a confirmação clínica do diagnóstico. Foram discutidos, por outro lado, o valor e as limitações das provas bacteriológica e sorológica no diagnóstico laboratorial da coqueluche.Cultures from cough plates and swabs were performed in 255 children with whooping-cough symptoms, hospitalized at "Hospital de Isolamento Emilio Ribas" in S. Paulo. Complement fixation and agglutination tests for Bordetella pertussis, agglutination tests for B. parapertussis and complement fixation test for adenovirus were performed in 46 children, using two blood samples taken one month apart; the isolation results for bacteria of genus Bordetella were correlated to the, period of illness, some of the major symptoms and the clinical diagnosis. A discussion of the efficiency and limitations of both bacteriological and serological tests in the laboratory diagnosis is presented.

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

    Science.gov (United States)

    Kallonen, Teemu; Gröndahl-Yli-Hannuksela, Kirsi; Elomaa, Annika; Lutyńska, Anna; Fry, Norman K; Mertsola, Jussi; He, Qiushui

    2011-12-01

    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

  11. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin.

    Science.gov (United States)

    Springer, Tzvia I; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L

    2014-10-10

    Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD's β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD's β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (Rh) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the β-hairpin's role in stabilizing interactions between CyaA-ACD and N-CaM.

  12. Genome Structural Diversity among 31 Bordetella pertussis Isolates from Two Recent U.S. Whooping Cough Statewide Epidemics.

    Science.gov (United States)

    Bowden, Katherine E; Weigand, Michael R; Peng, Yanhui; Cassiday, Pamela K; Sammons, Scott; Knipe, Kristen; Rowe, Lori A; Loparev, Vladimir; Sheth, Mili; Weening, Keeley; Tondella, M Lucia; Williams, Margaret M

    2016-01-01

    During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, single-contig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were verified with whole-genome optical mapping. Sixteen distinct genome rearrangement profiles were observed in epidemic isolate genomes, all of which were distinct from the genome structures of the two resequenced vaccine strains. These rearrangements appear to be mediated by repetitive sequence elements, such as high-copy-number mobile genetic elements and rRNA operons. Additionally, novel and previously identified single nucleotide polymorphisms were detected in 10 virulence-related genes in the epidemic isolates. Whole-genome variation analysis identified state-specific variants, and coding regions bearing nonsynonymous mutations were classified into functional annotated orthologous groups. Comprehensive studies on whole genomes are needed to understand the resurgence of pertussis and develop novel tools to better characterize the molecular epidemiology of evolving B. pertussis populations. IMPORTANCE Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B. pertussis strains circulating during epidemics exhibit diversity visible on a genome structural

  13. Real-Time PCR Measurement of Persistence of Bordetella pertussis DNA in Nasopharyngeal Secretions during Antibiotic Treatment of Young Children with Pertussis ▿

    Science.gov (United States)

    Bidet, Philippe; Liguori, Sandrine; De Lauzanne, Agathe; Caro, Valérie; Lorrot, Mathie; Carol, Agnès; Faye, Albert; Guiso, Nicole; Bingen, Edouard; Bonacorsi, Stéphane

    2008-01-01

    We used real-time PCR to examine the persistence of Bordetella pertussis DNA in serial nasopharyngeal aspirates from 22 children treated for pertussis. After 5 days of treatment, PCR was positive for all 21 assessable patients. After 14 and 21 days, PCR was still positive for 83% (10/12) and 66% (4/6) of assessable patients, respectively. One patient was tested 1 month after treatment initiation, and B. pertussis DNA was still detectable. Quantitative analysis showed that the DNA concentration diminished during treatment in all except one case. The PCR cycle threshold at which B. pertussis DNA became detectable increased by a mean of 1.7 cycles per day (range, 0.86 to 3.68 cycles per day). Real-time PCR can thus be used to diagnose pertussis in young children for up to 3 weeks after treatment initiation. Its potential value for assessing the treatment outcome remains to be determined. PMID:18768655

  14. Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W., E-mail: n.isaacs@chem.gla.ac.uk [Department of Chemistry and WestChem, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    2007-07-01

    P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6{sub 1}22, with unit-cell parameters a = b = 123.27, c = 134.43 Å.

  15. A Bordetella pertussis proteoliposome induces protection in mice without affecting the immunogenicity of diphtheria and tetanus toxoids in a trivalent formulation.

    Science.gov (United States)

    Castillo, Sonsire Fernández; Chovel, Mario Landys; Hernández, Niurka Gutiérrez; González, Lorena Corcho; Blanco, Amaya; Hernández, Daily Serrano; Medina, Mildrey Fariñas; Tito, Maydelis Álvarez; Quiñoy, José Luis Pérez

    2016-07-01

    In this study, a formulation of Bordetella pertussis proteoliposome (PLBp), diphtheria, and tetanus toxoids and alum (DT-PLBp) was evaluated as a trivalent vaccine candidate in BALB/c mice. Vaccine-induced protection was estimated using the intranasal challenge for pertussis and enzyme-linked immunosorbent assay fvto assess serological responses for diphtheria or tetanus. Both, diphtheria-tetanus-whole cell pertussis (DTP) and diphtheria-tetanus vaccines (DT) were used as controls. Animals immunized with DT-PLBp, PLBp alone, and DTP showed total reduction of CFU in lungs 7 days after intranasal challenge. Likewise, formulations DT-PLBp, DTP, and DT elicited antibody levels ≥2 IU/mL against tetanus and diphtheria, considered protective when neutralization tests are used. Overall, results showed that combination of PLBp with tetanus and diphtheria toxoids did not affect the immunogenicity of each antigen alone.

  16. Immunoregulatory effects of Taishan Pinus massoniana pollen polysaccharide on chicks co-infected with avian leukosis virus and Bordetella avium early in ovo.

    Science.gov (United States)

    Guo, Fanxia; Xue, Cong; Wu, Cun; Zhao, Xue; Qu, Tinghe; He, Xiaohua; Guo, Zhongkun; Zhu, Ruiliang

    2014-04-01

    In recent years, co-infection of chicken embryos with immunosuppressive viruses and bacteria occurs with an annually increasing frequency. Consequently, studies on new and safe immunoregulators, especially plant polysaccharides, have become a popular topic in the poultry industry. In the present study, we selected 300 specific pathogen free embryonated eggs, which were injected with subgroup B avian leukosis virus (ALV-B) and Bordetella avium (B. avium) to establish an artificial co-infection model. The chicks that hatched from these co-infected embryonated eggs were treated with Taishan Pinus massoniana pollen polysaccharide (TPPPS). Results indicated that relevant indices in the co-infection group were significantly lower than that in B. avium-only group. Furthermore, pathogenicity of B. avium was exacerbated, with the chicks exhibiting decreased body weights. The TPPPS groups exhibited gradual improvements in immune function and developmental status. Therefore, in terms of improving immunologic function and production performance, TPPPS could be used as immunoregulator for immune responses.

  17. A Bordetella pertussis proteoliposome induces protection in mice without affecting the immunogenicity of diphtheria and tetanus toxoids in a trivalent formulation

    Science.gov (United States)

    2016-01-01

    In this study, a formulation of Bordetella pertussis proteoliposome (PLBp), diphtheria, and tetanus toxoids and alum (DT-PLBp) was evaluated as a trivalent vaccine candidate in BALB/c mice. Vaccine-induced protection was estimated using the intranasal challenge for pertussis and enzyme-linked immunosorbent assay fvto assess serological responses for diphtheria or tetanus. Both, diphtheria-tetanus-whole cell pertussis (DTP) and diphtheria-tetanus vaccines (DT) were used as controls. Animals immunized with DT-PLBp, PLBp alone, and DTP showed total reduction of CFU in lungs 7 days after intranasal challenge. Likewise, formulations DT-PLBp, DTP, and DT elicited antibody levels ≥2 IU/mL against tetanus and diphtheria, considered protective when neutralization tests are used. Overall, results showed that combination of PLBp with tetanus and diphtheria toxoids did not affect the immunogenicity of each antigen alone. PMID:27489808

  18. [Comparative susceptibility of Ochrobactrum anthropi, Agrobacterium tumefaciens, Alcaligenes faecalis, Alcaligenes denitrificans subsp. denitrificans, Alcaligenes denitrificans subsp. xylosidans and Bordetella bronchiseptica against 35 antibiotics including 17 beta-lactams].

    Science.gov (United States)

    Bizet, C; Bizet, J

    1995-04-01

    Ochrobactrum anthropi, formerly known as "Achromobacter sp." or CDC group Vd has been isolated from water, hospital environment (antiseptic solutions, dialysis fluids ... ). O. anthropi is a Gram negative, motile, strictly aerobic, oxydase positive and non-fermentative bacteria with a strong urease activity. The susceptibility of 13 strains of O. anthropi was determined by agar diffusion method and compared to those of type strains of Agrobacterium tumefaciens, Alcaligenes faecalis, Alcaligenes denitrificans subsp. denitrificans, Alcaligenes denitrificans subsp. xylosoxydans and Bordetella bronchiseptica. The MICs of 20 antimicrobial agents confirmed the distinct phenotype susceptibility of O. anthropi. All the strains of O. anthropi are sensitive to imipenem, amikacin, gentamicin, netilmicin, nalidixic acid, pefloxacin, ciprofloxacin, tetracyclin, colistin, sulphonamides and rifampicin and resistant to ampicillin, amoxycillin + clavulanic acid, ticarcillin, mezlocillin, cefuroxime, cefamandol, cefoxitin, cefotaxime, cefoperazon, ceftazidime, cefsulodin, aztreonam, streptomycin, kanamycin, pipemidic acid, chloramphenicol, erythromicin, pristinamycin, trimethoprim and fosfomycin. O. anthropi is implicated in nosocomial infections. O. anthropi was the species with the greatest resistance to beta-lactamins.

  19. Real-time PCR measurement of persistence of Bordetella pertussis DNA in nasopharyngeal secretions during antibiotic treatment of young children with pertussis.

    Science.gov (United States)

    Bidet, Philippe; Liguori, Sandrine; De Lauzanne, Agathe; Caro, Valérie; Lorrot, Mathie; Carol, Agnès; Faye, Albert; Guiso, Nicole; Bingen, Edouard; Bonacorsi, Stéphane

    2008-11-01

    We used real-time PCR to examine the persistence of Bordetella pertussis DNA in serial nasopharyngeal aspirates from 22 children treated for pertussis. After 5 days of treatment, PCR was positive for all 21 assessable patients. After 14 and 21 days, PCR was still positive for 83% (10/12) and 66% (4/6) of assessable patients, respectively. One patient was tested 1 month after treatment initiation, and B. pertussis DNA was still detectable. Quantitative analysis showed that the DNA concentration diminished during treatment in all except one case. The PCR cycle threshold at which B. pertussis DNA became detectable increased by a mean of 1.7 cycles per day (range, 0.86 to 3.68 cycles per day). Real-time PCR can thus be used to diagnose pertussis in young children for up to 3 weeks after treatment initiation. Its potential value for assessing the treatment outcome remains to be determined.

  20. A Novel IgM-capture enzyme-linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection.

    Science.gov (United States)

    Otsuka, Nao; Gotoh, Kensei; Nishimura, Naoko; Ozaki, Takao; Nakamura, Yukitsugu; Haga, Kiyohito; Yamazaki, Makoto; Gondaira, Fumio; Okada, Kenji; Miyaji, Yusuke; Toyoizumi-Ajisaka, Hiromi; Shibayama, Keigo; Arakawa, Yoshichika; Kamachi, Kazunari

    2016-05-01

    An ELISA that measures anti-PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti-Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.

  1. Conservación por congelación de Bordetella pertussis y Corynebacterium diphtheriae, empleados en la producción de vacunas para uso humano

    Directory of Open Access Journals (Sweden)

    Yilian Plasencia,

    2000-11-01

    Full Text Available En el presente estudio se evaluó el método de congelación a –70ºC para la preservación de Bordetella pertussis y Corynebacterium diphtheriae. Para verificar el sustento de los cultivos se realizó un adecuado control de calidad, que incluyó comprobación de pureza, viabilidad y estabilidad de las propiedades de interés. En este trabajo se probaron diferentes formulaciones. Se seleccionó la que arrojó los mejores resultados y se realizó un estudio de mantenimiento de las características evaluadas durante el tiempo. Para medir determinados parámetros se realizaron procesos a escala industrial, empleándose para esto un biorreactor Chemap de 35 L. Se tomaron como referencia los valores obtenidos por las cepas conservadas por liofilización. De esta forma se buscaron alternativas y soluciones a problemas presentados en su conservación. Los resultados obtenidos sugieren la posible inclusión en el Programa de Mantenimiento establecido.

  2. Sorotipos e perfis genéticos de cepas de Bordetella pertussis isoladas na cidade de São Paulo, 2006-2008

    Directory of Open Access Journals (Sweden)

    Daniela Leite

    2012-08-01

    Full Text Available OBJETIVO: O conhecimento de Bordetella pertussis circulante na América Latina é limitado. Portanto, o objetivo deste estudo foi usar a técnica da eletroforese em campo pulsado e a sorotipagem para caracterizar cepas de B. pertussis isoladas na cidade de São Paulo (SP. MÉTODOS: Este estudo, conduzido entre 2006 e 2008, analisou 652 swabs de nasofaringe coletados de casos suspeitos e comunicantes de coqueluche, provenientes de 37 hospitais sentinela de São Paulo. Foram realizadas as técnicas da eletroforese em campo pulsado e sorotipagem em 91 (70% cepas de B. pertussis, escolhidas aleatoriamente. RESULTADOS: Noventa e sete por cento das cepas de São Paulo foram sorotipadas como Fim3. Foram identificados 14 perfis genéticos pela eletroforese em campo pulsado; o mais prevalente (57% também é o mais prevalente nos EUA. CONCLUSÕES: Esses dados, em conjunto com ações da vigilância, podem ter um impacto nas estratégias de prevenção e controle de coqueluche na região, oferecendo informações úteis para a introdução de estratégias novas de vacinação e redução do risco de transmissão para bebês menores de 6 meses de idade.

  3. Greater efficacy of the ECMS-oil adjuvant over other formulations on immune responses against Bordetella bronchiseptica in rabbits and the underlying mechanism.

    Science.gov (United States)

    Xiao, Chenwen; Bao, Guolian; Liu, Yan; Wei, Qiang; Ji, Quanan; Liu, Yaping; Pan, Lijun

    2016-09-01

    In this study, the adjuvant effects of the extract of Cochinchina momordica seed ECMS+oil, oil alone, ECMS alone, conventional alum adjuvant on inactivated Bordetella bronchiseptica (Bb) vaccine or control using antigen alone without adjuvant were evaluated along with the underlying mechanism. The results in experiment A demonstrated that antibody levels in Bb whole cell protein in the ECMS800μg+oil group were significantly higher than in the other adjuvant groups (pother groups (pother adjuvant groups, including control using antigen alone without adjuvant and the PBS group (pother groups in the lungs (pother groups except the ECMS(100μg/800μg)+oil groups (pother groups, including the control using antigen alone group (pother groups, except for the ECMS400μg+oil group (pother groups (p<0.05). GATA3 in the ECMS800μg+oil groups was significantly higher than the oil, ECMS800μg and control using antigen alone group (p<0.05). ECMS-oil adjuvant mixture could most effectively protect B. bronchiseptica immunized rabbits and, therefore, could be an alternative way of improving B. bronchiseptica vaccination in rabbits.

  4. Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis.

    Science.gov (United States)

    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, Tarsis F; Pávek, Petr; Trejtnar, František; Watts, Val J; Janeba, Zlatko

    2015-08-01

    Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

  5. Crystallization and preliminary X-ray diffraction analysis of two extracytoplasmic solute receptors of the DctP family from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Rucktooa, Prakash; Huvent, Isabelle [UMR8161 CNRS Institut de Biologie de Lille, Laboratoire de Cristallographie Macromoléculaire, 1 Rue du Professeur Calmette, BP 447, 59021 Lille CEDEX (France); IFR 142, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France); Antoine, Rudy; Lecher, Sophie; Jacob-Dubuisson, Françoise, E-mail: francoise.jacob@ibl.fr [IFR 142, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France); INSERM-U629, Lille (France); Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France); Villeret, Vincent, E-mail: francoise.jacob@ibl.fr; Bompard, Coralie [UMR8161 CNRS Institut de Biologie de Lille, Laboratoire de Cristallographie Macromoléculaire, 1 Rue du Professeur Calmette, BP 447, 59021 Lille CEDEX (France); IFR 142, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France)

    2006-10-01

    Sample preparation, crystallization and preliminary X-ray analysis are reported for two B. pertussis extracytoplasmic solute receptors. DctP6 and DctP7 are two Bordetella pertussis proteins which belong to the extracytoplasmic solute receptors (ESR) superfamily. ESRs are involved in the transport of substrates from the periplasm to the cytosol of Gram-negative bacteria. DctP6 and DctP7 have been crystallized and diffraction data were collected using a synchrotron-radiation source. DctP6 crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 108.39, b = 108.39, c = 63.09 Å, while selenomethionyl-derivatized DctP7 crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 64.87, b = 149.83, c = 170.65 Å. The three-dimensional structure of DctP7 will be determined by single-wavelength anomalous diffraction, while the DctP6 structure will be solved by molecular-replacement methods.

  6. Genome-wide gene expression analysis of Bordetella pertussis isolates associated with a resurgence in pertussis: elucidation of factors involved in the increased fitness of epidemic strains.

    Science.gov (United States)

    King, Audrey J; van der Lee, Saskia; Mohangoo, Archena; van Gent, Marjolein; van der Ark, Arno; van de Waterbeemd, Bas

    2013-01-01

    Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, which is a highly contagious disease in the human respiratory tract. Despite vaccination since the 1950s, pertussis remains the most prevalent vaccine-preventable disease in developed countries. A recent resurgence pertussis is associated with the expansion of B. pertussis strains with a novel allele for the pertussis toxin (ptx) promoter ptxP3 in place of resident ptxP1 strains. The recent expansion of ptxP3 strains suggests that these strains carry mutations that have increased their fitness. Compared to the ptxP1 strains, ptxP3 strains produce more Ptx, which results in increased virulence and immune suppression. In this study, we investigated the contribution of gene expression changes of various genes on the increased fitness of the ptxP3 strains. Using genome-wide gene expression profiling, we show that several virulence genes had higher expression levels in the ptxP3 strains compared to the ptxP1 strains. We provide the first evidence that wildtype ptxP3 strains are better colonizers in an intranasal mouse infection model. This study shows that the ptxP3 mutation and the genetic background of ptxP3 strains affect fitness by contributing to the ability to colonize in a mouse infection model. These results show that the genetic background of ptxP3 strains with a higher expression of virulence genes contribute to increased fitness.

  7. Differential effect of TLR2 and TLR4 on the immune response after immunization with a vaccine against Neisseria meningitidis or Bordetella pertussis.

    Directory of Open Access Journals (Sweden)

    Floris Fransen

    Full Text Available Neisseria meningitidis and Bordetella pertussis are Gram-negative bacterial pathogens that can cause serious diseases in humans. N. meningitidis outer membrane vesicle (OMV vaccines and whole cell pertussis vaccines have been successfully used in humans to control infections with these pathogens. The mechanisms behind their effectiveness are poorly defined. Here we investigated the role of Toll-like receptor (TLR 2 and TLR4 in the induction of immune responses in mice after immunization with these vaccines. Innate and adaptive immune responses were compared between wild type mice and mice deficient in TLR2, TLR4, or TRIF. TRIF-deficient and TLR4-deficient mice showed impaired immunity after immunization. In contrast, immune responses were not lower in TLR2-/- mice but tended even to be higher after immunization. Together our data demonstrate that TLR4 activation contributes to the immunogenicity of the N. meningitidis OMV vaccine and the whole cell pertussis vaccine, but that TLR2 activation is not required.

  8. Calcium, acylation, and molecular confinement favor folding of Bordetella pertussis adenylate cyclase CyaA toxin into a monomeric and cytotoxic form.

    Science.gov (United States)

    Karst, Johanna C; Ntsogo Enguéné, V Yvette; Cannella, Sara E; Subrini, Orso; Hessel, Audrey; Debard, Sylvain; Ladant, Daniel; Chenal, Alexandre

    2014-10-31

    The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

  9. Shotgun proteome analysis of Bordetella pertussis reveals a distinct influence of iron availability on the bacterial metabolism, virulence, and defense response.

    Science.gov (United States)

    Alvarez Hayes, Jimena; Lamberti, Yanina; Surmann, Kristin; Schmidt, Frank; Völker, Uwe; Rodriguez, Maria Eugenia

    2015-07-01

    One of the mechanisms involved in host immunity is the limitation of iron accessibility to pathogens, which in turn provokes the corresponding physiological adaptation of pathogens. This study reports a gel-free nanoLC-MS/MS-based comparative proteome analysis of Bordetella pertussis grown under iron-excess and iron-depleted conditions. Out of the 926 proteins covered 98 displayed a shift in their abundance in response to low iron availability. Forty-seven of them were found to be increased in level while 58 were found with decreased protein levels under iron starvation. In addition to proteins previously reported to be influenced by iron in B. pertussis, we observed changes in metabolic proteins involved in fatty acid utilization and poly-hydroxybutyrate production. Additionally, many bacterial virulence factors regulated by the BvgAS two-component system were found at decreased levels in response to iron limitation. These results, together with the increased production of proteins potentially involved in oxidative stress resistance, seem to indicate that iron starvation provokes changes in B. pertussis phenotype that might shape host-pathogen interaction.

  10. Caspase-1-independent interleukin-1β is required for clearance of Bordetella pertussis infections and whole-cell vaccine-mediated immunity.

    Science.gov (United States)

    Place, David E; Muse, Sarah J; Kirimanjeswara, Girish S; Harvill, Eric T

    2014-01-01

    Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1) response, which is required for the clearance of Bordetella pertussis. We show that IL-1β but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1β and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.

  11. B-cell responses after intranasal vaccination with the novel attenuated Bordetella pertussis vaccine strain BPZE1 in a randomized phase I clinical trial.

    Science.gov (United States)

    Jahnmatz, Maja; Amu, Sylvie; Ljungman, Margaretha; Wehlin, Lena; Chiodi, Francesca; Mielcarek, Nathalie; Locht, Camille; Thorstensson, Rigmor

    2014-06-05

    Despite high vaccination coverage, pertussis is still a global concern in infant morbidity and mortality, and improved pertussis vaccines are needed. A live attenuated Bordetella pertussis strain, named BPZE1, was designed as an intranasal vaccine candidate and has recently been tested in man in a phase I clinical trial. Here, we report the evaluation of the B-cell responses after vaccination with BPZE1. Forty-eight healthy males with no previous pertussis-vaccination were randomized into one of three dose-escalating groups or into a placebo group. Plasma blast- and memory B-cell responses were evaluated by ELISpot against three different pertussis antigens: pertussis toxin, filamentous haemagglutinin and pertactin. Seven out of the 36 subjects who had received the vaccine were colonized by BPZE1, and significant increases in the memory B-cell response were detected against all three tested antigens in the culture-positive subjects between days 0 and 28 post-vaccination. The culture-positive subjects also mounted a significant increase in the filamentous haemagglutinin-specific plasma blast response between days 7 and 14 post-vaccination. No response could be detected in the culture-negatives or in the placebo group post-vaccination. These data show that BPZE1 is immunogenic in humans and is therefore a promising candidate for a novel pertussis vaccine. This trial is registered at ClinicalTrials.gov (NCT01188512).

  12. A versatile, non genetically modified organism (GMO)-based strategy for controlling low-producer mutants in Bordetella pertussis cultures using antigenic modulation.

    Science.gov (United States)

    Goffin, Philippe; Slock, Thomas; Smessaert, Vincent; De Rop, Philippe; Dehottay, Philippe

    2015-08-01

    The uncontrolled presence of non-producer mutants negatively affects bioprocesses. In Bordetella pertussis cultures, avirulent mutants emerge spontaneously and accumulate. We characterized the dynamics of accumulation using high-throughput growth assays and competition experiments between virulent and avirulent (bvg(-) ) isolates. A fitness advantage of bvg(-) cells was identified as the main driver for bvg(-) accumulation under conditions of high virulence factor production. Conversely, under conditions that reduce their expression (antigenic modulation), bvg(-) takeover could be avoided. A control strategy was derived, which consists in applying modulating conditions whenever virulence factor production is not required. It has a wide range of applications, from routine laboratory operations to vaccine manufacturing, where pertussis toxin yields were increased 1.4-fold by performing early pre-culture steps in modulating conditions. Because it only requires subtle modifications of the culture medium and does not involve genetic modifications, this strategy is applicable to any B. pertussis isolate, and should facilitate regulatory acceptance of process changes for vaccine production. Strategies based on the same concept, could be derived for other industrially relevant micro-organisms. This study illustrates how a sound scientific understanding of physiological principles can be turned into a practical application for the bioprocess industry, in alignment with Quality by Design principles.

  13. Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme.

    Science.gov (United States)

    Masin, Jiri; Osicka, Radim; Bumba, Ladislav; Sebo, Peter

    2015-11-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. Among other effects, this yields activation of the tyrosine phosphatase SHP-1, BimEL accumulation and phagocyte apoptosis induction. In parallel, CyaA acts as a cytolysin that forms cation-selective pores in target membranes. Direct penetration of CyaA into the cytosol of professional antigen-presenting cells allows the use of an enzymatically inactive CyaA toxoid as a tool for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can be exploited for induction of antigen-specific CD8(+) cytotoxic T-lymphocyte immune responses.

  14. Functional significance of the highly conserved Glu(570) in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin.

    Science.gov (United States)

    Kurehong, Chattip; Powthongchin, Busaba; Thamwiriyasati, Niramon; Angsuthanasombat, Chanan

    2011-05-01

    Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3((570-593)) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu(570), Gln(574), Glu(581), Ser(584) and Ser(585)) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu(570), which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction.

  15. Both CD4+ and CD8+ Lymphocytes Participate in the IFN-γ Response to Filamentous Hemagglutinin from Bordetella pertussis in Infants, Children, and Adults

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    Violette Dirix

    2012-01-01

    Full Text Available Infant CD4+ T-cell responses to bacterial infections or vaccines have been extensively studied, whereas studies on CD8+ T-cell responses focused mainly on viral and intracellular parasite infections. Here we investigated CD8+ T-cell responses upon Bordetella pertussis infection in infants, children, and adults and pertussis vaccination in infants. Filamentous hemagglutinin-specific IFN-γ secretion by circulating lymphocytes was blocked by anti-MHC-I or -MHC-II antibodies, suggesting that CD4+ and CD8+ T lymphocytes are involved in IFN-γ production. Flow cytometry analyses confirmed that both cell types synthesized antigen-specific IFN-γ, although CD4+ lymphocytes were the major source of this cytokine. IFN-γ synthesis by CD8+ cells was CD4+ T cell dependent, as evidenced by selective depletion experiments. Furthermore, IFN-γ synthesis by CD4+ cells was sometimes inhibited by CD8+ lymphocytes, suggesting the presence of CD8+ regulatory T cells. The role of this dual IFN-γ secretion by CD4+ and CD8+ T lymphocytes in pertussis remains to be investigated.

  16. Caspase-1-independent interleukin-1β is required for clearance of Bordetella pertussis infections and whole-cell vaccine-mediated immunity.

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    David E Place

    Full Text Available Whooping cough remains a significant disease worldwide and its re-emergence in highly vaccinated populations has been attributed to a combination of imperfect vaccines and evolution of the pathogen. The focus of this study was to examine the role of IL-1α/β and the inflammasome in generation of the interleukin-1 (IL-1 response, which is required for the clearance of Bordetella pertussis. We show that IL-1β but not IL-1α is required for mediating the clearance of B. pertussis from the lungs of mice. We further found that IL-1β and IL-1R deficient mice, compared to wild-type, have similar but more persistent levels of inflammation, characterized by immune cell infiltration, with significantly increased IFNγ and a normal IL-17A response during B. pertussis infection. Contrary to expectations, the cleavage of precursor IL-1β to its mature form did not require caspase-1 during primary infections within the lung despite being required by bone marrow-derived macrophages exposed to live bacteria. We also found that the caspase-1 inflammasome was not required for protective immunity against a B. pertussis challenge following vaccination with heat-killed whole cell B. pertussis, despite IL-1R signaling being required. These findings demonstrate that caspase-1-independent host factors are involved in the processing of protective IL-1β responses that are critical for bacterial clearance and vaccine-mediated immunity.

  17. Cytokine and chemokine mRNA expression profiles in BALF cells isolated from pigs single infected or co-infected with swine influenza virus and Bordetella bronchiseptica.

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    Kowalczyk, Andrzej; Pomorska-Mól, Małgorzata; Kwit, Krzysztof; Pejsak, Zygmunt; Rachubik, Jarosław; Markowska-Daniel, Iwona

    2014-06-01

    Pigs serve as a valuable animal experimental model for several respiratory pathogens, including Swine Influenza Virus (SIV) and Bordetella bronchiseptica (Bbr). To investigate the effect of SIV and Bbr coinfection on cytokine and viral RNA expression, we performed a study in which pigs were inoculated with SIV, Bbr or both pathogens (SIV/Bbr). Our results indicate that Bbr infection alters SIV clearance. Pulmonary lesions in the SIV/Bbr group were more severe when compared to SIV or Bbr groups and Bbr did not cause significant lesions. Broncho-alveolar lavage fluid (BALF) was examined for inflammatory mediators by qPCR. Interferon (IFN)-α, interleukin IL-8, IL-1 peaked in BALF at 2 DPI, while the virus titres and severity of clinical signs were maximal at the same time. Despite its increased expression in co-infected pigs, interferon-α did not enhance SIV clearance, since the viral replication was detected at the same day as the highest IFN levels. The mRNA levels for IFN-α, IL-1β and IL-8 were significantly higher in BALF of co-infected pigs and correlated with enhanced viral RNA titers in lungs, trachea and nasal swabs. Transcription of mRNA for IL-1β was stable in SIV and SIV/Bbr groups throughout all the study. In Bbr group, the levels of mRNAs for IL-1β were significantly higher at 2, 4 and 9 DPI. The mean levels of mRNAs for TNF-α were lower than the levels of other chemokines and cytokines in all infected groups. Transcript levels of IL-10 and IL-4 did not increase at each time points. Overall, SIV replication was increased by Bbr presence and the enhanced production of pro-inflammatory mediators could contribute to the exacerbated pulmonary lesions.

  18. Bordetella pertussis Lipid A Recognition by Toll-like Receptor 4 and MD-2 Is Dependent on Distinct Charged and Uncharged Interfaces.

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    Maeshima, Nina; Evans-Atkinson, Tara; Hajjar, Adeline M; Fernandez, Rachel C

    2015-05-22

    Lipid A in LPS activates innate immunity through the Toll-like receptor 4 (TLR4)-MD-2 complex on host cells. Variation in lipid A has significant consequences for TLR4 activation and thus may be a means by which Gram-negative bacteria modulate host immunity. However, although even minor changes in lipid A structure have been shown to affect downstream immune responses, the mechanism by which the TLR4-MD-2 receptor complex recognizes these changes is not well understood. We previously showed that strain BP338 of the human pathogen Bordetella pertussis, the causative agent of whooping cough, modifies its lipid A by the addition of glucosamine moieties that promote TLR4 activation in human, but not mouse, macrophages. Using site-directed mutagenesis and an NFκB reporter assay screen, we have identified several charged amino acid residues in TLR4 and MD-2 that are important for these species-specific responses; some of these are novel for responses to penta-acyl B. pertussis LPS, and their mutation does not affect the response to hexa-acylated Escherichia coli LPS or tetra-acylated lipid IVA. We additionally show evidence that suggests that recognition of penta-acylated B. pertussis lipid A is dependent on uncharged amino acids in TLR4 and MD-2 and that this is true for both human and mouse TLR4-MD-2 receptors. Taken together, we have demonstrated that the TLR4-MD-2 receptor complex recognizes variation in lipid A molecules using multiple sites for receptor-ligand interaction and propose that host-specific immunity to a particular Gram-negative bacterium is, at least in part, mediated by very subtle tuning of one of the earliest interactions at the host-pathogen interface.

  19. Changes in the genomic content of circulating Bordetella pertussis strains isolated from the Netherlands, Sweden, Japan and Australia: adaptive evolution or drift?

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    van der Lee Saskia

    2010-01-01

    Full Text Available Abstract Background Bordetella pertussis is the causative agent of human whooping cough (pertussis and is particularly severe in infants. Despite worldwide vaccinations, whooping cough remains a public health problem. A significant increase in the incidence of whooping cough has been observed in many countries since the 1990s. Several reasons for the re-emergence of this highly contagious disease have been suggested. A particularly intriguing possibility is based on evidence indicating that pathogen adaptation may play a role in this process. In an attempt to gain insight into the genomic make-up of B. pertussis over the last 60 years, we used an oligonucleotide DNA microarray to compare the genomic contents of a collection of 171 strains of B. pertussis isolates from different countries. Results The CGH microarray analysis estimated the core genome of B. pertussis, to consist of 3,281 CDSs that are conserved among all B. pertussis strains, and represent 84.8% of all CDSs found in the 171 B. pertussis strains. A total of 64 regions of difference consisting of one or more contiguous CDSs were identified among the variable genes. CGH data also revealed that the genome size of B. pertussis strains is decreasing progressively over the past 60 years. Phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the strains. B. pertussis strains with the same gene content were found in several different countries. However, geographic specificity of the B. pertussis strains was not observed. The gene content was determined to highly correlate with the ptxP-type of the strains. Conclusions An overview of genomic contents of a large collection of isolates from different countries allowed us to derive a core genome and a phylogenetic structure of B. pertussis. Our results show that B. pertussis is a dynamic organism that continues to evolve.

