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Sample records for pertussis toxin sensitive

  1. Pertussis toxin

    SciTech Connect

    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.

  2. Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines.

    PubMed

    Jensen, Sten E; Illigen, Kristin E Engelhart; Badsberg, Jens Henrik; Hasløv, Kaare R

    2012-01-01

    Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.

  3. Lymphocyte receptors for pertussis toxin

    SciTech Connect

    Clark, C.G.; Armstrong, G.D. )

    1990-12-01

    We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.

  4. Pertussis toxin-sensitive G-protein mediates the alpha 2-adrenergic receptor inhibition of melatonin release in photoreceptive chick pineal cell cultures

    SciTech Connect

    Pratt, B.L.; Takahashi, J.S.

    1988-07-01

    The avian pineal gland is a photoreceptive organ that has been shown to contain postjunctional alpha 2-adrenoceptors that inhibit melatonin synthesis and/or release upon receptor activation. Physiological response and (32P)ADP ribosylation experiments were performed to investigate whether pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) were involved in the transduction of the alpha 2-adrenergic signal. For physiological response studies, the effects of pertussis toxin on melatonin release in dissociated cell cultures exposed to norepinephrine were assessed. Pertussis toxin blocked alpha 2-adrenergic receptor-mediated inhibition in a dose-dependent manner. Pertussis toxin-induced blockade appeared to be noncompetitive. One and 10 ng/ml doses of pertussis toxin partially blocked and a 100 ng/ml dose completely blocked norepinephrine-induced inhibition. Pertussis toxin-catalyzed (32P)ADP ribosylation of G-proteins in chick pineal cell membranes was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Membranes were prepared from cells that had been pretreated with 0, 1, 10, or 100 ng/ml pertussis toxin. In the absence of pertussis toxin pretreatment, two major proteins of 40K and 41K mol wt (Mr) were labeled by (32P)NAD. Pertussis toxin pretreatment of pineal cells abolished (32P) radiolabeling of the 40K Mr G-protein in a dose-dependent manner. The norepinephrine-induced inhibition of both cAMP efflux and melatonin release, as assessed by RIA of medium samples collected before membrane preparation, was also blocked in a dose-dependent manner by pertussis toxin. Collectively, these results suggest that a pertussis toxin-sensitive 40K Mr G-protein labeled by (32P)NAD may be functionally associated with alpha 2-adrenergic signal transduction in chick pineal cells.

  5. Characterization, localization and function of pertussis toxin-sensitive G proteins in the nervous systems of Aplysia and Loligo

    SciTech Connect

    Vogel, S.S.

    1989-01-01

    The author has characterized pertussis toxin-sensitive G proteins in the nervous systems of the gastropod mollusc Aplysia and the cephalopod Loligo using ({sup 32}P)ADP-ribosylation and immunoblotting with G protein specific antisera. As in vertebrates, this class of G protein is associated with membranes and enriched in nervous tissue in Aplysia. Analysis of dissected Aplysia ganglia reveal that it is enriched in neuropil, a region containing most of the central nervous system synapses. Because both Aplysia and Loligo synaptosomes are enriched in pertussis toxin-sensitive G proteins, it is likely that they are found in synaptic terminals. Fractionation of Aplysia synaptosomes into membrane and vesicle fractions reveals that, although the majority of G protein is recovered in the plasma membrane fraction, a small proportion is recovered in the vesicle fraction. He shows that G proteins are on intracellular membranes by ADP-ribosylating extruded axoplasm with pertussis toxin. A plausible explanation for vesicular localization of G protein in axoplasm is that G proteins are transported to terminals on vesicles. He has shown, using ligature experiments with Aplysia connectives and temperature block experiments in the giant axon of Loligo, that G proteins move by anterograde fast axonal transport. Injection of pertussis toxin into the identified Aplysia neuron L10 blocks histamine-induced presynaptic inhibition of transmitter release. This suggests that pertussis toxin sensitive G proteins play a role in modulating transmitter release at synaptic terminals. In the giant synapse of Loligo, he presents preliminary data that demonstrates that the activation of G proteins in the presynaptic terminal results in decreased transmitter release.

  6. Reporter cell lines for detection of pertussis toxin in acellular pertussis vaccines as a functional animal-free alternative to the in vivo histamine sensitization test.

    PubMed

    Hoonakker, Marieke E; Verhagen, Lisa M; van der Maas, Larissa; Sloots, Arjen; Hendriksen, Coenraad F M

    2017-02-22

    Detoxified pertussis toxin (pertussis toxoid) is a major antigen in acellular pertussis vaccines. Testing these vaccines on the presence of residual pertussis toxin (PTx) and reversion to toxicity is performed by the regulatory required in vivo Histamine Sensitization test (HIST). Lack of mechanistic understanding of the HIST, technical handicaps and animal welfare concerns, have promoted the development of alternative methods. As the majority of the cellular effects of PTx depend on its ability to activate intracellular pathways involving cAMP, the in vitro cAMP-PTx assay was developed. Although this assay could be used to detect PTx activity, it lacked sensitivity and robustness for use in a quality control setting. In the present study, novel reporter cell lines (CHO-CRE and A10-CRE) were generated that stably express a reporter construct responsive to changes in intracellular cAMP levels. These reporter cell lines were able to detect PTx in a concentration-dependent manner when combined with fixed amounts of forskolin. The CHO-CRE cell line enabled detection of PTx in the context of a multivalent vaccine containing aP, with a sensitivity equal to the HIST. However, the sensitivity of the A10-CRE cells was insufficient for this purpose. The experiments also suggest that the CHO-CRE reporter cell line might be suitable for assessment of cellular effects of PTd reverted to PTx. The CHO-CRE reporter cell line provides a platform that meets the criteria for specificity and sensitivity and is a promising in vitro model with potential to replace the HIST. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

    PubMed

    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.

  8. Sensitization to psychostimulants and stress after injection of pertussis toxin into the A10 dopamine region.

    PubMed

    Steketee, J D; Kalivas, P W

    1991-11-01

    An augmentation of psychostimulant-induced motor activity, termed sensitization, occurs with daily treatment and can last for months or years. At least in part, sensitization results from a long-term change in mesocorticolimbic dopamine transmission and may involve a disinhibition of dopamine neurons. Dopamine D2 autoreceptors and gamma-aminobutyric acidB (GABAB) receptors provide tonic inhibition to dopamine neurons via a G protein-mediated increase in K+ efflux. To evaluate the role of these inhibitory mechanisms in sensitization, pertussis toxin (PTX) was injected into the A10 dopamine region to uncouple the receptors via ADP-ribosylation of G proteins. In this study we demonstrated a significant augmentation in cocaine-stimulated motor activity, at doses greater than 3.0 mg/kg, 14 days after intra-A10 injection of PTX. Also, amphetamine-, but not morphine- or caffeine-stimulated motor activity was significantly augmented 2 weeks after PTX pretreatment. In vivo microdialysis revealed an augmentation of cocaine-induced increases in extracellular dopamine in the nucleus accumbens 14 days after PTX pretreatment. Pretreatment in the A10 region with the GABAB agonist baclofen, blocked cocaine-stimulated motor activity in control animals, but not in PTX-pretreated animals, indicating that the PTX treatment had uncoupled the GABAB receptor. Footshock stress activates mesocortical dopamine transmission, and postmortem tissue levels of dihydroxyphenylacetic acid and homovanillic acid in the prefrontal cortex were increased in PTX-pretreated animals. We hypothesize that the sensitized responses to cocaine, amphetamine and stress produced by PTX results from a decrease in dopamine D2 and GABAB-mediated inhibitory control of A10 dopamine neurons.

  9. Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils.

    PubMed

    Bylund, Johan; Pellmé, Sara; Fu, Huamei; Mellqvist, Ulf-Henrik; Hellstrand, Kristoffer; Karlsson, Anna; Dahlgren, Claes

    2004-05-24

    Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions. Tumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1. Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state. The TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.

  10. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin

    PubMed Central

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

    2016-01-01

    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. PMID:26812174

  11. Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors.

    PubMed

    Ninomiya, Hideki; Otani, Hajime; Lu, Kejie; Uchiyama, Takamichi; Kido, Masakuni; Imamura, Hiroji

    2002-05-01

    Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P(2y) purinoceptors. This study examined whether ATP-stimulated P(2y) purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 microM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 microM) or reactive blue (RB) (10 microM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 microM), ATP (30 microM), or UTP (50 microM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM). Although the protein kinase C inhibitor Ro318425 (0.3 microM) or tyrosine kinase inhibitor genistein (50 microM) had no significant effect on the functional protection afforded by adenosine

  12. Contribution of pertussis toxin to the pathogenesis of pertussis disease

    PubMed Central

    Carbonetti, Nicholas H.

    2015-01-01

    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. PMID:26394801

  13. Pancreastatin increases free cytosolic Ca2+ in rat hepatocytes, involving both pertussis-toxin-sensitive and -insensitive mechanisms.

    PubMed Central

    Sánchez-Margalet, V; Lucas, M; Goberna, R

    1993-01-01

    Freshly isolated rat hepatocytes, loaded with the Ca2+ probe Fluo-3, responded to homologous pancreastatin with a sudden increase in free cytosolic Ca2+ ([Ca2+]i) as well as glucose release. Addition of rat pancreastatin (0.1 microM) to hepatocytes resulted in an increase in [Ca2+]i from 150 nM to 700 nM, which declined back to nearly basal values within 2-3 min. Half-maximal and maximal effects were observed at 0.3 and 100 nM pancreastatin respectively. The increase in [Ca2+]i induced by vasopressin and noradrenaline was very similar in extent (from 150 to 800 nM) to that produced by pancreastatin. Neither the alpha 1-adrenergic blocker prazosin nor the vasopressin antagonist V1 modified the increase in [Ca2+]i induced by pancreastatin. Pig pancreastatin and its 33-49 C-terminal fragment produced about 65 and 75% of the effect of homologous pancreastatin respectively. Glucose production correlated with changes in [Ca2+]i in the same order of potency: vasopressin > rat pancreastatin > pig 33-49 pancreastatin > pig 1-49 pancreastatin. The effect of pancreastatin on [Ca2+]i was decreased by 50% when Ca2+ was omitted from the medium, and totally abolished when hepatocytes were depleted of internal Ca2+ stores by preincubation without Ca2+ and with 2 mM EGTA. When hepatocytes were preincubated for 5 min with PMA, the effects of ATP and noradrenaline were prevented, and those of vasopressin and pancreastatin remained unchanged. The pretreatment of hepatocytes with pertussis toxin diminished the response to pancreastatin and vasopressin. These results suggest that pancreastatin is a new Ca(2+)-mobilizing glycogenolytic hormone acting through a specific receptor which may involve both pertussis-toxin-sensitive and -insensitive GTP-binding regulatory proteins. PMID:8373359

  14. Contribution of pertussis toxin to the pathogenesis of pertussis disease.

    PubMed

    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. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Phytosphingosine-1-phosphate stimulates chemotactic migration of L2071 mouse fibroblasts via pertussis toxin-sensitive G-proteins.

    PubMed

    Kim, Mi-Kyoung; Park, Kyoung Sun; Lee, Hyuck; Kim, Young Dae; Yun, Jeanho; Bae, Yoe-Sik

    2007-04-30

    Phytosphingosine-1-phosphate (PhS1P) was found to stimulate an intracellular calcium increase via phospholipase C but not pertussis toxin (PTX)-sensitive G-proteins in L2071 mouse fibroblasts. PhS1P also activated ERK and p38 kinase, and these activations by PhS1P were inhibited by PTX. Moreover, PhS1P stimulated the chemotactic migration of L2071 cells via PTX-sensitive Gi protein(s). In addition, the PhS1P-induced chemotactic migration of L2071 cells was also dramatically inhibited by LY294002 and SB203580 (inhibitors of phosphoinositide 3-kinase and p38 kinase, respectively). L2071 cells are known to express four S1P receptors, i.e., S1P1, S1P2, S1P3, and S1P4, and pretreatment with an S1P1 and S1P3 antagonist (VPC 23019) did not affect on PhS1P-induced chemotaxis. This study demonstrates that PhS1P stimulates at least two different signaling cascades, one is a PTX-insensitive but phospholipase C dependent intracellular calcium increase, and the other is a PTX-sensitive chemotactic migration mediated by phosphoinositide 3-kinase and p38 kinase.

  16. Differential roles of spinal cholera toxin- and pertussis toxin-sensitive G proteins in nociceptive responses caused by formalin, capsaicin, and substance P in mice.

    PubMed

    Chung, K M; Lee, K C; Choi, S S; Suh, H W

    2001-03-15

    The aim of the present study is to characterize the roles of spinal cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G proteins in the regulation of various nociceptive responses. The effects of intrathecal (i.t.) pretreatments with CTX and PTX on the formalin (subcutaneous)-, capsaicin (i.t.)-, and substance P (SP; i.t.)-induced nociceptive behaviours were examined in mice. Pretreatment with CTX (i.t.; 24 h before) significantly and dose-dependently (0.05-0.5 microg) suppressed both the first and second phases of the formalin-induced nociceptive behaviour. On the other hand, pretreatment with PTX (i.t., 6 days before) at the same doses (0.05-N0.5 microg) did not affect the formalin-induced response. Capsaicin (i.t., 0.5 microg)- and SP (i.t., 0.7 microg)-induced nociceptive behaviours were attenuated by the pretreatment with CTX. In addition, SP-induced nociceptive response was also attenuated by the pretreatment with PTX. However, the capsaicin-induced nociceptive response was not influenced by PTX pretreatment. These findings suggest that, at the spinal cord level, CTX-sensitive G-proteins are involved in the formalin-, capsaicin-, and SP-induced nociceptive behavioural responses, whereas PTX-sensitive G proteins are involved in SP-induced nociceptive response.

  17. Pseudopterosin A inhibits phagocytosis and alters intracellular calcium turnover in a pertussis toxin sensitive site in Tetrahymena thermophila.

    PubMed

    Moya, Claudia E; Jacobs, Robert S

    2006-08-01

    The free living ciliate Tetrahymena thermophila was chosen as a cellular model in order to investigate the mode of action of the anti-inflammatory marine natural product Pseudopterosin A (PsA). In this paper we present evidence that PsA inhibits phagosome formation (KD=10.5 microM) and triggers a discrete intracellular calcium release (depletion) from a site in T. thermophila cells (KD=6.4 microM). Pre-treatment with the Gi/o protein inhibitor, pertussis toxin (PTX), inhibits PsA activity of both responses providing pharmacological evidence that the site of action for PsA is at a PTX sensitive G protein or a G protein coupled receptor (GPCR). Addition of extracellular calcium induced a concentration dependent increase in the incidence of phagosome formation (KD=30.3 microM) and was blocked by PsA pre-treatment. This particular effect of PsA on extracellular calcium was not blocked by PTX pre-treatment.

  18. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  19. Mechanical stimulation of skeletal muscle increases prostaglandin F2(alpha) synthesis and cyclooxygenase activity by a pertussis toxin sensitive mechanism

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.; Shansky, Janet; Solerssi, Rosa; Chromiak, Joseph

    1992-01-01

    Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increases the production of prostaglandin F(sub 2(alpha)), an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical activity, the activity of cyclooxygenase, a regulatory enzyme in prostaglandin synthesis, was increased 82% (P is less than .005), and this increase was maintained for at least 24 h. Kinetic analysis of the stretch-activated cyclooxygenase indicated a two to three-fold decrease in the enzyme's K(sub m) with no change in V(sub max). The stretch-induced increase in enzymatic activity was not inhibited by cycloheximide, was independent of cellular electrical activity (tetrodotoxin-insensitive), but was prevented by the G protein inhibitor pertussis toxin. Pertussis toxin also inhibited the stretch-induced increases in PGF(sub 2(alpha)) production, and cell growth. It is concluded that stretch of skeletal muscle increases the synthesis of the anabolic modulator PGF(sub 2(alpha)) by a G protein-dependent process which involves activation of cyclooxygenase by a posttranslational mechanism.

  20. Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools

    PubMed Central

    Carbonetti, Nicholas H

    2010-01-01

    Pertussis toxin and adenylate cyclase toxin are two important virulence factors of Bordetella pertussis, the bacterial cause of the respiratory disease pertussis or whooping cough. In addition to studies on the structure, function and role in pathogenesis of these two toxins, they are both used as cell biology tools for a variety of applications owing to their ability to enter mammalian cells, perform enzymatic activities and modify cell signaling events. In this article, recent data from the research literature that enhance our understanding of the nature of these two toxins, their role in the pathogenesis of B. pertussis infection and disease, particularly in modulating host immune responses, and their use as tools for other areas of research will be outlined. PMID:20210554

  1. Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein.

    PubMed Central

    Li, R Y; Gaits, F; Ragab, A; Ragab-Thomas, J M; Chap, H

    1995-01-01

    SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway. Images PMID:7781604

  2. Reduced Glutathione Is Required for Pertussis Toxin Secretion by Bordetella pertussis

    PubMed Central

    Stenson, Trevor H.; Patton, Angela K.; Weiss, Alison A.

    2003-01-01

    The abilities of cysteine-containing compounds to support growth of Bordetella pertussis and influence pertussis toxin transcription, assembly, and secretion were examined. Cysteine is an essential amino acid for B. pertussis and must be present for protein synthesis and bacterial growth. However, cysteine can be metabolized to sulfate, and high concentrations of sulfate can selectively inhibit transcription of the virulence factors, including pertussis toxin, via the BvgAS two-component regulatory system in a process called modulation. In addition, pertussis toxin possesses several disulfide bonds, and the cysteine-containing compound glutathione can influence oxidation-reduction reactions and perhaps disulfide bond formation. Bacterial growth was not observed in the absence of a source of cysteine. Oxidized glutathione, as a sole source of cysteine, also did not support bacterial growth. Cysteine, cystine, and reduced glutathione did support bacterial growth, and none of these compounds caused modulation at the concentrations tested. Similar amounts of periplasmic pertussis toxin were detected regardless of the source of cysteine; however, in the absence of reduced glutathione, pertussis toxin was not efficiently secreted. Addition of the reducing agent dithiothreitol was unable to compensate for the lack of reduced glutathione and did not promote secretion of pertussis toxin. These results suggest that reduced glutathione does not affect the accumulation of assembled active pertussis toxin in the periplasm but plays a role in efficient pertussis toxin secretion by the bacterium. PMID:12595447

  3. Natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries. Possible dysfunction of a pertussis toxin-sensitive G protein.

    PubMed

    Shimokawa, H; Flavahan, N A; Vanhoutte, P M

    1989-09-01

    caused by serotonin and aggregating platelets; this endothelium-dependent depression was markedly impaired in the previously denuded arteries throughout the study. Direct relaxation of the coronary smooth muscle to nitric oxide or sodium nitroprusside or direct contraction to KCl or serotonin were comparable between the control and previously denuded arteries. These experiments indicate that endothelium-dependent relaxations progressively worsen after regeneration of the endothelium and that the dysfunction of a pertussis toxin-sensitive G protein partly account for the endothelial dysfunction in the chronic regenerated state.

  4. Membrane Localization of the S1 Subunit of Pertussis Toxin in Bordetella pertussis and Implications for Pertussis Toxin Secretion

    PubMed Central

    Farizo, Karen M.; Fiddner, Stefanie; Cheung, Anissa M.; Burns, Drusilla L.

    2002-01-01

    Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system. PMID:11854200

  5. Pertussis toxin non-sensitive G protein mediates cholinergic stimulation for secretion of pancreastatin and somatostatin from QGP-1N cells.

    PubMed

    Funakoshi, A; Tateishi, K; Tsuru, M; Kono, A

    1992-01-02

    To clarify the possible role of a guanine nucleotide-binding protein (G-protein) in the signal transducing system activated by carbachol, actions of carbachol on human pancreastatin producing cell line (QGP-1N) were compared with those of fluoride, a well-known activator of stimulatory (Gs) or inhibitory (Gi) G protein. 10(-5) M of carbachol as well as 20 mM of NaF stimulated secretion of pancreastatin and somatostatin and intracellular Ca2+ mobilization. These secretion and Ca2+ mobilization were not modified by pertussis toxin, an inhibitor of Gi protein. These results suggest that pancreastatin and somatostatin secretions from QGP-1N are regulated by acetylcholine through a muscarinic receptor coupled to the activation of polyphosphoinositide breakdown by a G protein, which appears to be fluoride sensitive but is other than a Gi-like protein.

  6. Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism.

    PubMed

    Kaminski, N E; Koh, W S; Yang, K H; Lee, M; Kessler, F K

    1994-11-16

    of intracellular cAMP. These results indicate that inhibition of the sRBC AFC response by cannabinoids is mediated, at least in part, by inhibition of adenylate cyclase through a pertussis toxin-sensitive Gi-protein coupled cannabinoid receptor. Additionally, these studies further support the premise that cAMP is an important mediator of lymphocyte activation.

  7. Investigating pertussis toxin and its impact on vaccination.

    PubMed

    Coutte, Loic; Locht, Camille

    2015-01-01

    Whooping cough, caused by Bordetella pertussis, remains a major global health problem. Each year around 40 million of pertussis cases resulting in 200,000-400,000 annual deaths occur worldwide. Pertussis toxin is a major virulence factor of B. pertussis. Murine studies have shown its importance in bacterial colonization and in immunomodulation to evade innate or adaptive immunity. The toxin is composed of an A protomer expressing ADP-ribosyltransferase activity and a B oligomer, responsible for toxin binding to target cells. The toxin is also a major protective antigen in all currently available vaccines. However, vaccine escape mutants with altered toxin expression have recently been isolated in countries with high vaccination coverage illustrating the need for improved pertussis vaccines.

  8. CL316243 induces phosphatidylinositol 3,4,5-triphosphate production in rat adipocytes in an adenosine deaminase-, pertussis toxin-, or wortmannin-sensitive manner.

    PubMed

    Ohsaka, Y; Nomura, Y

    2016-07-18

    The effect of beta(3)-adrenoceptor (beta(3)-AR) agonists on adipocytes treated or not treated with signaling modulators has not been sufficiently elucidated. Using rat epididymal adipocytes (adipocytes) labeled with [(32)P]orthophosphate, we found that treatment with the selective beta(3)-AR agonist CL316243 (CL; 1 microM) induces phosphatidylinositol (PI) 3,4,5-triphosphate (PI[3,4,5]P(3)) production and that this response is inhibited by adenosine deaminase (ADA, an adenosine-degrading enzyme; 2 U/ml), pertussis toxin (PTX, an inactivator of inhibitory guanine-nucleotide-binding protein; 1 microg/ml), or wortmannin (WT, a PI-kinase inhibitor; 3 microM). The results showed that CL induced PI(3,4,5)P(3) production in intact adipocytes and that this production was affected by signaling modulators. Taken together, our findings indicate that CL produces PI(3,4,5)P(3) in an ADA-sensitive, PTX-sensitive, or WT-sensitive manner and will advance understanding of the effect of beta(3)-AR agonists on adipocytes.

  9. Molecular characterization of an operon required for pertussis toxin secretion.

    PubMed Central

    Weiss, A A; Johnson, F D; Burns, D L

    1993-01-01

    Mutants of Bordetella pertussis which are defective in secretion of pertussis toxin were isolated and characterized. The region of the B. pertussis chromosome identified by mutagenesis as playing a role in transport of pertussis toxin was sequenced. Analysis of this region revealed eight open reading frames, seven of which predict a protein exhibiting homology with one of the VirB proteins of Agrobacterium tumefaciens, which are involved in the transport of the T-DNA molecule across bacterial and plant membranes. Thus a set of accessory proteins are most likely involved in the secretion of pertussis toxin, and these proteins appear to be members of a family of proteins involved in the secretion of macromolecules from bacteria. PMID:8464913

  10. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels

    PubMed Central

    Tosetti, Patrizia; Parente, Valeria; Taglietti, Vanni; Dunlap, Kathleen; Toselli, Mauro

    2003-01-01

    In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity – such as regulator of G-protein-signalling (RGS) proteins – are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nm) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels. PMID:12651916

  11. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels.

    PubMed

    Tosetti, Patrizia; Parente, Valeria; Taglietti, Vanni; Dunlap, Kathleen; Toselli, Mauro

    2003-05-15

    In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity - such as regulator of G-protein-signalling (RGS) proteins - are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nM) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels.

  12. Development of a qualitative assay for screening of Bordetella pertussis isolates for pertussis toxin production.

    PubMed

    Gates, Irina; DuVall, Marina; Ju, Hong; Tondella, M Lucia; Pawloski, Lucia

    2017-01-01

    Bordetella pertussis infection has been increasing in the US, with reported cases reaching over 50,000 in 2012, a number last observed in the 1950s. Concurrently, B. pertussis lacking the pertactin protein, one of the immunogens included in the acellular vaccine formulations, has rapidly emerged since 2010, and has become the predominant circulating phenotype. Monitoring the production of the remaining acellular vaccine immunogens, such as pertussis toxin (Pt), is a critical next step. To date, methods for screening Pt have been either through genomic sequencing means or by conventional ELISAs. However, sequencing limits detection to the DNA level, missing potential disruptions in transcription or translation. Conventional ELISAs are beneficial for detecting the protein; however, they can often suffer from poor sensitivity and specificity. Here we describe a rapid, highly sensitive and specific electrochemiluminescent capture ELISA that can detect Pt production in prepared inactivated bacterial suspensions. Over 340 isolates were analyzed and analytical validation parameters, such as precision, reproducibility, and stability, were rigorously tested. Intra-plate and inter-plate variability measured at 9.8% and 11.5%, respectively. Refrigerated samples remained stable for two months and variability was unaffected (coefficient of variation was 12%). Interestingly, despite the intention of being a qualitative method, the assay was sensitive enough to detect a small, but statistically significant, difference in protein production between different pertussis promoter allelic groups of strains, ptxP1 and ptxP3. This technology has the ability to perform screening of multiple antigens at one time, thus, improving testing characteristics while minimizing costs, specimen volume, and testing time.

  13. σ-1 Receptor Inhibition of ASIC1a Channels is Dependent on a Pertussis Toxin-Sensitive G-Protein and an AKAP150/Calcineurin Complex.

    PubMed

    Mari, Yelenis; Katnik, Christopher; Cuevas, Javier

    2015-10-01

    ASIC1a channels play a major role in various pathophysiological conditions including depression, anxiety, epilepsy, and neurodegeneration following ischemic stroke. Sigma-1 (σ-1) receptor stimulation depresses the activity of ASIC1a channels in cortical neurons, but the mechanism(s) by which σ-1 receptors exert their influence on ASIC1a remains unknown. Experiments were undertaken to elucidate the signaling cascade linking σ-1 receptors to ASIC1a channels. Immunohistochemical studies showed that σ-1 receptors, ASIC1a and A-kinase anchoring peptide 150 colocalize in the plasma membrane of the cell body and processes of cortical neurons. Fluorometric Ca(2+) imaging experiments showed that disruption of the macromolecular complexes containing AKAP150 diminished the effects of the σ-1 on ASIC1a, as did application of the calcineurin inhibitors, cyclosporin A and FK-506. Moreover, whole-cell patch clamp experiments showed that σ-1 receptors were less effective at decreasing ASIC1a-mediated currents in the presence of the VIVIT peptide, which binds to calcineurin and prevents cellular effects dependent on AKAP150/calcineurin interaction. The coupling of σ-1 to ASIC1a was also disrupted by preincubation of the neurons in the G-protein inhibitor, pertussis toxin (PTX). Taken together, our data reveal that σ-1 receptor block of ASIC1a function is dependent on activation of a PTX-sensitive G-protein and stimulation of AKAP150 bound calcineurin.

  14. Seroprevalence of Bordetella pertussis antibodies to pertussis toxin among healthy children.

    PubMed

    Syed, Muhammad Ali; Ahmed, Waqas; Naeem, Aisha; Bokhari, Habib

    2012-10-01

    Pertussis or whooping cough is a highly communicable, vaccine-preventable acute respiratory tract disease, caused mainly by Bordetella pertussis. In this study, serosurveillance of IgG antibodies against pertussis toxin was carried out in healthy children, aged 3 months to 12 years, from the multiethnic cities of Islamabad and Rawalpindi, Pakistan. Kruskal- Wallis test was applied to determine the difference among groups. The results showed that the average values of pertussis toxin IgG dropped down significantly with age after getting the first dose in first 2 years (p < 0.001). Therefore, the researchers suggest introduction of booster vaccination with DTaP at second year of life and school going age to reduce the risk of getting B. pertussis infection.

  15. PURIFICATION OF HISTAMINE SENSITIZING FACTOR OF BORDETELLA PERTUSSIS.

    DTIC Science & Technology

    Histamine Sensitizing Factor ( HSF ) of Bordetella pertussis was studied. To improve the cultivation method for HSF preparation, growth conditions of...maintenance and surface bulk culture was devised. Intracellular localization of HSF and lethal toxin were studied. HSF was mainly localized in the cell...wall prepared by Mickle disintegration, and toxin was found in protoplasm Ribosomes prepared from sonicate contained considerable amount of HSF and

  16. Pertussis toxin promoter sequences involved in modulation.

    PubMed Central

    Gross, R; Rappuoli, R

    1989-01-01

    Previous analysis of the pertussis toxin (PT) promoter has shown that expression of PT requires a trans-activating factor encoded by the vir locus and a 170-base-pair DNA sequence upstream from the transcription start site containing a 21-base-pair direct repeat sequence crucial trans-activation (R. Gross and R. Rappuoli, Proc. Natl. Acad. Sci. USA 85:3913-3917, 1988). In this paper we extend the analysis to the modulative response to environmental stimuli. We show that modulation acts at the transcriptional level and occurs only in phase I bacteria. Modulation also requires a functional vir locus and the same promoter region of 170 base pairs. We show that, in addition to the previously identified direct repeat, even the sequences downstream from position -117 are required for trans-activation and modulation and that the deletion of four cytosine residues at position -31 causes the inactivation of the promoter. The kinetics of the change in transcription show that the PT promoter can be shut off very rapidly by adding 50 mM MgSO4 to the medium, whereas resumption of transcription after removal of the modulative agents from the medium is slow. Images PMID:2544567

  17. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells*

    PubMed Central

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-01-01

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gαi/o inhibitor, but not by NF449, a Gαs inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gαο1 and Gαi3 by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKeyTM assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gαi/o activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gαi/o upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants. PMID:25869129

  18. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

    PubMed

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-05-29

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.

  19. The interaction between pertussis toxin and 10 monoclonal antibodies.

    PubMed

    Schou, C; Au-Jensen, M; Heron, I

    1987-10-01

    Data on the epitope specificity of 10 monoclonal hybridoma antibodies (Mabs) that showed positive reaction in enzyme-linked immunosorbent assay (ELISA) towards pertussins toxin (Ptx) are presented. The relative functional affinity of the Mabs was determined in a catching ELISA system. The Mabs were tested for their ability to inhibit the biological activities of this toxin in two in vitro systems, viz. haemagglutination (HA) and Chinese Hamster Ovary cell (CHO) test, and in three in vivo assays: histamine sensitization (HS), leucocytosis-promoting activity (LP) and protection against intra-cerebral challenge (i.c.) with virulent B. pertussis organisms. Four Mabs were found inhibiting HA and three inhibited the effect on CHO cells. Two Mabs showed demonstrable protective effect on mice in i.c. test. The same two Mabs were also able to inhibit HS and LP activity of Ptx. Five of the ten Mabs reacted with Ptx subjected to blotting after separation of the toxin subunits in sodium-dodecyl sulphate polyacrylamide gel electrophoresis. The five Mabs all bound to more than one subunit. The epitopes defined by several of the Mabs might be useful in the context of a third-generation whooping cough vaccine.

  20. Modification of opiate agonist binding by pertussis toxin

    SciTech Connect

    Abood, M.E.; Lee, N.M.; Loh, H.H.

    1986-03-05

    Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in /sup 3/(H)-DADLE binding as compared with membranes treated identically without toxin. This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding.

  1. Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis.

    PubMed

    Barbier, Mariette; Boehm, Dylan T; Sen-Kilic, Emel; Bonnin, Claire; Pinheiro, Theo; Hoffman, Casey; Gray, Mary; Hewlett, Erik; Damron, F Heath

    2017-01-01

    Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions. Copyright © 2016 American Society for Microbiology.

  2. Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis

    PubMed Central

    Barbier, Mariette; Boehm, Dylan T.; Sen-Kilic, Emel; Bonnin, Claire; Pinheiro, Theo; Hoffman, Casey; Gray, Mary; Hewlett, Erik

    2016-01-01

    ABSTRACT Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions. PMID:27849178

  3. Multiple receptors mobilize calcium through a pertussis toxin (PT) sensitive GTP-binding protein in human neutrophils (PMN's)

    SciTech Connect

    Lad, P.M.; Olson, C.V.; Grewal, I.S.; Frolich, M.; Scott, S.J.

    1986-03-05

    Treatment of PMN's with PT causes an abolition of chemotaxis, enzyme release, superoxide generation and aggregation caused by f-met-leu-phe (FMLP),C5a and platelet activating factor (PAF). Lectin (Con-A) induced capping and receptor induced shape change are abolished, but phagocytosis is unaltered. In whole cells, calcium mobilization induced by FMLP, PAF and Con-A inhibited by PT although the FMLP-mediated effect is more susceptible to PT's effects. Treatment of PMN's with phorbol 12-myristate 13 acetate (PMA) causes an abolition of calcium mobilization by all agents in a range which also inhibits cap formation. Investigation of calcium uptake reveals PT sensitive and insensitive components. Reciprocal interactions between Ns and Ni proteins are also observed since pretreatment with FMLP and PAF causes a stimulation of Ns-mediated cyclic AMP enhancement while pretreatment with Ns linked receptors (PGE/sub 1/ and beta receptor agonists) inhibits calcium mobilization. Comparative peptide mapping studies indicate substantial similarity between Ni proteins in PMN's, platelets and human erythrocytes. The authors results suggest that the Ni linked calcium mobilization sensitive to PMA is important to the regulation of the human neutrophil.

  4. Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids.

    PubMed Central

    Lee, C K; Roberts, A; Perrin, S

    1989-01-01

    Pertussis toxin is produced only by strains of Bordetella pertussis. Cloned genes encoding pertussis toxin from B. pertussis were transferred into Bordetella bronchiseptica and Bordetella parapertussis by conjugation. These transconjugants expressed pertussis toxin at levels comparable to those expressed by B. pertussis. The toxin made by these strains was biologically active in the Chinese hamster cell clumping assay, contained all five subunits, and was mostly periplasmic. Toxin expression appeared to be modulated in the same way as are the vir-regulated genes of B. pertussis. Introduction of these plasmids into B. pertussis failed to produce hypertoxigenic strains. Instead, these transconjugants underwent plasmid loss, gene deletions, or conversion to the avirulent phase. Images PMID:2707851

  5. Pertussis seroprevalence in korean adolescents and adults using anti-pertussis toxin immunoglobulin G.

    PubMed

    Lee, Soo Young; Han, Seung Beom; Bae, E Young; Kim, Jong-Hyun; Kang, Jin Han; Park, Yeon-Joon; Ma, Sang Hyuk

    2014-05-01

    This study was conducted to evaluate age-specific seroprevalence of pertussis in Korea and to formulate a strategy to prevent and reduce the incidence of pertussis. Residual serum samples of healthy adolescents and adults 11 yr of age or older were collected between July 2012 and December 2012, and anti-pertussis toxin (PT) IgG titers were measured using a commercial ELISA kit. We compared the mean anti-PT IgG titers and seroprevalence of pertussis of the six age groups: 11-20, 21-30, 31-40, 41-50, 51-60, and ≥ 61 yr. A total of 1,192 subjects were enrolled. The mean anti-PT IgG titer and pertussis seroprevalence were 35.53 ± 62.91 EU/mL and 41.4%, respectively. The mean anti-PT IgG titers and seroprevalence were not significantly different between the age groups. However, the seroprevalence in individuals 51 yr of age or older was significantly higher than in individuals younger than 51 yr (46.5% vs 39.1%, P = 0.017). Based on these results, a new pertussis prevention strategy is necessary for older adults.

  6. Comparison of anti-pertussis toxin ELISA and agglutination assays to assess immune responses to pertussis.

    PubMed

    Khramtsov, Pavel; Bochkova, Maria; Timganova, Valeria; Zamorina, Svetlana; Rayev, Mikhail

    2017-08-01

    The goal of our study was to compare the following two methods of assessment of pertussis post-vaccination immunity: bacterial agglutination test and pertussis toxin enzyme-linked immunosorbent assay (ELISA). The study was carried out in Perm Region, Russia. We measured pertussis immunity using two serological methods: ELISA of IgG to pertussis toxin (PT) and the agglutination test (AT) among 135 children, in the age range from 2 months to 17 years old. The immunization schedule included four doses of DTwP: at 3, 4.5 and 6 months of age and a booster at 18 months. All participants were divided into six age groups. The percentage of samples with IgG level less than the detection limit in vaccinated children was 52.2%. The total seropositivity rate (the percent of children with agglutinin titres ≥1:160) in vaccinated children was 47.8%. Only a weak association was observed between agglutinin and anti-PT IgG titres (R = .3). Neither the primary nor the booster vaccination with DTwP influenced the IgG levels in children. Agglutinin titres significantly increased after vaccination and declined 5 years after the booster dose. Significant growth of IgG concentration was observed in 11-year-olds, indicating the presence of B. pertussis circulation in the childhood population. Based on the obtained results and the results of other authors, we summarize that anti-PT ELISA should be carefully used to assess the population immunity to pertussis. Currently, there is neither a serological test that accurately determines the protection against pertussis nor a distinctive criterion of protection that can be applied in seroepidemiological studies.

  7. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin.

    PubMed Central

    Gordon, V M; Leppla, S H; Hewlett, E L

    1988-01-01

    Bordetella pertussis and Bacillus anthracis produce extracytoplasmic adenylate cyclase toxins (AC toxins) with shared features including activation by calmodulin and the ability to enter target cells and catalyze intracellular cyclic AMP (cAMP) production from host ATP. The two AC toxins were evaluated for sensitivities to a series of inhibitors of known uptake mechanisms. Cytochalasin D, an inhibitor of microfilament function, abrogated the cAMP response to B. anthracis AC toxin (93%) but not the cAMP response elicited by B. pertussis AC toxin. B. anthracis-mediated intoxication of CHO cells was completely inhibited by ammonium chloride (30 mM) and chloroquine (0.1 mM), whereas the cAMP accumulation produced by B. pertussis AC toxin remained unchanged. The block of target cell intoxication by cytochalasin D could be bypassed when cells were first treated with anthrax AC toxin and then exposed to an acidic medium. These data indicate that despite enzymatic similarities, these two AC toxins intoxicate target cells by different mechanisms, with anthrax AC toxin entering by means of receptor-mediated endocytosis into acidic compartments and B. pertussis AC toxin using a separate, and as yet undefined, mechanism. PMID:2895741

  8. An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines.

    PubMed

    Xing, Dorothy; Gaines Das, Rose; Douglas-Bardsley, Alex; Asokanathan, Catpagavalli; Corbel, Michael

    2014-03-01

    Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose-response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.

  9. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. )

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  10. Bordetella pertussis Infection Exacerbates Influenza Virus Infection through Pertussis Toxin-Mediated Suppression of Innate Immunity

    PubMed Central

    Ayala, Victor I.; Teijaro, John R.; Farber, Donna L.; Dorsey, Susan G.; Carbonetti, Nicholas H.

    2011-01-01

    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. PMID:21533103

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

    PubMed

    Ayala, Victor I; Teijaro, John R; Farber, Donna L; Dorsey, Susan G; Carbonetti, Nicholas H

    2011-04-20

    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.

  12. [Production of pertussis toxin by Bordetella pertussis strains isolated from patients with whooping cough].

    PubMed

    Zaĭtsev, E M; Mertsalova, N U; Shinkarev, A S; Mazurova, I K; Zakharova, N S

    2011-01-01

    To assess level of pertussin toxin (PT) production by vaccine strains of Bordetella pertussis and strains isolated from patients with whooping cough. Concentration of PT in supernatants of microbial cultures of 3 vaccine strains and 25 strains of B. pertussis isolated from patients with pertussis in 2001 - 2005 was measured with enzyme immunoassay using gamma-globulin fractions of rabbit antiserum to PT as immunosorbent or included in peroxidase conjugates. Level of PT production by strains isolated from infected persons varied from 3 +/- 0.5 to 64.8 +/- 12.2 ng/MFU/ml: in 9 strains--from 3 +/- 0.5 to 9.4 +/- 2.1 ng/MFU/ml, in 7--10.5 +/- 1.8 to 18.4 +/- 2.6 ng/MFU/ml, and in 9--23.6 +/- 4.5 to 64.8 +/- 12.2 ng/MFU/ml. B. pertussis strains isolated from patients were heterogeneous on level of PT production. Difference in expression of PT between strains were as high as 20-fold. Conditionally low, moderate and high levels of PT production had 9 (36%), 7 (28%), and 9 (36%) of 25 studied strains. Three vaccine strains had levels of toxin production similar to recently isolated strains with moderate level of its production.

  13. ADP-ribosylation of transducin by pertussis toxin

    SciTech Connect

    Watkins, P.A.; Burns, D.L.; Kanaho, Y.; Liu, T.Y.; Hewlett, E.L.; Moss, J.

    1985-11-05

    Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is (TSP)ADP-ribosylated by pertussis toxin and (TSP)NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and TS kDa. The amino terminus of both 38- and TS-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The TS-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of (TSP)ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased (TSP)ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed (TSP)ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma.