  20. Study on Toxicity Reduction and Potency Induction in Whole-cell Pertussis Vaccine by Developing a New Optimal Inactivation Condition Processed on Bordetella pertussis

    Science.gov (United States)

    Mohammadpour Dounighi, Naser; Razzaghi-Abyane, Mehdi; Nofeli, Mojtaba; Zolfagharian, Hossein; Shahcheraghi, Fereshteh

    2016-01-01

    Background Whooping cough is caused by Bordetella pertussis, and it remains a public health concern. Whole-cell pertussis vaccines have been commonly employed for expanded immunization. There is no doubt of the efficacy of whole cell pertussis vaccine, but it is necessary to improve the vaccine to decrease its toxicity. Objectives In this study, an inactivation process of dealing with pertussis bacteria is optimized in order to decrease the bacteria content in human doses of vaccines and reduce the vaccine’s toxicity. Materials and Methods The bacterial suspensions of pertussis strains 509 and 134 were divided into 21 sample parts from F1 to F21 and inactivated under different conditions. The inactivated suspensions of both strains were tested for opacity, non-viability, agglutination, purity, and sterility; the same formulation samples that passed quality tests were then pooled together. The pool of inactivated suspensions were analyzed for sterility, agglutination, opacity, specific toxicity, and potency. Results The harvest of both bacterial strains showed purity. The opacity of various samples were lost under different treatment conditions by heat from 8% to 12%, formaldehyde 6% to 8%, glutaraldehyde 6% to 8%, and thimerosal 5% to 8%. Tests on suspensions after inactivation and on pooled suspensions showed inactivation conditions not degraded agglutinins of both strains. The samples of F2, F4, F8, F12, F15, and F17 passed the toxicity test. The potency (ED50) of these samples showed following order F17 > F12 > F8 > F15, F4 > F2, and F17 revealed higher potency compared to other formulations. Conclusions It can be concluded that F17 showed desirable outcomes in the toxicity test and good immunogenicity with a low bacterial number content. Consequently, lower adverse effects and good immunogenicity are foreseeable for vaccine preparation with this method. PMID:27679704

  1. Bordetella adenylate cyclase toxin differentially modulates toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells.

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    Irena Adkins

    Full Text Available Adenylate cyclase toxin (CyaA is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR-activated murine and human dendritic cells (DCs. cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4(+ and CD8(+ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4+ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4(+CD25(+Foxp3(+ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8(+ T cell proliferation and limited the induction of IFN-γ producing CD8(+ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.

  2. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.

    Science.gov (United States)

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-04-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity.

  3. Antibody responses to individual Bordetella pertussis fimbrial antigen Fim2 or Fim3 following immunization with the five-component acellular pertussis vaccine or to pertussis disease.

    Science.gov (United States)

    Alexander, Frances; Matheson, Mary; Fry, Norman K; Labram, Briony; Gorringe, Andrew R

    2012-11-01

    Bordetella pertussis expresses two serologically distinct fimbriae (Fim2 and Fim3) which are included in the Sanofi Pasteur 5-component acellular pertussis vaccine, and antibody responses to these antigens have been shown to be associated with protection. Studies to date have assessed the IgG response to this vaccine using a copurified mixture of Fim2 and Fim3, and the response to the individual antigens has not been characterized. We have purified separate Fim2 and Fim3 from strains that express either Fim2 or Fim3 and have used these antigens in an enzyme-linked immunosorbent assay (ELISA) to quantify IgG responses following immunization with 5-component acellular pertussis vaccine in 15-month-old, 4- to 6-year-old, and 11- to 18-year-old subjects. All individuals showed increases in Fim2 and Fim3 IgG concentrations following immunization, with 3-fold-greater Fim2 than Fim3 IgG concentrations seen in the younger two age groups. Fim2 IgG concentrations were 1.5-fold greater than Fim3 IgG concentrations in the 11- to 18-year-olds. We have also compared Fim2 and Fim3 IgG concentrations in individuals with prolonged cough who were diagnosed as having recent pertussis using a pertussis toxin (Ptx) IgG ELISA with individuals with prolonged cough but without elevated Ptx IgG concentrations. Individuals with evidence of recent pertussis had greater Fim3 IgG concentrations, consistent with the predominant serotype of isolates obtained in the United Kingdom. However, a surprising number of individuals had moderate Fim2 IgG concentrations despite very few isolates of that serotype obtained in the sampling period.

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

    Science.gov (United States)

    Elomaa, Annika; Advani, Abdolreza; Donnelly, Declan; Antila, Mia; Mertsola, Jussi; Hallander, Hans; He, Qiushui

    2005-08-01

    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.

  5. Strain Variation among Bordetella pertussis Isolates in Finland, Where the Whole-Cell Pertussis Vaccine Has Been Used for 50 Years

    Science.gov (United States)

    Elomaa, Annika; Advani, Abdolreza; Donnelly, Declan; Antila, Mia; Mertsola, Jussi; Hallander, Hans; He, Qiushui

    2005-01-01

    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. PMID:16081896

  6. Genome-wide gene expression analysis of Bordetella pertussis isolates associated with a resurgence in pertussis: elucidation of factors involved in the increased fitness of epidemic strains.

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    Audrey J King

    Full Text Available Bordetella pertussis (B. pertussis is the causative agent of whooping cough, which is a highly contagious disease in the human respiratory tract. Despite vaccination since the 1950s, pertussis remains the most prevalent vaccine-preventable disease in developed countries. A recent resurgence pertussis is associated with the expansion of B. pertussis strains with a novel allele for the pertussis toxin (ptx promoter ptxP3 in place of resident ptxP1 strains. The recent expansion of ptxP3 strains suggests that these strains carry mutations that have increased their fitness. Compared to the ptxP1 strains, ptxP3 strains produce more Ptx, which results in increased virulence and immune suppression. In this study, we investigated the contribution of gene expression changes of various genes on the increased fitness of the ptxP3 strains. Using genome-wide gene expression profiling, we show that several virulence genes had higher expression levels in the ptxP3 strains compared to the ptxP1 strains. We provide the first evidence that wildtype ptxP3 strains are better colonizers in an intranasal mouse infection model. This study shows that the ptxP3 mutation and the genetic background of ptxP3 strains affect fitness by contributing to the ability to colonize in a mouse infection model. These results show that the genetic background of ptxP3 strains with a higher expression of virulence genes contribute to increased fitness.

  7. Pesquisa de antigenos aglutinantes "major" 1, 2 e 3 em cepas de Bordetella pertussis, isoladas de crianças com coqueluche atendidas no Hospital de Isolamento Emílio Ribas de São Paulo, Brasil Determination of 1, 2 and 3 major antigens in Bordetella pertussis strains isolated from Brazilian children with whooping-cough

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    Sebastião Timo Iaria

    1977-09-01

    Full Text Available Em 30 cepas de Bordetella pertussis isoladas de crianças com coqueluche, atendidas no Hospital de Isolamento Emílio Ribas de São Paulo, foram pesquisados os antígenos aglutinantes ''major" 1, 2 e 3. Levando-se em conta a presença combinada dos três antígenos, as provas de soro-aglutinação rápida em lamina revelaram que 25 (83,3% cepas possuiam os fatores 1, 2 e 3, enquanto que 3 (10,0% e 2 (6,7% foram positivas, somente, para 1, 2 e 1, 3, respectivamente. Os resultados foram discutidos, considerando-se a importância deste antígeno no preparo de vacinas.The presence of major antigens, 1, 2 and 3 were determined in 30 strains of B. pertussis isolated from children with whooping-cough hospitalized at the Hospital Emílio Ribas, São Paulo Brazil. The method used was the slide-agglutination test. Tests showed that 25(83.3% of strains were positives for factors 1, 2 and 3. Factores 1 and 3 alone were present in 3 (10% of strains and 1 and 2 alone in 2 (6.7%.

  8. Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine.

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    Pádraig J Ross

    Full Text Available Whooping cough caused by Bordetella pertussis is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa. One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with B. pertussis and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with B. pertussis or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1β-induced IL-17A production, but also reveal that optimum protection against B. pertussis requires induction of Th1, but not Th2 cells.

  9. Infección por Bordetella pertussis en contactos domiciliarios de casos de tosferina en el suroriente de la ciudad de Cali, Colombia 2006-2007

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    Miryam Astudillo

    2011-06-01

    Full Text Available Introduction: Bordetella pertussis causes whooping cough or convulsive cough, a contagious and immune-preventable disease. It is one of the 10 leading causes of death among children younger than one year of age, when not completely immunized. It is considered reemerging in several countries, with high rates of complications and hospitalizations.Objective: to learn of the proportion of infection by B. pertussis among suspected cases of whooping cough and their household contacts among children from the southeast zone of Cali, a geographic area with great consultation demand due to this infection.Methodology: This is a cross-sectional descriptive study. Epidemiological data and nasopharyngeal samples were taken from 24 suspected cases and from their 109 household contacts. The samples were analyzed via real-time polymerase chain reaction (Q-PCR and through culture.Results: The proportion of positivity among the cases via the Q-PCR technique was at 50% (12/24 and at 40% via the culture technique (8/20, with good agreement between both techniques (Kappa 0.61. Regarding the household contacts, 30.3% (33/109 (CI 95%: 21.8%-39.8% tested positive. The sibling contacts (7/15 and the mothers (7/22 presented the greatest proportion of positivity. Regarding age, 60% were 4 years of age (3/5 and 50% were in the group comprised of individuals 45 to 64 years of age. No significant differences were found among the presence or absence of symptoms and the presence of B. pertussis infection, except for the presence of nasal secretions (runny nose (27% and coughing (36% during the last month.Conclusions: The study confirms the high prevalence of asymptomatic infection by B. pertussis among household contacts of children with whooping cough symptomatology and its household transmission. In Cali, health authorities need to review the effectiveness of implemented control strategies and the use of a vaccination scheme that does not cover adolescent and adult

  10. Bordetella pertussis infection in household contacts of cases of pertussis in the southeast zone of the city of Cali, Colombia, 2006-2007

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    Miryam Astudillo

    2011-06-01

    Full Text Available Introducción: Bordetella pertussis causa tos ferina o tos convulsiva, enfermedad contagiosa e inmunoprevenible, una de las primeras 10 causas de muerte entre niños menores de 1 año, al no estar completamente inmunizados. Se considera reemergente en varios países, con altas tasas de complicaciones y hospitalizaciones. Objetivo: conocer la proporción de infección por B. pertussis, entre casos sospechosos de tosferina y sus contactos domiciliarios entre niños del suroriente de Cali, área geográfica con mayor demanda de consulta por esta infección. Metodología: Estudio descriptivo de corte transversal. Se tomaron datos epidemiológicos y muestras nasofaringeas a 24 casos sospechosos y sus 109 contactos domiciliarios. Las muestras se analizaron por la reacción en cadena de la polimerasa en tiempo real (Q-PCR y por cultivo. Resultados: La proporción de positividad entre los casos por la técnica de Q-PCR fue de 50% (12/24 y 40% por la técnica de cultivo (8/20, con buena concordancia entre las dos técnicas (Kappa 0.61. En cuanto a los contactos, 30.3% (33/109 (IC 95%: 21.8%-39.8% resultaron positivos. Los contactos hermanos (7/15 y las madres (7/22 presentaron la mayor proporción de positividad. En cuanto a la edad, 60% con 4 años (3/5 y 50% en el grupo de 45- 64 años. No se encontraron diferencias significativas entre la presencia o ausencia de síntomas y la presencia de infección por B. pertussis, excepto en la presencia de flujo nasal (moquiadera (27% y tos (36% durante el último mes. Conclusiones: El estudio confirma la alta prevalencia de infección asintomática por B. pertussis entre contactos domiciliarios de niños con sintomatología de tosferina y la transmisión domiciliaria de la misma. En Cali es necesario revisar la efectividad de las estrategias de control implementadas y la utilización de un esquema de vacunación que no cubre a la población adolescente y adulta como control del foco de infección.

  11. 百日咳杆菌黏着素的纯化及生物学特性研究%Study on purification and biological characteristics of pertactin from Bordetella pertussis

    Institute of Scientific and Technical Information of China (English)

    王玲; 肖詹蓉; 叶娟; 韩俊杰; 孙翔宇

    2012-01-01

    目的 建立从天然百日咳杆菌中提纯百日咳杆菌黏着素( pertactin,PRN)的方法,研究PRN的生物学特性并确定PRN在无细胞百日咳疫苗中的作用.方法 百日咳杆菌CS株于发酵罐培养后,经热浸提、硫酸铵分级沉淀及离子交换层析得到纯化PRN,根据《中华人民共和国药典》2005年版三部的要求,对纯化PRN进行纯度检测及生物学特性研究.结果 各项检测显示,纯化PRN的纯度在95%以上,其相对分子质量和等电点与标准PRN一致.纯化PRN对小鼠具有保护效力,且其保护效力随PRN免疫浓度的增加而增强.未检出纯化PRN的特异性毒性和异常毒性.结论 建立了从天然百日咳杆菌中提纯PRN的方法,而且纯化PRN具有免疫原性和保护效力.%Objective To establish a method for purifying pertactin from Bordetella pertussis,study its biological characteristics and confirm its role in acellular pertussis vaccine.Methods Bordetella pertussis CS strain was cultured in a fermenter,and purified pertactin was obtained by hot lixiviation,ammonium sulfate fractional precipitation and ion-exchange chromatography.Purity and biological characteristics of the purified pertactin were studied according to requirements of Pharmacopoeia of the People's Republic of China 2005 Volume Ⅲ.Results The purity of the purified pertactin was above 95%,relative molecular mass and isoelectric point of the purified pertactin were identical with those of standard pertactin.Protective potency of the purified pertactin was observed in mice and enhanced with increase of its concentrations.No specific and abnormal toxicities were detected.Conclusions The method for purifying pertactin from Bordetella pertussis is established,and purified pertactin has immunogenicity and protective potency.

  12. 银川地区持续性咳嗽儿童百日咳博德特菌感染临床调查%Clinical investigation of Bordetella pertussis infection in children with persistent cough in Yinchuan

    Institute of Scientific and Technical Information of China (English)

    陈彦香; 王传清; 李乐; 赵忠; 黄列; 孙云

    2012-01-01

    Objective To understand current situation of Bordetella pertussis infection in children with persistent cough and analyze its clinical characteristics. Methods A total of 292 healthy children (≥2 years old) were randomly selected from Yinchuan area as the healthy group, and 157 children (≥6 years old) with persistent cough suspected of BordetelIa pertussis infection were assigned to patient group. Specific toxin antibody ofBordetella pertussis (FI'IgG) was detected by ELISA. Nasopha-ryngeal swabs specimens from ail subjects of patient group were examined by multiplex PCR and bacterial culture. Results The Bordetella pertussis infection rate was 2.6% (4/157) in patient group, 1.6% (1/63) in 6 - 8 years old, 2.8% (1/36) in 8 - 10 years old, 2.5% (1/34) in 10 - 12 years old and 4.2% (1/24) in 12-14 years old. There was a significant difference between each ages. In healthy group, PT TgG level was 1.3 FDA-U/ml. The PT TgG level was 2.5 FDA-U/ml in 2 - A years old, significantly higher than other ages (H=25.6, i'O.Ol). in 12-14 years old, the percentage of the children whose PT IgG level over 30 FDA-U/ml was 12.5%. There were no significant differences among deifferent ages f/'=6.66, P>0.05). Conclusions Bordetella pertussis infection is still one of the causes of children's persistent cough in Yinchuan area. It is necessary to encourage the pertussis vaccination because oflow levels or protective serum antibody in healthy children.%目的 了解银川地区持续性咳嗽儿童百日咳博德特菌感染现状及感染患儿的临床特点.方法随机选取银川地区≥2岁健康儿童292例为正常对照组,选择同期年龄≥6岁持续性咳嗽儿童157例作为可疑百日咳组,应用ELISA法测定两组儿童血清特异性百日咳博德特菌毒素IgG抗体(PT IgG),可疑百日咳组患儿留取鼻咽拭子进行百日咳博德特菌多重PCR检测及分离培养.结果在可疑百日咳患儿中百日咳博德特菌感染率为2.6%(4/157),其中6~8

  13. Phenotypical and genotypical characterization of Bordetella pertussis strains isolated in São Paulo, Brazil, 1988-2002 Caracterização fenotípica e genética de cepas de Bordetella pertussis isoladas em São Paulo, Brasil, 1988-2002

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    Célia R. Gonçalves

    2007-04-01

    Full Text Available Whooping cough or pertussis was a major cause of childhood morbidity and mortality in the world until the introduction of a whole-cell vaccine in the 1940's. However, since the early 1980's whooping cough cases have increased in many countries, becoming an important problem of public health. This increase may be due to accuracy of laboratory diagnosis and reporting of the disease, a decline in immunity over time, demographic changes, and adaptation of the bacterial population to vaccine-induced immunity. The purpose of this study was to analyze phenotypically and genotypically a collection of 67 Bordetella pertussis isolates recovered during the period 1988-2002 in São Paulo State, Brazil to determine their characteristics and relatedness. All isolates were submitted to susceptibility testing to erythromycin, serotyping, and 56 isolates were analyzed by Pulsed Field Gel Electrophoresis (PFGE. All isolates were susceptible to erythromycin and the majority of them belonged to serotype 1,3. The 56 isolates were classified into 11 PFGE profiles according to the differences in banding patterns. Although more than 60% of the isolates were recovered from patients aged less than three months, almost 15% of them were isolated from adolescents/adults evidencing the increase in the incidence of pertussis among this group of age.A coqueluche ou pertussis foi a maior causa de morbidade e mortalidade infantil em todo o mundo até a introdução de uma vacina na década de 1940. Entretanto, desde a década de 1980, a coqueluche tornou-se, em muitos países , um importante problema de saúde pública. Este acontecimento pode ser atribuído à melhoria do diagnóstico laboratorial e da notificação da doença, declínio da imunidade no decorrer do tempo, mudanças demográficas ou adaptação da população bacteriana à imunidade induzida pela vacina. O objetivo deste estudo foi analisar as características fenotípicas e genotípicas de uma coleção de 67

  14. Ca2+ influx and tyrosine kinases trigger Bordetella adenylate cyclase toxin (ACT endocytosis. Cell physiology and expression of the CD11b/CD18 integrin major determinants of the entry route.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3, its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

  15. Resistance to Erythromycin and Molecular Characteristics of Bordetella pertussis%百日咳鲍特菌红霉素耐药性及耐药相关分子特征分析

    Institute of Scientific and Technical Information of China (English)

    王增国; 刘莹; 韩锐郡; 栾阳; 杜全丽

    2016-01-01

    目的 分析百日咳鲍特菌(Bordetella pertussis,Bp)红霉素耐药性并对其耐药相关分子特征进行检测.方法 对分离自西安市2014年百日咳报告病例中的Bp通过E-Test检测其红霉素耐药性,通过聚合酶链反应(PCR)和PCR-测序方法分别检测可能出现的耐药基因及耐药相关位点改变.结果 10株Bp临床株分布于西安市6个区县,对红霉素的最小抑菌浓度(MIC)均>256μg/ml,所有菌株都出现23S rRNA基因的A2047G突变,未见其他耐药基因及位点改变.结论 西安市的红霉素耐药Bp已较为普遍,23S rRNA基因的A2047G突变可能是其最主要的耐药机制.应在百日咳监测的同时加强病原的抗生素耐药性监测及耐药相关分子的检测.

  16. D-alanine modification of a protease-susceptible outer membrane component by the Bordetella pertussis dra locus promotes resistance to antimicrobial peptides and polymorphonuclear leukocyte-mediated killing.

    Science.gov (United States)

    Taneja, Neetu Kumra; Ganguly, Tridib; Bakaletz, Lauren O; Nelson, Kimberly J; Dubey, Purnima; Poole, Leslie B; Deora, Rajendar

    2013-11-01

    Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite very high vaccine coverage, pertussis has reemerged as a serious threat in the United States and many developing countries. Thus, it is important to pursue research to discover unknown pathogenic mechanisms of B. pertussis. We have investigated a previously uncharacterized locus in B. pertussis, the dra locus, which is homologous to the dlt operons of Gram-positive bacteria. The absence of the dra locus resulted in increased sensitivity to the killing action of antimicrobial peptides (AMPs) and human phagocytes. Compared to the wild-type cells, the mutant cells bound higher levels of cationic proteins and peptides, suggesting that dra contributes to AMP resistance by decreasing the electronegativity of the cell surface. The presence of dra led to the incorporation of d-alanine into an outer membrane component that is susceptible to proteinase K cleavage. We conclude that dra encodes a virulence-associated determinant and contributes to the immune resistance of B. pertussis. With these findings, we have identified a new mechanism of surface modification in B. pertussis which may also be relevant in other Gram-negative pathogens.

  17. Development and Applification of TaqMan Fluorescence Quantitative PCR Assay to Detect Rabbit Bordetella Bronchiseptica%兔支气管败血波氏杆菌TaqMan荧光定量PCR检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    钱微; 刘燕; 肖琛闻; 韦强; 季权安; 鲍国连; 姚火春

    2013-01-01

    为了建立特异、敏感、快速检测兔支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)的TaqMan荧光定量PCR方法,本研究以Bb的毒力因子CyaA为目的基因设计特异性引物和探针,并将PCR扩增产物克隆测序,测序结果与GenBank上Bb的CyaA的同源性达100%.以阳性克隆质粒作为定量检测标准品建立标准曲线,以提取的Bb基因组DNA为模板,进行特异性、灵敏度和重复性实验.该方法对波氏杆菌基因组DNA检测最低限为0.32 pg,灵敏度是普通PCR的25倍,与临床常见细菌无交叉反应.对45份疑似感染兔波氏杆菌病料的检测表明,TaqMan荧光定量PCR和普通PCR检测阳性率分别为75.6%和66.7%,两者符合率88.2%.结果表明,建立的TaqMan荧光定量Bb检测方法具有较好的特异性、敏感性和重复性.该方法的建立对Bb的临床高效诊断,Bb的防控提供了有效手段.%A specific, sensitive and rapid TaqMan fluorescence quantitative PCR was established for testing Rabbit Bordetella bronchiseptica. In present study, a pair of primers and probes were designed from target gene of virulence factors CyaA of Bordetella bronchiseptica. Amplified PCR product was cloned and sequenced, the results showed that the homology was 100% compared with the reference sequence published in GenBank. The positive recombinant plasmids were served as quantitative detection of standards to establish standard curve. The detectable quantity of Bordetella bronchiseptica genomic DNA was 0.32 pg, which was 25 times sensitivity compared with common PCR, there was no cross reaction with common clinical bacteria by TaqMan fluorescence quantitative PCR. The 45 suspected samples were detected by TaqMan fluorescence quantitative PCR or routine PCR. The positive detection rates were 75.5% and 66.7%, respectively, the coincidence rate was 88.2%. The results showed that the TaqMan fluorescent quantitative Rabbit Bordetella bronchiseptica detection method was

  18. Characteristics of the antigenic genes of four clinical Bordetella pertussis strains%四株临床分离百日咳鲍特菌主要抗原基因序列分析

    Institute of Scientific and Technical Information of China (English)

    赵建宏; 魏红莲; 张庶民; 张柳; 孙素菊; 徐颖华; 李继红; 时东彦; 刘晓雷; 宋文杰; 王鑫

    2010-01-01

    Objectives To analyze the characteristics of antigenic genes of clinical Bordetella pertussis strains recently isolated by analyzing the sequence of pertussis toxin S1 subunit(ptxS1) , pertactin (Prn) , fimbriae 2 (Fim2) and fimbriae 3 (Fim3 ) genes of four clinical isolates. Methods The 4 clinical isolates were collected in 2002 in Shijiazhuang of Hebei province. Four strains were isolated from pertussis patient's nasopharyngeal aspirate. ptxS1, Prn, Fim2 and Fim3 genes of these strains were amplified and sequenced. The sequences of those genes were compared with those of the isolates in GenBank and the isoaltes used in the production of pertussis vaccine in China. Results The results of the gene sequencing showed the four clinical isolates belonged to ptxS1 A type, which were different from those in vaccine strains. In addition, three Prn and three Fim'3 variants were observed in the four clinical isolates. Sequence analysis showed that the nucleotide sequence and deduced amino acid sequence of those strains had more than 99% identity with those in vaccine strains. The phylogenetic trees of those genes also showed these strains had a higher level of similarity with other Bordetella pertussis strains. Conclusion The four clinical isolates are different from vaccine strains in four antigenic genes, which laid a foundation for further studies on pertussis epidemiology,quality control and development of pertussis vaccine in China.%目的 了解石家庄地区分离的百日咳鲍特菌4种抗原基因百日咳毒素S1亚单位(ptxS1)、外膜蛋白(Prn)、凝集素2(Fim2)和凝集素3(Fim3)的基因特征.方法 收集4株临床分离的百日咳鲍特菌,这4株菌于2002年分离自河北石家庄地区.分别对这4株茵的ptxS1、Prn、Fim2、Fim3主要抗原基因进行PCR扩增和测序,并与我国的百日咳鲍特菌疫苗生产株抗原基因序列以及GenBank中收录的百日咳基因序列进行比较、分析.结果 序列分析显示,新近分离的4

  19. 百日咳杆菌感染与成人不明原因慢性咳嗽相关性%Correlation of Bordetella pertussis infection with cryptogenic chronic cough in adults

    Institute of Scientific and Technical Information of China (English)

    文芳静; 徐志伟; 赵景景; 肖锦秀; 胡克

    2012-01-01

    Objective To determin the relationship between rryptogenir rhronir rough and Bordetella pertussis infertion by detect the concentration of pertussis toxin in serum of 64 rases of adult patients with rryptogenir rhronir rough. Methods From Derember 2010 to August 201 1 , we surressfully rollerted 64 rases who visited Renmin hospital of Wuhan university with the romplaint of rhronir rough ( All periods exreed 8 weeks, and the aetiology didnt inrluded any rauses from the guideline ). With the method of ELISA, we examine the ronrentration of pertussis toxin in serum of 64 rases of patients. Results For all 64 rases, only one rase was positive. The other 63 rases were negative. The rate of the positive one was 1. 56% . Conclusion As be different from the report in Europe, we found that%目的 探讨百日咳杆菌感染与成人不明原因慢性咳嗽的相关性.方法 采集2010年12月至2011年8月因慢性咳嗽(咳嗽时间>8周,但不符合咳嗽指南中的任何一种原因)就诊于武汉大学人民医院64例患者血清,置于-20℃保存,应用ELISA方法 测定患者血清中百日咳毒素的浓度.结果 64例患者中,仅1例为阳性,确诊为百日咳杆菌感染,另63例均呈阴性,百日咳杆菌感染阳性率为1.56%.结论 慢性咳嗽患者百日咳感染检出阳性率低.

  20. Minor modifications to the phosphate groups and the C3' acyl chain length of lipid A in two Bordetella pertussis strains, BP338 and 18-323, independently affect Toll-like receptor 4 protein activation.

    Science.gov (United States)

    Shah, Nita R; Albitar-Nehme, Sami; Kim, Emma; Marr, Nico; Novikov, Alexey; Caroff, Martine; Fernandez, Rachel C

    2013-04-26

    Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4'-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3' acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399-BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3' acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.

  1. Colonization of Bordetella pertussis clinical isolates that differ by pulsed field gel electrophoresis types in the lungs of naïve mice or mice immunized with the whole-cell pertussis vaccine used in Poland.

    Science.gov (United States)

    Polak, Maciej; Zawadka, Monika; Mosiej, Ewa; Rabczenko, Daniel; Augustynowicz, Ewa; Guiso, Nicole; Lutyńska, Anna

    2015-04-01

    The goal of our study was to compare the elimination of Bordetella pertussis clinical isolates that differ according to pulsed field gel electrophoresis (PFGE), serotypes and genes encoding virulence factors from the lungs of naïve mice or mice immunized with commercial diphtheria-tetanus-whole-cell pertussis vaccine used in Poland. When a mixture of four isolates, given in equal proportions and harboring different PFGE profiles, serotypes, and alleles encoding virulence factors, was used to infect non-immunized mice, a single isolate, characterized by PFGE type IVγ, Fim2 phenotype and ptxA1-prn2-tcfA2-fim2-1-ptxP1-ptxC1-fim3-1 alleles, was found to be significantly predominant compared to the others. This PFGE profile is commonly found in B. pertussis isolates circulating in some European countries since the late 1990s, confirming its high fitness. The Polish commercial whole-cell pertussis vaccine induced an immunity effective at eliminating the B. pertussis isolates from the lungs. However, the elimination of the isolate harboring PFGE type C profile, Fim2,3 phenotype and ptxA1-prn1-tcfA2-fim2-1-ptxP1-ptxC1-fim3-1 alleles was delayed as compared to the others, suggesting phenotypic differences with the other isolates and vaccine strains. Nevertheless, the same isolate, when challenged into mice in the defined mixture of strains, lost the competition with the others, as measured by lung colonization efficiency. This PFGE profile represents 15 % of the isolates circulating in Poland between 2001 and 2012.

  2. Biochemical and Structural Characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: Enzymes Required for the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Holden, Hazel M. (UW)

    2011-12-22

    The unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or ManNAc3NAcA, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic Gram-negative bacteria. It is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates UDP-ManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene and referred to as WlbA, catalyzes the NAD-dependent oxidation of the C-3' hydroxyl group of the UDP-linked sugar. Here we describe a combined structural and functional investigation of the WlbA enzymes from Bordetella pertussis and Chromobacterium violaceum. For this investigation, ternary structures were determined in the presence of NAD(H) and substrate to 2.13 and 1.5 {angstrom} resolution, respectively. Both of the enzymes display octameric quaternary structures with their active sites positioned far apart. The octamers can be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for these enzymes are sequential and that they do not require {alpha}-ketoglutarate for activity. These results are in sharp contrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermus thermophilus, which function via ping-pong mechanisms that involve {alpha}-ketoglutarate. Taken together, the results reported here demonstrate that there are two distinct families of WlbA enzymes, which differ with respect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms.

  3. Differences in purinergic amplification of osmotic cell lysis by the pore-forming RTX toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the role of pore size.

    Science.gov (United States)

    Masin, Jiri; Fiser, Radovan; Linhartova, Irena; Osicka, Radim; Bumba, Ladislav; Hewlett, Erik L; Benz, Roland; Sebo, Peter

    2013-12-01

    A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.

  4. Antibody responses to Bordetella pertussis Fim2 or Fim3 following immunization with a whole-cell, two-component, or five-component acellular pertussis vaccine and following pertussis disease in children in Sweden in 1997 and 2007.

    Science.gov (United States)

    Hallander, Hans; Advani, Abdolreza; Alexander, Frances; Gustafsson, Lennart; Ljungman, Margaretha; Pratt, Catherine; Hall, Ian; Gorringe, Andrew R

    2014-02-01

    Bordetella pertussis fimbriae (Fim2 and Fim3) are components of a five-component acellular pertussis vaccine (diphtheria-tetanus-acellular pertussis vaccine [DTaP5]), and antibody responses to fimbriae have been associated with protection. We analyzed the IgG responses to individual Fim2 and Fim3 in sera remaining from a Swedish placebo-controlled efficacy trial that compared a whole-cell vaccine (diphtheria-tetanus-whole-cell pertussis vaccine [DTwP]), a two-component acellular pertussis vaccine (DTaP2), and DTaP5. One month following three doses of the Fim-containing vaccines (DTwP or DTaP5), anti-Fim2 geometric mean IgG concentrations were higher than those for anti-Fim3, with a greater anti-Fim2/anti-Fim3 IgG ratio elicited by DTaP5. We also determined the responses in vaccinated children following an episode of pertussis. Those who received DTaP5 showed a large rise in anti-Fim2 IgG, reflecting the predominant Fim2 serotype at the time. In contrast, those who received DTwP showed an equal rise in anti-Fim2 and anti-Fim3 IgG concentrations, indicating that DTwP may provide a more efficient priming effect for a Fim3 response following contact with B. pertussis. Anti-Fim2 and anti-Fim3 IgG concentrations were also determined in samples from two seroprevalence studies conducted in Sweden in 1997, when no pertussis vaccine was used and Fim2 isolates predominated, and in 2007, when either DTaP2 or DTaP3 without fimbriae was used and Fim3 isolates predominated. Very similar distributions of anti-Fim2 and anti-Fim3 IgG concentrations were obtained in 1997 and 2007, except that anti-Fim3 concentrations in 1997 were lower. This observation, together with the numbers of individuals with both anti-Fim2 and anti-Fim3 IgG concentrations, strongly suggests that B. pertussis expresses both Fim2 and Fim3 during infection.

  5. 百日咳鲍特菌BP283基因的实时荧光定量PCR检测方法的建立%Development of real time fluorescent quantitative PCR for detection of Bordetella pertussis BP283 gene

    Institute of Scientific and Technical Information of China (English)

    罗炜; 王慧; 刘利东; 任筱兰; 赖克方

    2012-01-01

    目的 探讨检测百日咳鲍特菌BP283基因片段的有效方法.方法 以百日咳鲍特菌BP283序列为目的基因,设计探针和引物,构建质粒标准品;建立FQ-PCR体系,优化反应条件,并进行方法学评价.结果 成功构建了重组质粒pCR2.1-BP283;建立了检测BP283基因片段的FQ-PCR方法:标准曲线相关系数为0.998,最低检测浓度为102 copies/μL,对临床其他常见呼吸道病原体不出现特异性扩增曲线,批内及批间变异系数均<4%.结论 FQ-PCR方法可成功检测百日咳鲍特菌BP283基因,该方法具有敏感性好、特异性高及重复性好等优点.%Objective To evaluate the effective method for detection of Bordetella pertussis BP283 gene. Methods The primers and Taqman probe were designed according to the sequence BP283 of Bordetella pertussis, and the standards were constituted; then the real time fluorescent quantitative PCR(FQ-PCR) reaction system was optimized and evaluated. Results The recombinant vector (pCR2. 1-BP283) was cloned successfully; the FQ-PCR was well established; the corre-lation coefficient of standard curve was 0.998, the minimum detection limit concentration was 10 copies/μL , no specific amplification curve for the other common clinical pathogen in respiratory tract was found, both intra and inter assay coeffi-cients of variation were lower than 4% . Conclusion FQ-PCR can detect Bordetella pertussis BP283 gene successfully with good sensitivity, specificity and reproducibility.

  6. Establishment and Application of a Dual PCR Assay for Simultaneous Detection of Rabbit Pasteurella multocida and Bordetella Bronchiseptica%兔多杀性巴氏杆菌和支气管败血波氏杆菌双重PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    于新友; 李天芝; 王金良; 沈志强

    2015-01-01

    根椐GenBank公布的兔多杀性巴氏杆菌16S rRNA和支气管败血波氏杆菌fimN基因序列,设计两对引物,在建立两种细菌单项PCR检测方法的基础上,优化双重PCR反应条件,建立了两种细菌的双重PCR检测方法,用这两对引物对同一样品中的兔多杀性巴氏杆菌、支气管败血波氏杆菌核酸为模板进行双重PCR扩增,结果可同时扩增兔多杀性巴氏杆菌和支气管败血波氏杆菌的258bp和449bp的特异性片段,而对其他4种兔病原菌的扩增结果均为阴性。敏感性测定结果表明,对兔多杀性巴氏杆菌和支气管败血波氏杆菌的最低核酸检出限分别均为1pg。通过对78份临床病料检测,将建立的双重PCR技术和单项PCR方法进行对比验证,结果显示,两者的总符合率为100%。表明建立的双重PCR检测方法,具有特异、快速、准确的特点,可用于对这兔多杀性巴氏杆菌和支气管败血波氏杆菌的同时检测和鉴别诊断。%A dual PCR assay was optimized to simultaneously detect two pathogens of rabbit Pasteurella multo⁃cida and Bordetella Bronchiseptica in this article.According to the sequences of rabbit Pasteurella multocida 16S rRNA gene and Bordetella Bronchiseptica fimN gene in GenBank.Two pairs of primers were designed to amplify the unique fragments.By using two pairs of bacterias specific primers, two PCR assay were established to amplify the conservative regions of the two bacterias, respectively.Consequently,a dual PCR method to detect the two bacterias in one tube was developed.The dual PCR system would amplify a 258 bp fragment for rabbit Pasteurella multocida, a 449bp fargment for Bordetella Bronchiseptica simultaneously or separately in the sam⁃ples,depending on its infection status. But not specific band amplified from other four rabbit pathogenic bacte⁃rias.As little as 1pg of rabbit Pasteurella multocida, and 1pg of Bordetella Bronchiseptica DNA were detected

  7. Expression and characterization of the dermonecrotic toxingene of Bordetella bronchiseptica%支气管败血波氏杆菌皮肤坏死毒素的重组表达及其生物学特性

    Institute of Scientific and Technical Information of China (English)

    薛云; 赵战勤; 裴洁; 王臣; 丁轲; 程相朝

    2011-01-01

    Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-P-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates ofBL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. Bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.%皮肤坏死毒素(DNT)是支气管败血波氏杆菌的主要致病因子之一.通过PCR分段扩增和克隆获得了全长4 356 bp的dnt基因,并利用pET-28a/BL21系统对其进行了融合表达.Western blotting检测结果表明,表达产物具有良好的免疫学活性.使用His-band purification kit纯化试剂盒纯化后,得到纯度为93.2%的融合蛋白His6-DNT.在乳鼠皮肤坏死试验中,表达产物His6-DNT和天然DNT均能导致乳鼠皮肤产生坏死性病变.在乳鼠皮肤坏死阻断试验中,兔抗His6-DNT血清能中和天然DNT使其失去对乳鼠的皮肤坏死毒性.试验结果表明,重组蛋白具有天然DNT的生物学毒性和免疫原性,所产生的抗体具有中和活性.文中所获得的重组DNT蛋

  8. Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms

    Science.gov (United States)

    Gorgojo, Juan; Scharrig, Emilia; Gómez, Ricardo M.; Harvill, Eric T.; Rodríguez, Maria Eugenia

    2017-01-01

    B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts. PMID:28095485

  9. Immunomodulation by Proteins of Bordetella Pertussis

    Science.gov (United States)

    1985-08-30

    York, NT 10016 Dr. Alvin L. Winters Lyn Yaffe, M.D. Department of Microbiology Research Support Center University of Alabama Naval Medical Research Inst. University, AL 35486 National Naval Medical Center Bethesda, MD 20814

  10. Development and Application of a Real-Time Polymease Chain Reaction Method Based on the Duplex Target Gene for Detection of Bordetella Pertussis%百日咳鲍特菌双目标基因荧光定量聚合酶链反应检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    刘勇; 郭丽茹; 刘鹏; 黄海涛; 张颖; 高志刚; 苏旭; 陈锦英

    2012-01-01

    Objective To set up a molecular diagnostic method for Bordetella pertussis based on the duplex target gene real- time polymease chain reaction (PCR). Methods The assay based on primers and TaqMan-MGB probes were selected from highly conserved regions of the insertion sequence of IS481and IS1002 of Bordetella pertussis. Optimized in reactive system and condition ,the duplex target gene fragment from pertussis was cloned into the T vector generated from pertussis as template was set up to make the standard curve. Results The best concent ration of primers and probe is 600 mmol/ L and 200 mmol/ L rspectively, with good conservatism and specificity none of the negative control sample showed falsepositive reaction in duplication. The standard curve indicated the linear relationship between cycle threshold and template concentration. The detection limit of the assay was 10 copies/μl. A linear standard curve was obtained between 10 copies /μl and 10 copies /μl, the assay was simple and good reproducibility, 45 sample showed positive reaction in 50 suspected cases. Conclusion This realtime PCR assay provides a good method for quick and early detection to Bordetella pertussis.%目的 建立以百日咳鲍特菌重复插入序列(Insertion Sequences 481 and 1002,IS481,IS1002)双目标基因检测为特点的荧光定量聚合酶链反应(Polymease Chain Reaction,PCR),用于检测百日咳鲍特菌核酸,探讨在百日咳鲍特菌诊断中的应用意义.方法 针对百日咳鲍特菌的重复IS481及IS1002基因保守区域设计特异性引物、羧基荧光素与小沟结合基团(5-CarboxyFluorescein-Aminohexyl Amidite-Minor Groove Binder,TaqMan-MGB)荧光探针,做为双目标基因检测、筛选并优化荧光定量PCR反应体系与反应条件,并通过体外克隆技术建立百日咳鲍特菌双目标基因定量分析模型.结果 引物与探针的优化浓度分别为600毫摩尔/升(mmol/L)和200mmol/L,与其他呼吸道菌均无交叉反应,具有良好

  11. 迁延性咳嗽婴幼儿百日咳感染临床情况调查%Clinical research of Bordetella pertussis infection in infants with prolonged cough

    Institute of Scientific and Technical Information of China (English)

    米荣; 伏瑾; 王晓颖; 康利民; 李莉; 徐放生; 崔小岱

    2013-01-01

    Objective To explore the prevalence of Bordetella pertussis (B.pertussis) infection in unvaccinated or incomplete vaccinated infants with cough for a prolonged duration.Methods The serum samples and nasopharyngeal secretions were collected from 176 patients with cough for a prolonged duration (≥2 weeks) from 2011 to 2012 at Children's Hospital Affiliated to Capital Institute of Pediatrics.Multiplex PCR of nasopharyngeal secretion was employed to identify B.pertussis.And enzyme-linked immunosorbent assay(ELISA) was used to detect antibody to pertussis toxin (PT-IgG).Total bacterial DNA was exacted from nasopharyngeal secretion and two-target IS481/PT of B.pertussis was detected by PCR.The sera and nasopharyngeal secretions were also collected from household contacts with cough for a prolonged duration.Their clinical characteristic and epidemiological profiles were collected and analyzed.Results B.Pertussis infection was demonstrated in 51 cases (29.0%).The patients ages were from 23 days to 4 years.Among them,46 cases (90.2%) were aged under 12 months and 5 cases (9.8%) over 12 months.And40 cases were unvaccinated (31 cases < 3 months old,4 cases 3-12 months old,5 cases > 5 years old) and 11 cases incompletely vaccinated.There were 31 males and 20 females.More patients were found in spring and summer than those in autumn and winter.Nine infant cases had 12 household contacts.Among 12 household contacts,3 were PCR positive and 12 PT-IgG positive.Pertussis was remarkably critical in infants.Serious complications included failure to thrive,pneumonia,respiratory failure and seizures.Conclusions B.pertussis infection is an important cause in unvaccinated or incomplete vaccinated infants with prolonged cough.Peak seasons of pertussis are spring and summer.Undiagnosed adolescents and adults with pertussis may be a significant source for transmission of B.pertussis to other susceptible children.Infants aged under 1 year are at risk for severe pertussis and life

  12. Prokaryotic expression of pertactin of Bordetella pertussis and preparation of its monoclonal antibody%百日咳黏附素的原核表达及其单克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    徐颖华; 张华捷; 谭亚军; 吴丽洁; 王丽婵; 侯启明; 张庶民

    2012-01-01

    目的 原核表达百日咳黏附素(Pertactin,Prn),并制备抗Prn单克隆抗体.方法 从无细胞百日咳疫苗生产菌株CS株基因组DNA中克隆Prn基因,插入表达载体pQE-30中,构建重组表达质粒pQE-30-Prn,转化E.coli M15,IPTG诱导表达.表达的重组Prn蛋白经阳、阴离子交换层析纯化后,采用Western blot和ELISA法鉴定重组Prn蛋白的反应原性.以纯化的重组Prn蛋白为免疫原,利用杂交瘤技术制备单克隆抗体,并对制各的单抗进行鉴定.结果 重组表达质粒经双酶切和测序鉴定构建正确;表达的重组Prn蛋白相对分子质量约为69 000,主要以包涵体形式表达;纯化的重组Prn蛋白的纯度达95%以上,产量可达25 mg/L,能与不同来源的抗Prn-Ab血清特异性结合.获得2株分泌抗Prn单抗的杂交瘤细胞株,分泌的单抗均为IgG1类,轻链类型均为κ,效价均达1∶105以上,并能特异性识别Prn蛋白,而与百日咳杆菌其他抗原蛋白无交叉反应.结论 原核表达、纯化了重组Prn蛋白,并制备了2株效价高、特异性强的单抗,为无细胞百日咳疫苗的质量控制及百日咳杆菌致病机制的研究提供了材料.%Objective To express the pertactin (Prn) of Bordetella pertussis in prokaryotic cells and prepare the monoclonal antibody (McAb) against Prn. Methods Prn gene was cloned from the genomic DNA of CS strain for production of acellular pertussis vaccine and inserted into expression vector pQE-30. The constructed recombinanl plasmtd pQE-30-Prn was transformed to E. colt M15 for expression under induction of IPTG. The expressed recombinant protein was purified by anion and cation exchange chro-matography, and identified for reactogenicity by Western blot and ELISA. McAb was prepared by hybridoma technique using the purified Pm protein as immunogen and identified- Results Restriction analysis and sequencing proved thai Tecombinan! plasmid pQE-30-Prn was constructed correctly. The expressed Prn protein

  13. Cloning,Sequencing,Secondary Structure and B-cell Epitopes Prediction of Bordetella avium Outer Membrane Protein OmpA%禽波氏杆菌外膜蛋白A的克隆测序与生物信息分析

    Institute of Scientific and Technical Information of China (English)

    谭燕玲; 朱瑞良; 王慧; 王新建; 魏凯; 孙振红; 盛鹏程

    2011-01-01

    The aims of this study was to clone and sequence the ompA gene of Bordetella avium,and predict its secondary structure and B-cell epitopes, which provides a theoretical basis for the study of immune mechanism and B. avium subunit vaccine. The B. avium ompA gene was amplified, cloned, and sequenced. By using bioinformatics softwares and methods, the secondary structure and B-cell preponderant epitope of B. avium OmpA were predicted. The results indicated that B. avium ompA gene is 597 bp, encoding 199 amino acids. Using three methods, prediction of the secondary structure of B. avium OmpA indicated that random coils were the main structural type of the flexible region in secondary structure, and contain a small amount of α-helix, β-sheet and a rare β-turn. The OmpA protein was supposed contain 5 potential antigen epitopes and 2 potential glycosylated sites. These results provide a theoretical basis for the further study of immune mechanisms, monoclonal antibodies preparation and epitope vaccine design.%本文旨在克隆禽波氏杆菌外膜蛋白OmpA的编码基因,并预测OmpA蛋白二级结构和B细胞抗原表位,从而探讨禽波氏杆菌外膜蛋白OmpA在免疫保护中所起的作用.作者对禽波氏杆菌的外膜蛋白ompA基因进行PCR扩增、克隆及序列测定.应用生物信息学相关软件和方法,对禽波氏杆菌OmpA蛋白的二级结构和B细胞抗原表位进行预测.禽波氏杆菌ompA基因全长597 bp,编码199个氨基酸.二级结构以无规卷曲为主,有少量的α-螺旋和β-片层,少见β-转角;推测OmpA蛋白有5个B细胞优势抗原表位区域、2个糖基化位点.本研究为进一步分析禽波氏杆菌免疫机理、制备单克隆抗体和设计表位疫苗等奠定理论基础.

  14. 百日咳杆菌CpG-ODNs对大鼠变应性哮喘IgE生成的影响%The effect of CpG-ODNs extracted from Bordetella pertussis on IgE of rats with allergic asthma

    Institute of Scientific and Technical Information of China (English)

    胡婷; 王凤霞; 王骏; 孙云

    2011-01-01

    CpG-ODNs extracted from Bordetella pertussis (seqA) were administrated to allergic asthma models of rats by ip and ig route; These rats were used for detecting the concentration of IgE in serum by ELISA protocol Then, rats were administrated seqA for passive cutaneous anaphylaxis (PCA). PCA rats received the mixture of Even's blue and OVA by intravenous injection, the level of color which was leaked out of the back of rats was detected by spectrophotometer. Result: Compared with model group, seqA ip group inhibited the concentration of IgE in the serum and suppress the effusion of color significantly (P<0.001 ), and its inhibitory effect was better than that of seqA ig group (P<0. 01 ). SeqA ip group could descend the level of IgE and suppress PCA for preventing and therapeutic of allergic asthma effectively.%以百日咳杆菌CpG-ODNs有效序列seqA采用腹腔注射(ip组)和灌胃(ig组)2种途径干预变应性哮喘大鼠模型,ELISA法检测模型大鼠血清中IgE含量;使用seqA干预后,以分光光度法检测大鼠背部皮肤致敏部位伊文思蓝渗出程度.结果表明:seqA ip组能下调大鼠血清IgE,与模型组比较差异极显著(P<0.001);seqA ip组下调血清IgE的作用强于ig组;被动皮肤过敏反应试验中seqA ip干预组能有效下调致敏大鼠皮肤蓝斑吸光度,与模型组比较差异极显著(P<0.001),且seqA ip组的抑制作用强于ig组;说明效应序列seqA能有效下调IgE、抑制PCA,从而防治变应性哮喘.

  15. Systems vaccinology : molecular signatures of immunity to Bordetella pertussis

    NARCIS (Netherlands)

    Raeven, R.H.M.

    2016-01-01

    The worldwide resurgence of whooping cough (pertussis), even in highly vaccinated populations, demands improved pertussis vaccines. In this thesis a systems vaccinology approach is applied to deepen knowledge of the immune responses evoked by different pertussis vaccines and compare this with a Bord

  16. Modulation of Toll-like signal path of allergic asthma by CpG-ODNs from Bordetella pertussis%百日咳杆菌效应 CpG-ODNs 调控变应性哮喘 Toll 信号分子的实验研究

    Institute of Scientific and Technical Information of China (English)

    张宝袁; 迟深; 孙云

    2011-01-01

    This study focused on prevention and treatment of acute and chronic asthma by oligonucleotides containing unmethylated CpG motifs (CpG-ODNs). Acute and chronic asthma models of mice were made by sensitizing and inhaling ovalbumin (OVA); The number of white blood cells (WBC) and eosnophils (EOS) in bronchoalveolar lavage fluid (BALF) was counted and the concentration of cytokines and vascular endothelial growth factor (VEGF) was examined in BALF by ELISA kit. After that TLR-9 mRNA was detected in mice spleen cells by reverse transcription polymerase chain reaction (RT-PCR) and TLR-9 protein was determined in mice Iung tissues by Western blotting. Compared with acute asthma models of mice, WBC in BALF decreased obviously in the groups of Bordeiella pertussis, CpG-ODNs and seq A to seq I which were administrated by both of intragastric (ig) and intraperitoneal (ip) injection group, EOS decreased obviously in Bordetella pertussis, CpG+ and seq A to seq D ig groups, and in all ip administrating groups, although it was not effective in the groups of seq E to seq I. In chronic asthma models of mice, IFN-gamma increased(① control: 176.45± 23.46 pg·mL-1;② model: 174.11± 22.71 pg·mL-1;③ CpG+ ip: 220.56± 15.42 pg·mL-1;④ seq A ip: 225.23± 21.60 pg·mL-1) and IL-4 decreased obviously(① control: 66.91± 5.81 pg·mL-1;② model: 81.02± 11.24 pg·mL-1; ③ CpG+ ip: 63.99± 6.09 pg·mL-1;④ seq A ip: 62.75± 10.03 pg·mL-1) in the BALF of CpG+ and seq A ip group, although VEGF was not changed in this research. And also, TLR-9 mRNA in spleen cells (TLR-9/GAPDH: ① control: 0.62± 0.13;② model: 0.66± 0.17;③ CpG+ ip: 1.46± 0.26;④ seq A ip: 1.42± 0.34) and TLR-9 protein in lung tissues (TLR-9/β-actin:① control: 0.63± 0.16;② model: 0.61± 0.07;③ CpG+ ip: 1.15± 0.25; ④ seq A ip: 1.03± 0.29) both increased in ip groups, but the change was not significant in ig group. The study confirms that CpG-ODNs and seq A could inhibit airway inflammation

  17. Bordetella pertussis infection or vaccination substantially protects mice against B. bronchiseptica infection.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Goebel

    Full Text Available Although B. bronchiseptica efficiently infects a wide range of mammalian hosts and efficiently spreads among them, it is rarely observed in humans. In contrast to the many other hosts of B. bronchiseptica, humans are host to the apparently specialized pathogen B. pertussis, the great majority having immunity due to vaccination, infection or both. Here we explore whether immunity to B. pertussis protects against B. bronchiseptica infection. In a murine model, either infection or vaccination with B. pertussis induced antibodies that recognized antigens of B. bronchiseptica and protected the lower respiratory tract of mice against three phylogenetically disparate strains of B. bronchiseptica that efficiently infect naïve animals. Furthermore, vaccination with purified B. pertussis-derived pertactin, filamentous hemagglutinin or the human acellular vaccine, Adacel, conferred similar protection against B. bronchiseptica challenge. These data indicate that individual immunity to B. pertussis affects B. bronchiseptica infection, and suggest that the high levels of herd immunity against B. pertussis in humans could explain the lack of observed B. bronchiseptica transmission. This could also explain the apparent association of B. bronchiseptica infections with an immunocompromised state.

  18. Phenotypic and Genomic Analysis of Hypervirulent Human-associated Bordetella bronchiseptica

    Directory of Open Access Journals (Sweden)

    Ahuja Umesh

    2012-08-01

    Full Text Available Abstract Background B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Complex IV isolates appear to have a bias for infecting humans; however, little is known regarding their epidemiology, virulence properties, or comparative genomics. Results Here we report a characterization of the virulence of human-associated complex IV B. bronchiseptica strains. In in vitro cytotoxicity assays, complex IV strains showed increased cytotoxicity in comparison to a panel of complex I strains. Some complex IV isolates were remarkably cytotoxic, resulting in LDH release levels in A549 cells that were 10- to 20-fold greater than complex I strains. In vivo, a subset of complex IV strains was found to be hypervirulent, with an increased ability to cause lethal pulmonary infections in mice. Hypercytotoxicity in vitro and hypervirulence in vivo were both dependent on the activity of the bsc T3SS and the BteA effector. To clarify differences between lineages, representative complex IV isolates were sequenced and their genomes were compared to complex I isolates. Although our analysis showed there were no genomic sequences that can be considered unique to complex IV strains, there were several loci that were predominantly found in complex IV isolates. Conclusion Our observations reveal a T3SS-dependent hypervirulence phenotype in human-associated complex IV isolates, highlighting the need for further studies on the epidemiology and evolutionary dynamics of this B. bronchiseptica lineage.

  19. Purification of heat labile toxin from Bordetella pertussis vaccine strain 134 employed indigenous technology

    Directory of Open Access Journals (Sweden)

    K.C. Shivanandappa

    2016-06-01

    Conclusion: The B. pertussis HLT could be purified through two phase with G50 and DEAE, cost effective techniques, the G50 purification has reduced the bioburden problems during DEAE purification and at the same time the quality of the product was high.

  20. Bordetella pertussis: an underreported pathogen in pediatric respiratory infections, a prospective cohort study

    NARCIS (Netherlands)

    Brink, van den G.; Wishaupt, J.O.; Douma, J.C.; Hartwig, N.G.; Versteegh, F.G.A.

    2014-01-01

    Background: The incidence of pertussis has been increasing worldwide. In the Netherlands, the seroprevalence has risen higher than the reported cases, suggesting that laboratory tests for pertussis are considered infrequently and that even more pertussis cases are missed. The objective of our study

  1. Recent trends of antigenic variation in Bordetella pertussis isolates in Korea.

    Science.gov (United States)

    Kim, So-Hyun; Lee, Jin; Sung, Hwa Young; Yu, Jae Yon; Kim, Seong Han; Park, Mi Sun; Jung, Sang-Oun

    2014-03-01

    Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.

  2. [Insertional Inactivation of Virulence Operon in Population of Persistent Bordetella pertussis Bacteria].

    Science.gov (United States)

    Karataev, G I; Sinyashina, L N; Medkova, A Yu; Semin, E G; Shevtsova, Z V; Matua, A Z; Kondzariya, I G; Amichba, A A; Kubrava, D T; Mikvabia, Z Ya

    2016-04-01

    Avirulent B. pertussis bacteria containing IS elements in the bvgAS operon were detected during the study of whooping cough patients and bacilli carriers. The present work is devoted to the study of the accumulation dynamics and the mechanisms of generation of persistent forms of the B. pertussis bacteria in lower monkeys as the most adequate model for extrapolation ofthe experiment results to humans. By means of the real-time PCR method, it was established that the B. pertussis bacteria lived more than three months in the upper respiratory tract after a single intranasal monkey infection; the period was reduced to 14-28 days during repeated infection. An increase in the portion of B. pertussis Bvg mutants in the population to tens of percent from the total number of registered bacteria was registered. The experimental confirmation ofthe development and accumulation of avirulent B. pertussis Bvg mutants during the development of the infectious process was obtained. Further study of the composition of the B. pertussis persistent bacteria population at different stages of the disease will make it possible to formulate new approaches to the whooping cough diagnostics and prevention and creation of fundamentally new drugs.

  3. Immunoproteomic Profiling of Bordetella pertussis Outer Membrane Vesicle Vaccine Reveals Broad and Balanced Humoral Immunogenicity.

    Science.gov (United States)

    Raeven, René H M; van der Maas, Larissa; Tilstra, Wichard; Uittenbogaard, Joost P; Bindels, Tim H E; Kuipers, Betsy; van der Ark, Arno; Pennings, Jeroen L A; van Riet, Elly; Jiskoot, Wim; Kersten, Gideon F A; Metz, Bernard

    2015-07-01

    The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate.

  4. Changes in genetic diversity of the Bordetella pertussis population in Serbia between 1953 and 2011

    Directory of Open Access Journals (Sweden)

    Plješa Tatjana

    2014-01-01

    Full Text Available Mass vaccination has significantly reduced the incidence of pertussis, however, the disease is re-emerging, even in some countries with high vaccination coverage. In Serbia, whole cell pertussis vaccine was introduced in 1957. To monitor changes in bacterial population, 77 isolates collected from 1953 to 2011 were studied. The methods included serotyping of fimbriae (Fim, genotyping of pertactin (prn and pertussis toxin S1 subunit (ptxA. A shift from ptxA2 to ptxA1 has been observed in isolates since the late of 1960s. In the period 1961-1979, the genotype ptxA1 became as common as genotype ptxA2. After that, during the period 1980-1989, the predominant ptx genotype was ptxA1. The reappearance 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. The prn2 allele was only found in one strain isolated in 1984, two of the four strains isolated in 2000 and in three isolated strains from 2011. Serotype Fim2.3 disappeared before 1980 and serotype Fim2 became predominant thereafter. The results of this 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.

  5. [PERSISTENCE OF BORDETELLA PERTUSSIS BACTERIA AND A POSSIBLE MECHANISM OF ITS FORMATION].

    Science.gov (United States)

    Karataev, G I; Sinyashina, L N; Medkova, A Yu; Semin, E G

    2015-01-01

    A growth of pertussis morbidity is observed in many countries of the world against the background of mass vaccindtion. Forms of the disease course have changed. Atypical forms of pertussis occur predominately in adolescents and adults. Asymptomatic carriage of the causative agent has been established. Infection of infants with. BordetelIa pertussis bacteria in more than 90% of cases occurs from parents and relatives. A prolonged persistence of the causative agent has been identified. Morbidity increase in developed countries is associated with the use of acellular vaccines, that do not protect from the infection, but reduce severity of the disease. A change of genotypes of the circulating bacteria strains is observed ubiquitously. Formation of a persistent form of B. pertussis is possible due to a reversible integration of IS-elements into bvgAS operon and other virulence genes. The results of studies of invasion and survival of B. pertussis bacteria in eukaryotic cells, a change in B. pertussis bacteria population after experimental infection of laboratory mice and monkeys are presented, accumulation of avirulent insertion Bvg mutants of B. pertussis was detected. The data obtained are in accordance with the results of analysis of causative agent population in patients with typical and atypical forms of pertussis in humans. More than 50% of the population of B. pertussis bacteria in practically healthy carriers was shown to be presented by avirulent insertion Bvg mutants. B. pertussis virulence reducing as a result of inactivation of single or several virulence genes probably provide long-term persistence of bacteria in host organism and formation of apparently healthy vehicles. Follow-up studies on that front would help to formulate new attitudes to preventive measures of pertussis and lead to development of fundamentally new pharmaceuticals (vaccines) preventing formation of bacterial persistence.

  6. Bordetella pertussis epidemiology and evolution in the light of pertussis resurgence.

    Science.gov (United States)

    Sealey, Katie L; Belcher, Thomas; Preston, Andrew

    2016-06-01

    Whooping cough, or pertussis, is resurgent in many countries world-wide. This is linked to switching from the use of whole cell vaccines to acellular vaccines in developed countries. Current evidence suggests that this has resulted in the earlier waning of vaccine-induced immunity, an increase in asymptomatic infection with concomitant increases in transmission and increased selection pressure for Bordetellapertussis variants that are better able to evade vaccine-mediated immunity than older isolates. This review discusses recent findings in B. pertussis epidemiology and evolution in the light of pertussis resurgence, and highlights the important role for genomics-based studies in monitoring B. pertussis adaptation.

  7. Analysis of Bordetella pertussis populations in European countries with different vaccination policies.

    NARCIS (Netherlands)

    Amersfoorth, S C M van; Schouls, L M; Heide, H G J van der; Advani, A; Hallander, H O; Bondeson, K; König, C H W von; Riffelmann, M; Vahrenholz, C; Guiso, N; Caro, V; Njamkepo, E; He, Q; Mertsola, J; Mooi, F R

    2005-01-01

    Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studie

  8. Molecular characterization of a clinical Bordetella bronchiseptica strain isolated from a cystic fibrosis patient

    Science.gov (United States)

    While not considered a human pathogen, B. bronchiseptica infections in humans are reported and demonstrate zoonotic transmission into humans. An extensive characterization of these strains is needed to better understand the genetic and phenotypic traits associated with these zoonotic transmission ev...

  9. Bordetella bronchiseptica como un riesgo importante de salud publica. Estudio clínico patológico en conejos

    Directory of Open Access Journals (Sweden)

    Valladares-Carranza B.

    2011-10-01

    Full Text Available ResumenB. bronchiseptica es reconocida como un patógeno primario inicial del tracto respiratorio en animales domésticos, puede provocar tos de las perreras (perro, respiración ruidosa (en conejos y rinitis atrófica (en el cerdo.summary

  10. From process understanding to process control: application of PAT on the cultivation of Bordetella pertussis for a whole cell vaccine

    NARCIS (Netherlands)

    Streefland, M.

    2009-01-01

    De kwaliteit van farmaceutische producten wordt geborgd door een op Good Manufacturing Practice gebaseerd kwaliteits systeem, waarbij kwaliteitscontrole van het eindproduct een belangrijke rol vervult. Recentelijk is hierin verandering gekomen en vragen regelgevers bij overheden steeds meer om een k

  11. 百日咳流行病学研究进展%Research Progress of Bordetella Pertussis Epidemicology

    Institute of Scientific and Technical Information of China (English)

    张柳; 赵建宏; 张庶民

    2008-01-01

    百日咳足由百日咳鲍特菌感染引起的急性呼吸道传染病,人群普遍易感,尤以婴幼儿为重.全世界自20世纪50年代使用百日咳疫苗(Pertussis vaccine,PerV)以米,其发病率和死亡率有了很大程度的降低.但近些年,即使在PerV覆盖率较高的发达国家,百日咳的发病率又有了升高的趋势,而感染人群为已接种过PerV的青少年、成人为特点.产生这种现象的原因是多方面的,如:监测水平、免疫的持久性等.近睦年百日咳鲍特菌的主要毒力基因的抗原漂移现象,作为又一解释原因引起了各国学者的关注.该文就百日咳的致病机制、流行病学特点、百日咳"再现"的原因等进行探讨.

  12. Estimating the role of casual contact from the community in transmission of Bordetella pertussis to young infants

    Directory of Open Access Journals (Sweden)

    Poole Charles

    2007-10-01

    Full Text Available Abstract The proportion of infant pertussis cases due to transmission from casual contact in the community has not been estimated since before the introduction of pertussis vaccines in the 1950s. This study aimed to estimate the proportion of pertussis transmission due to casual contact using demographic and clinical data from a study of 95 infant pertussis cases and their close contacts enrolled at 14 hospitals in France, Germany, Canada, and the U.S. between February 2003 and September 2004. A complete case analysis was conducted as well as multiple imputation (MI to account for missing data for participants and close contacts who did not participate. By considering all possible close contacts, the MI analysis estimated 66% of source cases were close contacts, implying the minimum attributable proportion of infant cases due to transmission from casual contact with community members was 34% (95% CI = 24%, 44%. Estimates from the complete case analysis were comparable but less precise. Results were sensitive to changes in the operational definition of a source case, which broadened the range of MI point estimates of transmission from casual community contact to 20%–47%. We conclude that casual contact appears to be responsible for a substantial proportion of pertussis transmission to young infants. Medical subject headings (MeSH: multiple imputation, pertussis, transmission, casual contact, sensitivity analysis, missing data, community.

  13. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Science.gov (United States)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  14. Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes.

    Science.gov (United States)

    Soria-Guerra, Ruth E; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; López-Revilla, Rubén; Alpuche-Solís, Angel G

    2011-03-01

    DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.

  15. Disease: H01084 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available [Bordetella holmesii bacteremia in a child with sickle cell disease]. Enferm Infecc Microbiol Clin 29:779-80...Saint Jean M, Brun D, Guiso N, Bekal S, Ovetchkine P, Tapiero B Bordetella holmesii bacteremia in asplenic child

  16. Proteínas recombinantes útiles para la prevención de la infección por Bordetella pertussis

    OpenAIRE

    Olivera, María Noelia

    2014-01-01

    La tos convulsa es una enfermedad aguda, respiratoria y altamente contagiosa que a pesar de ser inmunoprevenible, hoy en día es considerada una enfermedad resurgente. Surge entonces la necesidad de aplicar refuerzos de inmunización de forma de prolongar la duración de la inmunidad contra la enfermedad, así como también de mejorar las vacunas actuales de manera tal que no solo ofrezcan una protección duradera en el tiempo sino que también protejan contra cepas circulantes antigénicamente disti...

  17. Cross-reactions in IgM ELISA tests to Legionella pneumophila sg1 and Bordetella pertussis among children suspected of legionellosis; potential impact of vaccination against pertussis?

    Science.gov (United States)

    Pancer, Katarzyna Wanda

    2015-01-01

    The objective of this study was preliminary evaluation of IgM cross-reaction in sera collected from children hospitalized because of suspected legionellosis. Sera with positive IgM results to L. pneumophila sgs1-7, B. pertussis or with simultaneous detection of IgM antibodies to L. pneumophila sgs1-7 and B. pertussis, or IgM to L. pneumophila sgs1-7 and M. pneumoniae in routine tests, were selected. In total, an adapted pre-absorption test was used for the serological confirmation of legionellosis in the sera of 19 children suspected of legionellosis, and also in 3 adult persons with confirmed Legionnaires' disease. Sera were pre-absorbed with antigens of L. pneumophila sg1, B. pertussis or both, and tested by ELISA tests. The reduction of IgM antibody level by pre-absorption with antigen/antigens was determined. Reduction of anti-Lpsgs1-7 IgM by pre-absorption with L.pneumophila sg1 antigen ranged from 1.5 to 80, and reduction of anti-Bp IgM by pre-absorption with B. pertussis ranged from 2.0 to 23.8. Reduction by both antigens varied depending on the age of the patients: among children tests. As a preliminary, we posed a hypothesis of a potential impact of an anti-pertussis vaccination on the results obtained in anti-L. pneumophila ELISA IgM tests among young children.

  18. Development of turbinate lesions and nasal colonization by Bordetella bronchiseptica and Pasteurella multocida during long-term exposure of healthy pigs to pigs affected by atrophic rhinitis.

    OpenAIRE

    Bäckström, L R; Brim, T A; Collins, M T

    1988-01-01

    Natural transmission of atrophic rhinitis from pigs from a herd with an endemic atrophic rhinitis problem to pigs from a herd free of atrophic rhinitis was demonstrated. Six replicates each with five pigs from the endemic atrophic rhinitis herd (Group A) and five pigs from the atrophic rhinitis-free herd (Group B) were housed together from 5 wk of age, with each replicate kept in isolation rooms maintained at optimal and controlled environmental conditions. Three replicates each with six pigs...

  19. Detection of Bordetella pertussis using a PCR test in infants younger than one year old hospitalized with whooping cough in five Peruvian hospitals

    Directory of Open Access Journals (Sweden)

    María Esther Castillo

    2015-12-01

    Conclusion: An increase in pertussis cases has been reported in recent years in Peru, despite national immunization efforts. Surveillance with PCR for B. pertussis is essential, especially in infants less than 1 year old, in whom a higher rate of disease-related complications and higher mortality have been reported.

  20. 百日咳鲍特菌黏附分子的研究进展%Progress in research on adhesive molecules of Bordetella pertussis

    Institute of Scientific and Technical Information of China (English)

    柯兵兵

    2013-01-01

    随着生物技术的发展,研究人员对百日咳鲍特菌致病机理进行了大量研究并取得一定的突破,尤其是黏附分子在细菌致病过程中所起的作用.本文就近年来在百日咳鲍特菌黏附分子的研究进展作一简要综述.