  14. Pertussis Toxin Exacerbates and Prolongs Airway Inflammatory Responses during Bordetella pertussis Infection

    PubMed Central

    Connelly, Carey E.; Sun, Yezhou

    2012-01-01

    Throughout infection, pathogenic bacteria induce dramatic changes in host transcriptional repertoires. An understanding of how bacterial factors influence host reprogramming will provide insight into disease pathogenesis. In the human respiratory pathogen Bordetella pertussis, the causative agent of whooping cough, pertussis toxin (PT) is a key virulence factor that promotes colonization, suppresses innate immune responses during early infection, and causes systemic disease symptoms. To determine the full extent of PT-associated gene regulation in the airways through the peak of infection, we measured global transcriptional profiles in the lungs of BALB/c mice infected with wild-type (WT) or PT-deficient (ΔPT) B. pertussis. ΔPT bacteria were inoculated at a dose equivalent to the WT dose and at a high dose (ΔPThigh) to distinguish effects caused by higher bacterial loads achieved in WT infection from effects associated with PT. The results demonstrated that PT was associated with a significant upregulation of immune and inflammatory response genes as well as several other genes implicated in airway pathology. In contrast to the early, transient responses observed for ΔPThigh infection, WT infection induced a prolonged expression of inflammatory genes and increased the extent and duration of lung histopathology. In addition, the administration of purified PT to ΔPThigh-infected mice 1 day after bacterial inoculation exacerbated and prolonged inflammatory responses and airway pathology. These data indicate that PT not only is associated with exacerbated host airway responses during peak B. pertussis infection but also may inhibit host mechanisms of attenuating and resolving inflammation in the airways, suggesting possible links between PT and pertussis disease symptoms. PMID:23027529

  15. Pertussis toxin exacerbates and prolongs airway inflammatory responses during Bordetella pertussis infection.

    PubMed

    Connelly, Carey E; Sun, Yezhou; Carbonetti, Nicholas H

    2012-12-01

    Throughout infection, pathogenic bacteria induce dramatic changes in host transcriptional repertoires. An understanding of how bacterial factors influence host reprogramming will provide insight into disease pathogenesis. In the human respiratory pathogen Bordetella pertussis, the causative agent of whooping cough, pertussis toxin (PT) is a key virulence factor that promotes colonization, suppresses innate immune responses during early infection, and causes systemic disease symptoms. To determine the full extent of PT-associated gene regulation in the airways through the peak of infection, we measured global transcriptional profiles in the lungs of BALB/c mice infected with wild-type (WT) or PT-deficient (ΔPT) B. pertussis. ΔPT bacteria were inoculated at a dose equivalent to the WT dose and at a high dose (ΔPT(high)) to distinguish effects caused by higher bacterial loads achieved in WT infection from effects associated with PT. The results demonstrated that PT was associated with a significant upregulation of immune and inflammatory response genes as well as several other genes implicated in airway pathology. In contrast to the early, transient responses observed for ΔPT(high) infection, WT infection induced a prolonged expression of inflammatory genes and increased the extent and duration of lung histopathology. In addition, the administration of purified PT to ΔPT(high)-infected mice 1 day after bacterial inoculation exacerbated and prolonged inflammatory responses and airway pathology. These data indicate that PT not only is associated with exacerbated host airway responses during peak B. pertussis infection but also may inhibit host mechanisms of attenuating and resolving inflammation in the airways, suggesting possible links between PT and pertussis disease symptoms.

  16. Expression of a synthetic pertussis toxin operon in Escherichia coli.

    PubMed

    Pozza, T D; Yan, H; Walker, M J

    1997-06-01

    Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A/B bacterial toxin consisting of five subunits S1-S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli beta-lactamase leader sequence. We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

  17. Serotonin decreases population spike amplitude in hippocampal cells through a pertussis toxin substrate.

    PubMed

    Clarke, W P; De Vivo, M; Beck, S G; Maayani, S; Goldfarb, J

    1987-05-05

    Activation of the serotonin1A receptor decreases CA1 population spike amplitude and inhibits forskolin-stimulated adenylate cyclase in rat hippocampus. Pretreatment of rats with pertussis toxin blocked both responses. Because the electrophysiological and biochemical responses to serotonin were correlated after pertussis toxin treatment, we conclude that both responses are mediated by a common regulatory protein, presumably Gi.

  18. Reduction of Pertussis Inflammatory Pathology by Therapeutic Treatment With Sphingosine-1-Phosphate Receptor Ligands by a Pertussis Toxin-Insensitive Mechanism.

    PubMed

    Skerry, Ciaran; Scanlon, Karen; Ardanuy, Jeremy; Roberts, Drew; Zhang, Li; Rosen, Hugh; Carbonetti, Nicholas H

    2017-01-15

    Recent data have demonstrated the potential of sphingosine 1-phosphate (S1P) receptor (S1PR) agonism in the treatment of infectious diseases. A previous study used a murine model of Bordetella pertussis infection to demonstrate that treatment with the S1PR agonist AAL-R reduces pulmonary inflammation during infection. In the current study, we showed that this effect is mediated via the S1PR1 on LysM+ (myeloid) cells. Signaling via this receptor results in reduced lung inflammation and cellular recruitment as well as reduced morbidity and mortality in a neonatal mouse model of disease. Despite the fact that S1PRs are pertussis toxin-sensitive G protein-coupled receptors, the effects of AAL-R were pertussis toxin insensitive in our model. Furthermore, our data demonstrate that S1PR agonist administration may be effective at therapeutic time points. These results indicate a role for S1P signaling in B. pertussis-mediated pathology and highlight the possibility of host-targeted therapy for pertussis. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  19. Pertussis toxin inhibits somatostatin-induced K/sup +/ conductance in human pituitary tumor cells

    SciTech Connect

    Yamashita, N.; Kojima, I.; Shibuya, N.; Ogata, E.

    1987-07-01

    The effect of pertussis toxin on somatostatin-induced K/sup +/ current was examined in dissociated human pituitary tumor cells obtained from two acromegalic patients. Somatostatin-induced hyperpolarization or K/sup +/ current was observed in 20 of 23 cells in adenoma 1 and 10 of 11 cells in adenoma 2. After treatment with pertussis toxin for 24 h, these responses were completely suppressed (0/14 in adenoma, 1, 0/10 in adenoma 2). Spontaneous action potentials, K/sup +/, Na/sup +/, and Ca/sup 2 +/ currents were well preserved after pertussis toxin treatment. When crude membrane fraction was incubated with (/sup 32/P)NAD, a 41K protein was ADP-ribosylated by pertussis toxin. Hormone release was inhibited by somatostatin and this inhibition was blocked by pertussis toxin treatment.

  20. Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family.

    PubMed

    Martín, César; Requero, M-Asunción; Masin, Jiri; Konopasek, Ivo; Goñi, Félix M; Sebo, Peter; Ostolaza, Helena

    2004-06-01

    Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

  1. Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis.

    PubMed

    Eby, Joshua C; Gray, Mary C; Warfel, Jason M; Merkel, Tod J; Hewlett, Erik L

    2017-01-01

    Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination. Copyright © 2017 American Society for Microbiology.

  2. A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

    PubMed

    Cyr, T; Menzies, A J; Calver, J; Whitehouse, L W

    2001-06-01

    The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that

  3. Dual pertussis toxin-sensitive pathway of zymosan-induced activation in guinea pig macrophages. An anti-CR3 antibody-inhibitable stimulation of phagocytosis and -resistant stimulation of O2- production and arachidonate release.

    PubMed

    Hazeki, K; Tamoto, K; Ui, M; Mori, Y

    1994-03-28

    Complement receptor type 3 (CR3)-mediated cellular responses in guinea pig macrophages were investigated by using zymosan and serum-opsonized zymosan (SOZ) as the multivalent ligand for CR3. The ingestion of zymosan and SOZ was accompanied by O2- generation and arachidonate release. These responses were suppressed by prior exposure of macrophages to pertussis toxin (PT). Opsonization of zymosan gave rise to more than 6-fold activation of the ingestion, whereas the magnitude of either arachidonate release or O2- generation was unchanged. The Fab' fragment of anti-Z-1, a monoclonal antibody specific for the alpha chain of guinea pig CR3, inhibited the ingestion of zymosan by 60% without affecting zymosan-induced arachidonate release and O2- generation. These data suggested that there might be at least two functionally distinct binding sites for zymosan. O2- generation and arachidonate release might be regulated through one site and phagocytosis another. Both sites should be coupled to PT-sensitive GTP binding protein.

  4. Pertussis toxins, other antigens become likely targets for genetic engineering

    SciTech Connect

    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.

  5. Mr 40,000 and Mr 39,000 pertussis toxin substrates are increased in surgically denervated dog ventricular myocardium

    SciTech Connect

    Hershberger, R.E.; Feldman, A.M.; Anderson, F.L.; Kimball, J.A.; Wynn, J.R.; Bristow, M.R. )

    1991-04-01

    To test the general hypothesis that cardiac innervation may participate in myocardial G protein regulation, we examined the effects of complete intrapericardial surgical denervation or sham operation in dogs. In particulate fractions of dog left ventricular (LV) myocardium harvested 28-33 days after denervation or sham operation, Mr 40,000 and Mr 39,000 pertussis toxin-sensitive substrates (G proteins) were increased by 31% (1.31 +/- 0.084 vs 1.00 +/- 0.058 OD, arbitrary units, p less than 0.01) and 40% (1.40 +/- 0.117 vs. 1.000 +/- 0.084 OD, arbitrary units, p less than 0.02), respectively, as compared with sham-operated controls. The Mr 40,000 pertussis toxin-sensitive band comigrated with a pertussis toxin-sensitive substrate in human erythrocyte membranes known to contain an alpha Gi species. In these same preparations basal, GTP and GppNHp stimulated adenylate cyclase activities were decreased in denervated heart by 20, 26, and 19%, respectively, consistent with increased activity of an inhibitory G protein. In contrast, Gs function was not altered, because cyc(-) membranes reconstituted with membrane extracts and fluoride and beta-receptor-stimulated adenylate cyclase activity were not different between groups. Furthermore, adenylate cyclase catalytic subunit function as assessed with forskolin and manganese stimulation was not different between preparations of control and denervated heart. We conclude that in preparations of surgically denervated dog myocardium Mr 40,000 and Mr 39,000 pertussis toxin-sensitive G proteins are increased by 31 and 40%, respectively, and that functional alterations in adenylate cyclase activity exist, consistent with increased inhibitory G-protein function.

  6. Antibody responses to pertussis toxin display different kinetics after clinical Bordetella pertussis infection than after vaccination with an acellular pertussis vaccine.

    PubMed

    Dalby, Tine; Petersen, Jesper Westphal; Harboe, Zitta B; Krogfelt, Karen Angeliki

    2010-09-01

    The measurement of IgG anti-pertussis toxin (IgG anti-PT) antibodies by ELISA is a frequently used method for studying the antibody responses after pertussis vaccination and after Bordetella pertussis infection. Such responses vary according to the different vaccines used as well as to the immunization and infection history of the participants. In the present study, the decay kinetics of the IgG anti-PT antibody response was determined for 71 Danish children and adults with bacteriologically confirmed B. pertussis infection and for 20 Danish adults booster-vaccinated with an acellular pertussis vaccine. For both groups, biphasic decay was seen, but the individual antibody responses varied greatly. No differences related to age were seen. Within each group, individual decay profiles showed parallel log-linear decay for the late part of the response. Antibody half-life was calculated for the late, slower part of the biphasic response curves for both groups (>5 months after diagnosis for individuals with confirmed infection; >3 months for vaccinated individuals). The median half-life for post-infection antibodies was 221 days [interquartile range (IQR) 159-314 days, 36 individuals], and the median half-life for post-vaccination antibodies was 508 days (IQR 428-616 days, 14 individuals). This difference was statistically significant (P<0.0001). Thus, in this setting, we found that the IgG anti-PT antibody decay after an infection with B. pertussis is more than twice as fast as the decay after booster vaccination with an acellular pertussis vaccine. Such knowledge of the IgG anti-PT decay kinetics is crucial for interpretation of serological data that will be used either for diagnosis or for epidemiological studies and surveillance of B. pertussis infections.

  7. Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes

    SciTech Connect

    Luttrell, L.M.; Hewlett, E.L.; Romero, G.; Rogol, A.D.

    1988-05-05

    The effects of pertussis toxin (PT) treatment on insulin-stimulated myristoyl-diacylglycerol (DAG) generation, hexose transport, and thymidine incorporation were studied in differentiated BC3H-1 mycocytes. Insulin treatment caused a biphasic increase in myristoyl-DAG production which was abolished in myocytes treated with PT. There was no effect of PT treatment on basal (nonstimulated) myristoyl-DAG production. Insulin-stimulated hydrolysis of a membrane phosphatidylinositol glycan was blocked by PT treatment. ADP-ribosylation of BC3H-1 plasma membranes with (/sup 32/P)NAD revealed a 40-kDa protein as the major PT substrate in vivo and in vitro. The time course and dose dependence of the effects of PT on diacylglycerol generation correlated with the in vivo ADP-ribosylation of the 40-kDa substrate. Pertussis toxin treatment resulted in a 71% attenuation of insulin-stimulated hexose uptake without effect on either basal or phorbol ester-stimulated uptake. The stimulatory effects of insulin and fetal calf serum on (/sup 3/H)thymidine incorporation into quiescent myocytes were attenuated by 61 and 59%, respectively, when PT was added coincidently with the growth factors. Nonstimulated and EGF-stimulated (/sup 3/H)thymidine incorporation was unaffected by PT treatment. These data suggest that a PT-sensitive G protein is involved in the cellular signaling mechanisms of insulin.

  8. Prospective evaluation of an Australian pertussis toxin IgG and IgA enzyme immunoassay.

    PubMed

    May, Meryta L; Doi, Suhail A; King, David; Evans, Jenny; Robson, Jennifer M

    2012-02-01

    Serological diagnosis of recent pertussis infection is an important part of both clinical assessment and epidemiological documentation of this disease. Standardization of serological testing and interpretation remains challenging despite international efforts to improve it. Currently, determining the anti-pertussis toxin (PT) IgG titer is recommended as the most accurate serological test in Europe and the United States, while Australia relies predominantly on measurement of Bordetella pertussis IgA antibody responses. Using B. pertussis PCR and the WHO clinical case definition as reference standards, the diagnostic utility of in-house anti-PT IgG and anti-PT IgA assays was evaluated prospectively in an Australian community-based cohort (n = 327). Patients provided up to four consecutive serum samples to document the kinetics of antibody response and decay. Previously validated cutoffs for positivity were converted to international units by using WHO-approved reference sera. At currently used cutoffs, both anti-PT IgG (>94 IU/ml) and anti-PT IgA (>20 IU/ml) assays had good specificity (80% [95% confidence interval {95% CI}, 68 to 88%] and 87% [95% CI, 77 to 94%]), but anti-PT IgG assay was consistently more sensitive than anti-PT IgA assay across a range of cutoffs (60 to 79% [95% CI, 53 to 84%] versus 41 to 62% [95% CI, 34 to 69%]). The combination of anti-PT IgG and anti-PT IgA assays performed no better than anti-PT IgG assay alone. The anti-PT IgA response in children under 12 years of age was poor. The accuracy of serology was optimal between 2 and 8 weeks after symptom onset. Cutoffs of >94 IU/ml for anti-PT IgG and >20 IU/ml for anti-PT IgA correlated well with recent pertussis infection and were consistent with recent recommendations from the EU Pertstrain group. Anti-PT IgG assay was superior to anti-PT IgA assay as the test of choice for the diagnosis of pertussis from a single sample.

  9. ADP-ribosylation of membrane components by pertussis and cholera toxin

    SciTech Connect

    Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.; Codina, J.; Field, J.B.; Birnbaumer, L.; Sekura, R.D.

    1985-01-01

    Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its ..cap alpha../sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its ..cap alpha..; subunit. By using (/sup 32/P)NAD/sup +/ and determining the transfer of its (/sup 32/P)ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/sub s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin.

  10. Accelerated maternal responding following intra-VTA pertussis toxin treatment.

    PubMed

    Byrnes, John J; Gleason, Erin D; Schoen, Matthew K; Schoen, Mathew T; Lovelock, Dennis F; Carini, Lindsay M; Byrnes, Elizabeth M; Bridges, Robert S

    2011-10-01

    Prior studies have supported a role for mesolimbic dopaminergic mechanisms in the regulation of maternal behavior. Accordingly, the ventral tegmental area (VTA) and its dopaminergic projections to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in both the onset and maintenance of normal maternal behavior. To date, studies of direct manipulation of VTA neurochemistry at the onset of maternal behavior have been limited. The current study was undertaken to directly test the hypothesis that enhancement of dopaminergic transmission in the mesolimbic dopamine system can stimulate maternal activity using a pup-induced virgin model. Nulliparous female rats were stereotaxically infused with pertussis toxin (PTX 0, 0.1, or 0.3 μg/hemisphere) into the VTA to chronically stimulate the activity of dopaminergic projection neurons. After 3 days of recovery, maternal responding to donor pups was tested daily, and latency (in days) to full maternal behavior was recorded. Intra-VTA PTX treatment produced a robust dose-dependent decrease in maternal behavior latency, and a long-lasting increase in locomotor activity. These effects were associated with significantly decreased dopamine D1 receptor mRNA expression in the NAc. No effects of PTX treatment on mesolimbic dopamine utilization or mPFC receptor expression were observed. The findings indicate that chronic neural activation in the VTA accelerates the onset of maternal behavior in virgin female rats via modification of the NAc dopamine D1 receptor.

  11. Accelerated Maternal Responding Following Intra-VTA Pertussis Toxin Treatment

    PubMed Central

    Byrnes, John J.; Gleason, Erin D.; Schoen, Mathew K.; Lovelock, Dennis F.; Carini, Lindsay M.; Byrnes, Elizabeth M.; Bridges, Robert S.

    2011-01-01

    Prior studies have supported a role for mesolimbic dopaminergic mechanisms in the regulation of maternal behavior. Accordingly, the ventral tegmental area (VTA) and its dopaminergic projections to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in both the onset and maintenance of normal maternal behavior. To date, studies of direct manipulation of VTA neurochemistry at the onset of maternal behavior have been limited. The current study was undertaken to directly test the hypothesis that enhancement of dopaminergic transmission in the mesolimbic dopamine system can stimulate maternal activity using a pup-induced virgin model. Nulliparous female rats were stereotaxically infused with pertussis toxin (PTX 0, 0.1, or 0.3 μg/hemisphere) into the VTA to chronically stimulate the activity of dopaminergic projection neurons. After 3 days of recovery, maternal responding to donor pups was tested daily, and latency (in days) to full maternal behavior was recorded. Intra-VTA PTX treatment produced a robust dose-dependent decrease in maternal behavior latency, and a long-lasting increase in locomotor activity. These effects were associated with significantly decreased dopamine D1 receptor mRNA expression in the NAc. No effects of PTX treatment on mesolimbic dopamine utilization or mPFC receptor expression were observed. The findings indicate that chronic neural activation in the VTA accelerates the onset of maternal behavior in virgin female rats via modification of the NAc dopamine D1 receptor. PMID:21571006

  12. Characterization of the carbohydrate binding and ADP-ribosyltransferase activities of chemically detoxified pertussis toxins.

    PubMed

    Oh, Hokyung; Kim, Byoung-Guk; Nam, Kyung-Tak; Hong, Seung-Hwa; Ahn, Dong-Ho; Choi, Gi-Sub; Kim, Hyungjin; Hong, Jin-Tae; Ahn, Byung-Yoon

    2013-06-24

    Pertussis toxin (PTx) is an essential component of the acellular pertussis (aP) vaccine. However, because PTx in its native form is considered too toxic for human vaccine use, it must be inactivated into a stable, nontoxic form by treatment with chemical detoxifying agents or by genetic modification. Therefore, testing for the residual PTx in the aP vaccine is a major quality control step for vaccine manufacturers and regulatory authorities. The histamine sensitization test is currently the standard safety test method for all aP vaccines, regardless of the vaccine formula or the detoxification process, except for those with genetically modified PTx. However, test result variability and ethical concerns regarding animal use necessitate an alternative method. In vitro assays based on the biochemical properties of PTx have been considered as potential alternatives to the histamine sensitization test. In this study, the suitability of assays based on the ADP-ribosyltransferase and carbohydrate binding activities of PTx was assessed for PTx after treatment with formaldehyde, glutaraldehyde or both denaturants in sequence. The results indicated a distinctive pattern of the biochemical activities depending on the detoxification methods and storage conditions. These results suggest that although a more careful study is needed, these in vitro biochemical assays can be considered potential alternatives to the histamine sensitization test, as they might provide more specific safety information of aP vaccines. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles.

    PubMed

    Donato, Gina M; Goldsmith, Cynthia S; Paddock, Christopher D; Eby, Joshua C; Gray, Mary C; Hewlett, Erik L

    2012-02-17

    Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

  14. Serological response to filamentous hemagglutinin and lymphocytosis-promoting toxin of Bordetella pertussis.

    PubMed Central

    Burstyn, D G; Baraff, L J; Peppler, M S; Leake, R D; St Geme, J; Manclark, C R

    1983-01-01

    Serum antibody responses to the filamentous hemagglutinin and the lymphocytosis-promoting toxin of Bordetella pertussis after vaccination with diphtheria and tetanus toxoids and pertussis vaccine, adsorbed, were assayed by using the enzyme-linked immunosorbent assay. The effect of early immunization, during the first week of life, on the antibody response also was determined. After vaccination, immunoglobulin G (IgG) and IgM directed against both the filamentous hemagglutinin and the lymphocytosis-promoting toxin were detected. Generally, antibody titers increased with subsequent injections and the age of the children. Maternal antibodies against filamentous hemagglutinin and lymphocytosis-promoting toxin were detected in cord blood. The ability of an infant to produce serum IgG anti-lymphocytosis-promoting toxin after vaccination with pertussis vaccine was inversely related to the cord blood serum IgG anti-lymphocytosis-promoting toxin titer at birth. A good antibody response was observed in infants with low cord blood titers, and a poor antibody response was seen in infants with high cord blood values. The IgM anti-lymphocytosis-promoting toxin response was good in groups with both low and high cord blood titer, with no significant difference observed between the two groups. No IgA anti-lymphocytosis-promoting toxin or IgA anti-filamentous hemagglutinin titers were observed in vaccines. IgA antibodies were observed in convalescent sera from two adults and may be presumptive evidence of infection with B. pertussis. PMID:6309662

  15. Seroprevalence of Antibodies to Pertussis Toxin among Different Age Groups in Thailand after 37 Years of Universal Whole-Cell Pertussis Vaccination.

    PubMed

    Wanlapakorn, Nasamon; Ngaovithunvong, Varisara; Thongmee, Thanunrat; Vichaiwattana, Preeyaporn; Vongpunsawad, Sompong; Poovorawan, Yong

    2016-01-01

    Despite the high coverage of prophylactic vaccine against Bordetella pertussis infection in many countries for more than three decades, pertussis remains a common vaccine-preventable disease. Infections have been detected more commonly in countries using acellular pertussis vaccine in their Expanded Program of Immunization. Thailand implemented a routine infant immunization program with whole-cell pertussis vaccine in 1977, and since 1992, the national vaccine policy has offered a five-dose whole-cell pertussis vaccine for children given at the ages of 2, 4, 6, 18, and 48 months. This study aimed to investigate the seroprevalence of antibodies to pertussis toxin among healthy people across all ages to determine the level of whole-cell vaccine-induced immunity in the population, and to identify which age group should be targeted for a booster dose. The lowest seronegative rate and highest geometric mean concentrations were found in the 0-10 years age group, corresponding to their recent pertussis vaccination. The proportion of people with undetectable IgG level was prominent, starting after 11 years of age onwards. Now that a reduced-dose pertussis vaccine with fewer adverse effects is available, a booster dose during adolescence should be considered in order to reduce the incidence of pertussis disease. Further studies exploring how long the reduced-dose pertussis vaccine can provide protective immunity against pertussis disease when administered to adults and adolescents should also be performed.

  16. Seroprevalence of Antibodies to Pertussis Toxin among Different Age Groups in Thailand after 37 Years of Universal Whole-Cell Pertussis Vaccination

    PubMed Central

    Wanlapakorn, Nasamon; Ngaovithunvong, Varisara; Thongmee, Thanunrat; Vichaiwattana, Preeyaporn; Vongpunsawad, Sompong; Poovorawan, Yong

    2016-01-01

    Despite the high coverage of prophylactic vaccine against Bordetella pertussis infection in many countries for more than three decades, pertussis remains a common vaccine-preventable disease. Infections have been detected more commonly in countries using acellular pertussis vaccine in their Expanded Program of Immunization. Thailand implemented a routine infant immunization program with whole-cell pertussis vaccine in 1977, and since 1992, the national vaccine policy has offered a five-dose whole-cell pertussis vaccine for children given at the ages of 2, 4, 6, 18, and 48 months. This study aimed to investigate the seroprevalence of antibodies to pertussis toxin among healthy people across all ages to determine the level of whole-cell vaccine-induced immunity in the population, and to identify which age group should be targeted for a booster dose. The lowest seronegative rate and highest geometric mean concentrations were found in the 0–10 years age group, corresponding to their recent pertussis vaccination. The proportion of people with undetectable IgG level was prominent, starting after 11 years of age onwards. Now that a reduced-dose pertussis vaccine with fewer adverse effects is available, a booster dose during adolescence should be considered in order to reduce the incidence of pertussis disease. Further studies exploring how long the reduced-dose pertussis vaccine can provide protective immunity against pertussis disease when administered to adults and adolescents should also be performed. PMID:26837004

  17. Pertussis toxin inhibits early chemokine production to delay neutrophil recruitment in response to Bordetella pertussis respiratory tract infection in mice.

    PubMed

    Andreasen, Charlotte; Carbonetti, Nicholas H

    2008-11-01

    Pertussis is an acute respiratory disease of humans caused by the bacterium Bordetella pertussis. Pertussis toxin (PT) plays a major role in the virulence of this pathogen, including important effects that it has soon after inoculation. Studies in our laboratory and other laboratories have indicated that PT inhibits early neutrophil influx to the lungs and airways in response to B. pertussis respiratory tract infection in mice. Previous in vitro and in vivo studies have shown that PT can affect neutrophils directly by ADP ribosylating G(i) proteins associated with surface chemokine receptors, thereby inhibiting neutrophil migration in response to chemokines. However, in this study, by comparing responses to wild-type (WT) and PT-deficient strains, we found that PT has an indirect inhibitory effect on neutrophil recruitment to the airways in response to infection. Analysis of lung chemokine expression indicated that PT suppresses early neutrophil recruitment by inhibiting chemokine upregulation in alveolar macrophages and other lung cells in response to B. pertussis infection. Enhancement of early neutrophil recruitment to the airways in response to WT infection by addition of exogenous keratinocyte-derived chemokine, one of the dominant neutrophil-attracting chemokines in mice, further revealed an indirect effect of PT on neutrophil chemotaxis. Additionally, we showed that intranasal administration of PT inhibits lipopolysaccharide-induced chemokine gene expression and neutrophil recruitment to the airways, presumably by modulation of signaling through Toll-like receptor 4. Collectively, these results demonstrate how PT inhibits early inflammatory responses in the respiratory tract, which reduces neutrophil influx in response to B. pertussis infection, potentially providing an advantage to the pathogen in this interaction.

  18. Pertussis

    PubMed Central

    Syed, Muhammad A.; Bana, Noureen F.

    2014-01-01

    Pertussis or whooping cough is a highly infectious, vaccine preventable disease. The incidence of the disease has greatly been reduced since the introduction of the diphtheria, tetanus, pertussis vaccine. Pertussis resurgence has been observed in highly vaccinated populations of Western countries since 1990s. Poor vaccine quality, waning vaccine induced immunity, pathogen adaptation, and enhanced surveillance as well as advancements in diagnostic facilities are some of the reasons considered responsible for the increased reporting of pertussis cases. Pertussis may have been ignored and unnoticed due to its atypical manifestations in partially immunized population or people with waning immunity. We review the reports of pertussis resurgence from different countries and attempt to investigate reasons behind the reappearance of the disease. Pertussis is still an under reported disease and the available data from the developing countries is not a true picture of the story. Therefore, developing countries need to improve their surveillance systems. PMID:25316461

  19. Toward a mechanism-based in vitro safety test for pertussis toxin.

    PubMed

    Vaessen, Stefan F C; Bruysters, Martijn W P; Vandebriel, Rob J; Verkoeijen, Saertje; Bos, Rogier; Krul, Cyrille A M; Akkermans, Arnoud M

    2014-01-01

    Pertussis vaccines are routinely administered to infants to protect them from whooping cough. Still, an adequate safety test for pertussis toxin (PT), one of the main antigens in these vaccines, is not available. The histamine sensitization test is currently the only assay accepted by regulatory authorities to test for the absence of active PT in vaccines. This is however, a lethal animal test with poor reproducibility. In addition, it is not clear whether the assumed underlying mechanism, i.e., ADP-ribosylation of G proteins, is the only effect that should be considered in safety evaluation of PT. The in vitro safety test for PT that we developed is based on the clinical effects of PT in humans. For this, human cell lines were chosen based on the cell types involved in the clinical effects of PT. These cell lines were exposed to PT and analyzed by microarray. In this review, we discuss the clinical effects of PT and the mechanisms that underlie them. The approach taken may provide as an example for other situations in which an in vitro assay based on clinical effects in humans is required.

  20. Toward a mechanism-based in vitro safety test for pertussis toxin

    PubMed Central

    Vaessen, Stefan FC; Bruysters, Martijn WP; Vandebriel, Rob J; Verkoeijen, Saertje; Bos, Rogier; Krul, Cyrille AM; Akkermans, Arnoud M

    2014-01-01

    Pertussis vaccines are routinely administered to infants to protect them from whooping cough. Still, an adequate safety test for pertussis toxin (PT), one of the main antigens in these vaccines, is not available. The histamine sensitization test is currently the only assay accepted by regulatory authorities to test for the absence of active PT in vaccines. This is however, a lethal animal test with poor reproducibility. In addition, it is not clear whether the assumed underlying mechanism, i.e., ADP-ribosylation of G proteins, is the only effect that should be considered in safety evaluation of PT. The in vitro safety test for PT that we developed is based on the clinical effects of PT in humans. For this, human cell lines were chosen based on the cell types involved in the clinical effects of PT. These cell lines were exposed to PT and analyzed by microarray. In this review, we discuss the clinical effects of PT and the mechanisms that underlie them. The approach taken may provide as an example for other situations in which an in vitro assay based on clinical effects in humans is required. PMID:24553455

  1. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha sub 1 -adrenergic system

    SciTech Connect

    Han, H.M.

    1989-01-01

    During development, the cardiac alpha{sub 1}-adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha{sub 1}-adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha{sub 1}-selective radioligand ({sup 125}I)-I-2-({beta}-(4-hydroxphenyl)ethylaminomethyl)tetralone (({sup 125}I)-IBE 2254) demonstrated that the alpha{sub 1}-adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha{sub 1}-adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha{sub 1}-adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha{sub 1}-mediated positive chronotropic response in the neonate, whereas in the adult the alpha{sub 1}-mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha{sub 1}-adrenergic receptor to its effector under certain circumstances such as high potassium (K{sup +}) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha{sub 1}-response.

  2. Concentrations of Immunoglobulin G Antibodies Against Pertussis Toxin Does Not Decrease Over a Long Period of Time in Japan.

    PubMed

    Sakakibara, Yumi; Ohtani, Yoshio; Jinta, Torahiko; Fujie, Toshihide; Miyazaki, Yasunari; Inase, Naohiko; Saito, Ryoichi; Akaza, Miho; Sasano, Tetsuo; Sumi, Yuki

    Objective Adult patients with pertussis rarely show typical symptoms, such as paroxysmal coughing, inspiratory "whoop", or post-tussive vomiting. While a culture is regarded as the gold standard for diagnosis, the sensitivity is very low. Therefore, the diagnosis of pertussis in adults in clinical practice is mostly based on single-sample serology using an enzyme-linked immunosorbent assay (ELISA) with the pertussis toxin antigen. Various cut-off values for the anti-pertussis toxin IgG (PT-IgG) have been proposed. It has been reported that concentrations of PT-IgG fall below the defined cut-off about 4.5 months after infection on average, and within 1 year in most patients. We investigated the distribution and time course of the PT-IgG levels. Methods The data were collected from the medical records. Patients The study retrospectively identified subjects who had visited Ikebukuro Otani Clinic, which is a specialized clinic for patients with cough. We retrospectively reviewed 406 patients with PT-IgG measurements to investigate the age distribution of PT-IgG levels. The changes in PT-IgG levels over time were assessed in the 205 patients who had more than one PT-IgG measurement. Results PT-IgG levels were ≥100 EU/mL in more than 15% of subjects. The PT-IgG levels of a few subjects had diminished over a long period of time. Conclusion A PT-IgG level greater than the defined cut-off value simply indicates past infection or immunization in most subjects. As such, a single measurement of PT-IgG using the cut-off values might lead to overdiagnosis of pertussis. Further data collection and analysis are required.

  3. Concentrations of Immunoglobulin G Antibodies Against Pertussis Toxin Does Not Decrease Over a Long Period of Time in Japan

    PubMed Central

    Sakakibara, Yumi; Ohtani, Yoshio; Jinta, Torahiko; Fujie, Toshihide; Miyazaki, Yasunari; Inase, Naohiko; Saito, Ryoichi; Akaza, Miho; Sasano, Tetsuo; Sumi, Yuki

    2016-01-01

    Objective Adult patients with pertussis rarely show typical symptoms, such as paroxysmal coughing, inspiratory “whoop”, or post-tussive vomiting. While a culture is regarded as the gold standard for diagnosis, the sensitivity is very low. Therefore, the diagnosis of pertussis in adults in clinical practice is mostly based on single-sample serology using an enzyme-linked immunosorbent assay (ELISA) with the pertussis toxin antigen. Various cut-off values for the anti-pertussis toxin IgG (PT-IgG) have been proposed. It has been reported that concentrations of PT-IgG fall below the defined cut-off about 4.5 months after infection on average, and within 1 year in most patients. We investigated the distribution and time course of the PT-IgG levels. Methods The data were collected from the medical records. Patients The study retrospectively identified subjects who had visited Ikebukuro Otani Clinic, which is a specialized clinic for patients with cough. We retrospectively reviewed 406 patients with PT-IgG measurements to investigate the age distribution of PT-IgG levels. The changes in PT-IgG levels over time were assessed in the 205 patients who had more than one PT-IgG measurement. Results PT-IgG levels were ≥100 EU/mL in more than 15% of subjects. The PT-IgG levels of a few subjects had diminished over a long period of time. Conclusion A PT-IgG level greater than the defined cut-off value simply indicates past infection or immunization in most subjects. As such, a single measurement of PT-IgG using the cut-off values might lead to overdiagnosis of pertussis. Further data collection and analysis are required. PMID:27853066

  4. Increased population prevalence of low pertussis toxin antibody levels in young children preceding a record pertussis epidemic in Australia.

    PubMed

    Campbell, Patricia; McIntyre, Peter; Quinn, Helen; Hueston, Linda; Gilbert, Gwendolyn L; McVernon, Jodie

    2012-01-01

    Cross-sectional serosurveys using IgG antibody to pertussis toxin (IgG-PT) are increasingly being used to estimate trends in recent infection independent of reporting biases. We compared the age-specific seroprevalence of various levels of IgG-PT in cross-sectional surveys using systematic collections of residual sera from Australian diagnostic laboratories in 1997/8, 2002 and 2007 with reference to both changes in the pertussis vaccine schedule and the epidemic cycle, as measured by disease notifications. A progressive decline in high-level (≥62.5 EU/ml) IgG-PT prevalence from 19% (95% CI 16-22%) in 1997/98 to 12% (95% CI 11-14%) in 2002 and 5% (95% CI 4-6%) in 2007 was consistent with patterns of pertussis notifications in the year prior to each collection. Concomitantly, the overall prevalence of undetectable (<5 EU/ml) levels increased from 17% (95% CI 14-20%) in 1997/98 to 38% (95% CI 36-40%) in 2007 but among children aged 1-4 years, from 25% (95% CI 17-34%) in 1997/98 to 62% (95% CI 56-68%) in 2007. This change followed withdrawal of the 18-month booster dose in 2003 and preceded record pertussis notifications from 2008 onwards. Population seroprevalence of high levels of IgG-PT is accepted as a reliable indicator of pertussis disease activity over time within and between countries with varying diagnostic practices, especially in unimmunised age groups. Our novel findings suggest that increased prevalence of undetectable IgG-PT is an indicator of waning immunity useful for population level monitoring following introduction of acellular vaccines and/or schedule changes.

  5. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

    PubMed Central

    Donato, Gina M.; Goldsmith, Cynthia S.; Paddock, Christopher D.; Eby, Joshua C.; Gray, Mary C.; Hewlett, Erik L.

    2012-01-01

    B.pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B.pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B.pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin’s receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. PMID:22289177

  6. Workshop on Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test.

    PubMed

    Bache, Christina; Hoonakker, Marieke; Hendriksen, Coenraad; Buchheit, Karl-Heinz; Spreitzer, Ingo; Montag, Thomas

    2012-07-01

    The Paul-Ehrlich-Institut (PEI), the Nederlands Vaccin Instituut (NVI) and the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised the international scientific workshop "Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test" at the PEI in Langen (Germany) on 09-10 June 2011. Twenty-seven experts (regulators, representatives from national control laboratories, vaccine manufacturers and academia) from 7 countries participated in this workshop. The meeting was triggered by the lack of satisfaction with the current safety testing for acellular pertussis vaccines, the "Histamine Sensitisation Test" (HIST) in mice, and the growing attention for the alternatives under development. The workshop objectives were: a) to review the current status of available alternative methods, b) to discuss the sensitivity that an alternative test needs, c) to plan experiments that allow for comparison of the alternative tests. The results of the workshop are summarised in this meeting report.

  7. Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells.

    PubMed Central

    Dickenson, J. M.; Hill, S. J.

    1995-01-01

    1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 n

  8. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

    PubMed

    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

    2015-12-02

    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.

  9. Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

    PubMed Central

    Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Devoisselle, Jean-Marie; Ladant, Daniel; Chopineau, Joel

    2013-01-01

    Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study’s in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions. PMID:24297899

  10. Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function

    PubMed Central

    Millen, Scott H.; Watanabe, Mineo; Komatsu, Eiji; Yamaguchi, Fuminori; Nagasawa, Yuki; Suzuki, Eri; Monaco, Haleigh; Weiss, Alison A.

    2015-01-01

    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. PMID:26375454

  11. Seroprevalence of IgG antibodies to pertussis toxin in children and adolescents in Estonia.

    PubMed

    Jõgi, Piia; Oona, Marje; Toompere, Karolin; Leedo, Sirje; Epstein, Jevgenia; Lutsar, Irja

    2014-09-15

    Despite high immunisation coverage and frequent booster doses, the national notification rates of pertussis in Estonia have been increasing. The peak of 97/100,000 was reached in 2010 which is the highest incidence rate since 1962 (210/100,000). We aimed to measure the prevalence of pertussis toxin (PT) IgG type antibodies in subjects of <18 years and to estimate the pertussis infection activity in a recently non-immunised cohort. In a cross-sectional serosurvey, all consecutive leftover sera were collected in the Tartu University Hospital during April-August 2012. Anti-PT IgG concentration was measured by commercial ELISA and analysed in yearly cohorts. The antibody concentrations ≥62.5 IU/mL was considered suggestive to pertussis in the last year among 9- to 14-year-olds. The GMC of the anti-PT-IgG was 7.4 IU/mL (95% CI 6.9-8.0). In the total of 1053 serum samples, the highest proportion of sera with high antibody titres ≥125 IU/mL and ≥62.5 IU/mL were at the ages when pertussis vaccine boosters were given: 7 years 10.9% (95% CI 4.1-22.3) and 2 years 36.9% (95% CI 25.3-49.8), respectively. Approximately half of all sera had undetectable anti-PT IgG levels. The estimated incidence of Bordetella pertussis infection among 9- to 14-year-olds in the year before serum sampling was 6.3% (95% CI 3.3-10.8), which is at least 60 times higher than the officially reported incidence of pertussis disease in respective years. The serologic method is not suitable for diagnosing pertussis in instances when the last pertussis immunisation was less than one year ago. The relatively high proportion of subjects with undetectable anti-PT IgG levels and the relatively low rate of officially reported pertussis cases suggest that low antibody levels do not necessarily indicate the absence of protection. The estimated incidence rate of pertussis is much higher than officially reported figures, which suggests that asymptomatic/mild B. pertussis infection remains unrecognised and

  12. Monoclonal antibodies that inhibit ADP-ribosyltransferase but not NAD-glycohydrolase activity of pertussis toxin.

    PubMed Central

    Kaslow, H R; Schlotterbeck, J D; Kenimer, J G

    1990-01-01

    Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identified three classes of monoclonal antibodies, termed A, B, and C, that recognize the S1 subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the S1 subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or S1 subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native S1 proteins and fails to distinguish between neutralizing and nonneutralizing antibodies. Images PMID:2155182

  13. Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.

    PubMed

    Hewlett, E L; Gray, M C; Ehrmann, I E; Maloney, N J; Otero, A S; Gray, L; Allietta, M; Szabo, G; Weiss, A A; Barry, E M

    1993-04-15

    Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin

  14. Genetically detoxified pertussis toxin (PT-9K/129G): implications for immunization and vaccines.

    PubMed

    Seubert, Anja; D'Oro, Ugo; Scarselli, Maria; Pizza, Mariagrazia

    2014-10-01

    Pertussis toxin (PT) is one of the major virulence factors of Bordetella pertussis and the primary component of all pertussis vaccines available to date. Because of its various noxious effects the toxin needs to be detoxified. In all currently available vaccines, detoxification is achieved by treatment with high quantity of chemical agents such as formaldehyde, glutaraldehyde or hydrogen peroxide. Although effective in detoxification, this chemical treatment alters dramatically the immunological properties of the toxin. In contrast, PT genetically detoxified through the substitution of two residues necessary for its enzymatic activity maintains all functional and immunological properties. This review describes in detail the characteristics of this PT-9K/129G mutant and shows that it is non-toxic and a superior immunogen compared with chemically detoxified PT. Importantly, data from an efficacy trial show that the PT-9K/129G-based vaccine induces earlier and longer-lasting protection, further supporting the hypothesis that PT-9K/129G represents an ideal candidate for future pertussis vaccine formulations.

  15. Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD

    SciTech Connect

    Barbieri, J.T.; Mende-Mueller, L.M.; Rappuoli, R.; Collier, R.J. )

    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. Differences in avidity of IgG antibodies to pertussis toxin after acellular pertussis booster vaccination and natural infection.