  1. Analysis of genomic polymorphisms of Bordetella pertussis isolates*%百日咳鲍特菌基因组多态性分析

    Institute of Scientific and Technical Information of China (English)

    徐颖华; 卫辰; 王丽婵; 骆鹏; 侯启明; 张庶民

    2012-01-01

    Objective To investigate the genomic characterizations of B .pertussis isolates .Methods Genomic polymorphisms of recent B .pertussis isolates were determined by two international universal molecular typing methods,allelic analysis and ML V A for B .pertussis .The diversity indexs of typing methods were analyzed statistically .Results The sequencing analysis of pertacitn(Prn), trachea! colonization factor(tcfA) and pertussis toxin promoter region(ptxP) showed that three allelic profiles Prnl/ptxPl/tcfA2, Prn2/PtxP3/tcfA2 and Prn3/ptxPl/tcf A2 wore; found with the; frequency of 71 .43%,19 .05% and 9 .52% in the; strains analyzed, respectively .21 isolates produced 12 different B .pertussis MLV A profiles,and a new profile was found .Main ML vA profiles were MLVA-136 and MLVA-152,accounted 28 .57% and for 19 .05% respectively .The results of statistical analysis suggested that ML-VA were better than allelic analysis for molecular typing of B .pertussis .Conclusion These results demonstrated that B .pertussis epidemic strains in some regions of China were different from those in European countries .These works could allow us to gain more insights into the molecular epidemiological patterns of the B .pertussis strains facilitating for a better control of pertussis spread and new vaccine prevention strategies in China .%目的 了解百日咳鲍特菌(简称百日咳杆菌)基因组特征.方法 应用百日咳杆菌等位基因分型法和多位点可变数量串联重复序列分析(MLVA)法分析百日咳杆菌基因多态性,同时对2种分型方法的多态性指数进行统计学分析.结果 百日咳黏附素(Prn)、支气管黏附因子(tcfA)和百日咳毒素基因上游启动子区域(ptxP)3种等位基因序列分析结果显示,共发现3种等位基因组合型:Prn1/ptxP1/tcfA2、Prn2/ptxP3/tcfA2和Prn3/ptxP1/tcfA2,所占比例分别为71.43%、19.05%和9.52%.MLVA法将21株分离菌株分成了12个不同的MLVA型,MLVA-136型和MLVA-152型为主要流行MLVA型,所占比例分别为28.57%和19.05%,并发现1种新MLVA型菌株.统计学分析表明MLVA法较等位基因分型法具有更高的分辨率.结论 国内部分地区百日咳杆菌流行菌株与欧洲其他国家不同,掌握百日咳杆菌分子流行病学的第一手资料,对有效预防和控制百日咳的流行以及制定新的百日咳疫苗免疫策略具有指导意义.

  2. Antimicrobial susceptibility testing of Bordetella pertussis : a surveillance study%百日咳杆菌抗生素敏感性检测结果分析

    Institute of Scientific and Technical Information of China (English)

    欧金玲; 袁林; 姚开虎; 沈叙庄; 杨永弘

    2008-01-01

    目的 了解百日咳杆菌对抗生素的敏感性情况,为临床合理用药提供依据.方法 以2000年7月至2007年4月北京儿童医院临床分离的16株和20世纪70年代的4株百日咳菌株为研究对象,采用琼脂稀释法、E test法和纸片扩散法检测菌株对红霉素的敏感性.其中琼脂稀释法还检测了菌株对阿齐霉素、克拉霉素、克林霉素和环丙沙星4种抗生素的敏感性.结果 3种方法检测2000年以后菌株对红霉素的敏感性结果显示,琼脂稀释法MIC均为0.125 μg/ml,E test MIC范围0.047~0.125μg/ml,纸片扩散法抑菌环直径都>42 mm(范围45~55 mm).琼脂稀释法检测2000年以后菌株阿齐霉素、克林霉素的MIC范围分别为0.031-0.063 μg/ml、0.25~2μg/ml;克拉霉素和环丙沙星的MIC分别为1μg/ml和2μg/ml.与20世纪70年代百日咳菌株相比,2000年以后菌株对5种抗生素的敏感性没有下降.结论 本研究未发现红霉素耐药株,2000年以后分离的16株百日咳菌株对红霉素均敏感,治疗百日咳仍可首选红霉素.

  3. Study on molecular epidemiology of Bordetella pertussis%百日咳杆菌的分子流行病学研究

    Institute of Scientific and Technical Information of China (English)

    欧金玲; 姚开虎; 袁林; 俞桑洁; 沈叙庄; 杨永弘

    2010-01-01

    目的 明确百日咳杆菌分离株的分子分型,比较不同年代分离株的型别差异.方法 共25株百日咳菌株,其中18株为2000年至2007年北京儿童医院临床分离株,7株为20世纪70年代分离株,采用脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)2种方法对菌株进行分型.结果 2000年以后菌株MIST分型均为ST2型,PFGE分型分为8种型;20世纪70年代菌株MLST分型均为ST1型,PFGE分为5种型.2000年后菌株与20世纪70年代菌株无相同的PFGE型和MLST型.结论 不同时期流行的百日咳杆菌具有不同的分子分型.

  4. Latent class analysis of diagnostic tests for adenovirus, Bordetella pertussis and influenza virus infections in German adults with longer lasting coughs.

    Science.gov (United States)

    Sobotzki, C; Riffelmann, M; Kennerknecht, N; Hülsse, C; Littmann, M; White, A; Von Kries, R; Wirsing VON König, C H

    2016-03-01

    Laboratory tests in adult outpatients with longer lasting coughs to identify a potential causal pathogen are rarely performed, and there is no gold standard for these diagnostic tests. While the diagnostic validity of serological tests for pertussis is well established their potential contribution for diagnosing adenovirus and influenza virus A and B infections is unclear. A sentinel study into the population-based incidence of longer lasting coughs in adults was done in Rostock (former East Germany) and Krefeld (former West Germany). A total of 971 outpatients who consulted general practitioners or internists were included. Inclusion criteria were coughing for ⩾1 week and no chronic respiratory diseases. We evaluated the performance of polymerase chain reaction (PCR) as well as IgG and IgA serology, applying a latent class model for diagnosing infections with adenovirus, B. pertussis, and influenza virus A and B. The adult outpatients first sought medical attention when they had been coughing for a median of 3 weeks. In this situation, direct detection of infectious agents by PCR had a low sensitivity. Modelling showed that additional serological tests equally improved sensitivity and specificity for diagnosis for adenovirus, B. pertussis and influenza virus A and B infections. The combination of serology and PCR may improve the overall performance of diagnostic tests for B. pertussis and also for adenovirus, and influenza virus A and B infections.

  5. A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase

    Science.gov (United States)

    2009-10-22

    ACKNOWLEDGMENT We thank Carol Li for technical help and Elliot Campbell for help with the stopped-flow fluorescence measurements. Dr. Ladant (Institute...387–401. 21. Ringler, P., and Schulz, G. E. (2003) Self-assembly of proteins into designed networks. Science 302, 106–109. 22. Reiersen, H., and Rees

  6. Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways.

    Directory of Open Access Journals (Sweden)

    Giorgio Fedele

    Full Text Available The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA, an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC, an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT or a mutant lacking CyaA (BpCyaA-, or the BpCyaA- strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.

  7. Consenso para el diagnóstico clínico y microbiológico y la prevención de la infección por Bordetella pertussis Consensus on the clinical and microbiologic diagnosis of Bordetella pertussis, and infection prevention

    Directory of Open Access Journals (Sweden)

    2011-02-01

    Full Text Available La tos ferina sigue siendo responsable de una carga de enfermedad importante en el mundo. Aunque la implementación del uso de la vacuna contra esta enfermedad ha disminuido en gran medida el número de casos en la población pediátrica, se ha observado que la inmunidad inducida por la vacuna y por la infeccion natural disminuye con el tiempo lo que hace nuevamente susceptibles a adolescentes y adultos jóvenes que pueden transmitir la enfermedad a lactantes no inmunizados o con esquema de vacunación incompleto. Este documento, resultado de la reunión de un grupo internacional de expertos en la Ciudad de México, ha analizado la información médica reciente para establecer el estado actual de la epidemiología, diagnóstico, vigilancia y, especialmente, el valor de la dosis de refuerzo con dTpa en adolescentes y adultos como estrategia de prevención de tos ferina en México.Pertussis continues to be responsible for a significant disease burden worldwide. Although immunization practices have reduced the occurrence of the disease among children, waning vaccine- and infection-induced immunity still allows the disease to affect adolescents and adults who, in turn, can transmit the disease to non-immunized or partially immunized infants. This document is the result of a meeting in Mexico City of international experts who analyzed recent medical information in order to establish the current status of the epidemiology, diagnosis and surveillance of pertussis and, especially, the value of the dTpa booster dose in adolescents and adults as a pertussis prevention strategy in Mexico.

  8. Disease: H01077 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available the genus Bordetella that is isolated from poultry with respiratory disease. B. hinzii may cause disease in...all PJ Bordetella hinzii sp. nov., isolated from poultry and humans. Int J Syst Bacteriol 45:37-45 (1995) ...

  9. NCBI nr-aa BLAST: CBRC-DNOV-01-1620 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-1620 ref|NP_887655.1| FMN-dependent dehydrogenase [Bordetella bronchiseptic...a RB50] emb|CAE31607.1| FMN-dependent dehydrogenase [Bordetella bronchiseptica RB50] NP_887655.1 9.9 27% ...

  10. NCBI nr-aa BLAST: CBRC-XTRO-01-0311 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0311 ref|NP_891493.1| hypothetical protein BB4959 [Bordetella bronchiseptic...a RB50] emb|CAE35323.1| putative membrane protein [Bordetella bronchiseptica RB50] NP_891493.1 1e-26 30% ...

  11. NCBI nr-aa BLAST: CBRC-DNOV-01-0780 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0780 ref|NP_889904.1| putative transmembrane transport protein [Bordetella bronchiseptic...a RB50] emb|CAE33862.1| putative transmembrane transport protein [Bordetella bronchiseptica RB50] NP_889904.1 4.0 32% ...

  12. NCBI nr-aa BLAST: CBRC-DNOV-01-0407 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0407 ref|NP_887655.1| FMN-dependent dehydrogenase [Bordetella bronchiseptic...a RB50] emb|CAE31607.1| FMN-dependent dehydrogenase [Bordetella bronchiseptica RB50] NP_887655.1 6.6 27% ...

  13. NCBI nr-aa BLAST: CBRC-DNOV-01-0816 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0816 ref|NP_887655.1| FMN-dependent dehydrogenase [Bordetella bronchiseptic...a RB50] emb|CAE31607.1| FMN-dependent dehydrogenase [Bordetella bronchiseptica RB50] NP_887655.1 9.0 27% ...

  14. NCBI nr-aa BLAST: CBRC-LAFR-01-2188 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-2188 ref|NP_887772.1| hypothetical protein BB1226 [Bordetella bronchiseptic...a RB50] emb|CAE31724.1| putative membrane protein [Bordetella bronchiseptica RB50] NP_887772.1 0.37 27% ...

  15. NCBI nr-aa BLAST: CBRC-DYAK-04-0110 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-04-0110 ref|NP_889241.1| hypothetical protein BB2704 [Bordetella bronchiseptic...a RB50] emb|CAE33197.1| Putative membrane protein [Bordetella bronchiseptica RB50] NP_889241.1 0.044 25% ...

  16. NCBI nr-aa BLAST: CBRC-XTRO-01-0648 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0648 ref|NP_890139.1| putative iron uptake protein [Bordetella bronchiseptic...a RB50] emb|CAE35577.1| putative iron uptake protein [Bordetella bronchiseptica RB50] NP_890139.1 2e-95 42% ...

  17. NCBI nr-aa BLAST: CBRC-XTRO-01-0575 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0575 ref|NP_890560.1| putative binding-protein-dependent permease [Bordetella bronchiseptic...a RB50] emb|CAE34389.1| putative binding-protein-dependent permease [Bordetella bronchiseptica RB50] NP_890560.1 1e-110 67% ...

  18. NCBI nr-aa BLAST: CBRC-DNOV-01-0156 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0156 ref|NP_887655.1| FMN-dependent dehydrogenase [Bordetella bronchiseptic...a RB50] emb|CAE31607.1| FMN-dependent dehydrogenase [Bordetella bronchiseptica RB50] NP_887655.1 8.8 34% ...

  19. NCBI nr-aa BLAST: CBRC-DDIS-04-0000 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DDIS-04-0000 ref|NP_889639.1| ABC transporter, ATP-binding protein [Bordetella bronchiseptic...a RB50] emb|CAE33595.1| ABC transporter, ATP-binding protein [Bordetella bronchiseptica RB50] NP_889639.1 4e-68 38% ...

  20. NCBI nr-aa BLAST: CBRC-TGUT-37-0075 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-37-0075 ref|NP_890205.1| hypothetical protein BB3670 [Bordetella bronchiseptic...a RB50] emb|CAE35643.1| putative membrane protein [Bordetella bronchiseptica RB50] NP_890205.1 0.80 35% ...

  1. NCBI nr-aa BLAST: CBRC-XTRO-01-0044 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0044 ref|NP_887857.1| putative membrane transport protein [Bordetella bronchiseptic...a RB50] emb|CAE31809.1| putative membrane transport protein [Bordetella bronchiseptica RB50] NP_887857.1 4e-99 68% ...

  2. NCBI nr-aa BLAST: CBRC-MMUS-11-0135 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MMUS-11-0135 ref|NP_889714.1| hypothetical protein BB3178 [Bordetella bronchiseptic...a RB50] emb|CAE33670.1| conserved hypothetical protein [Bordetella bronchiseptica RB50] NP_889714.1 3.0 26% ...

  3. NCBI nr-aa BLAST: CBRC-XTRO-01-0811 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0811 ref|NP_891493.1| hypothetical protein BB4959 [Bordetella bronchiseptic...a RB50] emb|CAE35323.1| putative membrane protein [Bordetella bronchiseptica RB50] NP_891493.1 2e-18 34% ...

  4. NCBI nr-aa BLAST: CBRC-DNOV-01-1857 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-1857 ref|NP_889799.1| multidrug resistance protein [Bordetella bronchiseptic...a RB50] emb|CAE33755.1| multidrug resistance protein [Bordetella bronchiseptica RB50] NP_889799.1 8.6 23% ...

  5. Proficiency Program for Real-Time PCR Diagnosis of Bordetella pertussis Infections in French Hospital Laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses▿ †

    Science.gov (United States)

    Caro, Valérie; Guiso, Nicole; Alberti, Corinne; Liguori, Sandrine; Burucoa, Christophe; Couetdic, Gérard; Doucet-Populaire, Florence; Ferroni, Agnès; Papin-Gibaud, Sophie; Grattard, Florence; Réglier-Poupet, Hélène; Raymond, Josette; Soler, Catherine; Bouchet, Sylvie; Charreau, Sandrine; Couzon, Brigitte; Leymarie, Isabelle; Tavares, Nicole; Choux, Mathilde; Bingen, Edouard; Bonacorsi, Stéphane

    2009-01-01

    With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/μl (0.2 to 2 CFU/μl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly. PMID:19692562

  6. Monitoring on antibodies against whole cell Bordetella pertussis in healthy adolescents in Hongkou District, Shanghai City, China%上海市虹口区青少年人群百日咳全抗体水平监测

    Institute of Scientific and Technical Information of China (English)

    钱晓华; 汤素文; 李淑华; 张莉; 张捷; 赵根明

    2015-01-01

    目的 分析上海市虹口区青少年人群中百日咳全抗体水平,为人群百日咳免疫策略提供依据.方法 2010年在上海市虹口区内随机选择6~10岁、11 ~ 14岁及15~19岁的健康青少年225人,对青少年百日咳疫苗免疫史和持续≥14 d咳嗽史进行调查.采集静脉血样,分离血清,用ELISA法检测百日咳全抗体IgG.结果 实际纳入研究的对象共214人,百日咳疫苗全程接种率为93.93%,持续2周以上咳嗽患病率为15.42%;青少年人群百日咳全抗体GMT水平为21.88 U/ml,全抗体阳性(IgG≥50 U/ml)率为26.64%,高抗体水平(IgG≥100 U/ml)阳性率为15.42%;各年龄组百日咳全抗体GMT(F=19.90)、抗体阳性率(x2=18.19)和高抗体水平阳性率(x2=9.73)差异均有统计学意义(P<0.01),随年龄增长均呈显著上升趋势(P<0.01);1年内有持续2周以上咳嗽史人群百日咳全抗体GMT(t=2.505)、抗体阳性率(x2=7.071)和高抗体水平阳性率(x2=6.626)均明显高于无2周以上咳嗽史人群(P均<0.05).结论 虹口区青少年人群中存在百日咳感染病例被漏诊或未发现;现行百日咳疫苗免疫程序对青少年不具有持久的保护作用.为虹口区青少年人群的百日咳疫苗免疫策略提供科学依据,有必要进一步开展医院/社区百日咳病例的前瞻性主动监测.

  7. Development and evaluation of real-time quantitative PCR assay for rapidly detecting Bordetella pertussis%实时荧光定量PCR快速检测百日咳鲍特菌方法学建立及评价

    Institute of Scientific and Technical Information of China (English)

    王慧; 罗炜; 赖克方

    2010-01-01

    目的 建立实时荧光定量PCR检测百日咳鲍特菌的方法.方法 以百日咳鲍特菌的重复插入序列IS481为目的 基因,设计探针和引物,以克隆的IS481基因片段为DNA模板,在荧光定量PCR仪上建立实时荧光定量PCR检测方法和标准曲线,并进行灵敏度、重复性、特异性实验.结果 建立的定量标准曲线阈值循环数(Ct)与模板拷贝数呈良好线性关系(r=0.998);最低检测浓度为102 copies/μl;批内及批间变异系数均小于4%;对临床其他常见呼吸道病原体不出现特异性扩增曲线.结论 实时荧光定量PCR法检测IS481基因可用于百日咳鲍特菌的快速检测,具有较好的灵敏度、特异性及重复性.

  8. 百日咳杆菌腺苷酸环化酶毒素基因的克隆及原核表达%Cloning and Prokaryotic Expression of Bordetella pertussis Adenylate Cyclase Toxin Gene

    Institute of Scientific and Technical Information of China (English)

    石碧珠; 张华捷; 孟民杰; 张庶民

    2010-01-01

    目的 克隆百日咳杆菌腺苷酸环化酶毒素(CyaA,ACT)基因,表达并纯化重组CyaA蛋白.方法 从百日咳杆菌CS株的基因组DNA中PCR扩增CyaA编码基因,克隆人载体pET30a,构建重组原核表达质粒pET30a/cyaA,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达.表达的重组蛋白经8 mol/L尿素变性、透析复性、DEAE阴离子交换柱纯化后,采用Western blot法鉴定其反应原性.结果 重组原核表达质粒pET30a/cyaA经PCR、双酶切及测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量约占菌体总蛋白的20%;纯化的重组蛋白纯度达90%左右,可与全细胞百日咳疫苗和无细胞百日咳疫苗免疫血清结合.结论 已成功克隆了百日咳杆菌cyaA基因,并在大肠杆菌中表达了重组CyaA蛋白,为进一步开展CyaA的应用研究奠定了基础.

  9. 中国百日咳鲍特菌血清型及其相关基因分析%Serotype and fimbriae-genotype analysis of Chinese Bordetella pertussis strains

    Institute of Scientific and Technical Information of China (English)

    徐颖华; 张柳; 王丽婵; 骆鹏; 卫辰; 侯启明; 张庶民

    2010-01-01

    目的 了解我国1955-2005年临床分离百日咳鲍特菌及疫苗株的血清型及其相关基因特征.方法 应用抗菌毛蛋白2(fim2)和3(fim3)单克隆抗体凝集反应分析我国不同时期分离的80株百日咳鲍特菌株和3株疫苗株的血清型,并与相应的多克隆抗体方法进行比较分析;同时应用PCR方法扩增这些菌株fim2和fimd基因,并对这些PCR产物进行DNA测序分析.结果 研究表明,与多克隆抗体分析结果相比较,单克隆抗体玻片凝集方法和微量凝集反应除一株临床分离株的血清型分析结果不同,其余菌株的结果均一致.研究菌株的血清型分析结果显示17株临床分离菌株和CS与P3S10疫苗株的血清型为fim2&3型、48株菌为fim2型、15株分离菌株与18530疫苗株为fim3型.在我国扩大免疫规划前分离菌株的血清型以fim2型和fim2&3型为主,随着大范围的百日咳疫苗接种,临床分离菌株中fimd型血清型细菌所占的比例逐渐增多.分析菌株中fim2和fimd的基因型以fim2-1(92.5%)和fim3-A(95.0%)亚型为主;18530疫苗株基因型为fim2-2和fim3-A,而其他两株疫苗株的基因型均为fim2-1和fim3-A.在2000年以后,分离出现含有fim3-B和fim3-D亚型菌株,这些结果表明随着我国大范围百日咳疫苗免疫接种,百日咳分离菌株表达的血清型及其基因序列也出现适应性变异.结论 本研究证实了抗fim2和fim3单克隆抗体在百日咳血清型分析的特异性和实用性.对我国不同时期分离菌株和疫苗株的血清型分析,为我国百日咳疫苗株的检定、流行病学和细菌进化的研究奠定了基础.%Objective To understand serotype and fimbriae-genotype of B. pertussis vaccine strains and isolates from different periods in China. Methods Serotype of eighty isolates and three vaccine strains were determined using anti-fim2 and fim3 monoclonal antibodies compared with polyclonal antisera. Fim2 and fim 3 genes were amplified by PCR and the amplified products were sequenced and analyzed . Results The serotype of three vaccine strains and all isolates but only one tested by the slide agglutination and micro-plate assay of anti-fim2 and fim3 monoclonal antibodies were the same in comparison with that of the slide agglutination of polyconal antisera. In this study, seventeen isolates and vaccine strains CS and P3S10 were fim2&3 serotype, and forty-eight isolates were tim2 serotype while fifteen isolates and vaccine strain 18530 were fim3 serotype. The predominant serotypes were fim2 and fim2&3 before Expanded Program on Immuni-zation in 1978, while the find became the most popular serotype after nation-wide pertussis vaccination in China. The fim2-1 and fim3-A genotype was the most common type, which was identified in 92.5% and 95.0% of the isolates, respectively. The genotype of vaccine strain 18530 was fim2-2 and fim3-A while oth-er vaccine strains were fim2-1 and fim3-A. The isolates contained fim3-B and fim3-D subtypes were found since 2000. These data indicated that the serotype and fimbriae genotype of B. pertussis isolates have been changed for immune environment of national-wide pertussis vaccination in China. Conclusion The validity and specificity of anti-fim2 and fim3 monoclonal antibodies have been validated for serotyping of B. pertussis strains. The information of serotype and fimbirae genotype of B. pertussis vaccine strains and isolates from dif-ferent time periods have been obtained. These data can facilitate the studies on quality control of vaccine strain, epidemiology and the evolution of B. pertussis in China.

  10. 中国百日咳鲍特菌ptxS1和prn基因多态性分析%Polymorphism of ptxS1 and prn Genes of Bordetella pertussis in China

    Institute of Scientific and Technical Information of China (English)

    张柳; 徐颖华; 赵建宏; 徐运强; 张庶民

    2010-01-01

    目的 分析我国近50年来分离的百日咳鲍特菌抗原百日咳毒素(Pertussis toxin,PT)S1亚基(ptxS1)和百日咳黏附素(Pertactin,prn)基因多态性.方法 收集85株百日咳杆菌,分别进行ptxS1和prn基因的PCR扩增和测序,并对序列进行比较分析.结果 共发现4个ptxS1基因型和6个prn基因型的百日咳杆菌.自20世纪60年代,非疫苗型ptxS1A菌株逐渐取代疫苗型菌株;含有prn2和prn3新基因型的菌株出现在2000年以后.基因的系统进化树显示,菌株ptxS1和prn基因型的核苷酸和氨基酸序列的同源性分别在97%以上.结论 我国近50年流行的百日咳临床分离菌株ptxSl和prn基因存在抗原漂移现象,本研究为加强我国百日咳的流行病学监控与新型无细胞百日咳疫苗的研发奠定了基础.

  11. 百日咳鲍特菌铁离子相关分泌蛋白研究%A study of iron-related proteins secreted by Bordetella pertussis

    Institute of Scientific and Technical Information of China (English)

    马广源; 季亚勇; 沙丹; 凌霞; 肖勇; 吴家林; 朱泳璋

    2014-01-01

    目的 探讨儿童呼吸道致病菌之一百日咳鲍特菌不同铁离子培养条件下分泌蛋白质组的差异以及与其致病的关系. 方法 百日咳鲍特菌分别在加铁、加铁螯合剂和加铁加镁的包-姜氏培养基中培养,于对数生长期(培养48h)收集细菌,分别用三氯乙酸丙酮沉淀法制备分泌蛋白,制备的分泌蛋白做MALDI-TOF MS质谱分析,并进行分泌蛋白的细菌亚细胞定位和分子信号肽预测. 结果 加铁培养从47 h开始细菌生长进入平台期,加铁离子螯合剂组细菌随铁离子螯合剂的增加而趋于不生长状态,加铁加镁组细菌在培养至47 h时缓慢生长;百日咳鲍特菌在3种培养条件下的蛋白亚细胞定位分别为:加铁组胞质蛋白326个,内膜蛋白61个,分泌蛋白3个,外膜蛋白13个,周质蛋白33个,未知蛋白201个;加铁螯合剂组胞质蛋白466个,外膜蛋白21个,周质蛋白63个,内膜蛋白66个,分泌蛋白3个,未知蛋白160个;加铁加镁组胞质蛋白601个,内膜蛋白97个,分泌蛋白5个,外膜蛋白26个,周质蛋白7个,未知蛋白185个. 结论 通过亚细胞定位和分子信号肽预测,锁定百日咳鲍特菌6个分泌蛋白,其中3个分泌蛋白核糖核苷二磷酸激酶(NDPK)、假定输出蛋白和血清三型菌毛亚单位是本研究首次发现的百日咳鲍特菌分泌蛋白.

  12. Research progress of Bordetella pertussis infection and chronic cough in adults%百日咳杆菌感染与成人不明原因慢性咳嗽相关性研究现状

    Institute of Scientific and Technical Information of China (English)

    文芳静; 肖锦秀; 胡克

    2012-01-01

    Although cough variant asthma,upper airway cough syndrome/postnasal drip syndrome,eosinophilic bronchitis and gastroesophageal reflux disease are generally considered as the most common causes of chronic cough,postinfectious cough is also an important factor.Mycoplasma pneumonia and Chlamydia pneumonia have been well known and we often empirically use macrolides for its treatment.Over the years,pertussis has been commonly considered infectious respiratory disease in children.But recent studies have demonstrated it's a common cause of persistent cough in adults and should be considered in differential diagnosis.%尽管咳嗽变异性哮喘、上气道咳嗽综合征/鼻后滴流综合征、嗜酸粒细胞性支气管炎和胃食管反流性咳嗽被普遍认为是不明原因慢性咳嗽(简称慢性咳嗽)最常见病因,但感染后咳嗽也是引起长期咳嗽的一个重要因素.由肺炎支原体和肺炎衣原体等引起的感染后咳嗽多为人们所熟知,且常经验性使用大环内酯类抗生素对其治疗.长期以来,百日咳被认为在儿童呼吸道感染性疾病中较为常见,但近年来的研究表明,百日咳杆菌感染也是引起成人慢性咳嗽的常见原因,需要加以重视和鉴别.

  13. Development of fluorescence quantitative PCR for detection of Bordetella pertussis and its application%百日咳杆菌荧光定量PCR检测方法的建立和应用

    Institute of Scientific and Technical Information of China (English)

    徐颖华; 徐运强; 张庶民; 王丽婵; 侯启明; 雷殿良

    2008-01-01

    Objective To establish a rapid,accurate,specific quantitative assay for detecting B.pertussis,and apply to clinical diagnosis.Methods According to the specific sequence of B.pertussis IS481 gene,the primers and the fluorescence probe were designed and synthesized.Then a fluorescence quantitative PCR for detecting B.pertussis was developed.The specificity,sensitivity and reproducibility of the method were evaluated.255 specimens including 225 nasopharyngeal swabs from suspected pertussis patients and 30 normal nasopharyngeal swabs were detected by fluorescence quantitative PCR.Results A rapid specific quantitative method for detecting B.pertussis was established.The standard curve of the method indicated that there was a good linear relationship between the CT value and the template concentration with the correlation coefficient being 0.998.The linear range of the system was from 102 to 108 copies/μl and the minimum was 102 copies.It had a high sensitivity and good specificity.The intra.and inter-assay coefficients of variation were 5.78%-16.7% and 8.25%-14.9% respectively.The fluorescence quantitative PCR identified 41 positive results for specimens from suspected pertussis patients and results of 30 normal specimens were all negative.Conclusions The method can quantitatively detect the B.pertussis rapidly with high sensitivity and specificity,it can be applied to clinical diagnosis.%目的 建立快速、准确、特异的定量检测百日咳杆菌的方法,并进行临床应用研究.方法 根据百日咳杆菌IS481基因序列,设计并合成引物和荧光探针,建立百日咳杆菌荧光定量PCR检测方法,并对方法的特异度、重复性和灵敏度进行评价,检测百日咳疑似患者咽拭子225份和健康者咽拭子30份.结果 该方法的标准曲线显示模板浓度在102~108拷贝/μl线性范围与其循环阈值(Ct)具有较好的线性关系,相关系数(r)为0.998,最小检出量为102拷贝.特异度较好,灵敏度较高,重复性实验结果显示在同一时间检测高低浓度阳性克隆质粒(2×107与3×103拷贝/μ1)10次,连续检测5次,变异系数(CV)为5.78%~16.7%.在不同时间检测高低浓度阳性克隆质粒(2×107与3×103拷贝/μl),连续7次,CV为8.25%~14.9%.经检测,疑似患者的咽拭子有41份样本为阳性,30份健康人咽拭子均为阴性.结论 由于本实验建立的荧光定量PCR方法快速、灵敏度高、特异度好,适合临床实验室进行临床标本的百日咳杆菌的定量检测,有较大的应用前景.

  14. Pertactin-negative Bordetella pertussis strains in Canada: characterization of a dozen isolates based on a survey of 224 samples collected in different parts of the country over the last 20 years

    Directory of Open Access Journals (Sweden)

    Raymond S.W. Tsang

    2014-11-01

    Conclusions: As reported elsewhere, pertactin-negative B. pertussis has emerged in Canada in recent years, notably in 2012. This coincided with an increase in pertussis activity in Canada. A further systematic study with a larger geographical representative sample is required to determine how these vaccine-negative strains may contribute to the overall changing epidemiology of pertussis in Canada.

  15. Survery of specific antibody against bordetella pertussis in healthy population of urumqi%乌鲁木齐市健康人群百日咳抗体水平调查

    Institute of Scientific and Technical Information of China (English)

    黄玉蓉; 张卫平

    2007-01-01

    目的:了解乌鲁木齐市健康人群及产妇针对百日咳的血清特异性IgG抗体水平,为制定免疫策略和防治措施提供科学的依据.方法:随机选取乌鲁木齐市505人作为检测对象,根据年龄分为9个组:0~5个月龄(50例)、6~11个月龄(47例)、12~17个月龄(53例)、18~23个月龄(60例)、2~3岁(56例)、4~6岁(60例)、7~11岁(60例)、12~14岁(59例)、≥15岁(60例);此外还随机选取了各60例的配对脐血组和产妇组.取检测对象血清,应用酶联免疫吸附试验(ELISA)测定特异性百日咳IgG抗体.结果:百日咳IgG抗体水平2~3岁最高,0~5月龄最低,12~14岁组及≥15岁组处于较低水平;同时,配对脐血及产妇组的百日咳IgG抗体水平存在很好的关联性(rs=0.79,P<0.05).结论:应该对年长儿及成人进行百日咳疫苗的加强免疫,从而根除低龄儿童百日咳的主要传染源.

  16. Disease: H01066 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available ordetella, B. petrii is an environmental species but sometimes can infect immunocompetent humans. Infectious...ith both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae.

  17. Bordetella Pertussis Infection and Its Clinical Symptoms and Moleculer Epidemiology in Children in Xinjiang Uygur Automonous Region%新疆地区小儿百日咳感染率、临床特点及分子流行病学特点

    Institute of Scientific and Technical Information of China (English)

    张卫平; 黄玉蓉; 罗琼; 多力坤; 何秋水

    2008-01-01

    目的 了解新疆地区健康儿童百日咳血清特异性IgG抗体水平.调查本地区小儿百口咳的感染率,探讨百日咳患儿的临床及分子流行病学特点.方法 随机选取新疆地区健康儿童445例作为对照,根据年龄分为8个组,取其血清,应用ELISA法测定特异性百口咳IgG抗体.同时选择新疆医科大学第一附属医院163例可疑百日咳患儿作为研究对象,分别取鼻咽拭子及血清标本,应用聚合酶链反应(PCR)法检测其百日咳杆菌的双目标基因(插入区域IS481以及百日咳毒素S1促进区域),ELISA法检测特异性IgG抗体以及碳琼脂培养基培养鲍特百日咳杆菌;应用脉冲场凝胶电泳(PFGE)对检测出的鲍特百日咳杆菌进行分子流行病学研究.结果 健康儿童百日咳IgG抗体水平以2~3岁最高,0~5个月最低,12~14岁组处于较低水平.确诊百日咳患儿26例中,百日咳特异性IgG抗体为(127.63±12.75)g/L,余137例非百日咳患儿为(7.71±16.32)g/L,确诊百日咳患儿百日咳特异性IsG抗体水平明显高于非百日咳患儿患儿(F=5.837 P<0.05);非百日咳患儿与健康对照组[(6.07±17.79)g/L]比较,差异无统计学意义.可疑患儿血清学阳性例数以7~15岁组最高(43/163例,占26.19%),1~11个月组21/163例(12.82%),1~3岁组为11/163例(6.67%),4~6岁组为8/163例(5.0%);≥7岁者血清学阳性占21.92%,<7岁者仅为9.68%,二者比较有显著差异(χ2=7.6975 P<0.05);未接种儿童百日咳特异性抗体阳性率(14/35例)明显高于接种过百日咳疫苗的儿童(22/128例),其差异有统计学意义(χ2=16.3358 P<0.001).可疑百日咳163患儿中26例百日咳特异性抗体阳性,其中24例(92.31%)PCR检测阳性,百日咳杆菌特异性培养无阳性结果;将PCR阳性标本经Xbal限制性内切酶消化后在48~630 kb产生9~14个DNA片段,共得14个PFGE核型.结论 应对年长儿进行百日咳疫苗的免疫.PCR是诊断百日咳感染的一项快速、敏感、易行、实用的工具,与血清学检查相结合能够提高所有年龄群体的百日咳感染的监测水平;百日咳杆菌的PFGE核型可以鉴别不同地区的流行菌株.