    PubMed

    Barkoff, Alex-Mikael; Gröndahl-Yli-Hannuksela, Kirsi; Vuononvirta, Juho; Mertsola, Jussi; Kallonen, Teemu; He, Qiushui

    2012-11-06

    Pertussis toxin (PT) is a specific virulence factor of Bordetella pertussis and it is included in all acellular pertussis vaccines (aP). Although immunity after infection seems to persist longer than that after vaccination, the exact mechanism(s) is not known. Primary aim of this study was to develop an ELISA method for measuring avidity index (AI) of IgG-anti-PT antibodies and to compare antibody responses after booster vaccination and infection. Secondary aim was to evaluate if the AI-ELISA has potential in the serodiagnosis of pertussis. Serum samples from a total of 409 subjects were included in the study. Paired sera were taken from 97 adolescents who received booster vaccine ten years ago (dTpa-004) and from 80 young adults who received a second booster dose ten years after the previous booster vaccine (dTpa-040). Thirty-two paired sera from culture-confirmed pertussis patients, 161 single sera from serologically diagnosed patients and 39 single sera from healthy controls were included. AI of IgG-anti-PT antibodies were determined with newly developed ELISA using diethylamine (DEA) as a bond breaking agent. The IgG-anti-PT antibodies were measured by standardized ELISA. A significant increase was found in antibody concentrations and AI between PRE and one month POST vaccination ten years ago [GMC for antibody: 7.9 IU/ml vs. 98.3 IU/ml (p=0.0001); for AI: 40.4% vs. 56.1% (p=0.0001)]. Similar result was observed after the second booster dose [GMC for antibody: 9.2 IU/ml vs. 92.4 IU/ml (p=0.0001); for AI: 36.1% vs. 59.5% (p=0.0001)] and between the first and second sera of culture-confirmed patients [GMC for antibody: 6.9 IU/ml vs. 285.1 IU/ml (p=0.0001); for AI: 40.5% vs. 68.4% (p=0.0001)]. Healthy controls showed lower levels of both antibodies and AI. Our results suggest that there may be difference in quality and quantity of antibodies to PT after vaccination and after infection. Furthermore, AI could be a help for vaccine studies. Copyright © 2012 Elsevier

  17. Pertussis

    PubMed Central

    Gabutti, Giovanni; Azzari, Chiara; Bonanni, Paolo; Prato, Rosa; Tozzi, Alberto E; Zanetti, Alessandro; Zuccotti, Gianvincenzo

    2014-01-01

    Pertussis continues to be an important public-health issue. The high immunization coverage rates achieved, mainly in industrialized countries, have certainly decreased the spread of the pathogen. However, as immunity wanes, adolescents and adults play an important role in the dynamics of the infection. The surveillance system has several limitations and the underestimation of pertussis in adolescents, young adults and adults is mainly related to the atypical clinical characteristics of cases and the lack of lab confirmation. The real epidemiological impact of pertussis is not always perceived. The unavailability of comprehensive data should not hamper the adoption of active prophylactic measures designed to avoid the impact of waning immunity against pertussis. Different immunization strategies have been suggested and/or already adopted such as immunization of newborns, pre-school and school children, adolescents, adults, healthcare workers, childcare workers, pregnant women, cocoon strategy. Prevention of pertussis requires an integrated approach and the adoption of different immunization strategies, with the objective of achieving and maintaining high coverage rates. PMID:25483523

  18. Seroprevalence of IgG antibodies to pertussis toxin in the Slovene population.

    PubMed

    Socan, Maja; Prosenc, Katarina; Vegnuti, Miljana

    2006-06-01

    The use of pertussis vaccines has reduced the morbidity and mortality of whooping cough. Immunity following the natural disease or vaccination is not life-long and reinfections causing an increase of pertussis antibodies can occur. In this study, the distribution of IgG antibodies to pertussis toxin (anti-PT IgG) among different age groups in Slovenia was determined. The seroprevalence of anti-PT IgG antibodies to Bordetella pertussis was investigated in 3418 persons (49.1% males). The population under study was stratified into 27 age groups. The serological results were assigned to five groups, according to their titer levels. The geometric mean titers (GMT) were calculated. In 11.5% sera tested, no IgG antibodies to pertussis toxin were detected. High titers (> or =125 U/ml) were confirmed in 2.3% sera. There were no statistically significant differences between age groups in the proportion of antibody levels. Pre-school children from three to five years of age had the lowest anti-PT IgG GMTs (9.6-10.7 U/ml). Vaccinated children (aged from one to two years) and adolescents from 17-18 years of age had the highest GMTs (>20 U/ml). GMTs were not statistically significantly different between males and females. The study demonstrated an early decline of anti-PT IgG after vaccination. According to the serological profile, school-age children and adolescents have the highest rate of infection. The large proportion of seropositive adults indicates that reinfection with B. pertussis is relatively common.

  19. Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo.

    PubMed

    Wagner, Ellen K; Wang, Xianzhe; Bui, Andre; Maynard, Jennifer A

    2016-11-01

    Bispecific antibodies are a rapidly growing class of therapeutic molecules, originally developed for the treatment of cancer but recently explored for the treatment of autoimmune and infectious diseases. Bordetella pertussis is a reemerging pathogen, and several of the key symptoms of infection are caused by the pertussis toxin (PTx). Two humanized antibodies, hu1B7 and hu11E6, bind distinct epitopes on PTx and, when coadministered, mitigate disease severity in murine and baboon models of infection. Here we describe the generation of a bispecific human IgG1 molecule combining the hu1B7 and hu11E6 binding sites via a knobs-in-holes design. The bispecific antibody showed binding activity equivalent to that of the antibody mixture in a competition enzyme-linked immunosorbent assay (ELISA). A CHO cell neutralization assay provided preliminary evidence for synergy between the two antibodies, while a murine model of PTx-induced leukocytosis definitively showed synergistic neutralization. Notably, the bispecific antibody retained the synergy observed for the antibody mixture, supporting the conclusion that synergy is due to simultaneous blockade of both the catalytic and receptor binding activities of pertussis toxin. These data suggest that a hu1B7/hu11E6 bispecific antibody is a viable alternative to an antibody mixture for pertussis treatment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Cyclic AMP-Mediated Suppression of Neutrophil Extracellular Trap Formation and Apoptosis by the Bordetella pertussis Adenylate Cyclase Toxin

    PubMed Central

    Gray, Mary C.; Hewlett, Erik L.

    2014-01-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis intoxicates target cells by generating supraphysiologic levels of intracellular cyclic AMP (cAMP). Since ACT kills macrophages rapidly and potently, we asked whether ACT would also kill neutrophils. In fact, ACT prolongs the neutrophil life span by inhibiting constitutive apoptosis and preventing apoptosis induced by exposure to live B. pertussis. Imaging of B. pertussis-exposed neutrophils revealed that B. pertussis lacking ACT induces formation of neutrophil extracellular traps (NETs), whereas wild-type B. pertussis does not, suggesting that ACT suppresses NET formation. Indeed, ACT inhibits formation of NETs by generating cAMP and consequently inhibiting the oxidative burst. Convalescent-phase serum from humans following clinical pertussis blocks the ACT-mediated suppression of NET formation. These studies provide novel insight into the phagocyte impotence caused by ACT, which not only impairs neutrophil function but also inhibits death of neutrophils by apoptosis and NETosis. PMID:25287922

  1. Low pertussis toxin antibody levels in two regional cohorts of Canadian pregnant women.

    PubMed

    Bigham, Mark; Konrad, Stephanie; Van Buynder, Paul; Van Buynder, Jan; Isaac-Renton, Judy; ElSherif, May; Halperin, Scott A

    2014-11-12

    Infants under 6 months of age are at greatest risk of mortality and severe morbidity from pertussis disease. Interventions that increase pertussis protection in newborns are therefore a clear public health imperative. The objective of this study was to assess maternal pertussis toxin antibody (anti-PT) level as a potential source of mother-to-child transfer of pertussis-associated antibodies that may reduce neonatal risk of pertussis disease. Anti-PT level was assessed in a 2013 cohort of pregnant women from two regions in two Canadian provinces, British Columbia and Nova Scotia. Basic demographics, health, and pertussis immunization history were collected, along with blood specimens. Anti-PT levels were compared for self-reported vaccination status and prior pertussis disease. To assess secular trend, a parallel analysis was also undertaken, using anonymized residual sera from a 1996-1997 cohort of pregnant women in British Columbia. A total of 169 pregnant women participated in the study - 50 from Nova Scotia and 119 from British Columbia. The mean and median age of participants from both sites was 31 years of age (range 16-42 years). The lower limit of quantification of the anti-PT assay was 10 ELISA units per milliliter (EU/ml). Overall, 59% of women had anti-PT levels less than 10 EU/ml and anti-PT level did not differ with time since last self-reported pertussis vaccination (χ(2)(2)=3.166, p=0.205). Among a 1996-1997 cohort of pregnant women in British Columbia, 101 of 200 (51%) had anti-PT levels less than 10 EU/ml. Our study found that most pregnant women in two geographically disparate health regions in Canada have low residual anti-PT levels, may be vulnerable to pertussis infection themselves, and would unlikely be a source of passive ante- or postnatal transfer of anti-PT to their newborn. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Role of pertussis toxin A subunit in neutrophil migration and vascular permeability.

    PubMed Central

    Brito, G A; Souza, M H; Melo-Filho, A A; Hewlett, E L; Lima, A A; Flores, C A; Ribeiro, R A

    1997-01-01

    The anti-inflammatory activity of pertussis toxin (Ptx) was compared to that of a noncatalytic mutant of pertussis toxin (9K/129G; Ptxm), which contains two amino acid substitutions in the A protomer, by using a rat model of inflammation. The toxins were administered intravenously 1 h prior to the injection of inflammatory stimuli. Ptx, but not Ptxm, inhibited neutrophil migration into peritoneal cavities in response to formyl-methionyl-leucyl-phenylalanine and lipopolysaccharide. The inhibitory effect of Ptx on neutrophil migration could not be explained by the ability of the toxin to induce leukopenia or neutropenia. The increase in skin vascular permeability induced by leukotriene B4, a powerful neutrophil chemotactic agent, was also inhibited only by Ptx. On the other hand, the increase in skin vascular permeability induced by histamine was potentiated by both toxins. These data show that Ptx inhibits neutrophil-mediated inflammation in vivo and that this effect is dependent on the ADP-ribosyltransferase activity of the A protomer. PMID:9038326

  3. Quantification of the adenylate cyclase toxin of Bordetella pertussis in vitro and during respiratory infection.

    PubMed

    Eby, Joshua C; Gray, Mary C; Warfel, Jason M; Paddock, Christopher D; Jones, Tara F; Day, Shandra R; Bowden, James; Poulter, Melinda D; Donato, Gina M; Merkel, Tod J; Hewlett, Erik L

    2013-05-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ≈ 10(8) CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ≈ 10(8) CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ≈ 60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

  4. Quantification of the Adenylate Cyclase Toxin of Bordetella pertussis In Vitro and during Respiratory Infection

    PubMed Central

    Eby, Joshua C.; Gray, Mary C.; Warfel, Jason M.; Paddock, Christopher D.; Jones, Tara F.; Day, Shandra R.; Bowden, James; Poulter, Melinda D.; Donato, Gina M.; Merkel, Tod J.

    2013-01-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis. PMID:23429530

  5. Adenylate cyclase toxin (ACT) from Bordetella hinzii: characterization and differences from ACT of Bordetella pertussis.

    PubMed

    Donato, Gina M; Hsia, Hung-Lun J; Green, Candace S; Hewlett, Erik L

    2005-11-01

    Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism.

  6. Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose

    SciTech Connect

    Armstrong, G.D.; Peppler, M.S.

    1987-05-01

    We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigations into the nature of the interaction and activity of PT in host tissues.

  7. Pertussis toxin-insensitive effects of mastoparan, a wasp venom peptide, in PC12 cells.

    PubMed

    Murayama, T; Oda, H; Nomura, Y

    1996-12-01

    Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, modifies the secretion of neurotransmitters and hormones from a variety of cell types. Mastoparan interacts with heterotrimeric guanine nucleotide-binding proteins (G proteins) such as Gi and G(o), which are ADP-ribosylated by pertussis toxin (PTX) and thereby uncoupled from receptors. Previously, some of the effects of mastoparan including secretion were reported to be modified selectively by PTX but not by cholera toxin (CTX). In the present study, we examined the influence of bacterial toxins on the effects of mastoparan in PC12 cells. Mastoparan stimulated [3H]noradrenaline (NA) release from prelabeled PC12 cells in the absence of CaCl2, although high K+ or ATP-stimulated the release in a Ca(2+)-dependent manner. Pretreatment with CTX, not PTX, for 24 h inhibited mastoparan-stimulated [3H]NA release. Mastoparan inhibited forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner, although mastoparan had no effect by itself. Pretreatment with PTX completely abolished the inhibitory effect of carbachol via Gi on cyclic AMP accumulation and partially reduced the effect of mastoparan. However, the inhibitory effect of 20 microM mastoparan was not modified by pretreatment with PTX. Thus, we investigated the effect of mastoparan on CTX-catalyzed [32P]ADP-ribosylation of proteins in PC12 cells. A subunit of CTX (CTX-A) catalyzed [32P]ADP-ribosylation of many proteins in the cytosolic fraction of PC12 cells. One of these was a 20 kDa protein, named ADP-ribosylating factor (ARF). The addition of mastoparan to assay mixtures inhibited ADP-ribosylation of many proteins including ARF and CTX-A in the presence of the cytosolic fraction. In the absence of the cytosolic fraction, however, mastoparan slightly enhanced ADP-ribosylation of bovine serum albumin and auto-ADP-ribosylation by CTX-A. Mastoparan did not inhibit ADP-ribosylation of the alpha subunit of Gs in the

  8. [Humoral reaction to Bordetella pertussis antigens: pertussis toxin, filamentous hemagglutinin and lipopolysaccharide in children with clinical symptoms of whooping cough. II. Occurence and level of B. pertussis antigens in children with suspected whooping cough].

    PubMed

    Chodorowska, M

    1999-01-01

    The aim of this study was to determine and evaluate IgG, IgM and IgA levels to pertussis toxin (PT), filamentous hemagglutinin (FHA) and endotoxin (LPS) of B. pertussis in children with clinical symptoms of whooping cough. The serum samples obtained from 265 children (age range: 2 months-16 years) suspected of pertussis were examined by indirect haemagglutination (IH) and ELISA tests. Higher antibody level was most frequently observed in IgA class to PT, FHA and LPS in 45.3%, 35.1% and 66% of pertussis patients sera respectively. The least positive results were obtained in IgM class to PT and FHA (in 9.8% and 2.6% of children sera respectively) but in the case of LPS applied as the antigen in ELISA, higher IgM level was determined in 46.8% of pertussis patients sera. The four times increase of antibody level to LPS determined by IH was observed in 86.7% of children suspected of pertussis. Humoral response to B. pertussis infection is mainly connected with higher IgA level to PT, FHA, LPS and IgM to LPS in children with clinical symptoms of whooping cough.

  9. Inhibition of epithelial Na sup + transport by atriopeptin, protein kinase c, and pertussis toxin

    SciTech Connect

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A. )

    1987-08-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na{sup +} by atrial natriuretic peptide and 8-bromoguanosine 3{prime},5{prime}-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK{sub i}. Using {sup 22}Na{sup +} fluxes, they further investigated the modulation of Na{sup +} transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na{sup +} uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na{sup +} uptake by 93 {plus minus} 13 and 51 {plus minus} 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK{sub i} cells, inhibits {sup 22}Na{sup +} influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na{sup +} uptake. These events may be sequentially involved in the action of atrial natriuretic peptide.

  10. Differential effects of pertussis toxin on insulin-stimulated phosphatidylcholine hydrolysis and glycerolipid synthesis de novo. Studies in BC3H-1 myocytes and rat adipocytes

    SciTech Connect

    Hoffman, J.M.; Standaert, M.L.; Nair, G.P.; Farese, R.V. )

    1991-04-02

    Insulin-induced increases in diacylglycerol (DAG) have been suggested to result from stimulation of de novo phosphatidic acid (PA) synthesis and phosphatidylcholine (PC) hydrolysis. Presently, the authors found that insulin decreased PC levels of BC3H-1 myocytes and rat adipocytes by approximately 10-25% within 30 s. These decreases were rapidly reversed in both cell types, apparently because of increased PC synthesis de novo. In BC3H-1 myocytes, pertussis toxin inhibited PC resynthesis and insulin effects on the pathway of de novo PA-DAG-PC synthesis, as evidenced by changes in ({sup 3}H)glycerol incorporation, but did not inhibit insulin-stimulated PC hydrolysis. Pertussis toxin also blocked the later, but not the initial, increase in DAG production in the myocytes. Phorbol esters activated PC hydrolysis in both myocytes and adipocytes, but insulin-induced stimulation of PC hydrolysis was not dependent upon activation of PKC, since this hydrolysis was not inhibited by 500 {mu}M sangivamycin, an effective PKC inhibitor. The results indicate that insulin increases DAG by pertussis toxin sensitive and insensitive (PC hydrolysis) mechanisms, which are mechanistically separate, but functionally interdependent and integrated. PC hydrolysis may contribute importantly to initial increases in DAG, but later sustained increases are apparently largely dependent on insulin-induced stimulation of the pathway of de novo phospholipid synthesis.

  11. Essential role of the consensus nucleotide-binding site of PtlH in secretion of pertussis toxin from Bordetella pertussis.

    PubMed Central

    Kotob, S I; Burns, D L

    1997-01-01

    PtlH is a member of a specialized set of transport proteins that is essential for secretion of pertussis toxin (PT) from Bordetella pertussis. Previously, PtlH was shown to contain a consensus nucleotide-binding motif. Here, we demonstrate that introduction of plasmids containing mutant forms of ptlH, altered in the putative nucleotide-binding region, into a wild-type strain of B. pertussis resulted in inhibition of PT secretion. Thus, this region of PtlH appears to be essential for protein function. Moreover, the observed dominant negative phenotype suggests that PtlH either functions as a multimer or interacts with another component necessary for secretion of PT. PMID:9393726

  12. Pertussis Toxin Is a Robust and Selective Inhibitor of High Grade Glioma Cell Migration and Invasion

    PubMed Central

    Wang, Lei; Natali, Letizia; Karimi-Mostowfi, Nicki; Brifault, Coralie; Gonias, Steven L.

    2016-01-01

    In high grade glioma (HGG), extensive tumor cell infiltration of normal brain typically precludes identifying effective margins for surgical resection or irradiation. Pertussis toxin (PT) is a multimeric complex that inactivates diverse Gi/o G-protein coupled receptors (GPCRs). Despite the broad continuum of regulatory events controlled by GPCRs, PT may be applicable as a therapeutic. We have shown that the urokinase receptor (uPAR) is a major driver of HGG cell migration. uPAR-initiated cell-signaling requires a Gi/o GPCR, N-formyl Peptide Receptor 2 (FPR2), as an essential co-receptor and is thus, PT-sensitive. Herein, we show that PT robustly inhibits migration of three separate HGG-like cell lines that express a mutated form of the EGF Receptor (EGFR), EGFRvIII, which is constitutively active. PT also almost completely blocked the ability of HGG cells to invade Matrigel. In the equivalent concentration range (0.01–1.0 μg/mL), PT had no effect on cell survival and only affected proliferation of one cell line. Neutralization of EGFRvIII expression in HGG cells, which is known to activate uPAR-initiated cell-signaling, promoted HGG cell migration. The increase in HGG cell migration, induced by EGFRvIII neutralization, was entirely blocked by silencing FPR2 gene expression or by treating the cells with PT. When U87MG HGG cells were cultured as suspended neurospheres in serum-free, growth factor-supplemented medium, uPAR expression was increased. HGG cells isolated from neurospheres migrated through Transwell membranes without loss of cell contacts; this process was inhibited by PT by >90%. PT also inhibited expression of vimentin by HGG cells; vimentin is associated with epithelial-mesenchymal transition and worsened prognosis. We conclude that PT may function as a selective inhibitor of HGG cell migration and invasion. PMID:27977780

  13. Effect of pertussis toxin pretreated centrally on blood glucose level induced by stress

    PubMed Central

    Suh, Hong-Won; Sim, Yun-Beom; Park, Soo-Hyun; Sharma, Naveen; Im, Hyun-Ju

    2016-01-01

    In the present study, we examined the effect of pertussis toxin (PTX) administered centrally in a variety of stress-induced blood glucose level. Mice were exposed to stress after the pretreatment of PTX (0.05 or 0.1 µg) i.c.v. or i.t. once for 6 days. Blood glucose level was measured at 0, 30, 60 and 120 min after stress stimulation. The blood glucose level was increased in all stress groups. The blood glucose level reached at maximum level after 30 min of stress stimulation and returned to a normal level after 2 h of stress stimulation in restraint stress, physical, and emotional stress groups. The blood glucose level induced by cold-water swimming stress was gradually increased up to 1 h and returned to the normal level. The intracerebroventricular (i.c.v.) or intrathecal (i.t.) pretreatment with PTX, a Gi inhibitor, alone produced a hypoglycemia and almost abolished the elevation of the blood level induced by stress stimulation. The central pretreatment with PTX caused a reduction of plasma insulin level, whereas plasma corticosterone level was further up-regulated in all stress models. Our results suggest that the hyperglycemia produced by physical stress, emotional stress, restraint stress, and the cold-water swimming stress appear to be mediated by activation of centrally located PTX-sensitive G proteins. The reduction of blood glucose level by PTX appears to due to the reduction of plasma insulin level. The reduction of blood glucose level by PTX was accompanied by the reduction of plasma insulin level. Plasma corticosterone level up-regulation by PTX in stress models may be due to a blood glucose homeostatic mechanism. PMID:27610033

  14. Pertussis-induced cough.

    PubMed

    Wang, Kay; Harnden, Anthony

    2011-06-01

    Pertussis (whooping cough) is one of the commonest vaccine preventable diseases in the UK, despite vaccination coverage being maintained for the last 15 years at over 90% among infants and the addition of a pre-school booster to the UK national immunisation programme in 2001. However, it is known that pertussis vaccine does not confer long-term immunity to clinical infection. Evidence of pertussis infection has been reported in 37% of children presenting in UK primary care and 20% of adolescents and adults presenting in Canadian health centres with persistent cough. In children and adults with persistent cough, paroxysmal coughing is the most sensitive indicator of pertussis, but has poor specificity and limited diagnostic value. Vomiting and whooping, particularly in combination, are stronger predictors of pertussis. Cough duration is longer in children than in adults with pertussis (median cough duration 112 days versus 42 days); individuals may take even longer to recover fully and regain previous levels of exercise tolerance. A diagnosis of pertussis may be confirmed by culture, Polymerase Chain Reaction (PCR) or serology. Single estimates of anti-pertussis toxin (PT) antibody titres in blood or oral fluid samples are highly specific. There are currently no proven efficacious treatments for pertussis-induced cough. Treatment with macrolide antibiotics reduces the duration of an individual's infectious period, but does not alter the duration of cough. Further research is needed to re-examine the epidemiology of pertussis in countries with different vaccination schedules, find efficacious treatments and develop methods of measuring cough frequency and severity in patients with pertussis-induced cough. Copyright © 2010. Published by Elsevier Ltd.

  15. 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.

    PubMed

    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.

  16. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

    PubMed Central

    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.