  18. 百日咳毒素S1亚单位突变体在大肠杆菌中的表达及纯化%Expression of Bordetella pertussis Toxin S1 Mutant in E.coli and Purification of Expressed Product

    Institute of Scientific and Technical Information of China (English)

    张晓丽; 邹全明; 章金勇; 张卫军

    2007-01-01

    目的 克隆百日咳毒素S1亚单位,获得其突变体(S1/9K-129G,rS1),构建原核表达系统,并鉴定融合蛋白的特异性.方法 采用PCR和重叠延伸扩增得到突变体,将其与pMD18-T连接,转化至大肠杆菌DH5α,筛选得到阳性重组克隆载体pMD18-T-rS1,经双酶切得到rS1活性结构域编码片段.将其插入pQE-30载体中,转化大肠杆菌M15株,经IPTG诱导表达.表达产物经镍离子柱纯化,并进行SDS-PAGE和Western blot鉴定.结果 PCR及重叠延伸获得了约700 bp的目的 基因片段,并获得阳性重组表达载体克隆,表达产物为相对分子质量27 000左右的蛋白,Western blot检测能与兔抗百日咳毒素多价抗血清发生反应.结论 成功表达并纯化了百日咳毒素S1亚单位突变体,为研制百日咳疫苗候补抗原的相关分子免疫学研究奠定了基础.

  19. 兴奋性氨基酸和特异性受体对大鼠百日咳杆菌脑损伤的作用%Effects of excitatory amino acids and its specific receptor on bordetella pertussis brain injury of rats

    Institute of Scientific and Technical Information of China (English)

    陈翔; 徐晔; 王霞; 陶永光; 虞佩兰; 杨于嘉

    2004-01-01

    目的:探讨兴奋性氨基酸、N-甲基-D-天冬氨酸受体(NMDAR)在大鼠百日咳杆菌脑损伤中的变化及作用机制.方法:用高效液相色谱法、[3H]MK-801放射配体结合分析法及Fura-2/Am分别检测大鼠百日咳杆菌脑损伤时大脑皮质内谷氨酸(Glu)和谷氨酰胺(Gln)含量、NMDAR钙通道的结合位点(最大结合位点Bmax)及活性(平衡解离常数Kd)、大脑皮质突触细胞内游离钙浓度([Ca2+]i)的变化.结果:与盐水组比较,注菌组Gln显著增高(P<0.05),Kd值减小(P<0.05)、Bmax值差异无显著性(P>0.05),[Ca2+]i较盐水组增高(P<0.01),并随注菌时间延长而显著增高(24 h vs 4 h,P<0.01);MK-801预处理组[Ca2+]i、脑含水量、伊文思蓝含量、Glu和Gln含量均较注菌组减少(F值分别为54.2436、15.628、14.425、10.2458、14.0234,P<0.01),24 h组[Ca2+]i减少最为显著(P<0.01).结论:EAAs介导的NMDAR激活及相应的钙内流增加在大鼠百日咳杆菌脑损伤中,尤其在百日咳杆菌致大鼠迟发性脑损伤的发病机制中可能起着重要作用.

  20. 百日咳丝状血凝素分子中强免疫原性片段的筛选、克隆与表达%Screening, Gene Cloning and Expression of A Highly Immunogenic Fragment of Filamentous Hemagglutinin of Bordetella pertussis

    Institute of Scientific and Technical Information of China (English)

    章金勇; 张晓丽; 张卫军; 邹全明

    2008-01-01

    目的 筛选百日咳丝状血凝素分子中强免疫原性的片段,并对其进行克隆及表达.方法 采用生物信息学软件对丝状血凝素分子的结构进行预测,从中筛选出具有强免疫原性的片段FHAs.经PCR扩增,T-A克隆后,构建表达质粒pET-22b(+)-FHAs,酶切鉴定正确后测序.将测序正确的质粒转化E.coli BL21(DE3),IPTG诱导目的蛋白的表达,并对表达产物进行Tricine-SDS-PAGE分析及Western blot鉴定.结果 从丝状血凝素分子中筛选出具有138个氨基酸的肽段FHAs,经PCR扩增,得到约400 bp的目的基因片段,构建的重组质粒经双酶切和测序鉴定,证明质粒构建正确,重组工程菌pET-22b(+)-FHAs/BL21经IPTG诱导,目的蛋白的表达量在50%以上,主要以包涵体形式存在.Western blot分析证实该蛋白能与抗FHA单抗发生反应.结论 筛选出的肽段FHAs具有强的免疫原性和免疫反应特异性,为百日咳基因工程疫苗的研究奠定了基础.

  1. Effects of sodium chloride in medium on growth of Bordetella pertussis and expression of pertussis toxin%培养基中氯化钠对百日咳杆菌CS株生长及百日咳毒素表达的影响

    Institute of Scientific and Technical Information of China (English)

    李应伟; 刘晓凤; 周晓舟; 夏永

    2015-01-01

    目的 探讨培养基中氯化钠(NaCl)对百日咳杆菌CS株生长及百日咳毒素(pertussis toxin, PT)表达的影响,确定最适PT表达的NaCl加入方式.方法 以MSS为起始培养基,补加不同浓度的NaCl溶液,接种生产用百日咳杆菌Ⅰ相CMCC58003(CS株)后,分别于不同时间取样;分别于接种菌种后不同时间补加NaCl至7.5 g/L,于培养结束时(39 h)取样;分别于接种菌种后0和25 h时补加NaCl至7.5 g/L,培养不同时间取样.上述样品分别测定菌浓度及PT表达量.结果 起始培养基中NaCl浓度越高,细菌生长越缓慢,菌浓度增加时间也越长,起始培养基中NaCl浓度为7.5 g/L时,培养上清中PT表达量最高,达6.61 μg/ml,比对照培养基(MSS)提高了15.6%.培养过程中在25 h时补加NaCl,培养结束时PT表达量最高,为8.64 μg/ml,比对照培养基(MSS)提高了43.28%,比0h时补加NaCl提高了26.13%.在25 h时补加NaCl后,细菌的菌浓度增长期比MSS培养基培养延长了2~3h,比0h时补加NaCl缩短了1-2h,菌浓度在培养的中后期明显高于0h时补加NaCl,PT表达周期与生长曲线基本平行,当细菌生长进入衰退期时,PT也随之开始逐步降解.结论 在培养前期,可采用含2.5 g/L NaCl的MSS培养基使菌体快速生长,当进入抗原PT表达期后,可通过补加NaCl来提高PT转录活性,从而提高PT的产量.

  2. Prevalence of pertussis and serum Bordetella pertussis antibody level among 1-15 years old children in Shenzhen city ,2009-2013%深圳市百日咳流行特征及1~15岁人群抗体水平调查

    Institute of Scientific and Technical Information of China (English)

    樊静洁

    2016-01-01

    目的 了解深圳市百日咳流行特征和疫情发生影响因素,检测1~15岁人群百日咳血清抗体水平,评价免疫效果并为免疫程序提供科学依据.方法 通过国家疾病监测信息报告管理系统收集深圳市2009-2013年百日咳病例资料,分析深圳市百日咳流行特征,用ELISA检测方法检测1~15岁人群百日咳血清抗体水平,采用病例对照研究方法分析百日咳影响因素.结果 深圳市2009-2013年共报告百日咳163例,年平均发病率为0.32/10万,发病高峰在7-8月,发病人群主要集中在5月龄以内,占全部发病85.27%;监测256人,百日咳抗体阳性率为38.30%,不同年龄百日咳抗体水平差异无统计学意义(x2=4.737,P>0.05).接触史、住房面积和家庭收入是影响发病的主要因素.结论 深圳百日咳以婴幼儿发病为主;不同年龄组百日咳血清抗体保护率呈上升趋势;接触是百日咳发病的重要危险因素.

  3. Detection of Specific Antibody against Bordetella Pertussis in 0-15 Year Old Healthy Children and the Puerpera%0~15岁健康人群及产妇血清百日咳抗体水平的检测

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    为了解0~15岁健康人群及产妇血清特异性百日咳IgG抗体水平,随机选取山西省太原市458人作为检测对象,根据年龄分为9个组:脐血组(58例)、0~5月龄(33例)、6~11月龄(62例)、1~2岁(50例)、3~5岁(50例)、6~8岁(50例)、9~12岁(49例)、13~15岁(48例)、产妇(58例).其中有54例为配对脐血组及产妇组.取血清应用酶联免疫吸附试验(ELISA)测定百日咳特异性IgG抗体.结果表明,百日咳IgG抗体水平1~2岁最高,13~15岁最低,0~5月龄处于较低水平.配对脐血组及产妇组的百日咳IgG抗体水平具有良好的相关性(r=0.741,P<0.01).百日咳抗体水平在13~15岁最低,<6月龄次之,1~2岁最高.为根除婴儿百日咳的主要传染源,对年长儿及成人进行百日咳加强免疫是必要的.

  4. Development and Validation of ELISA Assays for Determination of Serum Antibody against Pertussis Toxin, Filamentous Hemagglutinin, Pertactin of Bordetella Pertussis%百日咳毒素丝状血凝素和百日咳黏附素血清抗体检测方法的建立及验证

    Institute of Scientific and Technical Information of China (English)

    徐颖华; 骆鹏; 王丽婵; 卫辰; 侯启明; 张庶民

    2011-01-01

    目的 建立定量检测抗百日咳毒素抗体(Antibody to Pertussis Toxin,Anti-PT)、抗丝状血凝素抗体(Antibody to Filamentous Haemagglutinin,Anti-FHA)和抗百日咳黏附素抗体(Antibody to Pertactin,Anti-Prn)的酶联免疫吸附试验(Enzyme-linked Immunosorbent Assay,ELISA)方法.方法 分别应用纯化的百日咳毒素(PT)、丝状血凝素(FHA)和百日咳黏附素(Prn)作为包被抗原,百日咳血清抗体国际参考品为标准品,建立定量检测Anti-PT、Anti-FHA、Anti-Prn的ELISA方法.结果 建立了三种定量检测ELISA方法,并进行方法学验证.结果 显示,定量检测Anti-PT的ELISA方法的最低检测限度为1.31IU/ml(国际单位/毫升),批内和批间变异系数(Coefficient of Variability,CV)分别为9.99%和11.55%,回收率为97.19%;检测Anti-FHA方法的最低检测限度为1.02IU/ml,批内和批间CV分别为11.01%和12.76%,回收率为101.20%;检测Anti-Prn方法的最低检测限度为0.511U/ml,批内和批间CV分别为9.00%和11.18%,回收率为107.83%.用建立的ELISA方法与国外同类方法对30份血清样本进行分析,检测结果差异无统计学意义.应用上述建立的三种ELISA方法,检测分析了三组份无细胞百日咳疫苗(Acellular Pertussis Vaccine,aP)基础免疫后免疫原性.结论 所建立的百日咳血清抗体检测ELISA方法,准确度高和重复性好,无需特殊仪器,适合于一般实验室开展,可用于接种aP后血清学效果观察和百日咳流行病学研究.%Objective To develop three Enzyme-linked Immunosorbent Assay (ELISA ) methods for quantitative determination of IgG antibody against pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin(Prn)in human serum, respectively. Method Purified PT, FHA and Prn and international reference pertussis serum were used as coating antigen and standard for development of three ELISA assays. Results Three ELJSA assays had been established for determining anti-PT, FHA and Prn antibody, respectively. The limit of quantification of the assays had been demonstrated for anti-PT antibody with 1.31IU/ml, for anti-FHA antibody with 1.02IU/ml, for anti-Prn antibody with 0.51IU/ml. The average intra-assay coefficient of variation of three assays was 9.99%-. 11.01% and 9.00%. The average inter-assay coefficient of variation of three assays was 11.55% -. 12.76% and 11.18%, respectively. The average recovery rates of three assays were 97.19% ■. 101.20% and 107.83%, respectively. Three antibody levels in 30 serum sample were detected by ELISA methods developed in this study, which were not difference significantly in comparison with that of similar method from other country. These methods were also applied for immunogenicity analysis of three-component acellular pertussis vaccine in the clinical trial. Conclusion Three quantitative ELISA methods had been demonstrated to be simple, accurate and reproducible. They were used for evaluation of immunogenicy of acellular pertussis vaccine andinvestigation of pertussis epidemiology.

  5. 百日咳鲍特菌外膜蛋白体外促肥大细胞脱颗粒的作用%Outer membrane proteins of Bordetella pertussis function as a modulator on promoting degranulation of mastocyte in vitro

    Institute of Scientific and Technical Information of China (English)

    严杰; 毛亚飞; 邵浙新

    2004-01-01

    目的了解百日咳鲍特菌Ⅰ相菌株外膜蛋白(outer membrane proteins,OMP)生物学活性与IgE 介导哮喘的关系.方法采用去垢剂处理、DEAE-52和Sephadex G-150层析法制备百日咳鲍特菌Ⅰ相菌株OMP及其组分,以SDS-PAGE估计OMP组分的相对分子质量(Mr).采用ELISA检测百日咳全菌死疫苗诱导动物产生的总IgE和卵白蛋白(ovalbumin,OVA)特异的IgE量.采用肥大细胞脱颗粒试验观察OMP及其组分促进脱颗粒的作用、ELISA检测培养物上清中组胺水平.采用组胺敏感试验检测OMP及其组分增加小鼠对组胺致死敏感性的作用.结果从百日咳鲍特菌OMP中分离获得Mr为(30、32、38和69)×103的4个组分.1~100μg/ml OMP及其组分均未显示凝集鲎变形细胞溶解物的活性.OVA和百日咳鲍特菌联合免疫豚鼠的血清总IgE和OVA特异性IgE较单用OVA免疫有明显增加(t=2.75~6.59;P<0.05~0.01).OMP及其Mr 30×103、Mr 32×103组分有明显的促豚鼠肥大细胞脱颗粒的作用(t=4.914~9.137,P<0.01),但Mr 38×103、Mr 69×103组分无此活性.OMP及其Mr 30×103、Mr 32×103组分处理的上清中组胺水平明显高于阴性对照组(t=5.672~9.304,P<0.01),但Mr 38×103、Mr 69×103组分无此活性.结论百日咳鲍特菌有较强的IgE佐剂活性,某些OMP组分具有促进肥大细胞脱颗粒、组胺释放及增加小鼠对组胺致死敏感性的作用,提示百日咳鲍特菌感染或疫苗接种与IgE介导的哮喘有密切关系.

  6. Characterization of two Achromobacter xylosoxidans isolates from patients with pertussis-like symptoms

    Institute of Scientific and Technical Information of China (English)

    Fiorella Orellana-Peralta; Michelle Jacinto; Maria J Pons; Cludia Gomes; Carlos Bada; Isabel Reyes; Juana del Valle Mendoza; Joaquim Ruiz

    2015-01-01

    Objective:To characterize two Achromobacter xylosoxidans recovered from 2 patients diagnosed with pertussis during a Bordetella pertussis surveillance program. Methods:Nasopharyngeal swabs from 2 children under 1 year of age with clinical suspicion of pertussis were analyzed by culture and PCR. Results:Two Achromobacter xylosoxidans A8, closely related to Bordetella spp. were recovered from 2 patients diagnosed of pertussis, both carrying the ptxA gene and IS418 the pertussis toxin encoding gene. Subsequently, antibiotic susceptibility was evaluated by disk-diffusion method and by PCR. Conclusions:Although more detailed studies are needed, the present data highlight the possibility that Achromobacter xylosoxidans, closely related Bordetella pertussis microorganisms and not covered under the vaccine umbrella, might also result in cases of whooping cough. Thereby further surveillance is necessary to determine the extension and relevance of their pathogenic role in order to discriminate their real public health implication.

  7. The Role of the Aceullular Pertussis Vaccine and the Comeback of 'Pertussis Pete'?

    Directory of Open Access Journals (Sweden)

    John M Conly

    2001-01-01

    Full Text Available Pertussis or whooping cough is an acute infectious disease of the respiratory tract caused principally by Bordetella pertussis and less commonly by Bordetella parapertussis (1. Until two decades ago, pertussis in adults was a medical curiosity (2-4, but with the purification of specific Bordetella species antigens, the development of reliable enzyme immunoassays allowing accurate serological diagnosis and better understanding of the duration of immunity from vaccination, it has been clearly demonstrated that B pertussis is a common cause of prolonged cough in adults. Indeed, its incidence has been increasing gradually over the past decade in both adults and adolescents. Given the recognition of the importance of pertussis as a cause of prolonged cough in adults and the advent of the new acellular pertussis vaccines, it is timely to review current concepts of the pathogenesis of pertussis, its epidemiology in adults and the utility of the anticipated impact of the acellular vaccine.

  8. Pertussis seroprevalence in adults, post-partum women and umbilical cord blood.

    Science.gov (United States)

    Fallo, Aurelia; Manonelles, Gabriela; Hozbor, Daniela; Lara, Claudia; Huespe, Miguel; Mazzeo, Silvina; Canle, Oscar; Galas, Marcelo; López, Eduardo

    2014-08-01

    Pertussis is a vaccine-preventable disease that affects people of all ages. Young adults who have lost their immunity to pertussis are the major source of infection in infants. Given the steady increase of pertussis cases, new prevention strategies are required. Objective. To assess pertussis seroprevalence in adult blood donors, post-partum women, and umbilical cords. Metod. Measurement of total titers of anti-Bordetella spp. (Bordetella) antibodies using an enzyme-linked immunosorbent assay. Serum samples from 103 donors, 101 post-partum women and 100 umbilical cords were analyzed. Titers <80 were considered of low impact against the disease. The assessment included transplacental transfer of antibodies and the umbilical cord/maternal ratio of antibody titers. Results. Donors mean age was: 28 ± 6 years old. Mediananti-Bordetella titers: 320; interquartile range (IQR):160-320; 10% had titers <80. Post-partum women mean age was: 26 ± 6 years old. Median anti-Bordetella titers:160 (IQR:80-320), with titers significantly lower than in female donors (p= 0.00002). Titers <80 were found in 30% of post-partum women. Median anti-Bordetella titers in umbilical cords: 160 (IQR: 80-160). Titers <80 were more frequently found in umbilical cords than in mothers (44% versus 30%, p= 0.04). Transplacental transfer was 0.83. Umbilical cord titers were equal to maternal titers in 54% of cases, lower in 37%, and higher only in 8%. Conclusion. Titers of anti-Bordetella antibodies in post-partum women were significantly lower than in female blood donors. Titers <80 were found in 30% of post-partum women and 44% of umbilical cords. These data may account for the high rates of pertussis in young infants who have not yet completed their vaccination schedule.

  9. Optimizing polymerase chain reaction testing for the diagnosis of pertussis: current perspectives

    Directory of Open Access Journals (Sweden)

    Arbefeville S

    2015-09-01

    Full Text Available Sophie Arbefeville, Patricia Ferrieri Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN, USA Abstract: Nucleic acid testing has revolutionized the diagnosis of pertussis in the clinical microbiology laboratory and has become the main avenue of testing for pertussis infection. Real-time polymerase chain reaction (RT-PCR is an important tool for timely diagnosis of pertussis and is more sensitive than culture. The most commonly amplified targets are the insertion-sequence (IS genes, which are found in multiple copies in the genome of Bordetella species. Some strains of Bordetella pertussis have more than 200 copies of IS481 in their genome. This high number of repeats allows RT-PCR assays to be very sensitive and makes nucleic acid testing two to three times more sensitive than culture. Despite these advantages, RT-PCR can give inaccurate results due to contamination or lack of specificity. Contamination can easily happen during specimen collection, DNA extraction, or nucleic acid amplification steps. To avoid contamination, laboratories need to have quality controls and good workflows in place. The poor specificity of the nucleic acid assays amplifying the IS genes is because they are found in various Bordetella species and, thus, not unique to a specific species. Bordetella holmesii, a more recently described Bordetella species found to be responsible for respiratory symptoms similar to pertussis in adolescents and adults, can be misidentified as B. pertussis in RT-PCR assays that amplify only the IS481 target. Use of multiple targets may improve specificity of RT-PCR assays for pertussis. In the past few years, the US Food and Drug Administration has cleared three commercial assays for the detection of B. pertussis in respiratory specimens. Several commercial assays and analyte-specific reagents, which are not US Food and Drug Administration cleared, are available for the detection of one

  10. Cellular and humoral immunity after infection with B. pertussis : the role of age, antigen and vaccination history

    NARCIS (Netherlands)

    van Twillert, I

    2017-01-01

    Pertussis (whooping cough), is a bacterial disease of the respiratory tract, caused by the human pathogen Bordetella pertussis. Vaccination against pertussis has dramatically lowered pertussis incidence and mortality rates; however pertussis still occurs. The duration of immunity to B. pertussis aft

  11. Chlamydia pneumoniae Infection Among Basic Underwater Demolition/SEAL (BUD/S) Trainees, Coronado, California, July 2008

    Science.gov (United States)

    2011-03-01

    including Streptococcus pneumoniae, Chlamydophila pneu- moniae, Mycoplasma pneumoniae, Legionella pneumophila , and Bordetella pertussis . 3 CAP...pneumoniae , L pneumophila , or M pneumoniae . Although the samples proved to be negative for viral etiologies, testing against the bacterial panel dem...controls for L pneumophila (487 bp), M pneumoniae (360 bp), C pneumoniae (283 bp), and B pertussis (122 bp). Delivered by Publishing Technology to

  12. Pertussis specific T-cell immunity in Dutch children: Differences after whole-cell versus acellular vaccination

    NARCIS (Netherlands)

    Schure, R.M.

    2014-01-01

    Bordetella pertussis is the causative bacteria of whooping cough. Whooping cough is a highly contagious infection, which is characterized by coughing with whooping and post-tussive vomiting. In particular, infants under 6 months of age who have not been fully vaccinated, are at risk for serious comp

  13. Treatment Failure of Nosocomial Pertussis Infection in a Very-Low-Birth-Weight Neonate

    Science.gov (United States)

    Bonacorsi, Stéphane; Farnoux, Caroline; Bidet, Philippe; Caro, Valérie; Aizenfisz, Sophie; Benhayoun, Mounir; Aujard, Yannick; Guiso, Nicole; Bingen, Edouard

    2006-01-01

    We describe a case of nosocomial maternal transmission of Bordetella pertussis to a very-low-birth-weight (VLBW) neonate in whom treatment was unsuccessful. This case underscores the need for rapid and sensitive PCR diagnosis in VLBW neonates and in parents with clinical signs of pertussis and suggests that standard treatment may not be appropriate for VLBW neonates. PMID:17021121

  14. Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality : A possible complementation to the Pertussis Serological Potency test

    NARCIS (Netherlands)

    Hoonakker, M E; Verhagen, L M; van der Maas, L; Metz, B; Uittenbogaard, J P; van de Waterbeemd, B; van Els, C A C M; van Eden, W; Hendriksen, C F M; Sloots, A; Han, W G H

    2016-01-01

    Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunolog

  15. Pertussis toxin

    Energy Technology Data Exchange (ETDEWEB)

    Sekura, R.D.; Moss, J.; Vaughan, M.

    1985-01-01

    This book contains 13 selections. Some of the titles are: Genetic and Functional Studies of Pertussis Toxin Substrates; Effect of Pertussis Toxin on the Hormonal Responsiveness of Different Tissues; Extracellular Adenylate Cyclase of Bordetella pertussis; and GTP-Regulatory Proteins are Introcellular Messagers: A Model for Hormone Action.

  16. Migration of antigen-presenting B cells from peripheral to mucosal lymphoid tissues may induce intestinal antigen-specific IgA following parenteral immunization

    NARCIS (Netherlands)

    Coffin, SE; Clark, SL; Bos, NA; Brubaker, JO; Offit, PA

    1999-01-01

    Parenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymp

  17. Läkaköha - aktuaalne uurimisteema / Marje Oona

    Index Scriptorium Estoniae

    Oona, Marje, 1963-

    2012-01-01

    TÜ peremeditsiini õppetooli töötajate poolt algatatud uurimistööst, mille eesmärgiks on uurida Bordetella spp. infektsioonide epidemioloogiat, molekulaargeneetikat ja kliinilisi eripärasid ning selgitada läkaköha sagedasema diagnsimise põhjusi Eestis

  18. Association of Interacting Genes in the Toll-Like Receptor Signaling Pathway and the Antibody Response to Pertussis Vaccination

    NARCIS (Netherlands)

    Kimman, Tjeerd G.; Banus, Sander; Reijmerink, Naomi; Reimerink, Johan; Stelma, Foekje F.; Koppelman, Gerard H.; Thijs, Carel; Postma, Dirkje S.; Kerkhof, Marjan

    2008-01-01

    Background: Activation of the Toll-like receptor (TLR) signaling pathway through TLR4 may be important in the induction of protective immunity against Bordetella pertussis with TLR4-mediated activation of dendritic and B cells, induction of cytokine expression, and reversal of tolerance as crucial s

  19. AcEST: BP915880 [AcEST

    Lifescience Database Archive (English)

    Full Text Available oxide stress-activated histidine kinase ... 53 9e-07 >sp... bvgS OS=Bordetella... 55 2e-07 sp|P33529|PHY_MOUSC Phytochrome OS=Mougeotia scalaris GN=PHY PE=... 54 7e-07 sp|Q9P7Q7|MAK1_SCHPO Per

  20. 77 FR 52333 - International Workshop on Alternatives to the Murine Histamine Sensitization Test (HIST) for...

    Science.gov (United States)

    2012-08-29

    ... Pertussis, also known as whooping cough, is a highly contagious disease caused by the bacterium Bordetella pertussis. Pertussis was one of the most common childhood diseases of the early 20th century and was once a.... pertussis bacteria (e.g., inactivated pertussis toxin , pertactin, and fimbriae) and are less...

  1. Outbreak of Pneumonia in the Setting of Fatal Pneumococcal Meningitis among US Army Trainees: Potential Role of Chlamydia pneumoniae Infection

    Science.gov (United States)

    2011-06-02

    physical stress may contribute to an increased risk for infections with Streptococcus pneumoniae , Streptococcus pyogenes, Mycoplasma pneumoniae ...Chlamydia pneumoniae , Mycoplasma pneumoniae , Streptococcus pneumoniae , Bordetella pertussis, and Legionella pneumophila[10] in addition to undergoing...postexposure chemoprophylaxis. Mil Med 2003;168:1-6 7. Balicer RD, Zarka S, Levine H, et al. Control of Streptococcus pneumoniae serotype 5 epidemic of

  2. PAT for vaccines: The first stage of PAT implementation for development of a well-defined whole-cell vaccine against whooping cough disease

    NARCIS (Netherlands)

    Streefland, M.; Waterbeemd, van de B.; Happe, R.P.; Pol, van der L.A.; Beuvery, E.C.; Tramper, J.; Martens, D.E.

    2007-01-01

    Since variation in process time and process output is commonly accepted to be inevitable for biological processes, application of Process Analytical Technologies (PAT) on these processes is challenging. In this paper the applicability of PAT on the cultivation of Bordetella pertussis bacteria as par

  3. Improving pertussis vaccines by lipopolysaccharide engineering

    NARCIS (Netherlands)

    Geurtsen, J.J.G.

    2007-01-01

    Pertussis or whooping cough is a highly contagious respiratory tract disease that is caused by the Gram-negative bacterium Bordetella pertussis. Introduction of whole-cell pertussis (wP) vaccines in the 1940s and 1950s, and later of acellular pertussis (aP) vaccines in the 1980s and 1990s, led to a

  4. Respiratory Diseases of Poultry

    Science.gov (United States)

    A new Respiratory Diseases of Poultry CRIS will be established effective October 1, 2006. Initially, the disease agents to be studied will include Ornithobacterium rhinotracheale (ORT), Bordetella avium (BART) and Pasteurella multocida. The research will focus on development of more effective vacc...

  5. Performance of a Handheld PCR Instrument in the Detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis: Sensitivity, Specificity, and Effect of Interferents on Assay Results

    Science.gov (United States)

    2004-12-01

    A Bordetella pertussis 105 0/3 N/A Campylobacter jejuni 105 0/3 N/A Clostridium perfringens 105 1/5 50 Clostridium tetani ...ATCC 9797 105 0/3 N/A Campylobacter jejuni ATCC 33560 105 0/3 N/A Clostridium perfringens 105 0/3 N/A Escherichia coli ATCC

  6. [Pertussis (Whooping cough)--an update].

    Science.gov (United States)

    Stock, Ingo

    2015-12-01

    Whooping cough is a highly contagious respiratory disease which is caused predominantly by the gram-negative bacterium Bordetella pertussis. Further Bordetella species such as B. parapertussis and the recently discovered species B. holmesii are also involved in whooping cough-like diseases. Depending on age, vaccination status and distance to pre-infection with B. pertussis, whooping cough shows a wide range of symptoms. The disease occurs at any age, leaving only short time immunity. During the last 15 years, in industrialized countries the number of reported pertussis cases has been increased markedly. The reason for this observation is still unclear Macrolides such as azithromycin and clarithromycin are regarded as antibiotics of first choice. In Germany, combination vaccines containing acellular pertussis vaccines is the most important strategy of prevention. To ensure the best possible protection against pertussis, booster doses at determined times should be given after primary vaccination in infancy.

  7. Whooping cough in South-East Romania: a 1-year study.

    Science.gov (United States)

    Dinu, Sorin; Guillot, Sophie; Dragomirescu, Cristiana Cerasella; Brun, Delphine; Lazăr, Stefan; Vancea, Geta; Ionescu, Biatrice Mariana; Gherman, Mariana Felicia; Bjerkestrand, Andreea-Florina-Dana; Ungureanu, Vasilica; Guiso, Nicole; Damian, Maria

    2014-03-01

    The incidence of whooping cough in Romania is substantially underestimated, and, as noted by the health authorities, this is mostly due to the lack of both awareness and biological diagnosis. We conducted a 1-year study in Bucharest in order to assess the circulation of Bordetella pertussis, the main etiological agent of whooping cough. Fifty-one subjects suspected of whooping cough were enrolled. Culture, real-time PCR, and enzyme-linked immunosorbent assay were used for laboratory diagnosis. Whooping cough patients (63%) were distributed among all age groups, and most were unvaccinated, incompletely vaccinated, or had been vaccinated more than 5 years previously. Bordetella holmesii DNA was detected in 22% of the bordetellosis cases; these patients included adults; teenagers; and, surprisingly, young children. B. pertussis isolates were similar to the clinical isolates currently circulating elsewhere in Europe. One isolate does not express pertactin, an antigen included in some acellular pertussis vaccines.

  8. The dlt operon of Bacillus cereus is required for resistance to cationic antimicrobial peptides and for virulence in insects.

    Science.gov (United States)

    Abi Khattar, Z; Rejasse, A; Destoumieux-Garzón, D; Escoubas, J M; Sanchis, V; Lereclus, D; Givaudan, A; Kallassy, M; Nielsen-Leroux, C; Gaudriault, S

    2009-11-01

    The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Delta dlt(Bc) mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Delta dlt(Bc) was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Delta dlt(Bc)). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

  9. Pertussis toxins, other antigens become likely targets for genetic engineering

    Energy Technology Data Exchange (ETDEWEB)

    Marwick, C.

    1990-11-14

    Genetically engineered pertussis vaccines have yet to be fully tested clinically. But early human, animal, and in vitro studies indicate effectiveness in reducing toxic effects due to Bordetella pertussis. The licensed pertussis vaccines consists of inactivated whole cells of the organism. Although highly effective, they have been associated with neurologic complications. While the evidence continues to mount that these complications are extremely rare, if they occur at all, it has affected the public's acceptance of pertussis immunization.

  10. Whooping cough in a renal transplant recipient.

    Science.gov (United States)

    Garbiras, M; Shabaka, A; Calvo, N; Martin, L; Moreno, M A; Lopez de la Manzanara, V; Sanchez-Fructuoso, A I

    2016-04-01

    Whooping cough is a respiratory infection with a severity that varies with age, immune status, and probably with other factors such as the degree of exposure and the virulence of the organism. The most frequent microorganism responsible for whooping cough is Bordetella pertussis. We present the case of a 62-year-old renal transplant recipient presenting with typical and severe manifestations of whooping cough caused by B. pertussis.

  11. Limitaciones del ensayo de toxicidad específica para el componente pertussis de células completas

    OpenAIRE

    Vicente Perdomo; Juan Miguel Figueroa; Mario Landys Chovel

    2008-01-01

    Las vacunas que contienen células inactivadas de Bordetella pertussis se han utilizado con efectividad en los Programas Nacionales de Inmunización de todo el mundo. Pese a su reconocida eficacia, ellas se caracterizan por su elevada reactogenicidad, atribuible a la presencia de componentes como toxina pertussis y endotoxinas. Para monitorear la seguridad de estas vacunas existe el ensayo de ganancia en peso en ratones, el cual ha sido criticado por su inespecificidad, poca sensibilidad y alta...

  12. Measuring Immunoglobulin G Antibodies to Tetanus Toxin, Diphtheria Toxin, and Pertussis Toxin with Single-Antigen Enzyme-Linked Immunosorbent Assays and a Bead-Based Multiplex Assay▿

    OpenAIRE

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz

    2008-01-01

    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assa...

  13. Synthetic Nanovaccines Against Respiratory Pathogens (SYNARP)

    Science.gov (United States)

    2011-07-01

    mice (5 mice/group). H5HA specific antibodies in blood sera were detected by enzyme-linked immunosorbent assay ( ELISA ). Blood sera were collected on...formulations induced low but detectable levels of total IgG antibodies as measured by ELISA plates coated with H5HA protein (Fig. 19). The levels of virus...simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus. J Immunol Methods 2008;335:79-89. 8. Stenger RM et al

  14. Bacillus anthracis Edema Toxin Inhibits Staphylococcus aureus Enterotoxin B Effects in Vitro: A Potential Protein Therapeutic?

    Science.gov (United States)

    2005-10-01

    shown that the adverse effects of the SEs and TSST-1 are naturally poten- tiated by a ubiquitous component of all gram-negative bacte- ria, namely...5). Inherent characteristics of edema toxin and other procaryotic adenylate cyclases from Bordetella pertussis, Pseudomonas aeruginosa, and Yersinia...various groups (11, 28), and this effect is linked to gene transcription (9). As evidenced with other cell types (18), the cAMP levels in human

  15. Comparison of molecular detection methods for pertussis in children during a state-wide outbreak.

    Science.gov (United States)

    Qin, X; Zerr, D M; Kronman, M P; Adler, A L; Berry, J E; Rich, S; Buccat, A M; Xu, M; Englund, J A

    2016-04-27

    A state-wide pertussis outbreak occurred in Washington during the winter-spring months of 2012, concurrent with respiratory viral season. We compared performance characteristics of a laboratory-developed pertussis PCR (LD-PCR for Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii) and rapid multiplex PCR (RM-PCR) for respiratory viruses (FilmArray™, BioFire, B. pertussis data unblinded following FDA approval post outbreak). We analyzed three cohorts of patients using physician testing orders as a proxy for clinical suspicion for pertussis or respiratory viruses: Cohort 1, tested by LD-PCR for pertussis pathogens only by nasopharyngeal swab; Cohort 2, by RM-PCR for respiratory viruses only by mid-nasal turbinate swab; and Cohort 3, by both methods. B. pertussis was detected in a total of 25 of the 490 patients in Cohort 3 in which LD-PCR detected 20/25 (80 %) cases and the RM-PCR detected 24/25 (96 %; p = 0.2). Pertussis pathogens were detected in 21/584 (3.6 %) of samples from Cohort 1 where clinicians had a relatively strong suspicion for pertussis. In contrast, B. pertussis was detected in only 4/3071 (0.1 %) specimens from Cohort 2 where suspicion for pertussis was lower (p < 0.001 for comparison with Cohort 1). In summary, the two laboratory methods were comparable for the detection of B. pertussis.

  16. Epidemiology, reemergence of pertussis and vaccine development in Latin America: an overview

    Directory of Open Access Journals (Sweden)

    Celso Pérez-Bolaños

    2011-01-01

    Full Text Available Pertussis o tosferina es una enfermedad bacteriana aguda del tracto respiratorio, causada principalmente por Bordetella pertussis y en menor medida por Bordetella parapertussis. Bordetella pertussis ocupa el quinto lugar en la lista de muertes atribuidas a enfermedades prevenibles por vacunas en niños menores de cinco años en todo el mundo. Se ha reportado que provoca morbilidad y mortalidad significativa, tanto en países desarrollados como en países en desarrollo. La enfermedad es más severa en niños pequeños, pero su prevalencia ha sido observada en todo el mundo en todos los grupos de edad, aún después del desarrollo de vacunas a partir de células completas contra pertussis en los años cuarenta del pasado siglo. Desde la última década ha sido reportada una re-emergencia de pertussis en muchos países desarrollados, incluidos aquellos con una elevada cobertura de vacunación por años. Varios factores pudieran provocar la re-emergencia de pertussis, por ejemplo, una mayor conciencia del problema, un mejor diagnóstico mediante la implementación de técnicas de PCR, disminución de la cobertura de vacunación, la utilización de vacunas de baja protección, baja inmunidad inducida por vacunas y adaptación del patógeno. Este trabajo revisa el estado actual de la epidemiología y la re-emergencia de la enfermedad en América Latina y enfoca la situación actual en Cuba, para dar un panorama de la aplicación de estrategias novedosas en el desarrollo de vacunas futuras, así como medidas generales, recomendadas para el tratamiento de la enfermedad.

  17. Pertussis in adulthood: report of two cases and review of the literature.

    Science.gov (United States)

    Poirrier, Anne-Lise; Gillard-Tromme, Noelle; Lefebvre, Philippe P; El-Shazly, Amr

    2009-09-01

    Whooping cough is resurgent in the developed world. Systematic vaccination has changed its epidemiology, with the majority of cases now primarily affecting adolescents and adults. A 46-year-old male physiotherapist presented with a 1-week history of bothersome cough and respiratory difficulties, and a 51-year-old man was admitted to the emergency department with a 4-week history of increasing cough and dyspnea. Polymerase chain reaction of nasopharyngeal swab were positive for Bordetella pertussis. These cases illustrate pertussis in adulthood. We review the clinical features, the prevalence, the diagnostic tools, and the management of the patients and their relatives to increase awareness of this highly contagious disease.

  18. Dicty_cDB: Contig-U16436-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available :none) Burkholderia vietnamiensis G4 c... 271 8e-71 AM902716_277( AM902716 |pid:none) Bordetella petrii... synthase; ... 244 1e-62 CP000614_1059( CP000614 |pid:none) Burkholderia vietnami...6 0.007 1 ( CO013215 ) EST801550 Coccidioides posadasii spherule cDNA li... 56 0.007 1 ( AC117075 ) Dictyostelium discoid...001157_4095( CP001157 |pid:none) Azotobacter vinelandii DJ, comp... 253 2e-65 AL646053_1106( AL646053 |pid:none) Ralstonia solana...:none) Francisella tularensis subsp. ho... 38 1.5 AL939120_104( AL939120 |pid:none) Streptomyces coelico

  19. Internamento por tosse convulsa – casuística de 10 anos de um hospital de nível III

    Directory of Open Access Journals (Sweden)

    Inês Ferreira

    2017-02-01

    Discussão e Conclusões: À semelhança de outros estudos, verificou-se um pico de incidência no ano de 2012. Os lactentes foram o grupo mais vulnerável para infeção por Bordetella pertussis, com maior número de internamentos. Parecem ser necessárias novas estratégias de prevenção complementares às existentes para reduzir a ocorrência desta infeção neste grupo etário.

  20. Health effects from the inhalation of oxidant air pollutants as related to the immune system. Final report. Research report

    Energy Technology Data Exchange (ETDEWEB)

    Osebold, J.W.; Zee, Y.C.

    1985-07-01

    The investigations dealt with links between ozone inhalation and lung diseases. Mice exposed to ozone both continuously and intermittently in four-day cycles showed allergic sensitization to the test antigen (ovalbumin) when compaired to mice maintained in ambeint air. An adjuvant (inactivated Bordetella pertussis cells) administered to mice augmented the immunological response to the test antigen with periodic exposure to ozone. Guinea pigs were tested for allergic sensitization from ozone exposure similar to human asthma, but this trial was not definitive. Ozone inhalation reduced the severity of pneumonia in mice from influenza virus infection, even when ozone and virus exposures were timed to produce maximal edema in the lung.

  1. Deciphering Parameter Sensitivity in the BvgAS Signal Transduction.

    Directory of Open Access Journals (Sweden)

    Tarunendu Mapder

    Full Text Available To understand the switching of different phenotypic phases of Bordetella pertussis, we propose an optimized mathematical framework for signal transduction through BvgAS two-component system. The response of the network output to the sensory input has been demonstrated in steady state. An analysis in terms of local sensitivity amplification characterizes the nature of the molecular switch. The sensitivity analysis of the model parameters within the framework of various correlation coefficients helps to decipher the contribution of the modular structure in signal propagation. Once classified, the model parameters are tuned to generate the behavior of some novel strains using simulated annealing, a stochastic optimization technique.

  2. Deciphering Parameter Sensitivity in the BvgAS Signal Transduction.

    Science.gov (United States)

    Mapder, Tarunendu; Talukder, Srijeeta; Chattopadhyay, Sudip; Banik, Suman K

    2016-01-01

    To understand the switching of different phenotypic phases of Bordetella pertussis, we propose an optimized mathematical framework for signal transduction through BvgAS two-component system. The response of the network output to the sensory input has been demonstrated in steady state. An analysis in terms of local sensitivity amplification characterizes the nature of the molecular switch. The sensitivity analysis of the model parameters within the framework of various correlation coefficients helps to decipher the contribution of the modular structure in signal propagation. Once classified, the model parameters are tuned to generate the behavior of some novel strains using simulated annealing, a stochastic optimization technique.

  3. The role of B. pertussis vaccine antigen gene variants in pertussis resurgence and possible consequences for vaccine development.

    Science.gov (United States)

    Preston, Andrew

    2016-05-01

    Whooping cough, or pertussis, caused by Bordetella pertussis is considered resurgent in a number of countries world-wide, despite continued high level vaccine coverage. Among a number of causes for this that have been proposed, is the emergence of B. pertussis strains expressing variants of the antigens contained in acellular pertussis vaccines; i.e. the evolution of B. pertussis toward vaccine escape. This commentary highlights the contradictory nature of evidence for this but also discusses the importance of understanding the role of B. pertussis adaptation to vaccine-mediated immune selection pressures for vaccine-mediated pertussis control strategies.

  4. Forekomst af resistente bakterier og forbrug af antibiotika til hunde

    DEFF Research Database (Denmark)

    Pedersen, Karl; Pedersen, Kristina; Jensen, Helene;

    2007-01-01

    ), Pasteurella multocida (n=25), Bordetella bronchiseptica (n=14), Proteus spp. (n=29), og E. coli (n=449). I undersøgelsen anvendtes data fra VetStat databasen. Størstedelen af de antibiotika, der bruges til hunde er bredspektrede. Penicilliner med udvidet spektrum, cephalosporiner samt sulphonamider...... penicillin, 30,2% overfor fucidin og 27,9% overfor macrolider. E. coli isolaterne var mest resistente overfor ampicillin, sulphonamider, trimethoprim, tetracykliner og streptomycin, og anhæmolytiske isolater var oftere resistente overfor tetracykliner, trimethoprim og chloramphenikol end hæmolytiske isolater...

  5. Deciphering Parameter Sensitivity in the BvgAS Signal Transduction

    Science.gov (United States)

    Mapder, Tarunendu; Talukder, Srijeeta; Chattopadhyay, Sudip; Banik, Suman K.

    2016-01-01

    To understand the switching of different phenotypic phases of Bordetella pertussis, we propose an optimized mathematical framework for signal transduction through BvgAS two-component system. The response of the network output to the sensory input has been demonstrated in steady state. An analysis in terms of local sensitivity amplification characterizes the nature of the molecular switch. The sensitivity analysis of the model parameters within the framework of various correlation coefficients helps to decipher the contribution of the modular structure in signal propagation. Once classified, the model parameters are tuned to generate the behavior of some novel strains using simulated annealing, a stochastic optimization technique. PMID:26812153

  6. Avaliação sorológica de vacinações preventivas da difteria, do tétano e da coqueluche, efetuadas em crianças prematuras

    Directory of Open Access Journals (Sweden)

    Vicente Amato Neto

    1977-08-01

    Full Text Available Vinte crianças prematuras receberam, no primeiro ano de vida. vacinas que habitualmente fazem parte do esquema básico de imunizações ativas. Em amostra de soro obtida quando elas atingiram a idade de 12 meses, foram dosados os teores de antitoxina diftérica, de antitoxina tetânica e de aglutininas anti Bordetella pertussis. Valores plenamente satisfatórios de anticorpos relativos à difteria e ao tétano puderam ser encontrados e, quanto à coqueluche, nunca notaram os Autores ausência de aglutininas, mas conclusão mais decisiva não ocorreu, em virtude da falta de melhor conhecimento da cifra indicativa de proteção. O estudd em questão representa subsídio no sentido de arrefecer o temor e o cepticismo, bastante divulgados, acerca da vacinação de prematuros.Twenty premature-born children received, during their first year of life, vaccines routinely apptied as part of a basic immunization schedule. Sera obtained at the age of 12 months were titered for antibodies against diphteria, tetanus and pertussis. Values considered protective were observed for diphteria and tetanus. Anti - Bordetella pertussis agglutinins were always present, however, in the absence of a consensus as to what are protective levels, no conclusion could be drawn. The present study contributes towards erasing the prejudice and scepticism concerning the immunization of the premature-born.

  7. Antimicrobial properties ofTeucrium polium against some clinical pathogens

    Institute of Scientific and Technical Information of China (English)

    Esmaeil Darabpour; Hossein Motamedi; Seyyed Mansour Seyyed Nejad

    2010-01-01

    Objective:To explore the antibacterial effect of the alcoholic extracts of aerial parts ofTeucrium polium, native in Iran on some pathogenic bacteria.Methods: Antibacterial activity of ethanolic extract (50 to 400 mg/mL) and methanolic extract (400 and 600 mg/mL) was evaluated by disc diffusion method.Results: The ethanolic extract results showed thatBacillus anthracis was the most sensitive species, whileEscherichia coli andProteus mirabilis were more resistant than others. In the case of the methanolic extract,Bordetella bronchiseptica was the most sensitive andProteus mirabilis andArcanobacterium pyogenes were the most resistant species. The hydroalcoholic extract ofTeucrium polium had a relatively satisfactory effect onSalmonella typhi. The minimal inhibitory concentration (MIC) ofStaphylococcus aureus andSalmonella typhiwas 40 mg/mL andBordetella bronchiseptica andBacillus anthracis was 10 mg/mL. The minimal bactericidal concentration (MBC) againstBacillus anthracis was 10 mg/mL while against other species were not found (>200 mg/mL). The methanolic extract had also synergistic effect with methicillin, vancomycin againstStaphylococcus aureus and with novobiocin against Salmonella typhi.Conclusions: These results suggest that this plant contains relatively good antibacterial activity and it can be used as a source of antiseptic compounds for medicinal uses.

  8. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Directory of Open Access Journals (Sweden)

    César Martín

    Full Text Available Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  9. BtcA, A class IA type III chaperone, interacts with the BteA N-terminal domain through a globular/non-globular mechanism.

    Directory of Open Access Journals (Sweden)

    Chen Guttman

    Full Text Available Bordetella pertussis, the etiological agent of "whooping cough" disease, utilizes the type III secretion system (T3SS to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.

  10. Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB Affect Protein Function.

    Directory of Open Access Journals (Sweden)

    Scott H Millen

    Full Text Available Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one μg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice.

  11. Shattering a myth - Whooping cough susceptible to antibiotics.

    Science.gov (United States)

    Syed, Muhammad Ali; Jamil, Bushra; Bokhari, Habib

    2016-05-01

    Bordetella parapertussis is the causative agent of a milder form of pertussis or whooping cough. Little is reported about the antibiotic resistance patterns and mechanism of drug resistance of Bordetella parapertussis. The objective of this study has been to investigate antimicrobial resistance, distribution of integrons and presence of gene cassettes to quinolones (qnr) and sulfonamides (sul) among B. parapertussis strains' isolated from Pakistan. Thirty-five (35) samples were collected from various hospitals of Pakistan from children (median age 3 years) with pertussis-like symptoms, all were tested and confirmed to be B. Parapertussis. Resistance profile of Ampicillin, Cephalexin, Sulphamethoxazole, Chloramphenicol, Ofloxacin, Nalidixic acid, Gentamycin and Erythromycin were investigated through all samples. Majority of the isolates were found to be resistant to the afore-mentioned antibiotics except erythromycin. All isolates were resistant to quinolones phenotypically, but qnr genes were detected in only 25.7% (9/35) of isolates. On the other hand, 71.4% (25/35) isolates were resistant to sulfonamides phenotypically. From these 71% strains showing phenotypical resistance, 96% (24/25) were found to possess sul genes. Only two isolates were carrying class 1 integrons, which also harbored sul gene and qnr gene cassettes. It can be safely concluded that the phenotypic resistance patterns seemed mostly independent of presence of integrons. However, interestingly both integrons harboring strains were resistant to quinolones and sulfonamides and also possessed qnr and sul genes.

  12. Pertussis: clinical and bacteriological diagnosis of six cases

    Directory of Open Access Journals (Sweden)

    Arellano Penagos Mario

    2014-07-01

    Full Text Available ertussis is an endemic disease in our population. Every 3 to 4 years, pertussis has an epidemic pattern even in countries with good health conditions. Antipertussis vaccine first dose is adminis- tered at the age of 2 months; a second and third dose are given at 4 and 6 months of age. This vaccine has an 8 to 10 year protective effect, for which reason it is suggested that pregnant women in the third trimester should be vaccinated in order to prevent pertussis in newborns. It should also be administered to older people to avoid turning them into asymptomatic carriers. Clinic manifestations are easily identifiable due to respiratory symptoms, especially to the particular characteristics of the cough. The diagnosis is supported by the presence of leukocytosis (predominantly lymphocytes and by certain thoracic radiologic findings. The diagnosis is confirmed with a positive culture for Bordetella pertussis or with a polymerase chain reaction (PCR. In a non complicated clinic course macrolides are still the best therapeutic choice. Nonetheless clinic observation is highly recom- mended in order to avoid complications. Redefinition of vaccine programs against Bordetella pertussis in Mexican population is recommended and also to notify the presence of the disease to the corresponding health authorities.

  13. Pertussis vaccination and whooping cough: and now what?

    Science.gov (United States)

    Guiso, Nicole

    2014-10-01

    Pertussis or whooping cough is a respiratory disease caused by Bordetella pertussis or Bordetella parapertussis that are only known to infect humans. This severe and acute respiratory disease presents epidemic cycles and became a vaccine-preventable disease in the 1940s/1950s when developed countries introduced vaccination. The first type of vaccine developed against this disease was a whole-cell pertussis (wP) vaccine containing inactivated B. pertussis bacteria. Most developed countries produced their own vaccine and given the pediatric nature of the disease at the time of licensure, infants and toddlers were the primary targets and were thus massively vaccinated. The characterization of few virulence factors produced by B. pertussis enabled the development of second-generation pertussis vaccines called the acellular pertussis (aP) vaccines. These only contain 1-5 purified, detoxified B. pertussis proteins and were first introduced in Japan around 30 years ago. Australia, Europe and North America introduced aP vaccines approximately 15 years later, which replaced wP vaccines since then.

  14. Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough.

    Directory of Open Access Journals (Sweden)

    Nathalie Mielcarek

    2006-07-01

    Full Text Available Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth.

  15. [Diagnosis of whooping cough by serology and real-time PCR].

    Science.gov (United States)

    Mikešová, Romana; Stiborová, Ivana; Richter, Josef; Rajnohová Dobiášová, Lucie; Král, Vlastimil

    2013-09-01

    The goal of this study is to summarize the results of the detection of Bordetella pertussis (BP) and Bordetella parapertussis (BPP) by a real-time polymerase chain reaction (RT-PCR) assay and serological methods. In 2008-2010, 73 patients of the Department of Clinical Immunology and Allergology of the Centre for Immunology and Microbiology, Public Health Institute in Ústí nad Labem were screened for pertussis. They were selected according to the WHO and ECDC criteria, i. e. they presented with a persistent cough lasting more than two weeks. Direct detection of BP and BPP DNA from nasopharyngeal wash specimens was performed using a RT PCR assay. The serological responses were evaluated by a direct agglutination test for the detection of total antibodies and by enzyme-linked immunosobent assay (ELISA) for the detection of IgG, IgA, and IgM antibodies against pertussis toxin. Forty-two patients were positive for BP and/or BPP, 19 of them by RT-PCR (group A) and 23 by serology (group B). Ten group A patients (52.6%) were also positive by serology. Our results show that pertussis needs to be a consideration in persistent cough. We believe that increased awareness of the medical community, along with improved laboratory tests will result in increased detection of pertussis that is still considered by many physicians as a childhood infection.

  16. Genome implosion elicits host-confinement in Alcaligenaceae: evidence from the comparative genomics of Tetrathiobacter kashmirensis, a pathogen in the making.

    Directory of Open Access Journals (Sweden)

    Wriddhiman Ghosh

    Full Text Available This study elucidates the genomic basis of the evolution of pathogens alongside free-living organisms within the family Alcaligenaceae of Betaproteobacteria. Towards that end, the complete genome sequence of the sulfur-chemolithoautotroph Tetrathiobacter kashmirensis WT001(T was determined and compared with the soil isolate Achromobacter xylosoxidans A8 and the two pathogens Bordetella bronchiseptica RB50 and Taylorella equigenitalis MCE9. All analyses comprehensively indicated that the RB50 and MCE9 genomes were almost the subsets of A8 and WT001(T, respectively. In the immediate evolutionary past Achromobacter and Bordetella shared a common ancestor, which was distinct from the other contemporary stock that gave rise to Tetrathiobacter and Taylorella. The Achromobacter-Bordetella precursor, after diverging from the family ancestor, evolved through extensive genome inflation, subsequent to which the two genera separated via differential gene losses and acquisitions. Tetrathiobacter, meanwhile, retained the core characteristics of the family ancestor, and Taylorella underwent massive genome degeneration to reach an evolutionary dead-end. Interestingly, the WT001(T genome, despite its conserved architecture, had only 85% coding density, besides which 578 out of its 4452 protein-coding sequences were found to be pseudogenized. Translational impairment of several DNA repair-recombination genes in the first place seemed to have ushered the rampant and indiscriminate frame-shift mutations across the WT001(T genome. Presumably, this strain has just come out of a recent evolutionary bottleneck, representing a unique transition state where genome self-degeneration has started comprehensively but selective host-confinement has not yet set in. In the light of this evolutionary link, host-adaptation of Taylorella clearly appears to be the aftereffect of genome implosion in another member of the same bottleneck. Remarkably again, potent virulence factors

  17. Is pertussis actually reemerging? Insights from an individual-based model

    Directory of Open Access Journals (Sweden)

    Codeço Cláudia Torres

    2001-01-01

    Full Text Available In this paper, we introduce a spatially explicit, individual-based model developed to simulate the dynamics of pertussis in a small population. With this simulation approach, complex epidemic systems can be built using information on parasite population structure (strain diversity, virulence diversity, etc., human population structure (individual risk, age structure, interaction matrices, immune response, etc., as well as mechanisms of evolution and learning. We parameterized our model to describe pertussis in an age-structured community. Pertussis or whooping cough is an acute infection of the respiratory tract caused by Bordetella pertussis. Despite wide-scale vaccination in many countries, this disease is reemerging throughout the world in both adults and children. Emergence has been explained by many factors: wane of vaccine and natural immunity, increase of asymptomatic carriers, and/or natural selection of non-vaccine strains. Here, we model these hypotheses and analyze their potential impact on the observed increase of pertussis notification.

  18. Dicty_cDB: Contig-U15648-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e--fructose-6-phosphate aminotr... 284 1e-74 CP000615_662( CP000615 |pid:none) Burkholderia vietna... CP001581_3640( CP001581 |pid:none) Clostridium botulinum A2 str. K... 347 2e-93 AL939114_9( AL939114 |pid:none) Streptomyces coeli...eum gene for protein kinase. 44 0.007 2 ( BJ407955 ) Dictyostelium discoideu...2 0.007 5 ( CF569167 ) EST028 Subtracted, Clontech (cat. # K1804-1) Trit... 42 0.007 2 ( AF362373 ) Dictyostelium discoideum histidi...:none) Shewanella frigidimarina NCIMB ... 311 8e-83 AM902716_186( AM902716 |pid:none) Bordetella petrii strain

  19. Dicty_cDB: Contig-U10122-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CP000616_572( CP000616 |pid:none) Burkholderia vietnamiensis G4 ch... 63 7e-11 CP001016_1170( CP001016 |pid:none) Beijerinckia indi...molyticus putative c... 87 1e-33 CP000614_2313( CP000614 |pid:none) Burkholderia vietnamiensis G...:none) Streptomyces coelicolor A3(2) co... 48 7e-04 BX640429_129( BX640429 |pid:none) Bordetella parapertussis strain...a subsp. multocida str. Pm70 ... 58 1e-05 3 ( CP000447 ) Shewanella frigidimarina NCIMB 400, compl...a carbonic anhydrase (... 69 3e-10 AL939126_50( AL939126 |pid:none) Streptomyces coelicolor A3(2) com... 69

  20. Immunological Links to Nonspecific Effects of DTwP and BCG Vaccines on Infant Mortality

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg

    2011-01-01

    A number of mainly observational studies suggest that many African females below the age of one year die each year from the nonspecific effects of vaccination with diphtheria-tetanus toxoids and killed (whole-cell) Bordetella pertussis (DTwP). In contrast, similar studies suggest that many African...... females and males may have their lives saved each year by the nonspecific immunological benefits of Bacillus Calmette-Guerin (BCG) vaccination. From an immunological point of view, we hypothesise that the adverse effects of DTwP vaccine may occur because of the Th2-polarising effect of the aluminium...... phosphate adjuvant in the vaccine and because intramuscular administration of the vaccine may cause chronic inflammation at the site of injection. However, the Th1-polarising effect of BCG is likely to be beneficial. Sexual dimorphism affecting immune functions and vitamin A supplementation may influence...

  1. Broad-Range Detection of Microorganisms Directly from Bronchoalveolar Lavage Specimens by PCR/Electrospray Ionization-Mass Spectrometry

    Science.gov (United States)

    Ullberg, Måns; Lüthje, Petra; Mölling, Paula; Strålin, Kristoffer

    2017-01-01

    The clinical demand on rapid microbiological diagnostic is constantly increasing. PCR coupled to electrospray ionization-mass spectrometry, PCR/ESI-MS, offers detection and identification of over 750 bacteria and Candida species directly from clinical specimens within 6 hours. In this study, we investigated the clinical performance of the IRIDICA BAC LRT Assay for detection of bacterial pathogens in 121 bronchoalveolar lavage (BAL) samples that were received consecutively at our bacterial laboratory for BAL culture. Commensal or pathogenic microorganisms were detected in 118/121 (98%) BAL samples by PCR/ESI-MS, while in 104/121 (86%) samples by routine culture (PLegionella pneumophila, Bordetella pertussis, Norcadia species and Mycoplasma pneumoniae. In conclusion, PCR/ESI-MS detected a broad range of potential pathogens with equal or superior sensitivity compared to conventional methods within few hours directly from BAL samples. This novel method might thus provide a relevant tool for diagnostics in critically ill patients. PMID:28085931

  2. Avaliação sorológica de vacinações preventivas da difteria, do tétano e da coqueluche, efetuadas em crianças prematuras

    Directory of Open Access Journals (Sweden)

    Vicente Amato Neto

    1977-08-01

    Full Text Available Vinte crianças prematuras receberam, no primeiro ano de vida. vacinas que habitualmente fazem parte do esquema básico de imunizações ativas. Em amostra de soro obtida quando elas atingiram a idade de 12 meses, foram dosados os teores de antitoxina diftérica, de antitoxina tetânica e de aglutininas anti Bordetella pertussis. Valores plenamente satisfatórios de anticorpos relativos à difteria e ao tétano puderam ser encontrados e, quanto à coqueluche, nunca notaram os Autores ausência de aglutininas, mas conclusão mais decisiva não ocorreu, em virtude da falta de melhor conhecimento da cifra indicativa de proteção. O estudd em questão representa subsídio no sentido de arrefecer o temor e o cepticismo, bastante divulgados, acerca da vacinação de prematuros.

  3. Is pertussis actually reemerging? Insights from an individual-based model

    Directory of Open Access Journals (Sweden)

    Cláudia Torres Codeço

    2001-06-01

    Full Text Available In this paper, we introduce a spatially explicit, individual-based model developed to simulate the dynamics of pertussis in a small population. With this simulation approach, complex epidemic systems can be built using information on parasite population structure (strain diversity, virulence diversity, etc., human population structure (individual risk, age structure, interaction matrices, immune response, etc., as well as mechanisms of evolution and learning. We parameterized our model to describe pertussis in an age-structured community. Pertussis or whooping cough is an acute infection of the respiratory tract caused by Bordetella pertussis. Despite wide-scale vaccination in many countries, this disease is reemerging throughout the world in both adults and children. Emergence has been explained by many factors: wane of vaccine and natural immunity, increase of asymptomatic carriers, and/or natural selection of non-vaccine strains. Here, we model these hypotheses and analyze their potential impact on the observed increase of pertussis notification.

  4. The pI determination and purification of pertussis toxin%百日咳毒素的等电点测定及层析分离

    Institute of Scientific and Technical Information of China (English)

    刘国安; 杨晓明; 葛瑞昌; 何长民

    2004-01-01

    百日咳毒素是百日咳杆菌(Bordetella pertussis)产生的一种主要毒力因子,也是百日咳菌苗的主要成分之一.用等电聚焦法测得其等电点为pH 6.8.将百日咳杆菌液体培养液在等电点盐析后, 通过DEAE-Sephadex-A50 阴离子交换剂进一步分离纯化了百日咳毒素.ELISA测定其比活力表明,3批样品的提纯倍数分别为1.47,1.37和1.36.

  5. Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Cronin, M.J.; Evans, W.S.; Rogol, A.D.; Weiss, A.A.; Thorner, M.O.; Orth, D.N.; Nicholson, W.E.; Yasumoto, T.; Hewlett, E.L.

    1986-08-01

    Bordetella pertussis synthesis a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changes the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones. The authors conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is the response that explains the subsequent acceleration of hormone release.

  6. Renal effects of renal x irradiation and induced autoallergic glomerulonephritis

    Energy Technology Data Exchange (ETDEWEB)

    Rappaport, D.S.; Casarett, G.W.

    1979-09-01

    This study was conducted to determine what influence a single large x-ray exposure of kidney has on the development and course of an experimental autoallergic glomerulonephritis (EAG) in rats. EAG was induced in female Sprague-Dawley rats by immunization with Bordetella pertussis vaccine and homogenate of homologous kidney tissue and Freund's complete adjuvant. Progressive arteriolonephrosclerosis (ANS) was observed in right (irradiated) kidneys following unilateral renal irradiation (1500 rad). Rats were either immunized, sham-immunized, irradiated, sham-irradiated, or both immunized and irradiated. Light and immunofluorescent microscopic observation, urine protein content, and kidney weights were evaluated. In immunized-irradiated animals the effects of irradiation and immunization were largely additive. Immunization did not considerably influence the development and course of ANS and irradiation did not considerably influence the development and course of EAG.

  7. Update on pertussis and pertussis immunization

    Directory of Open Access Journals (Sweden)

    Jung Yun Hong

    2010-05-01

    Full Text Available Pertussis is a highly contagious respiratory tract disease caused by Bordetella pertussis infection. The clinical manifestation of this infection can be severe enough to cause death. Although pertussis has been supposed to be a vaccine-preventable disease ever since the widespread vaccination of children against pertussis was started, since the 1990s, cases of pertussis and related fatalities are on the rise, especially in countries with high vaccination coverage. In Korea, there have been no deaths due to pertussis since 1990, and the vaccination rate continues to be approximately 94%. However, the number of pertussis cases reported to the Korea Center for Disease Control and Prevention has tended to increase in the 2000s, and in 2009, there was an obvious increase in the number of pertussis cases reported. This review aims to present the latest information about the pathogenesis, diagnosis, treatment, and prevention of pertussis.

  8. Adenylate cyclase toxin-mediated delivery of the S1 subunit of pertussis toxin into mammalian cells.

    Science.gov (United States)

    Iwaki, Masaaki; Konda, Toshifumi

    2016-02-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis internalizes its catalytic domain into target cells. ACT can function as a tool for delivering foreign protein antigen moieties into immune effector cells to induce a cytotoxic T lymphocyte response. In this study, we replaced the catalytic domain of ACT with an enzymatically active protein moiety, the S1 (ADP-ribosyltransferase) subunit of pertussis toxin (PT). The S1 moiety was successfully internalized independent of endocytosis into sheep erythrocytes. The introduced polypeptide exhibited ADP-ribosyltransferase activity in CHO cells and induced clustering typical to PT. The results indicate that ACT can act as a vehicle for not only epitopes but also enzymatically active peptides to mammalian cells.

  9. Monitoring of an antigen manufacturing process.

    Science.gov (United States)

    Zavatti, Vanessa; Budman, Hector; Legge, Raymond; Tamer, Melih

    2016-06-01

    Fluorescence spectroscopy in combination with multivariate statistical methods was employed as a tool for monitoring the manufacturing process of pertactin (PRN), one of the virulence factors of Bordetella pertussis utilized in whopping cough vaccines. Fluorophores such as amino acids and co-enzymes were detected throughout the process. The fluorescence data collected at different stages of the fermentation and purification process were treated employing principal component analysis (PCA). Through PCA, it was feasible to identify sources of variability in PRN production. Then, partial least square (PLS) was employed to correlate the fluorescence spectra obtained from pure PRN samples and the final protein content measured by a Kjeldahl test from these samples. In view that a statistically significant correlation was found between fluorescence and PRN levels, this approach could be further used as a method to predict the final protein content.

  10. Dark fermentative hydrogen production by defined mixed microbial cultures immobilized on ligno-cellulosic waste materials

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Sanjay K.S. [Microbial Biotechnology and Genomics, Institute of Genomics and Integrative Biology (IGIB), CSIR, Delhi University Campus, Mall Road, Delhi 110007 (India); Department of Biotechnology, University of Pune, Pune 411007 (India); Purohit, Hemant J. [Environmental Genomics Unit, National Environmental Engineering Research Institute (NEERI), CSIR, Nehru Marg, Nagpur 440020 (India); Kalia, Vipin C. [Microbial Biotechnology and Genomics, Institute of Genomics and Integrative Biology (IGIB), CSIR, Delhi University Campus, Mall Road, Delhi 110007 (India)

    2010-10-15

    Mixed microbial cultures (MMCs) based on 11 isolates belonging to Bacillus spp. (Firmicutes), Bordetella avium, Enterobacter aerogenes and Proteus mirabilis (Proteobacteria) were employed to produce hydrogen (H{sub 2}) under dark fermentative conditions. Under daily fed culture conditions (hydraulic retention time of 2 days), MMC6 and MMC4, immobilized on ligno-cellulosic wastes - banana leaves and coconut coir evolved 300-330 mL H{sub 2}/day. Here, H{sub 2} constituted 58-62% of the total biogas evolved. It amounted to a H{sub 2} yield of 1.54-1.65 mol/mol glucose utilized over a period of 60 days of fermentation. The involvement of various Bacillus spp. -Bacillus sp., Bacillus cereus, Bacillus megaterium, Bacillus pumilus and Bacillus thuringiensis as components of the defined MMCs for H{sub 2} production has been reported here for the first time. (author)

  11. Genomic analysis of isolates from the United Kingdom 2012 pertussis outbreak reveals that vaccine antigen genes are unusually fast evolving.

    Science.gov (United States)

    Sealey, Katie L; Harris, Simon R; Fry, Norman K; Hurst, Laurence D; Gorringe, Andrew R; Parkhill, Julian; Preston, Andrew

    2015-07-15

    A major outbreak of whooping cough, or pertussis, occurred in 2012 in the United Kingdom (UK), with nearly 10 000 laboratory-confirmed cases and 14 infant deaths attributed to pertussis. A worldwide resurgence of pertussis has been linked to switch to the use of acellular pertussis vaccines and the evolution of Bordetella pertussis away from vaccine-mediated immunity. We have conducted genomic analyses of multiple strains from the UK outbreak. We show that the UK outbreak was polyclonal in nature, caused by multiple distinct but closely related strains. Importantly, we demonstrate that acellular vaccine antigen-encoding genes are evolving at higher rates than other surface protein-encoding genes. This was true even prior to the introduction of pertussis vaccines but has become more pronounced since the introduction of the current acellular vaccines. The fast evolution of vaccine antigen-encoding genes has serious consequences for the ability of current vaccines to continue to control pertussis.

  12. From pertussis to meningococcal disease and back.

    Science.gov (United States)

    Gorringe, Andrew

    2011-04-01

    From pertussis to meningococcal disease and back represents nearly 30 years of research at Porton, first at the Centre for Applied Microbiology and Research and latterly as part of the Health Protection Agency. I joined the group lead by Andy Robinson developing an acellular pertussis vaccine and was part of an exciting period that encompassed basic antigen characterisation and pathogenesis studies with the development of an acellular vaccine containing fimbriae. Research then changed to focus on serogroup B meningococcal disease, studying the vaccine potential of iron-regulated proteins and then Neisseria lactamica. The resurgence of pertussis seen in some countries alerted me to the lack of understanding of protective immune responses to Bordetella pertussis infection and disease and this is now an active area of research.