    2016-01-01

    In spite of wide-spread vaccination, pertussis rates are rising in industrialized countries and remain high world-wide. 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. Here, we humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human IgG1 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 B. pertussis-induced rise in white blood cell count and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated but not control animals experienced a blunted rise in white blood cell count 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. PMID:26631634

  17. Effect of different detoxification procedures on the residual pertussis toxin activities in vaccines.

    PubMed

    Yuen, Chun-Ting; Asokanathan, Catpagavalli; Cook, Sarah; Lin, Naomi; Xing, Dorothy

    2016-04-19

    Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and its detoxified form is one of the major protective antigens in vaccines against whooping cough. Ideally, PTx in the vaccine should be completely detoxified while still preserving immunogenicity. However, this may not always be the case. Due to multilevel reaction mechanisms of chemical detoxification that act on different molecular sites and with different production processes, it is difficult to define a molecular characteristic of a pertussis toxoid. PTx has two functional distinctive domains: the ADP-ribosyltransferase enzymatic subunit S1 (A-protomer) and the host cell binding carbohydrate-binding subunits S2-5 (B-oligomer); and in this study, we investigated the effect of different detoxification processes on these two functional activities of the residual PTx in toxoids and vaccines currently marketed worldwide using a recently developed in vitro biochemical assay system. The patho-physiological activities in these samples were also estimated using the in vivo official histamine sensitisation tests. Different types of vaccines, detoxified by formaldehyde, glutaraldehyde or by both, have different residual functional and individual baseline activities. Of the vaccines tested, PT toxoid detoxified by formaldehyde had the lowest residual PTx ADP-ribosyltransferase activity. The carbohydrate binding results detected by anti-PTx polyclonal (pAb) and anti-PTx subunits monoclonal antibodies (mAb) showed specific binding profiles for toxoids and vaccines produced from different detoxification methods. In addition, we also demonstrated that using pAb or mAb S2/3 as detection antibodies would give a better differential difference between these vaccine lots than using mAbs S1 or S4. In summary, we showed for the first time that by measuring the activities of the two functional domains of PTx, we could characterise pertussis toxoids prepared from different chemical detoxification

  18. [Serum immunoglobulin IgG subclass distribution of antibody responses to pertussis toxin and filamentous hemagglutinin of Bordetella pertussis in patients with whooping cough].

    PubMed

    Rastawicki, Waldemar; Smietańska, Karolina; Rokosz-Chudziak, Natalia; Jagielski, Marek

    2013-01-01

    The present study was aimed at determining the IgG subclass distribution against pertussis toxin (PT) and filamentous hemagglutinin (FHA) of Bordetella pertussis in patients with whooping cough. The total number of 222 serum samples obtained from patients suspected in clinical investigation for pertussis were tested separately by in-house ELISA for the presence of IgG antibodies to pertussis toxin and filamentous hemagglutinin. The percentage distribution of specific anti-PT and anti-FHA IgG subclass response was calculated only on the basis of group of sera confirmed in the present study as positive for total IgG antibodies (183 sera to PT antigen and 129 to FHA antigen). Paired serum specimens were obtained from 36 patients. Based on the results of determining the level of antibodies in the sera of 40 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus two standard deviations. Antibodies of IgG1 to pertussis toxin and filamentous hemagglutinin were diagnosed in 151 (82.5%) and 99 (76.7%), IgG2 in 72 (39.0%) and 50 (38.8%), IgG3 in 17 (9.3%) and 43 (33.3%), IgG4 in 55 (30.1%) and 53 (41.1%) serum samples, respectively. There were no significant differences in percentage of sera with IgG1, IgG2 and IgG3 in relation to age of the patients. However, the frequency of occurrence of IgG4 antibodies was highest in the group of the youngest children to the age of 6 years old (61.8% for PT and 68.0% for FHA), and decrease with age, reaching the minimum in the group of patients above 40 years old (13.2% and 4.2% for PT and FHA, respectively). We also found significantly higher frequency of IgG4 to PT and FHA antigens in men than in women. Statistically significant, essential changes in the pattern of IgG subclass during the course of infection were not found. In conclusion, this study showed that all four subclasses of IgG antibodies to pertussis toxin and filamentous hemagglutinin are produced during whooping cough.

  19. Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

    SciTech Connect

    Kim, M.H.; Neubig, R.R.

    1986-03-05

    High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonist (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.

  20. Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence.

    PubMed Central

    Gross, M K; Au, D C; Smith, A L; Storm, D R

    1992-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. One of these putative virulence factors is the adenylate cyclase (AC) toxin that elevates intracellular cAMP in eukaryotic cells to cytotoxic levels. This toxin is a bifunctional protein comprising both AC and hemolysin (HLY) enzymatic domains. The gene encoding the AC toxin (cyaA) is expressed as part of an operon that includes genes required for secretion or activation of the toxin. Because of this genetic organization, it is difficult to create B. pertussis mutants of cyaA that are ablations of a single enzyme function by conventional means, such as transposon mutagenesis. Therefore, to clarify the role of individual toxin functions in the virulence of B. pertussis, we have used site-directed or deletion mutagenesis and genetic recombination to specifically target the cyaA gene of B. pertussis to produce mutants that lack only the AC or HLY activity of this toxin. A point mutant of B. pertussis with abolished AC catalytic activity was greater than 1000 times less pathogenic to newborn mice than wild-type bacteria, directly demonstrating the importance of the AC toxin in pertussis virulence. Similarly, an in-frame deletion mutant of B. pertussis that lacks HLY is equally avirulent, supporting observations that the HLY domain plays a critical role in AC toxin entry into cells. Furthermore, the genetically inactivated AC toxin produced by the point mutant is antigenically similar to the native toxin, suggesting that this strain may be useful in the development of pertussis component vaccines. Images PMID:1594590

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

    PubMed

    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.

  2. Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

    SciTech Connect

    Mahy, N.; Woolkalis, M.; Thermos, K.; Carlson, K.; Manning, D.; Reisine, T.

    1988-08-01

    The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog (125I)CGP 23996. (125I)CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was to a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity (125I)CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of (125I)CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect (125I)CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with (125I) CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to (125I)CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors.

  3. Bordetella pertussis infection of human respiratory epithelial cells up-regulates intercellular adhesion molecule-1 expression: role of filamentous hemagglutinin and pertussis toxin.

    PubMed

    Ishibashi, Yoshio; Nishikawa, Akemi

    2002-09-01

    Adhesion molecules on respiratory epithelial cells play a critical role in inflammatory cell recruitment and accumulation at sites of inflammation. Bordetella pertussis colonizes the human respiratory tract by infecting epithelial cells, leading to an inflammatory response. In this study, the role of bacterial factors in the expression of intercellular adhesion molecule-1 (ICAM-1) on human respiratory epithelial cells was investigated in response to B. pertussis. Flow cytometry and real time RT-PCR analysis showed that BEAS-2B human bronchial epithelial cells expressed increased levels of ICAM-1 mRNA and surface protein in response to B. pertussis infection. Filamentous hemagglutinin (FHA) played a role in this response because of the impaired capability of a FHA-deficient isogenic strain. A mutant strain in which an Arg-Gly-Asp (RGD) site of FHA had been changed to Arg-Ala-Asp had diminished ability to up-regulate ICAM-1 expression. RGD sequence-associated up-regulation of ICAM-1 expression was also observed in primary normal human bronchial epithelial cells. Pretreatment of cells with integrin antagonists such as RGD-containing peptide and antibody against very late antigen-5 (VLA-5) inhibited the up-regulation of ICAM-1 expression, suggesting the participation of VLA-5 integrin in this response. Pertussis toxin (PT) prevented the up-regulation of ICAM-1 expression because a PT-deficient mutant strain induced higher levels of ICAM-1 mRNA and surface protein than the parental strain. Consistent with this, purified PT suppressed the up-regulation of epithelial ICAM-1 expression. These findings demonstrate that B. pertussis FHA up-regulates ICAM-1 expression on respiratory epithelial cells through interaction of its RGD site with host cell VLA-5 integrin, and that PT impairs this response.

  4. Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli.

    PubMed Central

    Barbieri, J T; Moloney, B K; Mende-Mueller, L M

    1989-01-01

    The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C180I-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C180I-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction. Images PMID:2546919

  5. NADP/sup +/ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

    SciTech Connect

    Kawai, Y.; Whitsel, C.; Arinze, I.J.

    1986-05-01

    Cholera or pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD/sup +/, by endogenous enzymes such as NAD/sup +/-glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP/sup +/. The effect is concentration dependent; with 20 ..mu..M (/sup 32/P)NAD/sup +/ as substrate maximal enhancement is obtained at 0.5-1.0 mM NADP/sup +/. The enhancement of (/sup 32/P)ADP-ribosylation by NADP/sup +/ was much greater than that by other known effectors such as Mg/sup 2 +/, phosphate or isoniazid. The effect of NADP/sup +/ on ADP-ribosylation may occur by inhibition of the degradation of NAD/sup +/ probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP/sup +/, isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl/sub 2/) to suppress NADase activity, NADP/sup +/ was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP/sup +/ in the assay is necessary to obtain maximal ADP-ribosylation.

  6. Structure–function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins

    PubMed Central

    Littler, Dene R.; Ang, Sheng Y.; Moriel, Danilo G.; Kocan, Martina; Kleifeld, Oded; Johnson, Matthew D.; Tran, Mai T.; Paton, Adrienne W.; Paton, James C.; Summers, Roger J.; Schembri, Mark A.; Rossjohn, Jamie; Beddoe, Travis

    2017-01-01

    Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity. PMID:28663369

  7. Structure-function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins.

    PubMed

    Littler, Dene R; Ang, Sheng Y; Moriel, Danilo G; Kocan, Martina; Kleifeld, Oded; Johnson, Matthew D; Tran, Mai T; Paton, Adrienne W; Paton, James C; Summers, Roger J; Schembri, Mark A; Rossjohn, Jamie; Beddoe, Travis

    2017-09-08

    Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD(+)-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells.

    PubMed Central

    Walker, M J; Wehland, J; Timmis, K N; Raupach, B; Schmidt, M A

    1991-01-01

    Three murine monoclonal antibodies (MAb), E19, E205, and E251, raised against pertussis toxin reacted in Western blots (immunoblots) with the S1, S4, and S2-S3 subunits, respectively, and neutralized the Chinese hamster ovary cell-clustering activity of pertussis toxin. MAb E251 recognized a linear synthetic peptide corresponding to amino acids 107 to 120 of the S2 subunit, suggesting a role for this region in receptor binding. Images PMID:1718872

  9. Analogous IgG subclass response to pertussis toxin in vaccinated children, healthy or affected by whooping cough.

    PubMed

    Giammanco, A; Taormina, S; Chiarini, A; Dardanoni, G; Stefanelli, P; Salmaso, S; Mastrantonio, P

    2003-05-16

    The study of antigen specific IgG subclass distribution during disease, or during any other natural or artificial immunisation, can provide useful information on the kind of the immune response and the expected levels of protection. This is particularly true for diseases, such as pertussis in which the mechanisms underlying specific defence are still not completely understood. An investigation was therefore performed to evaluate the IgG subclass response to pertussis toxin (PT) in sera from 89 healthy vaccinated children and 131 vaccinated or unvaccinated children convalescent after a confirmed B. pertussis symptomatic infection. Antibody titres were expressed in arbitrary ELISA units/ml, and statistical analyses were performed. In unvaccinated convalescent children IgG1 and IgG3 were prevalent whereas in children immunised with two different acellular pertussis (aP) vaccines, both healthy and convalescent, IgG1, IgG2 and IgG4 antibodies were mainly produced. Maintenance of the same anti-PT antibody response pattern in healthy acellular pertussis vaccine recipients and in vaccinated children who later acquire the disease is an interesting result indicative of the priming effect induced by these vaccines in the direction of a relatively higher Th2 cell-polarisation of the immune response.

  10. Development and validation of an Australian in-house anti-pertussis toxin IgG and IgA enzyme immunoassay.

    PubMed

    May, Meryta L; Evans, Jenny; Riley, Jennifer; Lambkin, Geoffrey; Robson, Jennifer M

    2013-02-01

    Although anti-pertussis toxin (PT) immunoglobulin G (IgG) is considered one of the most specific serological markers for Bordetella pertussis infection, there are few commercial kits available in Australia. We aimed to present the process of development, quality control and on-going clinical validation of an anti-PT IgG and IgA enzyme immunoassay (EIA) in use since 1999, and discuss the application of such tests in the diagnosis of B. pertussis infections. A total of 1311 serum samples were used during multiple clinical validations from 1998 to 2010. The samples were drawn from healthy adults, children, patients with other respiratory infections, and patients with confirmed pertussis. Assay reproducibility, accuracy and precision criteria conformed to National Pathology Accreditation Advisory Council (NPAAC) guidelines. Using the World Health Organization clinical and/or laboratory definition of a definite case as the comparative standard, sensitivity was 84% [95% confidence interval (CI) 75-93] and specificity was 98% (95%CI: 90-100) for anti-PT IgG. Sensitivity was 72% (95%CI 64-80) and specificity was 98% (95%CI 90-100) for anti-PT IgA. There was minimal background positivity in either healthy adults or children using the established cut-offs. There was no appreciable effect of immunisation or cross reactions with other respiratory pathogens. Serological evaluation of various populations enabled the development of an anti-PT IgG and IgA EIA assay which was suitable for the diagnosis of acute infection in convalescent samples from clinically confirmed cases. Repeated evaluations of population-based cut-offs are required for in-house assays to ensure they remain clinically relevant. The subsequent validation of the cut-offs with WHO international standards has been published in a recent prospective study.

  11. Effect of phorbol ester and pertussis toxin on the enhancement of noradrenaline release by angiotensin II in mouse atria.

    PubMed Central

    Musgrave, I. F.; Majewski, H.

    1989-01-01

    1. Mouse atria were incubated with [3H]-noradrenaline, and the outflow of radioactivity due to electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (0.01 and 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity. 2. Phorbol 12-myristate 13-acetate (0.001, 0.03, 0.1 and 1.0 microM), a protein kinase C activating phorbol ester, significantly enhanced the S-I outflow of radioactivity. When angiotensin II (0.1 microM) was present with the concentration of phorbol 12-myristate 13-acetate that was maximally effective in increasing the S-I outflow (0.1 microM), the enhancement of S-I outflow produced by angiotensin II was maintained. 3. Polymyxin B (70 microM), an inhibitor of protein kinase C, significantly inhibited the S-I outflow. Polymyxin B also inhibited the enhancement of the S-I outflow produced by angiotensin II (0.1 microM). 4. In another series of experiments mice were injected with pertussis toxin (1.5 micrograms per mouse), 4 days before their atria were removed. The effectiveness of pertussis toxin pretreatment was determined indirectly using carbachol. Carbachol caused a concentration-dependent fall in both the rate and force of beating of isolated spontaneously beating atria from mice pretreated with vehicle. This effect of carbachol was not seen with atria from mice pretreated with pertussis toxin. 5. Pertussis toxin pretreatment did not alter the enhancement of the S-I outflow of radioactivity produced by angiotensin II (0.01 and 0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2720295

  12. Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins.

    PubMed Central

    Fields, T A; Casey, P J

    1997-01-01

    Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein alpha subunits of the Gi family, and this modification prevents the occurrence of the receptor-G-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as 'PTX-resistant' due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivo have not been clearly established. Since two of these proteins, G12 alpha and Gz alpha, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC. PMID:9032437

  13. The use of microcalorimetry to characterize tetanus neurotoxin, pertussis toxin and filamentous haemagglutinin.

    PubMed

    Krell, Tino; Greco, Frédéric; Nicolaï, Marie-Claire; Dubayle, Jean; Renauld-Mongénie, Geneviève; Poisson, Nadège; Bernard, Isabelle

    2003-12-01

    Tetanus neurotoxin (TeNT), pertussis toxin (PT) and pertussis filamentous haemagglutinin (FHA) are major virulence factors of Clostridium tetani and Bordetella pertussis, which are the causative agents of tetanus and whooping cough respectively. Inactivated forms of these virulence factors are the protein components of vaccines against these diseases. Here we report microcalorimetric studies to characterize these proteins. The microcalorimetric titration curves of TeNT with micelles of gangliosides GD1b, GT1b and GQ1b were biphasic. For these gangliosides a high-affinity binding site (KD 45-277 nM) can be distinguished from a lower-affinity binding event (KD 666-1190 nM). This is direct evidence for multiple binding sites for gangliosides of the 1b series at TeNT as proposed by Emsley et al. [Emsley, Fotinou, Black, Fairweather, Charles, Watts, Hewitt and Isaacs (2000) J. Biol. Chem. 275, 8889-8894]. In agreement with previous reports, no binding was observed for gangliosides GM1, GM2, GM3 and GD2. The thermal denaturation of TeNT was characterized by two unfolding transitions centred around 57.4 and 62.4 degrees C. The conversion of TeNT into the toxoid form by formaldehyde treatment was accompanied by a large increase in Tm (the midpoint of protein unfolding transition, that is, the temperature at which half the protein is denatured and the other half is still present in its native form). Fetuin and asialofetuin bound to PT with similar affinities (KD 420 and 335 nM respectively). Binding was largely enthalpy-driven and counterbalanced by an unfavourable entropy change, indicating a loss of conformational flexibility. The latter could account for the observed inhibition of ATP binding after binding to fetuin. Furthermore, the molecular limits of mature PT subunit S5 were defined by MS and N-terminal peptide sequencing. The differential-scanning-calorimetry thermogram of FHA shows four well-resolved unfolding transitions, a finding consistent with the sequential

  14. Bradykinin modulates potassium and calcium currents in neuroblastoma hybrid cells via different pertussis toxin-insensitive pathways.

    PubMed

    Wilk-Blaszczak, M A; Gutowski, S; Sternweis, P C; Belardetti, F

    1994-01-01

    In NG108-15 cells, bradykinin (BK) activates a potassium current (IK,BK) and inhibits the voltage-dependent calcium current (ICa,V). BK also stimulates a phosphatidylinositol-specific phospholipase C (PI-PLC). The subsequent release of inositol 1,4,5-trisphosphate and increase in intracellular calcium contribute to IK,BK, through activation of a calcium-dependent potassium current. In membranes from these cells, stimulation of PI-PLC by BK is mediated by Gq and/or G11, two homologous, pertussis toxin-insensitive G proteins. Here, we have investigated the role of Gq/11 in the electrical responses to BK. GTP gamma S mimicked and occluded both actions of BK, and both effects were insensitive to pertussis toxin. Perfusion of an anti-Gq/11 alpha antibody into the pipette suppressed IK,BK, but not the inhibition of ICa,V by BK. Thus, BK couples to IK,BK via Gq/11, but coupling to ICa,V is most likely via a different, pertussis toxin-insensitive G protein.

  15. Pertussis toxin B-oligomer inhibits HIV infection and replication in hu-PBL-SCID mice.

    PubMed

    Lapenta, Caterina; Spada, Massimo; Santini, Stefano M; Racca, Sara; Dorigatti, Fernanda; Poli, Guido; Belardelli, Filippo; Alfano, Massimo

    2005-04-01

    Bordetella pertussis toxin B-oligomer (PTX-B) has been shown to inhibit HIV infection and replication in vitro. The potential anti-viral effect of PTX-B was tested here in an in vivo surrogate model of HIV infection, i.e. SCID mice reconstituted with human peripheral blood leukocytes (PBL) (hu-PBL-SCID) and infected with a CCR5-dependent (R5) HIV-1 strain. SCID mice inoculated intra-peritoneal (i.p.) with PTX-B and then infected with the R5 strain SF-162 were sacrificed 7 days later and analyzed for human PBL (hu-PBL) lymphoid tissue reconstitution, infection of hu-PBL, plasma viremia and viral rescue from ex vivo-cultivated i.p. hu-PBL. Unlike mice treated with 500 ng per animal of PTX-B showing no evidence of viral inhibition, daily administration of PTX-B (50 ng per mouse) strongly inhibited virus infection and replication, as determined by undetectable viremia, absence of infected hu-PBL and lack of rescue of infectious HIV in most animals. Furthermore, PTX-B injection 2 h before and twice after infection prevented HIV-1 infection and replication in all (10/10) tested animals. Thus, PTX-B potently inhibited virus infection and replication in hu-PBL-SCID mice, supporting the hypothesis that it may represent a new pharmacological agent against HIV-1 infection.

  16. Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin*

    PubMed Central

    Subrini, Orso; Sotomayor-Pérez, Ana-Cristina; Hessel, Audrey; Spiaczka-Karst, Johanna; Selwa, Edithe; Sapay, Nicolas; Veneziano, Rémi; Pansieri, Jonathan; Chopineau, Joel; Ladant, Daniel; Chenal, Alexandre

    2013-01-01

    Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1–384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1–489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375–485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454–485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454–485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane. PMID:24064217

  17. Pertussis toxin activates adult and neonatal naive human CD4+ T lymphocytes.

    PubMed

    Tonon, Sandrine; Badran, Bassam; Benghiat, Fleur Samantha; Goriely, Stanislas; Flamand, Véronique; Willard-Gallo, Karen; Willems, Fabienne; Goldman, Michel; De Wit, Dominique

    2006-07-01

    Pertussis toxin (PTX) is known to be mitogenic for T lymphocytes, but its direct action on naive human T cells has not been specified. Herein, we show that PTX induces the proliferation of purified adult CD45RA(+)CD4(+) T cells independently of its ADP-ribosyltransferase activity. PTX directly induces TNF-alpha and IL-2 mRNA expression, modulates the level of several cell surface receptors and induces Forkhead box p3 (Foxp3) protein accumulation in naive CD4(+) T cells. Addition of autologous dendritic cells was found to be required for the production of high levels of IFN-gamma by PTX-stimulated naive T cells. These effects of PTX occurred in conjunction with activation of NF-kappaB and NFAT transcription factors. Overall, responses of neonatal CD4(+) T cells to PTX were similar to those of adult CD45RA(+)CD4(+) naive T cells except for their blunted CD40 ligand up-regulation. We suggest that the adjuvant properties of PTX during primary cell-mediated immune responses involve a direct action on naive T lymphocytes in addition to activation of antigen-presenting cells.