  13. Is pertussis actually reemerging? Insights from an individual-based model.

    Science.gov (United States)

    Codeço, C T; Luz, P M

    2001-01-01

    In this paper, we introduce a spatially explicit, individual-based model developed to simulate the dynamics of pertussis in a small population. With this simulation approach, complex epidemic systems can be built using information on parasite population structure (strain diversity, virulence diversity, etc.), human population structure (individual risk, age structure, interaction matrices, immune response, etc.), as well as mechanisms of evolution and learning. We parameterized our model to describe pertussis in an age-structured community. Pertussis or whooping cough is an acute infection of the respiratory tract caused by Bordetella pertussis. Despite wide-scale vaccination in many countries, this disease is reemerging throughout the world in both adults and children. Emergence has been explained by many factors: wane of vaccine and natural immunity, increase of asymptomatic carriers, and/or natural selection of non-vaccine strains. Here, we model these hypotheses and analyze their potential impact on the observed increase of pertussis notification.

  14. Public fear of vaccination: separating fact from fiction.

    Science.gov (United States)

    Amanna, Ian; Slifka, Mark K

    2005-01-01

    During the last two centuries, the world has seen a substantial increase in the number and availability of vaccines for the prevention of infectious disease. Smallpox vaccine remains the most celebrated vaccine-related achievement in human history, but worldwide reductions in many other diseases including measles, mumps, rubella, polio, diphtheria, and whooping cough (Bordetella pertussis) also illustrate the power of vaccination in controlling outbreaks of contagious diseases. Ironically, as advances in vaccination successfully limit disease outbreaks, the impact that these infectious agents once had on society becomes marginalized. Public confidence in vaccination may erode because of real or perceived risks associated with immunization, and this in turn may lead to lower vaccination coverage and loss of herd immunity. Here, we will discuss some of the elements associated with public perceptions and fear of vaccination and place these into the context of how deadly several vaccine-preventable childhood diseases can be if vaccination coverage is insufficient.

  15. Immunological Links to Nonspecific Effects of DTwP and BCG Vaccines on Infant Mortality

    Directory of Open Access Journals (Sweden)

    Mogens Helweg Claesson

    2011-01-01

    Full Text Available A number of mainly observational studies suggest that many African females below the age of one year die each year from the nonspecific effects of vaccination with diphtheria-tetanus toxoids and killed (whole-cell Bordetella pertussis (DTwP. In contrast, similar studies suggest that many African females and males may have their lives saved each year by the nonspecific immunological benefits of Bacillus Calmette-Guerin (BCG vaccination. From an immunological point of view, we hypothesise that the adverse effects of DTwP vaccine may occur because of the Th2-polarising effect of the aluminium phosphate adjuvant in the vaccine and because intramuscular administration of the vaccine may cause chronic inflammation at the site of injection. However, the Th1-polarising effect of BCG is likely to be beneficial. Sexual dimorphism affecting immune functions and vitamin A supplementation may influence both the deleterious and beneficial nonspecific effects of immunisation.

  16. Septic arthritis due to Roseomonas gilardii in an immunocompetent adolescent.

    Science.gov (United States)

    Fanella, Sergio; Schantz, Daryl; Karlowsky, James; Rubinstein, Ethan

    2009-11-01

    The genus Roseomonas comprises groups of slow-growing, Gram-negative coccobacilli, which only infrequently cause infection in humans. When identified, they are associated with immunocompromised adults, often causing bacteraemia. Due to their rarity, members of this genus can be overlooked or misidentified using automated laboratory identification systems. We report on an immunocompetent adolescent patient who developed septic arthritis due to Roseomonas gilardii following surgery for a sports injury. The isolate was initially misidentified as Bordetella bronchiseptica using the Vitek 2 system, but confirmed as R. gilardii based on 16S rRNA gene sequencing. To the best of our knowledge, this is the first case of a healthy paediatric patient with septic arthritis due to R. gilardii.

  17. Immunization of teenagers with a fifth dose of reduced DTaP-IPV induces high levels of pertussis antibodies with a significant increase in opsonophagocytic activity.

    Science.gov (United States)

    Aase, Audun; Herstad, Tove Karin; Merino, Samuel; Bolstad, Merete; Sandbu, Synne; Bakke, Hilde; Aaberge, Ingeborg S

    2011-08-01

    Waning vaccine-induced immunity against Bordetella pertussis is observed among adolescents and adults. A high incidence of pertussis has been reported in this population, which serves as a reservoir for B. pertussis. A fifth dose of reduced antigen of diphtheria-tetanus-acellular-pertussis and inactivated polio vaccine was given as a booster dose to healthy teenagers. The antibody activity against B. pertussis antigens was measured prior to and 4 to 8 weeks after the booster by different assays: enzyme-linked immunosorbent assays (ELISAs) of IgG and IgA against pertussis toxin (PT) and filamentous hemagglutinin (FHA), IgG against pertactin (PRN), opsonophagocytic activity (OPA), and IgG binding to live B. pertussis. There was a significant increase in the IgG activity against PT, FHA, and PRN following the booster immunization (P antibodies against FHA and PRN contribute the most to the OPA and IgG binding.

  18. Filamentous-haemagglutinin-like protein genes encoded on a plasmid of Moraxella bovis.

    Science.gov (United States)

    Kakuda, Tsutomu; Sarataphan, Nopporn; Tanaka, Tetsuya; Takai, Shinji

    2006-11-26

    The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.

  19. Motor activity as an unbiased variable to assess anaphylaxis in allergic rats

    Science.gov (United States)

    Abril-Gil, Mar; Garcia-Just, Alba; Cambras, Trinitat; Pérez-Cano, Francisco J; Castellote, Cristina; Franch, Àngels

    2015-01-01

    The release of mediators by mast cells triggers allergic symptoms involving various physiological systems and, in the most severe cases, the development of anaphylactic shock compromising mainly the nervous and cardiovascular systems. We aimed to establish variables to objectively study the anaphylactic response (AR) after an oral challenge in an allergy model. Brown Norway rats were immunized by intraperitoneal injection of ovalbumin with alum and toxin from Bordetella pertussis. Specific immunoglobulin (Ig) E antibodies were developed in immunized animals. Forty days after immunization, the rats were orally challenged with the allergen, and motor activity, body temperature and serum mast cell protease concentration were determined. The anaphylaxis induced a reduction in body temperature and a decrease in the number of animal movements, which was inversely correlated with serum mast cell protease release. In summary, motor activity is a reliable tool for assessing AR and also an unbiased method for screening new anti-allergic drugs. PMID:25716015

  20. 幼兔细菌性感染症的检验与分析%EXAMINATION AND ANALYSIS ON THE BACTERIA INFECTION OF YOUNG RABBIT

    Institute of Scientific and Technical Information of China (English)

    陈翠珍; 刘铮铸; 姚国安

    2003-01-01

    从自然发生的2月龄幼兔肺脏中分离到3种类型的细菌(记作:a,b,c),经对所做的纯培养30株分离菌的形态特征、理化特性等检验,结果表明a菌(17株)为支气管炎博德特氏菌(Bordetella bronchiseptica);b菌(8株)为铜绿假单胞菌(Pseudomonas aeruginosa);c菌(6株)为大肠埃希氏菌(Escherichia coli).通过人工感染试验,表明其为相应的致病菌.

  1. Dicty_cDB: Contig-U16232-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ent WO2007060... 65 3e-09 (Q9D1Q6) RecName: Full=Endoplasmic reticulum resident protein ER... 64 4e-09 (Q6IU...l=Endoplasmic reticulum resident protein ER... 67 7e-10 CT573072_936( CT573072 |pid:none) Kuenenia stuttgart... |pid:none) Bordetella pertussis strain Toha... 66 1e-09 (Q3T0L2) RecName: Full=Endoplasmic reticulum resi...dent protein ER... 66 1e-09 AL954747_1035( AL954747 |pid:none) Nitrosomonas europae...2 |pid:none) Macaca fascicularis brain cDNA clo... 67 7e-10 (Q9BS26) RecName: Ful

  2. Whooping cough: identification, assessment and management.

    Science.gov (United States)

    Bentley, Jackie; Pinfield, Jenny; Rouse, Jo

    Although there is a high uptake of vaccinations providing protection against Bordetella pertussis, the main cause of whooping cough, there has been an increase in the incidence of notifications of the disease in the UK and other developed countries in recent years. The increase in cases of whooping cough is mainly evident in older children and adults. While these individuals may experience persistent and unpleasant symptoms, most notably prolonged cough, symptoms may be mild, in part, because most older children and adults have been vaccinated against the disease. The most significant public health concern relating to whooping cough is that infected older children and adults may transmit the disease to unvaccinated infants who are most vulnerable to the symptoms. This article aims to develop the reader's understanding of whooping cough, including its prevention and management.

  3. What to do about pertussis vaccines? Linking what we know about pertussis vaccine effectiveness, immunology and disease transmission to create a better vaccine.

    Science.gov (United States)

    Bolotin, Shelly; Harvill, Eric T; Crowcroft, Natasha S

    2015-11-01

    Pertussis (whooping cough) is a respiratory disease caused by the bacterium Bordetella pertussis. Despite the implementation of immunization programs and high vaccine coverage in most jurisdictions, pertussis is still one of the most common vaccine-preventable diseases, suggesting that the current vaccines and immunization schedules have not been sufficiently effective. Several factors are thought to contribute to this. The acellular pertussis vaccine that has been used in many jurisdictions since the 1990s is less effective than the previously used whole-cell vaccine, with immunity waning over time. Both whole-cell and acellular pertussis vaccines are effective at reducing disease severity but not transmission, resulting in outbreaks in vaccinated cohorts. In this review, we discuss various limitations of the current approaches to protection from pertussis and outline various options for reducing the burden of pertussis on a population level.

  4. Contribution of pertussis toxin to the pathogenesis of pertussis disease.

    Science.gov (United States)

    Carbonetti, Nicholas H

    2015-11-01

    Pertussis toxin (PT) is a multisubunit protein toxin secreted by Bordetella pertussis, the bacterial agent of the disease pertussis or whooping cough. PT in detoxified form is a component of all licensed acellular pertussis vaccines, since it is considered to be an important virulence factor for this pathogen. PT inhibits G protein-coupled receptor signaling through Gi proteins in mammalian cells, an activity that has led to its widespread use as a cell biology tool. But how does this activity of PT contribute to pertussis, including the severe respiratory symptoms of this disease? In this minireview, the contribution of PT to the pathogenesis of pertussis disease will be considered based on evidence from both human infections and animal model studies. Although definitive proof of the role of PT in humans is lacking, substantial evidence supports the idea that PT is a major contributor to pertussis pathology, including the severe respiratory symptoms associated with this disease.

  5. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

    Science.gov (United States)

    Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A

    2015-12-01

    Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

  6. Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

    Directory of Open Access Journals (Sweden)

    Elian Dupré

    2015-03-01

    Full Text Available Two-component systems (TCS represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

  7. 犬/传/染/性/气/管/支/气/管/炎

    Institute of Scientific and Technical Information of China (English)

    鲁文

    2001-01-01

    @@ 犬传染性气管支气管炎(Infectious tracheobronchitis, ITB)又名犬窝咳.早在19世纪初期,文献上即有本病与犬瘟热一起发病的病例报道,是犬的一种重要的、世界范围的呼吸道传染病,常在犬集中饲养的群体中传染流行. 1 病原 在ITB病犬呼吸道中,支气管败血波氏杆菌(Bordetella Bronchiseptica)是最常被分离到的革兰氏阴性、需氧球杆菌,被认为是本病的主要病原菌.与人百日咳的病原菌(百日咳波氏杆菌,Bordetella Pertussis)同为一属,但免疫性不同.在人类身上虽然也能分离到支气管败血波氏杆菌,但它不是人兽共患病的病原菌,仅对那些免疫力差的人产生危害,对犬则能单独致病,不需要先有病毒的呼吸道感染.但是,一个病毒的激发因素常常在本病的早期发生作用,并也可能成为本病的病原,如犬副流感病毒(CPIV)、犬腺病毒Ⅱ型(CAV-Ⅱ)、犬疱疹病毒和呼肠孤病毒.

  8. Molecular identification of bacteria by total sequence screening: determining the cause of death in ancient human subjects.

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    Catherine Thèves

    Full Text Available Research of ancient pathogens in ancient human skeletons has been mainly carried out on the basis of one essential historical or archaeological observation, permitting specific pathogens to be targeted. Detection of ancient human pathogens without such evidence is more difficult, since the quantity and quality of ancient DNA, as well as the environmental bacteria potentially present in the sample, limit the analyses possible. Using human lung tissue and/or teeth samples from burials in eastern Siberia, dating from the end of 17(th to the 19(th century, we propose a methodology that includes the: 1 amplification of all 16S rDNA gene sequences present in each sample; 2 identification of all bacterial DNA sequences with a degree of identity ≥ 95%, according to quality criteria; 3 identification and confirmation of bacterial pathogens by the amplification of the rpoB gene; and 4 establishment of authenticity criteria for ancient DNA. This study demonstrates that from teeth samples originating from ancient human subjects, we can realise: 1 the correct identification of bacterial molecular sequence signatures by quality criteria; 2 the separation of environmental and pathogenic bacterial 16S rDNA sequences; 3 the distribution of bacterial species for each subject and for each burial; and 4 the characterisation of bacteria specific to the permafrost. Moreover, we identified three pathogens in different teeth samples by 16S rDNA sequence amplification: Bordetella sp., Streptococcus pneumoniae and Shigella dysenteriae. We tested for the presence of these pathogens by amplifying the rpoB gene. For the first time, we confirmed sequences from Bordetella pertussis in the lungs of an ancient male Siberian subject, whose grave dated from the end of the 17(th century to the early 18(th century.

  9. Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Holden, Hazel M. (UW)

    2010-09-08

    The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 {angstrom} resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the L{beta}H superfamily of N-acyltransferases. Each subunit contains 27 {beta}-strands, 23 of which form the canonical left-handed {beta}-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O{sup {delta}1} of Asn 84 and the sugar C-3{prime} amino group and the second between the backbone amide group of Arg 94 and the sugar C-5{prime} carboxylate. The sugar C-3{prime} amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.

  10. Safety evaluation of a vaccine: Effect in maternal reproductive outcome and fetal anomaly frequency in rats using a leishmanial vaccine as a model

    Science.gov (United States)

    Soares, Thaigra S.; Silva, Ana Luiza T.; Giunchetti, Rodolfo C.; Takano, Maria A. S.; Akamatsu, Milena A.; Kubrusly, Flávia S.; Lúcio-Macarini, Fernanda; Raw, Isaias; Iourtov, Dmitri; Ho, Paulo Lee; Bueno, Lilian L.; Fujiwara, Ricardo T.; Volpato, Gustavo T.

    2017-01-01

    While the immunogenic potential of the vaccination against infectious diseases was extensively shown, data on the safety assessment of recombinant proteins in vaccine formulations administered during pregnancy are still scarce. In the current study, the antigenicity of a vaccine against leishmaniasis (based on Leishmania braziliensis recombinant protein peroxidoxin) during pregnancy and possible maternal reproductive outcomes and fetal anomalies after immunization with a leishmanial vaccine or adjuvant alone (Bordetella pertussis derived MPLA adjuvant) were assessed. Rats were mated and allocated in three groups: Control—rats received saline; Adjuvant—rats received the adjuvant MPLA, and Vaccine—rats received the combination of MPLA and peroxidoxin. The administration was subcutaneously at the dorsal region, three times (days 0, 7, 14 of pregnancy). On day 21 of pregnancy, all rats were bled for biochemical and immunological measurements. The gravid uterus was weighed with its contents, and the fetuses were analyzed. The immunization with peroxidoxin induced a significant production of circulating IgG levels compared to other groups but caused a significant in post-implantation loss (14.7%) when compared to Control (5.0%) and Adjuvant (4.4%) groups. Furthermore, a significantly high rate of fetal visceral anomalies, such as hydronephrosis and convoluted ureter, was also observed in animals that received vaccine when compared to Control or Adjuvant groups. These data indicate the importance of safety evaluation of vaccines during pregnancy and the limited use of peroxidoxin administration during pregnancy. More importantly, the safety monitoring of immunization with MPLA derived from Bordetella pertussis demonstrated no reproductive outcomes associated with adjuvant administration, suggesting its safe use during pregnancy. PMID:28249007

  11. Measuring immunoglobulin g antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay.

    Science.gov (United States)

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz

    2008-05-01

    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an "in-house" IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.

  12. Multi-functional characteristics of the Pseudomonas aeruginosa type III needle-tip protein, PcrV; comparison to orthologs in other gram negative bacteria

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    Hiromi eSato

    2011-07-01

    Full Text Available Pseudomonas aeruginosa possesses a type III secretion system (T3SS to intoxicate host cells and evade innate immunity. This virulence-related machinery consists of a molecular syringe and needle assembled on the bacterial surface, which allows delivery of T3 effector proteins into infected cells. To accomplish a one-step effector translocation, a tip protein is required at the top end of the T3 needle structure. Strains lacking expression of the functional tip protein fail to intoxicate host cells.P. aeruginosa encodes a T3S that is highly homologous to the proteins encoded by Yersinia species. The needle tip proteins of Yersinia, LcrV, and P. aeruginosa, PcrV, share 37% identity and 65% similarity. Other known tip proteins are AcrV (Aeromonas, IpaD (Shigella, SipD (Salmonella, BipD (Burkholderia, EspA (EPEC, EHEC, Bsp22 (Bordetella, with additional proteins identified from various Gram negative species, such as Vibrio and Bordetella. The tip proteins can serve as a protective antigen or may be critical for sensing host cells and evading innate immune responses. Recognition of the host microenvironment transcriptionally activates synthesis of T3SS components. The machinery appears to be mechanically controlled by the assemblage of specific junctions within the apparatus. These junctions include the tip and base of the T3 apparatus, the needle proteins and components within the bacterial cytoplasm. The tip proteins likely have chaperone functions for translocon proteins, allowing the proper assembly of translocation channels in the host membrane and completing vectorial delivery of effector proteins into the host cytoplasm. Multifunctional features of the needle-tip proteins appear to be intricately controlled. In this review, we highlight the functional aspects and complex controls of T3 needle-tip proteins with particular emphasis on PcrV and LcrV.

  13. Is pertussis actually reemerging? Insights from an individual-based model A coqueluche realmente está reermegindo? Reflexões a partir de um modelo baseado no indivíduo

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    Cláudia Torres Codeço

    2001-06-01

    Full Text Available In this paper, we introduce a spatially explicit, individual-based model developed to simulate the dynamics of pertussis in a small population. With this simulation approach, complex epidemic systems can be built using information on parasite population structure (strain diversity, virulence diversity, etc., human population structure (individual risk, age structure, interaction matrices, immune response, etc., as well as mechanisms of evolution and learning. We parameterized our model to describe pertussis in an age-structured community. Pertussis or whooping cough is an acute infection of the respiratory tract caused by Bordetella pertussis. Despite wide-scale vaccination in many countries, this disease is reemerging throughout the world in both adults and children. Emergence has been explained by many factors: wane of vaccine and natural immunity, increase of asymptomatic carriers, and/or natural selection of non-vaccine strains. Here, we model these hypotheses and analyze their potential impact on the observed increase of pertussis notification.Neste trabalho, nós apresentamos um modelo de indivíduos, cuja representação espacial é explícita, para simular a dinâmica da coqueluche numa pequena população. Utilizando esta abordagem de simulação, podemos construir modelos complexos utilizando informações sobre a estrutura populacional dos parasitas (diversidade fenotípica, de virulência, etc sobre a estrutura populacional humana (risco individual, estrutura etária, matrizes de interação, resposta imunológica, etc assim como processos evolutivos e de aprendizagem. Nós parametrizamos este modelo para representar a dinâmica da coqueluche numa população com estrutura etária. Coqueluche é uma infecção aguda do trato respiratório, causada por Bordetella pertussis. Apesar da vacinação em larga escala em vários países, esta infecção está reemergindo por todo o mundo, atacando adultos e crianças. Reemergência tem sido

  14. Serum IgA responses against pertussis proteins in infected and Dutch wP or aP vaccinated children: an additional role in pertussis diagnostics.

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    Lotte H Hendrikx

    Full Text Available BACKGROUND: Whooping cough is a respiratory disease caused by Bordetella pertussis, which induces mucosal IgA antibodies that appear to be relevant in protection. Serum IgA responses are measured after pertussis infection and might provide an additional role in pertussis diagnostics. However, the possible interfering role for pertussis vaccinations in the induction of serum IgA antibodies is largely unknown. METHODS/PRINCIPAL FINDINGS: We compared serum IgA responses in healthy vaccinated children between 1 and 10 years of age with those in children who despite vaccinations recently were infected with Bordetella pertussis. All children have been vaccinated at 2, 3, 4 and 11 months of age with either the Dutch whole-cell pertussis (wP vaccine or an acellular pertussis (aP vaccine and additionally received an aP booster vaccination at 4 years of age. Serum IgA responses to pertussis toxin (PT, filamentous heamagglutinin (FHA and pertactin (Prn were measured with a fluorescent multiplex bead-based immuno-assay. An ELISPOT-assay was used for the detection of IgA-memory B-cells specific to these antigens. Serum IgA levels to all pertussis vaccine antigens were significantly higher in infected children compared with healthy children. High correlations between anti-PT, anti-FHA or anti-Prn IgA and IgG levels were found in infected children and to some degree in wP primed children, but not at all in aP primed children. Highest numbers of IgA-pertussis-specific memory B-cells were observed after infection and generally comparable numbers were found after wP and aP vaccination. CONCLUSIONS: This study provides new insight in the diagnostic role for serum IgA responses against PT in vaccinated children. Since aP vaccines induce high serum IgG levels that interfere with pertussis diagnostics, serum IgA-PT levels will provide an additional diagnostic role. High levels of serum IgA for PT proved specific for recent pertussis infection with reasonable

  15. 鸡胚胎性病原茵多重PCR检测方法的建立%Establishment of multiplex PCR detection for pathogens of chicken embryos

    Institute of Scientific and Technical Information of China (English)

    谭燕玲; 朱瑞良; 王慧; 王新建; 魏凯; 孙振红; 盛鹏程

    2011-01-01

    To establish a multiplex PCR assay for simultaneous detecation of Bordetella avium, Escherichia coli, Salmonella and Pseudomonas aeruginosa in infected embryos, four pairs of specific primers were designed and synthesized based on the genes of ompA in B. ayium,invA in Salmonella, phoA in E. coli and toxR in P. aeruginosa for detection of these pathogenic bacteria by multiplex PCR. Test results indicated that the PCR products were 597 bp for B. ayium, 724 bp for Salmonella, 372 bp for E. coli,and 278 bp for P. aeruginosa, and the limit detections were approximate l04 cfu/mL for B. avium, E. coli and P. aeruginosa and 103 cfu/mL for Salmonella, respectively, without any amplification from other related bacteria. The detection results from clinical embrvos samples showed that the multiplex PCR were consistent with that of bacteria culture isolation. The multiplex PCR assay could be used for rapid detection of pathogenic bactera.%为建立同时检测禽波氏杆菌(Bordetella avium)、沙门氏茵(Salmonella)、大肠杆菌(Escherichia coli)和绿脓杆菌(Pseudomonas aeruginosa)4种导致鸡胚死亡病原菌的多重PCR方法,本研究根据B.aviun的ompA基因、Salmonella的invA基因、E.coli的phoA基因和P.aeruginosa的toxR基因序列,各设计一对特异性引物进行多重PCR反应,并对反应体系和条件进行优化.结果显示,4对引物分别扩增出597bp、724bp、372bp和278bp的目的条带;并且特异性强不与其他非目的茵发生反应.经优化B.aviun、P.aeruginosa和E.coli多重PCR检测灵敏度达到104cfu/mL,而Salmonella为103cfu/mL.本研究建立的多重PCR方法为相关病原茵的快速检测提供方法.

  16. Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol Resposta imune em camundongos imunizados com a fração ácida antigênica do Citosol do Trypanosoma cruzi

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    Susana Gea

    1992-10-01

    Full Text Available The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG aginst T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytothropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.As respostas humorais e celulares, assim como a resistência à infecção a formas sanguícolas do T. cruzi foram estudadas em camundongos imunizados com as frações antigênicas ácidas do Citosol parasitário, F III e F IV com Bordetella pertussis como adjuvante. A imunização com F III induziu respostas positivas ITH e DTH aos antígenos homólogos. Em camundongos imunizados com F IV o ITH foi negativo e quatro dos seis animais apresentaram reações DTH positivas. Em ambos os grupos de camundongos a análise de IgG contra T. cruzi mostrou que o principal isotipo produzido foi IgG1. IgE específico foi também detectado em soros de camundongos imunizados com F III, portanto confirmando a presença de anticorpos homocitotrópicos. As parasitemias atingidas pelos camundongos imunizados por F III e F IV após desafio foram mais baixas do que aquelas dos controles mostrando proteção parcial contra a infec

  17. Use of immunomodulators in infectious diseases of domestic animals/ Uso de imunomoduladores nas enfermidades infecciosas dos animais domésticos

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    Jane Megid

    2007-08-01

    Full Text Available Immunomodulators are substances that act in the immune system providing, increase of the organic answer against microorganisms, including virus, bacteria and protozoa, by inducing the production of interferon and its inducers. There are a lot of situations in veterinary medicine where it is usefull to potencialize the immune response of individuals, mainly when is desired to increase the resistance to infections and the treatment of immunossupressing or multifactorials infectious diseases. In veterinary medicine some of more used immunomodulators are interferons and interferon inducers, interleukines, Baccilus of Calmett-Guérin (BCG and its derivated, Propionibacterium acnes (Corynebacterium parvum, mixed bacterial vaccine, PIND-ORF, Phosprenyl, Quillja Saponis, Bordetella pertussis, avridine and the levamizole. The present work review the available scientific literature, regarding the use of different immunomodulators in the prophylaxis and in the therapeutics of infectious diseases in domestic animals.Imunomoduladores são substâncias que atuam no sistema imunológico conferindo aumento da resposta orgânica contra determinados microorganismos, incluindo vírus, bactérias e protozoários, mediante à produção de interferon e seus indutores. Existem muitas situações na medicina veterinária em que se torna desejável potencializar a resposta imune, principalmente quando se pretende aumentar a resistência às infecções e no tratamento de enfermidades imunossupressoras ou de doenças infecciosas multifatorias, ou seja, nas quais vários agentes estão envolvidos e devido a isso, dificilmente obtêm-se sucesso no emprego de tratamentos convencionais. Na medicina veterinária alguns imunomoduladores utilizados são interferons , interleucinas, Bacilo de Calmett-Guérin (BCG e seus derivados, Propionibacterium acnes (Corynebacterium parvum, vacina bacteriana mista, PIND-ORF, Phosprenyl, Quillaja saponis, Bordetella pertussis, avridina e

  18. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics.

    Science.gov (United States)

    Linnes, J C; Rodriguez, N M; Liu, L; Klapperich, C M

    2016-04-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications.

  19. cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects.

    Science.gov (United States)

    Beckert, Ulrike; Grundmann, Manuel; Wolter, Sabine; Schwede, Frank; Rehmann, Holger; Kaever, Volkhard; Kostenis, Evi; Seifert, Roland

    2014-09-05

    In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.

  20. Temperature-accelerated molecular dynamics gives insights into globular conformations sampled in the free state of the AC catalytic domain.

    Science.gov (United States)

    Selwa, Edithe; Huynh, Tru; Ciccotti, Giovanni; Maragliano, Luca; Malliavin, Thérèse E

    2014-10-01

    The catalytic domain of the adenyl cyclase (AC) toxin from Bordetella pertussis is activated by interaction with calmodulin (CaM), resulting in cAMP overproduction in the infected cell. In the X-ray crystallographic structure of the complex between AC and the C terminal lobe of CaM, the toxin displays a markedly elongated shape. As for the structure of the isolated protein, experimental results support the hypothesis that more globular conformations are sampled, but information at atomic resolution is still lacking. Here, we use temperature-accelerated molecular dynamics (TAMD) simulations to generate putative all-atom models of globular conformations sampled by CaM-free AC. As collective variables, we use centers of mass coordinates of groups of residues selected from the analysis of standard molecular dynamics (MD) simulations. Results show that TAMD allows extended conformational sampling and generates AC conformations that are more globular than in the complexed state. These structures are then refined via energy minimization and further unrestrained MD simulations to optimize inter-domain packing interactions, thus resulting in the identification of a set of hydrogen bonds present in the globular conformations.

  1. Impact of vaccination and birth rate on the epidemiology of pertussis: a comparative study in 64 countries.

    Science.gov (United States)

    Broutin, H; Viboud, C; Grenfell, B T; Miller, M A; Rohani, P

    2010-11-01

    Bordetella pertussis infection remains an important public health problem worldwide despite decades of routine vaccination. A key indicator of the impact of vaccination programmes is the inter-epidemic period, which is expected to increase with vaccine uptake if there is significant herd immunity. Based on empirical data from 64 countries across the five continents over the past 30-70 years, we document the observed relationship between the average inter-epidemic period, birth rate and vaccine coverage. We then use a mathematical model to explore the range of scenarios for duration of immunity and transmission resulting from repeat infections that are consistent with empirical evidence. Estimates of pertussis periodicity ranged between 2 and 4.6 years, with a strong association with susceptible recruitment rate, defined as birth rate × (1 - vaccine coverage). Periodicity increased by 1.27 years on average after the introduction of national vaccination programmes (95% CI: 1.13, 1.41 years), indicative of increased herd immunity. Mathematical models suggest that the observed patterns of pertussis periodicity are equally consistent with loss of immunity that is not as rapid as currently thought, or with negligible transmission generated by repeat infections. We conclude that both vaccine coverage and birth rate drive pertussis periodicity globally and that vaccination induces strong herd immunity effects. A better understanding of the role of repeat infections in pertussis transmission is critical to refine existing control strategies.

  2. Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and iso-octylthioglycolic acid by aerobic gram-negative bacteria.

    Science.gov (United States)

    Toups, Mario; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2010-04-01

    Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater's toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.

  3. Disorder-to-Order Transition in the CyaA Toxin RTX Domain: Implications for Toxin Secretion

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    Ana-Cristina Sotomayor-Pérez

    2014-12-01

    Full Text Available The past decade has seen a fundamental reappraisal of the protein structure-to-function paradigm because it became evident that a significant fraction of polypeptides are lacking ordered structures under physiological conditions. Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. This trait is exhibited by RTX (Repeat in ToXin motifs found in more than 250 virulence factors secreted by Gram-negative pathogenic bacteria. We have investigated several RTX-containing polypeptides of different lengths, all derived from the Bordetella pertussis adenylate cyclase toxin, CyaA. Using a combination of experimental approaches, we showed that the RTX proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium. This intrinsic disorder mainly results from internal electrostatic repulsions between negatively charged residues of the RTX motifs. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the RTX proteins. We propose that the intrinsically disordered character of the RTX proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. These results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing RTX motifs, could be finely regulated by the calcium gradient across bacterial cell wall.

  4. Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

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    Michael J Lodes

    Full Text Available Bacterial and viral upper respiratory infections (URI produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

  5. Limitaciones del ensayo de toxicidad específica para el componente pertussis de células completas

    Directory of Open Access Journals (Sweden)

    Vicente Perdomo

    2008-01-01

    Full Text Available Las vacunas que contienen células inactivadas de Bordetella pertussis se han utilizado con efectividad en los Programas Nacionales de Inmunización de todo el mundo. Pese a su reconocida eficacia, ellas se caracterizan por su elevada reactogenicidad, atribuible a la presencia de componentes como toxina pertussis y endotoxinas. Paramonitorear la seguridad de estas vacunas existe el ensayo de ganancia en peso en ratones, el cual ha sido criticado por su inespecificidad, poca sensibilidad y alta variabilidad. Basado en lo anterior, el Laboratorio Nacional de Biológicos del Centro Estatal para el Control de de la Calidad de los Medicamentos en Cuba, decidió evaluar la relevancia de esta prueba para la liberación de los lotes de la vacuna DPT. Para ello se estimó la sensibilidad del método para detectar diferentes concentraciones de endotoxinas y toxina pertussis, así como la variabilidad entre ensayos. Los resultados de este trabajo mostraron que sólo altas concentraciones de endotoxinas y toxina pertussis, muy superiores a las habituales en las vacunas DPT, provocan una disminución de la ganancia en peso promedio y un fallo en la especificación de la prueba. Este elemento y la inherente variabilidad de este método resultaron claves en la decisión de no utilizarlo para la liberación nacional de lotes de la vacuna DPT.

  6. Limitaciones del ensayo de toxicidad específica para el componente pertussis de células completas

    Directory of Open Access Journals (Sweden)

    Mario Landys Chovel

    2008-12-01

    Full Text Available Las vacunas que contienen células inactivadas de Bordetella pertussis se han utilizado con efectividad en los Programas Nacionales de Inmunización de todo el mundo. Pese a su reconocida eficacia, ellas se caracterizan por su elevada reactogenicidad, atribuible a la presencia de componentes como toxina pertussis y endotoxinas. Para monitorear la seguridad de estas vacunas existe el ensayo de ganancia en peso en ratones, el cual ha sido criticado por su inespecificidad, poca sensibilidad y alta variabilidad. Basado en lo anterior, el Laboratorio Nacional de Biológicos del Centro Estatal para el Control de de la Calidad de los Medicamentos en Cuba, decidió evaluar la relevancia de esta prueba para la liberación de los lotes de la vacuna DPT. Para ello se estimó la sensibilidad del método para detectar diferentes concentraciones de endotoxinas y toxina pertussis, así como la variabilidad entre ensayos. Los resultados de este trabajo mostraron que sólo altas concentraciones de endotoxinas y toxina pertussis, muy superiores a las habituales en las vacunas DPT, provocan una disminución de la ganancia en peso promedio y un fallo en la especificación de la prueba. Este elemento y la inherente variabilidad de este método resultaron claves en la decisión de no utilizarlo para la liberación nacional de lotes de la vacuna DPT.

  7. Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis

    Science.gov (United States)

    Kerkhof, L.; Santoro, M.; Garland, J.