  18. Role of Bordetella pertussis RseA in the cell envelope stress response and adenylate cyclase toxin release

    PubMed Central

    Hanawa, Tomoko; Yonezawa, Hideo; Kawakami, Hayato; Kamiya, Shigeru; Armstrong, Sandra K.

    2013-01-01

    Bordetella pertussis is the bacterial agent of the human disease, whooping cough. In many bacteria, the extracellular function sigma factor σE is central to the response to envelope stress, and its activity is negatively controlled by the RseA anti-sigma factor. In this study, the role of RseA in B. pertussis envelope stress responses was investigated. Compared with the wild-type strain, an rseA mutant showed elevated resistance to envelope stress and enhanced growth at 25°C. rpoH and other predicted σE target genes demonstrated increased transcription in the rseA mutant compared with the wild type parent. Transcription of those genes was also increased in wild type B. pertussis and Escherichia coli under envelope stress, whereas no stress-induced increase in transcription was observed in the rseA mutant. rseA inactivation was also associated with altered levels of certain proteins in culture supernatant fluids, which showed increased adenylate cyclase toxin (CyaA) levels. The increased CyaA in the mutant was correlated with an apparent increased stability of the extracellular toxin and increased production of CyaA-containing outer membrane vesicles. Consistent with this, compared with the wild type strain, rseA mutant cells produced increased numbers of large surface-associated vesicles. PMID:23821542

  19. Clustered cases of Bordetella pertussis infection cause high levels of IgG antibodies against pertussis toxin in adolescents in Gaobeidian city, China.

    PubMed

    Wu, Y; Zhu, B; Gao, Y; Shi, Z; Wang, J; Wang, H; Shao, Z

    2014-04-01

    SUMMARY In a large serosurvey performed in Gaobeidian city, China, 1032 children and adolescents aged 3-18 years were divided into three age groups: 3-6, 7-11, and 12-18 years. The geometric mean of IgG antibodies against pertussis toxin (anti-PT IgG) varied from 3.47 IU/ml (3-6 years) to 8.71 IU/ml (12-18 years) and the seroprevalence (≥ 40 IU/ml) varied from 4.48% (7-11 years) to 11.76% (12-18 years). Infections were clustered in three secondary schools (schools 1-3). However, excluding secondary schools 1-3, the IgG PT levels, seroprevalence and rates of recent infections (>100 IU/ml) in secondary schools 4-7 (where infections were not clustered) were very close to those in the 3-6 and 7-11 years age groups. This suggests that the high seroprevalence and anti-PT IgG levels in adolescents in Gaobeidian city, China were caused by the clustered cases in certain secondary schools. Our results also suggested that pertussis was still circulating in China, and the reported incidence might be underestimated.

  20. Sensitivity and specificity of the World Health Organization pertussis clinical case definition.

    PubMed

    Ghanaie, Roxana Mansour; Karimi, Abdollah; Sadeghi, Hossein; Esteghamti, Abdolreza; Falah, Fateme; Armin, Shahnaz; Fahimzad, Alireza; Shamshiri, Ahmadreza; Kahbazi, Manige; Shiva, Faride

    2010-12-01

    Bordetella pertussis continues to circulate even in countries with good childhood vaccination coverage. This study was undertaken to define the relationship between documented disease and the clinical criteria proposed by the World Health Organization (WHO). Nasopharyngeal swab samples were collected from previously healthy 6-14-year-old school children in Tehran, presenting with persistent cough of at least 2- week duration. Specimens were examined for Bordetella pertussis and Bordetella parapertussis by culture and polymerase chain reaction (PCR). Out of 6601 students, 328 (5.0%) had been coughing for at least 2 weeks. Of these children with cough, 182 (55.5%) experienced whooping, 194 (59.1%) suffered a paroxysmal cough, and 73 (22.3%) had post-tussive vomiting. Twenty-one (6.4%) samples tested positive for B. pertussis and six (1.8%) for B. parapertussis by PCR. Culture of four (1.2%) specimens was positive for B. pertussis. In comparison to PCR, the sensitivity and the specificity of the WHO clinical criteria (year 2000) were 95.2% and 15.0%, respectively. Pertussis remains one of the etiologies of prolonged cough, even in communities with high immunization in children. The specificity of the WHO criteria is low in diagnosing pertussis compared with PCR. Copyright © 2010 International Society for Infectious Diseases. All rights reserved.

  1. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli

    PubMed Central

    Jobling, Michael G.

    2016-01-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB5 toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins. PMID:26755534

  2. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli.

    PubMed

    Jobling, Michael G

    2016-04-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB(5) toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins.

  3. Resurgence of Pertussis and Emergence of the Ptxp3 Toxin Promoter Allele in South Italy.

    PubMed

    Loconsole, Daniela; De Robertis, Anna Lisa; Morea, Anna; Metallo, Angela; Lopalco, Pier Luigi; Chironna, Maria

    2017-09-21

    Despite universal immunization programs, pertussis remains a major public health concern. This study aimed to describe the pertussis epidemiology in the Puglia region in 2006-2015 and to identify recent polymorphisms in Bordetella pertussis (BP) virulence-associated genes. The pertussis cases in 2006-2015 were identified from the National Hospital Discharge Database and the Information System of Infectious Diseases. Samples of pertussis cases in 2014-2016 that were confirmed by the Regional Reference Laboratory were subjected to ptxA, ptxP, and prn gene sequencing and, in 10 cases, multiple-locus variable-number tandem repeat analysis (MLVA). In Puglia in 2006-2015, the pertussis incidence rose from an average of 1.39/100,000 inhabitants in 2006-2013 to 2.56-2.54/100,000 in 2014-2015. In infants <1 year of age, the incidence rose from an average of 60.4/100,000 infants in 2006-2013 to 149.9/100,000 in 2015. Of the 661 cases recorded in 2006-2015, 80.3% required hospitalization; of these, 45.4% were aged <1 year. Of the 80 sequenced samples, the allelic profile ptxA1-ptxP3-prn2 was detected in 74. This variant was detected in both vaccinated and unvaccinated people. Six BP samples were prn-deficient. The MLVA cases exhibited MLVA-type 27. The pertussis incidence in Puglia has risen. The hypervirulent strain was also found in vaccinated people. This suggests bacterial adaptation to the vaccine and raises questions about acellular vaccine effectiveness. Prevention of infant pertussis cases is best achieved by immunizing the pregnant mother. Enhanced surveillance and systematic laboratory confirmation of pertussis should be improved in Italy.

  4. Immunization with the recombinant Cholera toxin B fused to Fimbria 2 protein protects against Bordetella pertussis infection.

    PubMed

    Olivera, Noelia; Castuma, Celina E; Hozbor, Daniela; Gaillard, María E; Rumbo, Martín; Gómez, Ricardo M

    2014-01-01

    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.

  5. Expression and immunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908.

    PubMed Central

    Barry, E M; Gomez-Duarte, O; Chatfield, S; Rappuoli, R; Pizza, M; Losonsky, G; Galen, J; Levine, M M

    1996-01-01

    Salmonella typhi vaccine strain CVD 908 can deliver heterologous antigens to the host immune system following mucosal immunization. Stable expression of foreign proteins in Salmonella cells often requires antigen-specific engineering strategies. Fusion of antigens to stabilizing proteins has proven to be a successful strategy for rescuing otherwise unstable proteins. We designed plasmids to allow the fusion of antigens to the amino terminus or carboxyl terminus of fragment C of tetanus toxin, separated by a 4-amino-acid hinge region. Towards the ultimate goal of developing a live oral diphtheria-pertussis-tetanus vaccine, we used these plasmids to stably express the S1 subunit of pertussis toxin in CVD 908. Driven by the anaerobically inducible nirB promoter, the S1 subunit alone was expressed poorly in Salmonella cytoplasm. In contrast, hybrid proteins with S1 fused to either the amino or carboxyl terminus of fragment C were expressed at a high level in CVD 908 and were recognized in Western blot (immunoblot) analysis by monoclonal antibodies directed to S1 and to fragment C. Mice were immunized by the oral or intranasal routes with CVD 908 derivatives harboring these recombinant plasmids. All fusion proteins elicited serum antibody responses to fragment C following intranasal immunization, whereas oral inoculation did not. The configuration of antigens constituting the fusion was critical; S1 fused to the amino terminus of fragment C was less effective than S1 fused to the carboxyl terminus in generating anti-fragment C antibodies. CVD 908 expressing truncated S1 fused to the carboxyl terminus of fragment C elicited neutralizing serum pertussis antitoxin following intranasal immunization of mice. PMID:8926085

  6. Two-component cluster analysis of a large serodiagnostic database for specificity of increases of IgG antibodies against pertussis toxin in paired serum samples and of absolute values in single serum samples.

    PubMed

    de Greeff, Sabine C; Teunis, Peter; de Melker, Hester E; Mooi, Frits R; Notermans, Daan W; Elvers, Bert; Schellekens, Joop F P

    2012-09-01

    Measuring IgG antibodies against pertussis toxin (IgG-Ptx) with an enzyme-linked immunosorbent assay (ELISA) can be used to diagnose pertussis infection; however, the cutoff points are not unanimously defined. To determine the diagnostic specificity of increases of IgG-Ptx in paired sera and of absolute values in single serum samples, we applied a two-component cluster analysis to serum samples of patients suspected for pertussis, whose sera had been submitted to a routine diagnostic laboratory between 2003 and 2009, and had been assayed with an in-house IgG-Ptx ELISA calibrated with the international FDA lot 3 IgG-Ptx reference serum. Children eligible for the acellular pertussis vaccination were excluded to avoid interference from a vaccine-induced IgG-Ptx rise. Binary distribution mixtures were fitted to the data. Receiver operating characteristic (ROC) curves were calculated for absolute values in single samples (n = 14,452) and increases in paired samples (n = 2,455). For both parameters, two subpopulations could be identified: a population with high reactivity (persons with pertussis infection) and a population with low reactivity (persons without pertussis infection). For absolute values in single samples, the area under the curve (AUC) of the ROC curve was 0.993 and the optimum cutoff (with the highest cumulative value of specificity plus sensitivity) was 67.7 IU/ml (95% confidence interval, 63.9 to 74.1; sensitivity, 96.4%; specificity, 95.7%). A previously determined diagnostic cutoff of 125 IU/ml was associated with a sensitivity of 88.1% and a specificity of 98.8%. For increases in paired sera, the AUC was 0.999 and the optimum cutoff was 3.1-fold (95% CI, 2.8 to 3.4; sensitivity, 99.6%; specificity, 99.2%). Given the methodology of this study, estimates of sensitivity probably are overrated (because pertussis patients without IgG-Ptx response are not detected), but estimates of specificities can be considered very accurate.

  7. Decline of IgG pertussis toxin measured in umbilical cord blood, and neonatal and early infant serum.

    PubMed

    Smallenburg, L C S; van Welie, N A; Elvers, L H; van Huisseling, J C M; Teunis, P F M; Versteegh, F G A

    2014-09-01

    Maternal pertussis-specific antibodies are passively acquired by infants during pregnancy. An IgG pertussis toxin (IgG-PT) concentration of >20 U/ml is considered to protect neonates against pertussis. To evaluate the IgG concentration at birth and during the first two months of life, we examined the IgG-PT concentration in the umbilical cord blood and three times during the neonatal and early infant period. IgG-PT was measured by validated IgG-specific enzyme-linked immunosorbent assays (ELISA) in umbilical cord blood and in Guthrie card blood samples of umbilical cord blood in 2,790 children, born between 1 August 2006 and 1 December 2008. These measurements were comparable. All children with concentrations of IgG-PT >30 U/ml were included. IgG-PT was also measured in Guthrie card blood samples, when the neonates or early infants were 5 days, 1 month and 2 months old. The mean concentrations of IgG-PT were calculated. The mean concentration of IgG-PT in umbilical cord blood was 60.1 U/ml (LN 4.1; 0.6 SD; n = 103). At the age of 5 days, 1 month and 2 months, the mean concentration of IgG-PT was 40.6 U/ml (LN 3.7; 0.5 SD; n = 103), 20.7 U/ml (LN 3.0; 0.7 SD; n = 62) and 16.7 U/ml (LN 2.8; 0.9 SD; n = 61), respectively. Four percent of the neonates had a concentration of IgG-PT >30 U/ml in umbilical cord blood, which declined to levels around the concentration needed for protection against pertussis (>20 U/ml) in the first two months of life. Hence, it is of great importance to further investigate the safety of maternal immunisation during pregnancy to prevent life-threatening pertussis in newborns.

  8. Cholera toxin and pertussis toxin regulate the Fc receptor-mediated phagocytic response of human neutrophils in a manner analogous to regulation by monoclonal antibody 1C2.

    PubMed

    Gresham, H D; Clement, L T; Volanakis, J E; Brown, E J

    1987-12-15

    Data presented in this paper indicate that polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis can be markedly augmented and that this augmentation is under regulatory control. Stimulation of PMN with either a low m.w., heat-labile cytokine(s) (the culture supernatant effluent from a YM-10 Centricon unit, YM-10E), phorbol esters (phorbol dibutyrate), or the polyene antibiotic, amphotericin B, enhances Fc-mediated ingestion in a dose-dependent manner. YM-10 effluent- and amphotericin B-stimulated ingestion is completely abrogated by treating the PMN with either pertussis toxin (PT), cholera toxin (CT), or a monoclonal antibody (mAb), 1C2. However, neither toxin nor mAb 1C2 affects nonstimulated ingestion or phagocytosis stimulated by phorbol esters or synthetic diacylglycerol. Increasing intracellular cyclic adenosine monophosphate levels by stimulation with prostaglandin E1 and the phosphodiesterase inhibitor, isobutylmethylxanthine, does not mimic the effect of either toxin or mAb 1C2. In addition, toxin-mediated inhibition is not due to loss of either the Fc receptor recognized by mAb 3G8 or the antigen recognized by mAb 1C2. These data indicate that both CT and PT regulate the phagocytic response of PMN, in a manner like mAb 1C2, probably by affecting a guanosine 5'-triphosphate-binding protein distinct from those that regulate adenylate cyclase. Since phorbol ester-stimulated ingestion is not inhibited by either PT, CT, or mAb 1C2 and phorbol esters activate protein kinase C directly, phagocytosis amplification regulated by PT, CT, and mAb 1C2 may involve protein kinase C activation.

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

    SciTech Connect

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

    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

  10. [Survey of pertussis infection in infants aged under 3 months with persistent cough].

    PubMed

    Mi, Rong; Fu, Jin; Kang, Li-min; Cui, Xiao-dai; Wang, Xiao-ying; Li, Li; Xu, Fang-sheng

    2012-07-24

    To explore the prevalence of pertussis in hospitalized infants aged under 3 months with persistent cough. The nasopharyngeal secretions and serum samples were collected from hospitalized infants aged under 3 months with cough for over 2 weeks from January 2011 to January 2012. The samples of nasopharyngeal secretion were suctioned and collected. Multiplex PCR assay was employed to identify Bordetella pertussis (B. pertussis) and enzyme-linked immunosorbent assay 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. Fifty-nine infants (32 boys and 27 girls) were enrolled. None of them was pre-immunized with diphtheria-pertussis-tetanus vaccine. Seventeen infants (28.8%) were B. Pertussis positive. Among 17 cases, 3 infants under 1 month, 4 infants 1 -2 months, and 10 infants 2 - 3 months. Three infants had household contacts with persistent cough and their serum antibodies to pertussis toxin were positive. Sixteen infants with pertussis had the paroxysms of frequent and rapid coughs while another 5 with pertussis had long inspiratory effort accompanied by a high-pitched "whoop" at the end of paroxysms. Seven infants with pertussis had conjunctiva bleeding, a special sign of pertussis. Ten infants had lymphocytosis with a predominant elevation of lymphocytes. B. pertussis is an important pathogen for the infants under 3 months with persistent cough. Multiplex PCR may be used to identify B. pertussis with a high sensitivity. The unrecognized close family members of the infants with pertussis are probably an important source of infection.

  11. Detection of antibodies against fimbria type 3 (Fim3) is useful diagnostic assay for pertussis.

    PubMed

    Oguchi, Kaoru; Miyata, Akiko; Kazuyama, Yukimasa; Noda, Atsuya; Suzuki, Eri; Watanabe, Mineo; Nakayama, Tetsuo

    2015-09-01

    Isolation of Bordetella pertussis and detection of the pertussis genome are not always successful because of low bacterial loads in adult patients with pertussis. Antibodies against pertussis toxin (PT) are measured but have low sensitivity in vaccinated subjects. There is no reliable diagnostic method at present. In this study, a fluorescent-EIA against several pertussis antigens and genome detection were investigated to establish clinical laboratory diagnostic methods for pertussis. The study was conducted in an outpatient clinic between September 2007 and 2013. Subjects consisted of 209 patients including adults suspected of pertussis and 35 staff members of the clinic. Loop-mediated isothermal amplification (LAMP) was performed to detect the pertussis genome in 5' UTR of the pertussis toxin (PT) gene. The catalytic region of the adenylate cyclase toxin (catACT), C-terminal of filamentous hemagglutinin (cFHA), and type 3 fimbria (Fim3) were selected, which are not pertussis vaccine component. Conventional PT and FHA antibodies were examined together with type 2 fimbria (Fim2) antibodies, and these are vaccine antigens. Pertussis DNA was detected in 23 (11%) out of 209. Detection sensitivity was high in young infants. Antibodies against Fim3 showed a higher positive rate in all age groups. Staff members at the pediatric outpatient clinic showed serological booster responses in Fim2 and Fim3 antibodies more sensitively than those in PT antibodies during outbreaks. LAMP was useful for detecting the pertussis genome in young infants, whereas a serological assay for fluorescent-EIA against Fim2 and Fim3 was preferable for adolescents and adults. Copyright © 2015. Published by Elsevier Ltd.

  12. Pharmacological differentiation by pertussis toxin of the in vivo acute responses to fMLP and PAF in guinea-pig lungs.

    PubMed Central

    Arreto, C. D.; Bureau, M. F.; Imaizumi, A.; Vargaftig, B. B.

    1993-01-01

    1. The effects of pertussis toxin on the N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and platelet-activating factor (PAF)-induced variations in pulmonary capillary albumin exchanges, blood volume, leucocyte or platelet sequestration were studied in the guinea-pig, by use of radioactive tracers. The effects of pertussis toxin on pulmonary insufflation pressure were studied in parallel. 2. The i.v. administration of fMLP and PAF to the guinea-pig was followed by bronchoconstriction, increased lung capillary albumin exchanges (vasopermeability) sequestration of leucocytes, leucopenia and reduction of blood volume (vasoconstriction). PAF also induced platelet sequestration in lungs and thrombocytopenia. 3. Pertussis toxin (10 micrograms kg-1, i.v., 72 h before the experiment) prevented all the studied fMLP-induced effects, but failed to modify PAF-induced bronchoconstriction, lung vasoconstriction, platelet sequestration, thrombocytopenia and the increased capillary vasopermeability. In the same conditions the lung leucocyte sequestration was not significantly affected when leucopenia was partially reduced. 4. It is suggested that the effects of fMLP, but not those of PAF, involve a Gi-like protein. PMID:8448592

  13. Evaluation of culture, immunofluorescence, and serology for the diagnosis of pertussis.

    PubMed Central

    Halperin, S A; Bortolussi, R; Wort, A J

    1989-01-01

    Nasopharyngeal culture, direct immunofluorescence, and serology of acute-phase and paired serum specimens were compared for the laboratory diagnosis of infections due to Bordetella pertussis in a community-based pediatric population with both high vaccine usage and high pertussis incidence. In 77 (37%) of 210 patients evaluated, one or more tests were positive for pertussis. A clinical illness compatible with pertussis was present in 52 (71%) of 73 pertussis test-positive and 42 (35%) of 119 test-negative patients (P less than 0.001). Nasopharyngeal culture was of low sensitivity (20 [26%] of 77 positive tests) but was most commonly confirmed by another positive pertussis test (85%). Direct immunofluorescence was both insensitive and nonspecific; only 6 (30%) of 20 cases positive by culture were positive by immunofluorescence, and only 4 (33%) of 12 of the culture-negative, immunofluorescence-positive cases could be confirmed by another positive pertussis test. Although serology by enzyme immunoassay proved to be the most sensitive of the laboratory tests (87%), this sensitivity could be achieved only by assaying both acute-phase and paired serum specimens and measuring immunoglobulin G (IgG), IgA, and IgM antibodies to two pertussis antigens (pertussis toxin and filamentous hemagglutinin). Loss of sensitivity occurred with any reduction in the number of these serologic assays performed. Optimal laboratory diagnosis of endemic pertussis in a pediatric population requires both nasopharyngeal culture and serology by enzyme immunoassay. PMID:2542366

  14. Epidermal growth factor (EGF)-stimulated inositol phosphate formation in hepatocytes is abolished by pertussis toxin and phorbol esters

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1987-05-01

    The EGF-stimulated rise in intracellular Ca/sup 2 +/ (Ca/sup 2 +/)/sub i/ and Ca/sup 2 +/-dependent protein phosphorylation events in isolated hepatocytes are blocked by pertussis toxin and phorbol ester pretreatment. The present study characterized the EGF-stimulated formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P/sub 3/) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P/sub 3/) in hepatocytes using HPLC methodology to separate the InsP/sub 3/ isomers. Both 66 nM EGF and 10 nM angiotensin II (ANG II) caused a rapid increase in the Ins(1,4,5)P/sub 3/ isomer although EGF-stimulated formation was smaller. At a concentration of ANG II (0.1 nM) which gave an equivalent rise in (Ca/sup 2 +/)/sub i/ as 66 nM EGF, the kinetics and magnitude of Ins(1,4,5)P/sub 3/ formation were similar. EGF or ANG II-stimulated formation of the Ins(1,3,4)P/sub 3/ isomer was more gradual and increased beyond the level of Ins(1,4,5)P/sub 3/ after 60 sec. The initial EGF and ANG II-stimulated increase in both InsP/sub 3/ isomers was not affected by removing external Ca/sup 2 +/ with a 10-fold excess of EGTA. Pretreatment of rats with pertussis toxin for 72 hrs blocked the ability of EGF to increase Ins(1,4,5)P/sub 3/ but did not affect the increase due to ANG II. Three main pretreatment of cells with 1 ..mu..g/ml phorbol 12-myristate-13-acetate (PMA) also inhibited the EGF-stimulated Ins(1,4,5)P/sub 3/ formation. PMA slightly attenuated Ins(1,4,5)P/sub 3/ formation stimulated by 0.1 nM ANG II but not enough to affect the Ca/sup 2 +/ signal. These data suggest that the signal transduction system used by EGF receptors to increase Ins (1,4,5)P/sub 3/ in hepatocytes is somehow different from that used by ANG II receptors.