    2000-01-01

    In this report, we describe an experiment conducted at Kennedy Space Center in the biomass production chamber (BPC) using soybean plants for purification and processing of human hygiene water. Specifically, we tested whether it was possible to detect changes in the root-associated bacterial assemblage of the plants and ultimately to identify the specific microorganism(s) which differed when plants were exposed to hygiene water and other hydroponic media. Plants were grown in hydroponics media corresponding to four different treatments: control (Hoagland's solution), artificial gray water (Hoagland's+surfactant), filtered gray water collected from human subjects on site, and unfiltered gray water. Differences in rhizosphere microbial populations in all experimental treatments were observed when compared to the control treatment using both community level physiological profiles (BIOLOG) and molecular fingerprinting of 16S rRNA genes by terminal restriction fragment length polymorphism analysis (TRFLP). Furthermore, screening of a clonal library of 16S rRNA genes by TRFLP yielded nearly full length SSU genes associated with the various treatments. Most 16S rRNA genes were affiliated with the Klebsiella, Pseudomonas, Variovorax, Burkholderia, Bordetella and Isosphaera groups. This molecular approach demonstrated the ability to rapidly detect and identify microorganisms unique to experimental treatments and provides a means to fingerprint microbial communities in the biosystems being developed at NASA for optimizing advanced life support operations.

  8. Influence of Chicken Manure Fertilization on Antibiotic-Resistant Bacteria in Soil and the Endophytic Bacteria of Pakchoi.

    Science.gov (United States)

    Yang, Qingxiang; Zhang, Hao; Guo, Yuhui; Tian, Tiantian

    2016-01-01

    Animal manure is commonly used as fertilizer for agricultural crops worldwide, even though it is believed to contribute to the spread of antibiotic resistance from animal intestines to the soil environment. However, it is unclear whether and how there is any impact of manure fertilization on populations and community structure of antibiotic-resistant endophytic bacteria (AREB) in plant tissues. To investigate the effect of manure and organic fertilizer on endophytic bacterial communities, pot experiments were performed with pakchoi grown with the following treatments: (1) non-treated; (2) chicken manure-treated and (3) organic fertilizer-treated. Manure or organic fertilizer significantly increased the abundances of total cultivable endophytic bacteria (TCEB) and AREB in pakchoi, and the effect of chicken manure was greater than that of organic fertilizer. Further, 16S rDNA sequencing and the phylogenetic analysis indicated that chicken manure or organic fertilizer application increased the populations of multiple antibiotic-resistant bacteria (MARB) in soil and multiple antibiotic-resistant endophytic bacteria (MAREB) in pakchoi. The identical multiple antibiotic-resistant bacterial populations detected in chicken manure, manure- or organic fertilizer-amended soil and the vegetable endophytic system were Brevundimonas diminuta, Brachybacterium sp. and Bordetella sp., suggesting that MARB from manure could enter and colonize the vegetable tissues through manure fertilization. The fact that some human pathogens with multiple antibiotic resistance were detected in harvested vegetables after growing in manure-amended soil demonstrated a potential threat to human health.

  9. DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas

    Science.gov (United States)

    Tahir, Shaista; Badshah, Amin; Hussain, Raja Azadar; Tahir, Muhammad Nawaz; Tabassum, Saira; Patujo, Jahangir Ali; Rauf, Muhammad Khawar

    2015-11-01

    Four new nitrosubstituted acylthioureas i.e. 1-acetyl-3-(4-nitrophenyl)thiourea (TU1), 1-acetyl-3-(2-methyl-4-nitrophenyl)thiourea (TU2), 1-acetyl-3-(2-methoxy-4-nitrophenyl)thiourea (TU3) and 1-acetyl-3-(4-chloro-3-nitrophenyl)thiourea (TU4) have been synthesized and characterized (by C13 and H1 nuclear magnetic resonance, Fourier transform infrared spectroscopy and single crystal X-ray diffraction). As a preliminary investigation of the anti-cancer potencies of the said compounds, DNA interaction studies have been carried out using cyclic voltammetry and UV-vis spectroscopy along with verification from computational studies. The drug-DNA binding constants are found to be in the order, KTU3 9.04 × 106 M-1 > KTU4 8.57 × 106 M-1 > KTU2 6.05 × 106 M-1 > KTU1 1.16 × 106 M-1. Furthermore, the antioxidant, cytotoxic, antibacterial and antifungal activities have been carried out against DPPH (1,1-diphenyl-2-dipicrylhydrazyl), Brine shrimp eggs, gram positive (Micrococcus luteus, Staphylococcus aureus) and gram negative (Bordetella bronchiseptica, Salmonella typhimurium, Enterobacter aerogens) and fungal cultures (Aspergillus fumigatus, Mucor species, Aspergillus niger, Aspergillus flavus) respectively.

  10. Staphylococcus epidermidis Biofilms: Functional Molecules, Relation to Virulence, and Vaccine Potential

    Science.gov (United States)

    Mack, Dietrich; Davies, Angharad P.; Harris, Llinos G.; Knobloch, Johannes K. M.; Rohde, Holger

    Medical device-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. The formation of adherent, multilayered bacterial biofilms is crucial in the pathogenesis of these infections. Polysaccharide intercellular adhesin (PIA), a homoglycan of β-1,6-linked 2-acetamido-2-deoxy-d-glucopyranosyl residues, of which about 15% are non-N-acetylated, is central to biofilm accumulation in staphylococci. It transpires that polysaccharides - structurally very similar to PIA - are also key to biofilm formation in a number of other organisms including the important human pathogens Escherichia coli, Aggregatibacter (Actinobacillus) actinomycetemcomitans, Yersinia pestis, and Bordetella spp. Apparently, synthesis of PIA and related polysaccharides is a general feature important for biofilm formation in diverse bacterial genera. Current knowledge about the structure and biosynthesis of PIA and related polysaccharides is reviewed. Additionally, information on their role in pathogenesis of biomaterial-related and other type of infections and the potential use of PIA and related compounds for prevention of infection is evaluated.

  11. Polyphasic analysis of a middle ages coprolite microbiota, Belgium.

    Directory of Open Access Journals (Sweden)

    Sandra Appelt

    Full Text Available Paleomicrobiological investigations of a 14(th-century coprolite found inside a barrel in Namur, Belgium were done using microscopy, a culture-dependent approach and metagenomics. Results were confirmed by ad hoc PCR--sequencing. Investigations yielded evidence for flora from ancient environment preserved inside the coprolite, indicated by microscopic observation of amoebal cysts, plant fibers, seeds, pollens and mold remains. Seventeen different bacterial species were cultured from the coprolite, mixing organisms known to originate from the environment and organisms known to be gut inhabitants. Metagenomic analyses yielded 107,470 reads, of which known sequences (31.9% comprised 98.98% bacterial, 0.52% eukaryotic, 0.44% archaeal and 0.06% viral assigned reads. Most abundant bacterial phyla were Proteobacteria, Gemmatimonadetes, Actinobacteria and Bacteroidetes. The 16 S rRNA gene dataset yielded 132,000 trimmed reads and 673 Operational Taxonomic Units. Most abundant bacterial phyla observed in the 16 S rRNA gene dataset belonged to Proteobacteria, Firmicutes, Actinobacteria and Chlamydia. The Namur coprolite yielded typical gut microbiota inhabitants, intestinal parasites Trichuris and Ascaris and systemic pathogens Bartonella and Bordetella. This study adds knowledge to gut microbiota in medieval times.

  12. Pathogenic and opportunistic gram-negative bacteria in soil, leachate and air in San Nicolás landfill at Aguascalientes, Mexico.

    Science.gov (United States)

    Flores-Tena, Francisco J; Guerrero-Barrera, Alma L; Avelar-González, Francisco J; Ramírez-López, E Marcela; Martínez-Saldaña, M Consolación

    2007-01-01

    The occurrence of Gram-negative pathogenic and opportunistic species, was studied for two years on air. soil and leachate from the San Nicolás Landfill. Aguascalientes, Mexico. For soil and leachate four samplings were done, two during the dry season and two during the rainy season. For soil there were 15 sampling points, the leachate samples were taken on the leachate tank. For air, twelve sampling were done in three points of the landfill. Twenty pathogenic and/or opportunistic bacteria were identified from air, twenty from soil and eleven from leachate. Most of them were enteric; however respiratory tract pathogenic bacteria were also identified. Pasteurella haemolytica were isolated in all air samples. Nine species were found in the a half of the soil samples. The most frequent species in leachate were Acinetobacter baumanii, Bordetella sp, Brucella sp. and Escherichia coli var II. The occurrence of pathogenic and opportunistic species points out to the nosocomial and domestic clinical wastes discharged in the landfill as a potential risk for public and occupational health.

  13. Biodegradation of phenanthrene and pyrene in compost-amended soil.

    Science.gov (United States)

    Yuan, Shaw Y; Su, Lai M; Chang, Bea V

    2009-06-01

    This study investigated the biodegradation of the polycyclic aromatic hydrocarbons (PAHs) phenanthrene and pyrene in compost and compost-amended soil. The degradation rates of the two PAHs were phenanthrene>pyrene. The degradation of PAH was enhanced when the two PAHs were present simultaneously in the soil. The addition of either of the two types of compost (straw and animal manure) individually enhanced PAH degradation. Compost samples were separated into fractions with various particle size ranges, which spanned 2-50 microm, 50-105 microm, 105-500 microm, and 500-2000 microm. We observed that the compost fractions with smaller particle sizes demonstrated higher PAH degradation rates. However, when the different compost fractions were added to soil, compost particle size had no significant effect on the rate of PAH degradation. Of the micro-organisms isolated from the soil-compost mixtures, strains S1, S2, and S8, which were identified as Arthrobacter nicotianae, Pseudomonas fluorescens, and Bordetella Petrii, respectively, demonstrated the best degradation ability.

  14. Discovery of Novel Secreted Virulence Factors from Salmonella enterica Serovar Typhimurium by Proteomic Analysis of Culture Supernatants

    Energy Technology Data Exchange (ETDEWEB)

    Niemann, George; Brown, Roslyn N.; Gustin, Jean K.; Stufkens, Afke; Shaikh-Kidwai, Afshan S.; Li, Jie; McDermott, Jason E.; Brewer, Heather M.; Schepmoes, Athena A.; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2011-01-01

    The intracellular pathogen Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis in the world. This pathogen has two type-III secretion systems (TTSS) necessary for virulence that are encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) and are expressed during extracellular or intracellular infectious states, respectively, to deliver virulence factors (effectors) to the host cell cytoplasm. While many have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this mass spectrometry-based proteomics study, we identified effector proteins secreted under minimal acidic medium growth conditions that induced the SPI-2 TTSS and its effectors, and compared the secretome from the parent strain to the secretome from strains missing either essential (SsaK) or regulatory components (SsaL) of the SPI-2 secretion apparatus. We identified 75% of the known TTSS effector repertoire. Excluding translocon components, 95% of the known effectors were biased for identification in the ssaL mutant background, which demonstrated that SsaL regulates SPI-2 type III secretion. To confirm secretion to animal cells, we made translational fusions of several of the best candidates to the calmodulin-dependent adenylate cyclase of Bordetella pertussis and assayed cAMP levels of infected J774 macrophage-like cells. From these infected cells we identified six new TTSS effectors and two others that are secreted independent of TTSS. Our results substantiate reports of additional secretion systems encoded by Salmonella other than TTSS.

  15. Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD

    Energy Technology Data Exchange (ETDEWEB)

    Barbieri, J.T.; Mende-Mueller, L.M.; Rappuoli, R.; Collier, R.J. (Medical College of Wisconsin, Milwaukee (USA))

    1989-11-01

    UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from (3H-nicotinamide)NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from (32P-adenylate)NAD (0.2 mol/mol of protein). Label from (3H-nicotinamide)NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with (3H-nicotinamide)NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observed with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.

  16. DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates

    Science.gov (United States)

    Braselmann, Esther; Chaney, Julie L.; Champion, Matthew M.

    2016-01-01

    The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress. PMID:27626276

  17. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

  18. Establishment of and Effects of BPV on Experimental Allergic Encephalomyelitis in Lewis Rats.%Lewis大鼠实验性变态反应性脑脊髓炎动物模型的建立及百日咳疫苗的影响

    Institute of Scientific and Technical Information of China (English)

    朱振国; 郑荣远; 李剑敏; 韩钊

    2008-01-01

    目的 建立可靠的Lewis大鼠实验性过敏性脑脊髓炎(experimental allergic encephalomyelitis, EAE)动物模型;方法 采用Lewis大鼠脚垫皮下注射豚鼠全脊髓匀浆(spinal cords homogenate of guinea pigs, GPSCH)和含有卡介苗(bacille calmette guerin, BCG) 10mg/ml的完全福氏佐剂(complete Freund's adjuvant, CFA)诱导EAE,从临床症状评分、光镜、电镜下病理学改变对模型进行评价.结果 Lewis大鼠EAE的发病率为9/10,潜伏期为12.6±1.01天.百日咳疫苗(bordetella pertussis vaccine, BPV)可提高EAE的发病率,缩短EAE的潜伏期(P<0.05),加重其临床症状和病理改变(P<0.05).结论 GPSCH和CFA免疫Lewis大鼠建立的EAE是成功可靠的;BPV对EAE有显著的促发作用,可诱导形成超急性EAE.

  19. 无细胞百日咳疫苗特异性毒性检测方法现状及展望%Current status and prospect of specific methods for determination of toxicity of an acellular pertussis vaccine

    Institute of Scientific and Technical Information of China (English)

    夏德菊; 郭林达

    2014-01-01

    百日咳毒素是百日咳杆菌重要的保护性抗原,经化学方法脱毒成为百日咳类毒素后制成的无细胞百日咳疫苗(acellular pertussis vaccine,APV)具有残余毒性和毒性逆转的可能性,所以建立特异性毒性检测方法对于保证APV的安全性是十分必要的.此文就国内外APV特异性毒性检测方法的现状做一综述,并结合近几年国外研究的毒性检测新方法进行展望.%Pertussis toxin (PT) is a major protective antigen of Bordetella pertussis and its detoxified form obtained by a chemical process is an important component of an acellular pertussis vaccine (APV).In view of the possibility of residual toxicity or toxicity reversion of PT in the vaccines,specific tests are required to assure safety of the vaccines.This paper reviews specific methods for determination of toxicity of APV and prospect of new tests in the world.

  20. Old Disease and New Challenges: Major Obstacles of Current Strategies in the Prevention of Pertussis

    Science.gov (United States)

    Sedighi, Iraj; Karimi, Abdollah; Amanati, Ali

    2016-01-01

    Context Universal immunization against Bordetella pertussis has partially controlled the burden of the disease and its transmission. However, according to recent data, the epidemiology of this vaccine-preventable disease has changed. Now, younger infants, adolescents, and adults are at greater risk of infection. This article has studied the interaction between the various factors involved in the changing epidemiology of pertussis and the major obstacles faced by the current strategies in its prevention. Evidence Acquisition In this narrative review, the most recently published sources of information on pertussis control measures, consisting of textbooks and articles, have been reviewed. We focused on the more recent data about the changing epidemiology or pertussis in Scopus through the use of the MeSH-term words [pertussis] or [whooping cough] and [epidemiology] or [outbreak] or [resurgence], but our search was not restricted to this particular strategy; we also tried to find all of the most recent available data in the general field through other means. Results Primary and booster doses of the pertussis vaccine seem to partially control transmission of the disease, but despite the different preventive strategies available, pertussis continues to cause mortality and morbidity among high-risk groups. Conclusions Adding booster doses of acellular pertussis vaccine to the current national immunization practices with whole-cell vaccines for young adults and pregnant women seems to be a good option for controlling mortality and morbidity among high-risk groups such as very young infants. PMID:27729960

  1. Enterovirus D68 Infection in Children with Acute Flaccid Myelitis, Colorado, USA, 2014

    Science.gov (United States)

    Messacar, Kevin; Pastula, Daniel M.; Robinson, Christine C.; Leshem, Eyal; Sejvar, James J.; Nix, W. Allan; Oberste, M. Steven; Feikin, Daniel R.; Dominguez, Samuel R.

    2016-01-01

    During August 8, 2014–October 14, 2014, a total of 11 children with acute flaccid myelitis and distinctive neuroimaging changes were identified near Denver, Colorado, USA. A respiratory prodrome was experienced by 10, and nasopharyngeal specimens were positive for enterovirus D68 (EV-D68) for 4. To determine whether an association exists between EV-D68 infection and acute flaccid myelitis, we conducted a retrospective case–control study comparing these patients with 2 groups of outpatient control children (1 group tested for acute respiratory illness and 1 for Bordetella pertussis infection). Adjusted analyses indicated that, for children with acute flaccid myelitis, the odds of having EV-D68 infection were 10.3 times greater than for those tested for acute respiratory infection and 4.5 times greater than for those tested for B. pertussis infection. No statistical association was seen between acute flaccid myelitis and non–EV-D68 enterovirus or rhinovirus infection. These findings support an association between EV-D68 infection and acute flaccid myelitis. PMID:27434186

  2. Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

    Directory of Open Access Journals (Sweden)

    Kerstin Y Beste

    Full Text Available Guanylyl cyclases (GCs regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs and a nitric oxide-activated soluble GC (sGC. Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF from Bacillus anthracis possess nucleotidyl cyclase (NC activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

  3. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

    Science.gov (United States)

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The ‘AC to Hly-linking segment’ thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

  4. Loss of multi-epitope specificity in memory CD4(+ T cell responses to B. pertussis with age.

    Directory of Open Access Journals (Sweden)

    Wanda G H Han

    Full Text Available Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4(+ T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4(+ T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4(+ T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.

  5. DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates.

    Science.gov (United States)

    Braselmann, Esther; Chaney, Julie L; Champion, Matthew M; Clark, Patricia L

    2016-01-01

    The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress.

  6. Intracellular disassembly and activity of pertussis toxin require interaction with ATP.

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    Plaut, Roger D; Scanlon, Karen M; Taylor, Michael; Teter, Ken; Carbonetti, Nicholas H

    2016-08-01

    The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.

  7. Comparison of point-of-care-compatible lysis methods for bacteria and viruses.

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    Heiniger, Erin K; Buser, Joshua R; Mireles, Lillian; Zhang, Xiaohong; Ladd, Paula D; Lutz, Barry R; Yager, Paul

    2016-09-01

    Nucleic acid sample preparation has been an especially challenging barrier to point-of-care nucleic acid amplification tests in low-resource settings. Here we provide a head-to-head comparison of methods for lysis of, and nucleic acid release from, several pathogenic bacteria and viruses-methods that are adaptable to point-of-care usage in low-resource settings. Digestion with achromopeptidase, a mixture of proteases and peptidoglycan-specific hydrolases, followed by thermal deactivation in a boiling water bath, effectively released amplifiable nucleic acid from Staphylococcus aureus, Bordetella pertussis, respiratory syncytial virus, and influenza virus. Achromopeptidase was functional after dehydration and reconstitution, even after eleven months of dry storage without refrigeration. Mechanical lysis methods proved to be effective against a hard-to-lyse Mycobacterium species, and a miniature bead-mill, the AudioLyse, is shown to be capable of releasing amplifiable DNA and RNA from this species. We conclude that point-of-care-compatible sample preparation methods for nucleic acid tests need not introduce amplification inhibitors, and can provide amplification-ready lysates from a wide range of bacterial and viral pathogens.

  8. Antibacterial activity of selected Egyptian ethnomedicinal plants

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    Mashait, M.

    2013-01-01

    Full Text Available Aims: Medicinal plants have recently received the attention of the antimicrobial activity of plants and their metabolites due to the challenge of growing incidences of drug-resistant pathogens. The aims of this study were to determine the antibacterial activities of plant extracts used as ethnomedicinal in Egypt. Methodology and Results: Investigations were carried out to assess the antibacterial efficiency of 11 plant extracts used as ethnopharmacological among Egyptian native people against infectious diseases. Crude methanol, ethanol,chloroform, hexane, acetone and aqueous extract of plants were tested for antibacterial activity in vitro against ten bacterial isolates using the disc diffusion method test. Discs were impregnated with 2 mg/mL of different solvent extracts. Among all the crude extracts, the methanol extract showed the highest activity than other extracts. P. harmala and S. officinalis exhibited highest antibacterial activity against gram positive and negative bacteria while the remainingplants extracts showed less activity. All the plant extracts showed no significant effect against the Bordetella bronchisepta ATCC 4617 except the extracts of M. fragrans and L. sativum. E. coli is the most sensitive microorganism tested, with the lowest MIC value (0.5 mg/mL in the presence of the plant extract of P. harmala and S. officinalis.Conclusion, significance and impact of study: Results obtained herein, may suggest that the ethnomedicinal Egyptian plants possess antimicrobial activity and therefore, they can be used in biotechnological fields as natural preservative ingredients in food and/or pharmaceutical industry.

  9. BACTERIAL FLORA OF HATCHERY ENVIRONMENT AND THEIR IN-VITRO SUSCEPTIBILITY TO ANTIMICROBIAL AGENTS

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    F. M. Khan, H. Afzal and F. Deeba

    2003-04-01

    Full Text Available Four hatcheries, located in and around Faisalabad, were sampled a day before hatch out in six batches for environmental bacterial flora. Hatchery air, egg-shell surface, surfaces of selected locations and water supply samples were taken for this purpose. The percent (relative occurrence of various bacterial species recovered from hatchery environment revealed that Bacillus subtilis was the predominant isolate (26.93%. followed by Escherichia coli (24.08%, Staphylococcus epidermidis (16.32%, Staphylococcus aureus (8.16%, Paratyphoid salmonellae (6.93%, Pseudomonas aeruginosa (4.48%, Citrobacter jreundii (4.08%, Enterococcus faecalis (3.26%, Klebsiella pneumoniae (3.26%, Bordetella avium (1.63% and Proteus vulgaris (0.81%. In second part of the study, bacterial isolates were subjected to in-vitro antibiotic sensitivity to 8 antibiotics of common poultry use. It was found that 98.92, 79.56. 65.59, 61.29, 61.29, 61.29, 53.76 and 38.70 percent of bacterial isolates were sensitive to Norfloxacin, Gentamicin, Neomycin, Chloramphenicol, Doxycycline, Flumequine, Erythromycin, and Ampicillin, respectively. In the final part of the study, bacterial isolates were tested for resistance to 3 commerical hatchery disinfectants (TH4®, Aldekol Des® 0.2, and Bromosept 10% soln. ®. Only 3.22% of the isolates showed resistance at manufacturer's recommended dilution (MRD levels while 11.82% of the isolates showed resistance at concentrations below the MRD levels.

  10. Synthesis and biochemical analysis of 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-N-hydroxy-octanediamides as inhibitors of human histone deacetylases.

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    Henkes, Leonhard M; Haus, Patricia; Jäger, Felix; Ludwig, Joachim; Meyer-Almes, Franz-Josef

    2012-01-15

    Inhibition of human histone deacetylases (HDACs) has emerged as a novel concept in the chemotherapeutic treatment of cancer. Two chemical entities, SAHA (ZOLINZA, Merck) and romidepsin (Istodax, Celgene) have been recently approved by the FDA as first-in-class drugs against cutaneous T-cell lymphoma. Clinical use of these drugs revealed several side effects including gastro-intestinal symptoms, fatigue, thrombocytopenia, thrombosis. Romidepsin is associated with an yet unresolved cardiotoxicity issue. A general hypothesis for the diminishment of unwanted adverse effects and an improved therapeutical window suggests the development of more isotype selective inhibitors. In this study the first time HDAC inhibitors with perfluorinated spacers between the zinc chelating moiety and the aromatic capping group were synthesized and tested against representatives of HDAC classes I, IIa and IIb. Competitive binding assays and a combined approach by using blind docking and molecular dynamics support binding of the perfluorinated analogs of SAHA to the active site of the HDAC-like amidohydrolase from Bordetella/Alcaligenes and presumably also to human HDACs. In contrast to the alkyl spacer of SAHA and derivatives, the perfluorinated alkyl spacer seems to contribute to or facilitate the induction of selectivity for class II, particularly class IIa, HDACs even though the overall potency of the perfluorinated SAHA analogs in this study against human HDACs remained still rather moderate in the micromolar range.

  11. Search for antibacterial and antifungal agents from selected Indian medicinal plants.

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    Kumar, V Prashanth; Chauhan, Neelam S; Padh, Harish; Rajani, M

    2006-09-19

    A series of 61 Indian medicinal plants belonging to 33 different families used in various infectious disorders, were screened for their antimicrobial properties. Screening was carried out at 1000 and 500 microg/ml concentrations by agar dilution method against Bacillus cereus var mycoides, Bacillus pumilus, Bacillus subtilis, Bordetella bronchiseptica, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus faecalis, Candida albicans, Aspergillus niger and Saccharomyces cerevisiae. Twenty-eight plant extracts showed activity against at least one of the test organisms used in the screening. On the basis of the results obtained, we conclude that the crude extracts of Dorema ammoniacum, Sphaeranthus indicus, Dracaena cinnabari, Mallotus philippinensis, Jatropha gossypifolia, Aristolochia indica, Lantana camara, Nardostachys jatamansi, Randia dumetorum and Cassia fistula exhibited significant antimicrobial activity and properties that support folkloric use in the treatment of some diseases as broad-spectrum antimicrobial agents. This probably explains the use of these plants by the indigenous people against a number of infections.

  12. Swine infectious agents in Tayassu pecari and Pecari tajacu tissue samples from Brazil.

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    de Castro, Alessandra Marnie Martins Gomes; Brombila, Talita; Bersano, Josete Garcia; Soares, Herbert Sousa; Silva, Sheila Oliveira de Souza; Minervino, Antonio Humberto Hamad; Ogata, Renato Akio; Gennari, Solange Maria; Richtzenhain, Leonardo Jose

    2014-04-01

    Peccaries and pigs, Tayassuidae and Suidae respectively, diverged approximately one million years ago from a common ancestor. Because these families share some pathogens, peccaries can act as reservoirs of infectious pathogens for domestic and wild swine. We evaluated the presence of swine infectious agents in the spleen and lung tissues of white-lipped peccaries (WLP; Tayassu pecari) and collared peccaries (CP; Pecari tajacu) in Brazil. Samples from 10 adult CP and three WLP, which had been hunted by locals or hit by motor vehicles, were obtained from two free-ranging Brazilian populations. The samples were tested by PCR for Mycoplasma hyopneumoniae, Bordetella bronchiseptica, Pasteurella multocida, porcine circovirus 2 (PCV2), Suid herpesvirus 1 (SuHV-1), and porcine parvovirus (PPV). Positive samples were sequenced. Both species were negative for PPV and B. bronchiseptica and positive for PCV2 and SuHV-1. The lungs of two animals were positive for M. hyopneumoniae and P. multocida. This report is the first demonstration of PCV2 and SuHV-1 swine viruses and of M. hyopneumoniae and P. multocida bacteria in peccaries. One factor contributing to this detection was access to tissue samples, which is uncommon. The role of these infectious agents in peccaries is unknown and further epidemiologic studies should be performed. This study identified several infectious agents in peccaries and highlighted the importance of the tissue type used to detect pathogens.

  13. Pharmacophore Selection and Redesign of Non-nucleotide Inhibitors of Anthrax Edema Factor

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    Maria Estrella Jimenez

    2012-11-01

    Full Text Available Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF, an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin’s basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.

  14. Isolation and characterization of facultative mixotrophic ammonia-oxidizing bacteria from constructed wetlands

    Institute of Scientific and Technical Information of China (English)

    Soulwène Kouki; Neila Saidi; Fadhel M'hiri; Houda Nasr; Hanène Cherif; Hadda Ouzari; Abdermaceur Hassen

    2011-01-01

    Autotrophic ammonia-oxidizing bacteria (AOB) have been widely studied in constructed wetlands systems,while mixotrophic AOB have been less thoroughly examined.Heterotrophic bacteria were isolated from wastewater and rhizospheres of macrophytes of constructed wetlands,and then cultivated in a mixotrophic medium containing ammonium and acetic acid.A molecular characterization was accomplished using ITS-PCR amplification,and phylogenetic analysis based on 16S rRNA gene sequences.Results showed the presence of 35 bacteria,among 400 initially heterotrophic isolates,that were able to remove ammonia.These 35 isolates were classified into 10 genetically different groups based on ITS pattern.Then,a collection of 10 isolates were selected because of their relatively high ammonia removal efficiencies (ARE ≥ 80%) and their phylogenetic diversity.In conditions of mixotrophy,these strains were shown to be able to grow (increase of optical density OD660 during incubation with assimilation of nitrogen into cellular biomass) and to oxidize ammonia (important ammonia oxidation efficiencies,AOE between 79% and 87%).Among these facultative mixotrophic AOB,four isolates were genetically related to Firmicutes (Bacillus and Exiguobacterium),three isolates were affiliated to Actinobacteria (Arthrobacter) and three other isolates were associated with Proteobacteria (Pseudomonas,Ochrobactrum and Bordetella).

  15. Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

    Science.gov (United States)

    Soria-Guerra, Ruth Elena; Rosales-Mendoza, Sergio; Márquez-Mercado, Crisóforo; López-Revilla, Rubén; Castillo-Collazo, Rosalba; Alpuche-Solís, Angel Gabriel

    2007-07-01

    A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

  16. Antibiotic resistance and extended-spectrum β-lactamases in isolated bacteria from seawater of Algiers beaches (Algeria).

    Science.gov (United States)

    Alouache, Souhila; Kada, Mohamed; Messai, Yamina; Estepa, Vanesa; Torres, Carmen; Bakour, Rabah

    2012-01-01

    The aim of the study was to evaluate bacterial antibiotic resistance in seawater from four beaches in Algiers. The most significant resistance rates were observed for amoxicillin and ticarcillin, whereas they were relatively low for ceftazidime, cefotaxime and imipenem. According to sampling sites, the highest resistance rates were recorded for 2 sites subjected to chemical and microbiological inputs (amoxicillin, 43% and 52%; ticarcillin, 19.6% and 47.7%), and for 2 sites relatively preserved from anthropogenic influence, resistance rates were lowest (amoxicillin, 1.5% and 16%; ticarcillin, 0.8% and 2.6%). Thirty-four bacteria resistant to imipenem (n=14) or cefotaxime (n=20) were identified as Pseudomonas aeruginosa (n=15), Pseudomonas fluorescens (7), Stenotrophomonas maltophilia (4), Burkholderia cepacia (2), Bordetella sp. (1), Pantoea sp. (1), Acinetobacter baumannii (1), Chryseomonas luteola (1), Ochrobactrum anthropi (1) and Escherichia coli (1). Screening for extended spectrum β-lactamase showed the presence of CTX-M-15 β-lactamase in the E. coli isolate, and the encoding gene was transferable in association with the IncI1 plasmid of about 50 kbp. Insertion sequence ISEcp1B was located upstream of the CTX-M-15 gene. This work showed a significant level of resistance to antibiotics, mainly among environmental saprophytic bacteria. Transmissible CTX-M-15 was detected in E. coli; this may mean that contamination of the environment by resistant bacteria may cause the spread of resistance genes.

  17. Healthcare providers as sources of vaccine-preventable diseases.

    Science.gov (United States)

    Sydnor, Emily; Perl, Trish M

    2014-08-27

    Vaccine-preventable infectious diseases may be introduced into the healthcare setting and pose a serious risk to vulnerable populations including immunocompromised patients. Healthcare providers (HCPs) are exposed to these pathogens through their daily tasks and may serve as a reservoir for ongoing disease transmission in the healthcare setting. The primary method of protection from work-related infection risk is vaccination that protects not only an individual HCP from disease, but also subsequent patients in contact with that HCP. Individual HCPs and healthcare institutions must balance the ethical and professional responsibility to protect their patients from nosocomial transmission of preventable infections with HCP autonomy. This article reviews known cases of HCP-to-patient transmission of the most common vaccine-preventable infections encountered in the healthcare setting including hepatitis B virus, influenza virus, Bordetella pertussis, varicella-zoster virus, measles, mumps and rubella virus. The impact of HCP vaccination on patient care and current recommendations for HCP vaccination against vaccine-preventable infectious diseases are also reviewed.

  18. Usage of Latent Class Analysis in Diagnostic Microbiology in the Absence of Gold Standard Test

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    Gul Bayram Abiha

    2016-12-01

    Full Text Available The evaluation of performance of various tests diagnostic tests in the absence of gold standard is an important problem. Latent class analysis (LCA is a statistical analysis method known for many years, especially in the absence of a gold standard for evaluation of diagnostic tests so that LCA has found its wide application area. During the last decade, LCA method has widely used in for determining sensivity and specifity of different microbiological tests. It has investigated in the diagnosis of mycobacterium tuberculosis, mycobacterium bovis, human papilloma virus, bordetella pertussis, influenza viruses, hepatitis E virus (HEV, hepatitis C virus (HCV and other various viral infections. Researchers have compared several diagnostic tests for the diagnosis of different pathogens with LCA. We aimed to evaluate performance of latent class analysis method used microbiological diagnosis in various diseases in several researches. When we took into account all of these tests' results, we suppose that LCA is a good statistical analysis method to assess different test performances in the absence of gold standard. [Archives Medical Review Journal 2016; 25(4.000: 467-488

  19. Pharmacodynamics and pharmacokinetics of flumequine in pigs after single intravenous and intramuscular administration.

    Science.gov (United States)

    Villa, R; Cagnardi, P; Acocella, F; Massi, P; Anfossi, P; Asta, F; Carli, S

    2005-07-01

    The pharmacokinetics and intramuscular (IM) bioavailability of flumequine (15 mgkg(-1)) were investigated in healthy pigs and the findings related to published minimal inhibitory concentrations (MICs) for susceptible bacteria of animal origin, and to experimentally determined MICs for susceptible strains of porcine origin. We found MICs for Escherichia coli, Salmonella spp., Pasteurella spp. and Bordetella spp. in the range 0.5 to >64 microg mL(-1) isolated from infected pigs in the Forli area of Italy; only the Pasteurella multocida strains were sensitive (MIC(90)=0.5 microg mL(-1)). After intravenous (IV) injection, flumequine was slowly distributed and eliminated (t(1/2lambda(1))1.40+/-0.16 h and t(1/2lambda(2))6.35+/-1.69 h). The distribution volume at steady state (V(dss)) was 752.59+/-84.03 mL kg(-1) and clearance (Cl(B)) was 237.19+/-17.88 mL kg(-1)h(-1). After IM administration, peak serum concentration (4.99+/-0.92 microg mL(-1)) was reached between the 2nd and the 3rd hour. The results on MIC of isolated bacteria, although only indicative, suggest that the efficacy of flumequine on Gram-negative bacteria may be impaired by the emergence of less sensitive or resistant strains.

  20. Depletion of regulatory T cells augments a vaccine-induced T effector cell response against the liver-stage of malaria but fails to increase memory.

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    Maria del Rosario Espinoza Mora

    Full Text Available Regulatory T cells (T(reg have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4(+CD25(+ T(reg were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3(+CD25(- T(reg. To obtain more insights in the specific function of T(reg during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG. As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP of Plasmodium berghei (Pb was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC. Using this system we demonstrate here that the number of CSP-specific T cells increases when T(reg are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.