  15. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

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

    PubMed Central

    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

  17. Pulsed-Field Gel Electrophoresis, Pertactin, Pertussis Toxin S1 Subunit Polymorphisms, and Surfaceome Analysis of Vaccine and Clinical Bordetella pertussis Strains▿

    PubMed Central

    Bottero, Daniela; Gaillard, María Emilia; Fingermann, Matías; Weltman, Gabriela; Fernández, Julieta; Sisti, Federico; Graieb, Augusto; Roberts, Roy; Rico, Osvaldo; Ríos, Gustavo; Regueira, Mabel; Binsztein, Norma; Hozbor, Daniela

    2007-01-01

    To add new insight to our previous work on the molecular epidemiology of Bordetella pertussis in Argentina, the prn and ptxS1 gene sequences and pulsed-field gel electrophoresis (PFGE) profiles of 57 clinical isolates obtained during two periods, 1969 to 1989 and 1997 to 2006, were analyzed. Non-vaccine-type ptxS1A was detected in isolates obtained since 1969. From 1989 on, a shift of predominance from the vaccine prn1 type to the nonvaccine prn2 type was observed. This was also reflected in a transition of PFGE group IV to group VI. These results show that nonvaccine B. pertussis strains are currently circulating. To analyze whether the observed genomic divergences between vaccine strains and clinical isolates have functional implications, protection assays using the intranasal mouse challenge model were performed. For such experiments, the clinical isolate B. pertussis 106 was selected as representative of circulating bacteria, since it came from the major group of the PFGE dendrogram (PFGE group VI). Groups of mice were immunized either with diphtheria-tetanus-whole-cell pertussis vaccine (ptxS1B prn1) or a vaccine prepared by us containing B. pertussis 106. Immunized mice were then challenged with a B. pertussis vaccine strain (Tohama, harboring ptxS1B and prn1) or the clinical isolate B. pertussis 106 (ptxS1A prn2). An adequate bacterial-elimination rate was observed only when mice were immunized and challenged with the same kind of strain. For further characterization, comparative proteomic profiling of enriched membrane proteins was done using three vaccine strains and the selected B. pertussis 106 clinical isolate. By matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, a total of 54 proteins were identified. This methodology allowed us to detect differing proteins among the four strains studied and, in particular, to distinguish the three vaccine strains from each other, as well as the vaccine strains from the clinical

  18. Crawling phagocytes recruited in the brain vasculature after pertussis toxin exposure through IL6, ICAM1 and ITGαM.

    PubMed

    Richard, Jean-François; Roy, Monica; Audoy-Rémus, Julie; Tremblay, Pierrot; Vallières, Luc

    2011-11-01

    The cerebral vasculature is constantly patrolled by rod-shaped leukocytes crawling on the luminal endothelial surface. These cells are recruited in greater numbers after exposure to bacterial lipopolysaccharide (LPS) by a mechanism involving tumor necrosis factor (TNF), interleukin-1β (IL1β) and angiopoietin-2 (Angpt2). Here, we report that the population of crawling leukocytes, consisting mainly of granulocytes, is also increased in the brains of mice suffering from experimental autoimmune encephalomyelitis (EAE) or injected with pertussis toxin (PTX), which is commonly used to induce EAE. However, this recruitment occurs through an alternative mechanism, independent of Angpt2. In a series of experiments using DNA microarrays, knockout mice and neutralizing antibodies, we found that PTX acts indirectly on the endothelium in part through IL6, which is essential for the post-transcriptional upregulation of intercellular adhesion molecule 1 (ICAM1) in response to PTX but not to LPS. We also found that phagocytes adhere to brain capillaries through the interaction of integrin αM (ITGαM) with ICAM1 and an unidentified ligand. In conclusion, this study supports the concept that PTX promotes EAE, at least in part, by inducing vascular changes necessary for the recruitment of patrolling leukocytes. © 2011 The Authors; Brain Pathology © 2011 International Society of Neuropathology.

  19. Murine hypothalamic destruction with vascular cell apoptosis subsequent to combined administration of human papilloma virus vaccine and pertussis toxin

    PubMed Central

    Aratani, Satoko; Fujita, Hidetoshi; Kuroiwa, Yoshiyuki; Usui, Chie; Yokota, Shumpei; Nakamura, Ikuro; Nishioka, Kusuki; Nakajima, Toshihiro

    2016-01-01

    Vaccination is the most powerful way to prevent human beings from contracting infectious diseases including viruses. In the case of the human papillomavirus (HPV) vaccine, an unexpectedly novel disease entity, HPV vaccination associated neuro-immunopathetic syndrome (HANS), has been reported and remains to be carefully verified. To elucidate the mechanism of HANS, we applied a strategy similar to the active experimental autoimmune encephalitis (EAE) model - one of the most popular animal models used to induce maximum immunological change in the central nervous system. Surprisingly, mice vaccinated with pertussis toxin showed neurological phenotypes that include low responsiveness of the tail reflex and locomotive mobility. Pathological analyses revealed the damage to the hypothalamus and circumventricular regions around the third ventricle, and these regions contained apoptotic vascular endothelial cells. These data suggested that HPV-vaccinated donners that are susceptible to the HPV vaccine might develop HANS under certain environmental factors. These results will give us the new insight into the murine pathological model of HANS and help us to find a way to treat of patients suffering from HANS. PMID:27833142

  20. The inflammasome pyrin contributes to pertussis toxin-induced IL-1β synthesis, neutrophil intravascular crawling and autoimmune encephalomyelitis.

    PubMed

    Dumas, Aline; Amiable, Nathalie; de Rivero Vaccari, Juan Pablo; Chae, Jae Jin; Keane, Robert W; Lacroix, Steve; Vallières, Luc

    2014-05-01

    Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1β synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1β into its active form. In turn, IL-1β stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.

  1. Preliminary studies on the use of pertussis toxin for the modulation of intravaginal SIV transmission in rhesus macaques.

    PubMed

    Ortiz, Kristina; Sampathkumar, Rebecca S; Ansari, Aftab A; Byrareddy, Siddappa N

    2017-09-21

    Pertussis toxin (PTX) blocks GPCR signaling resulting in the inhibition of chemotaxis/cell adhesion. It was reasoned that inhibition of cell trafficking may be an approach to prevent HIV/SIV transmission. In this study, PTX in HEC gel was applied to the vaginal wall of monkeys that were then challenged intravaginally with SIVmac251. Results of these studies showed that 2 of 4 animals were resistant to infection. Furthermore, infection was correlated with a marked increase in the plasma and cervicovaginal lavage levels of select chemokines and cytokines. Results from this preliminary feasibility study dictate that further studies that include a larger number of animals are required to optimize this protocol and establish the efficacy of this approach. In addition, such future studies will provide important information on the role of specific chemokines that play a role in lymphocyte trafficking within the genital tract and serve as additional therapeutic targets. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Osteopontin Expression in the Brain Triggers Localized Inflammation and Cell Death When Immune Cells Are Activated by Pertussis Toxin

    PubMed Central

    Marcondes, Maria Cecilia Garibaldi; Ojakian, Ryan; Bortell, Nikki; Flynn, Claudia; Conti, Bruno; Fox, Howard S.

    2014-01-01

    Upregulation of osteopontin (OPN) is a characteristic of central nervous system pathologies. However, the role of OPN in inflammation is still controversial, since it can both prevent cell death and induce the migration of potentially damaging inflammatory cells. To understand the role of OPN in inflammation and cell survival, we expressed OPN, utilizing an adenoviral vector, in the caudoputamen of mice deficient in OPN, using beta-galactosidase- (β-gal-) expressing vector as control. The tissue pathology and the expression of proinflammatory genes were compared in both treatments. Interestingly, inflammatory infiltrate was only found when the OPN-vector was combined with a peripheral treatment with pertussis toxin (Ptx), which activated peripheral cells to express the OPN receptor CD44v6. Relative to β-gal, OPN increased the levels of inflammatory markers, including IL13Rα1, CXCR3, and CD40L. In Ptx-treated OPN KOs, apoptotic TUNEL+ cells surrounding the OPN expression site increased, compared to β-gal. Together, these results show that local OPN expression combined with a peripheral inflammatory stimulus, such as Ptx, may be implicated in the development of brain inflammation and induction of cell death, by driving a molecular pattern characteristic of cytotoxicity. These are characteristics of inflammatory pathologies of the CNS in which OPN upregulation is a hallmark. PMID:25525298

  3. Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

    PubMed Central

    Palazzo, Raffaella; Nasso, Maria; Cheung, Gordon Yiu Chong; Coote, John Graham; Ausiello, Clara Maria

    2010-01-01

    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. PMID:20090944

  4. Pertussis Tests

    MedlinePlus

    ... less specific and sensitive than either the pertussis culture or PCR. ^ Back to top Proudly sponsored by ... Learn more about ... Understanding Your Tests Inside the Lab In the News Article Index About This Site Send Us Your Comments For Health Professionals Get the Mobile ...

  5. Report on the international workshop on alternatives to the murine histamine sensitization test (HIST) for acellular pertussis vaccines: state of the science and the path forward.

    PubMed

    Isbrucker, Richard; Arciniega, Juan; McFarland, Richard; Chapsal, Jean-Michel; Xing, Dorothy; Bache, Christina; Nelson, Sue; Costanzo, Angele; Hoonakker, Marieke; Castiaux, Amélie; Halder, Marlies; Casey, Warren; Johnson, Nelson; Jones, Brett; Doelling, Vivian; Sprankle, Cathy; Rinckel, Lori; Stokes, William

    2014-03-01

    Regulatory authorities require safety and potency testing prior to the release of each production lot of acellular pertussis (aP)-containing vaccines. Currently, the murine histamine sensitization test (HIST) is used to evaluate the presence of residual pertussis toxin in aP containing vaccines. However, the testing requires the use of a significant number of mice and results in unrelieved pain and distress. NICEATM, ICCVAM, their partners in the International Cooperation on Alternative Test Methods, and the International Working Group for Alternatives to HIST organized a workshop to discuss recent developments in alternative assays to the HIST, review data from an international collaborative study on non-animal alternative tests that might replace the HIST, and address the path toward global acceptance of this type of method. Currently, there are three potential alternative methods to HIST. Participants agreed that no single in vitro method was sufficiently developed for harmonized validation studies at this time. It is unlikely that any single in vitro method would be applicable to all aP vaccines without modification, due to differences between vaccines. Workshop participants recommended further optimization of cell-based assays under development. Participants agreed that the next international collaborative studies should commence in 2013 based on discussions during this workshop.

  6. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  7. Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors of Bordetella pertussis Adenylyl Cyclase Toxin

    PubMed Central

    Geduhn, Jens; Dove, Stefan; Shen, Yuequan; Tang, Wei-Jen; König, Burkhard

    2011-01-01

    Whooping cough is caused by Bordetella pertussis and still constitutes one of the top five causes of death in young children, particularly in developing countries. The calmodulin-activated adenylyl cyclase (AC) toxin CyaA substantially contributes to disease development. Thus, potent and selective CyaA inhibitors would be valuable drugs for the treatment of whooping cough. However, it has been difficult to obtain potent CyaA inhibitors with selectivity relative to mammalian ACs. Selectivity is important for reducing potential toxic effects. In a previous study we serendipitously found that bis-methylanthraniloyl (bis-MANT)-IMP is a more potent CyaA inhibitor than MANT-IMP (Mol Pharmacol 72:526–535, 2007). These data prompted us to study the effects of a series of 32 bulky mono- and bis-anthraniloyl (ANT)-substituted nucleotides on CyaA and mammalian ACs. The novel nucleotides differentially inhibited CyaA and ACs 1, 2, and 5. Bis-ANT nucleotides inhibited CyaA competitively. Most strikingly, bis-Cl-ANT-ATP inhibited CyaA with a potency ≥100-fold higher than ACs 1, 2, and 5. In contrast to MANT-ATP, bis-MANT-ATP exhibited low intrinsic fluorescence, thereby substantially enhancing the signal-to noise ratio for the analysis of nucleotide binding to CyaA. The high sensitivity of the fluorescence assay revealed that bis-MANT-ATP binds to CyaA already in the absence of calmodulin. Molecular modeling showed that the catalytic site of CyaA is sufficiently spacious to accommodate both MANT substituents. Collectively, we have identified the first potent CyaA inhibitor with high selectivity relative to mammalian ACs. The fluorescence properties of bis-ANT nucleotides facilitate development of a high-throughput screening assay. PMID:20962032

  8. Pertussis is under diagnosed in infants hospitalized with lower respiratory tract infection in the pediatric intensive care unit.

    PubMed

    Greenberg, David; Bamberger, Ellen; Ben-Shimol, Shalom; Gershtein, Rosa; Golan, Daniel; Srugo, Isaac

    2007-11-01

    To determine the rate of pertussis in infants aged <1 year hospitalized at the pediatric intensive care unit (PICU) with lower respiratory tract infection (LRTI) and to characterize the clinical aspects of these patients. Infants <12 months hospitalized with LRTI in the PICU were identified retrospectively. Demographic, clinical and laboratory data were obtained. Stored nasopharyngeal washes (NPW) were tested by polymerase chain reaction (PCR) for the pertussis toxin gene (ptxA) and insertion element IS481 sequences. Eleven of seventy-four (15%) NPW were positive for pertussis, none of them was clinically suspected. Of the positive patients, 82% suffered from paroxysmal cough vs. 32% of the negative patients and from prolonged cough, 45.5% vs. 8%, respectively (P<0.01). Thirty (41%) patients needed mechanical ventilation with no differences between positive and negative pertussis patients. Infiltrates in x-rays were more common among pertussis negative than among pertussis positive patients, 29% vs. 0% (P=0.05). Lower respiratory tract infections caused by pertussis are a more frequent cause of admission to the PICU than generally recognized. Patient's history with paroxysmal cough suggests the possibility of pertussis and the lack of whooping cough does not rule out pertussis. Pediatricians should be aware of the limitations of their diagnostic tools when ruling out pertussis especially in infants with a diagnosis of bronchiolitis. Using new diagnostic methods add considerably to the sensitivity of pertussis diagnosis in PICU and PCR techniques should be used routinely, at least in a PICU setting.

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

    PubMed

    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.

  10. Relationship of immunogenicity to protective potency in acellular pertussis vaccines.

    PubMed

    Xing, Dorothy; Asokanathan, Catpagavalli; Xu, Ying Hua; Bolgiano, Barbara; Douglas-Bardsley, Alex; Zhang, Shumin; Wang, Junzhi; Corbel, Michael

    2014-01-01

    Comparison of the immunogenicity response and resistance to challenge in the modified intracerebral challenge assay induced by various acellular pertussis vaccines showed that these were not closely linked. The immunogenicity assay was effective for confirming the presence of specific antigenic components and was invaluable for detecting minor components present in co-purified vaccines. However, the magnitude of antibody responses was not consistently related to antigen concentration nor did it correlate with protection in the modified intracerebral challenge assay. The immunogenicity assay detected degradation of pertussis toxin and pertactin components but not of filamentous haemagglutinin or fimbriae 2 and 3 in denatured acellular pertussis vaccines. The modified intracerebral challenge assay was effective in detecting antigen degradation in all types of acellular pertussis vaccines including those of European/North American origin but was dominated by the response to pertussis toxin. Aerosol challenge was more sensitive in detecting denaturation of filamentous haemagglutinin or fimbriae. The modified intracerebral challenge assay was the only assay that provided a quantitative indication of protective activity. Both immunogenicity and challenge assays provided useful data on acellular pertussis vaccine properties but were complementary and not alternatives.

  11. Relationship of immunogenicity to protective potency in acellular pertussis vaccines

    PubMed Central

    Xing, Dorothy; Asokanathan, Catpagavalli; Xu, Ying Hua; Bolgiano, Barbara; Douglas-Bardsley, Alex; Zhang, Shumin; Wang, Junzhi; Corbel, Michael

    2014-01-01

    Comparison of the immunogenicity response and resistance to challenge in the modified intracerebral challenge assay induced by various acellular pertussis vaccines showed that these were not closely linked. The immunogenicity assay was effective for confirming the presence of specific antigenic components and was invaluable for detecting minor components present in co-purified vaccines. However, the magnitude of antibody responses was not consistently related to antigen concentration nor did it correlate with protection in the modified intracerebral challenge assay. The immunogenicity assay detected degradation of pertussis toxin and pertactin components but not of filamentous haemagglutinin or fimbriae 2 and 3 in denatured acellular pertussis vaccines. The modified intracerebral challenge assay was effective in detecting antigen degradation in all types of acellular pertussis vaccines including those of European/North American origin but was dominated by the response to pertussis toxin. Aerosol challenge was more sensitive in detecting denaturation of filamentous haemagglutinin or fimbriae. The modified intracerebral challenge assay was the only assay that provided a quantitative indication of protective activity. Both immunogenicity and challenge assays provided useful data on acellular pertussis vaccine properties but were complementary and not alternatives. PMID:25424817

  12. Laboratory Diagnosis of Pertussis.

    PubMed

    van der Zee, Anneke; Schellekens, Joop F P; Mooi, Frits R

    2015-10-01

    The introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. During an outbreak, timely diagnosis of pertussis allows prophylactic treatment of infants too young to be (fully) vaccinated, for whom pertussis is a severe, sometimes fatal disease. Finally, reliable diagnosis of pertussis is required to reveal trends in the (age-specific) disease incidence, which may point to changes in vaccine efficacy, waning immunity, and the emergence of vaccine-adapted strains. Here we review current approaches to the diagnosis of pertussis and discuss their limitations and strengths. In particular, we emphasize that the optimal diagnostic procedure depends on the stage of the disease, the age of the patient, and the vaccination status of the patient.

  13. Laboratory Diagnosis of Pertussis

    PubMed Central

    Schellekens, Joop F. P.; Mooi, Frits R.

    2015-01-01

    SUMMARY The introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. During an outbreak, timely diagnosis of pertussis allows prophylactic treatment of infants too young to be (fully) vaccinated, for whom pertussis is a severe, sometimes fatal disease. Finally, reliable diagnosis of pertussis is required to reveal trends in the (age-specific) disease incidence, which may point to changes in vaccine efficacy, waning immunity, and the emergence of vaccine-adapted strains. Here we review current approaches to the diagnosis of pertussis and discuss their limitations and strengths. In particular, we emphasize that the optimal diagnostic procedure depends on the stage of the disease, the age of the patient, and the vaccination status of the patient. PMID:26354823

  14. Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    SciTech Connect

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-05-01

    Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/sub z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

  15. [Highly sensitive detection technology for biological toxins applying sugar epitopes].

    PubMed

    Uzawa, Hirotaka

    2009-01-01

    The Shiga toxin is a highly poisonous protein produced by enterohemorrhagic Escherichia coli O157. This bacterial toxin causes the hemolytic uremic syndrome. Another plant toxin from castor beans, ricin, is also highly toxic. The toxin was used for assassination in London. Recently, there were several cases of postal matter containing ricin. Both toxins are categorized as biological warfare agents by the Centers of Disease Control and Prevention. Conventional detection methods based on the antigen-antibody reaction, PCR and other cell-free assays have been proposed. However, those approaches have drawbacks in terms of sensitivity, analytical time, or stability of the detection reagents. Therefore, development of a facile and sensitive detection method is essential. Here we describe new detection methods applying carbohydrate epitopes as the toxin ligands, which is based on the fact that the toxins bind cell-surface oligosaccharides. Namely, the Shiga toxin has an affinity for globobiosyl (Gb(2)) disaccharide, and ricin binds the beta-D-galactose residue. For Shiga toxin detection, surface plasmon resonance (SPR) was applied. A polyanionic Gb(2)-glycopolymer was designed for this purpose, and it was used for the assembly of Gb(2)-chips using alternating layer-by-layer technology. The method allowed us to detect the toxin at a low concentration of LD(50). A synthetic carbohydrate ligand for ricin was designed and immobilized on the chips. SPR analysis with the chips allows us to detect ricin in a highly sensitive and facile manner (10 pg/ml, 5 min). Our present approaches provide a highly effective way to counter bioterrorism.

  16. Prevalence of Bordetella holmesii and Bordetella bronchiseptica in respiratory tract samples from Belgian patients with pertussis-like symptoms by sensitive culture method and mass spectrometry.

    PubMed

    Van den Bossche, D; De Bel, A; De Smet, D; Heylen, O; Vekens, E; Vandoorslaer, K; Soetens, O; Piérard, D

    2013-01-01

    Insertion sequences IS481 and IS1001 are targets for molecular detection of respectively Bordetella pertussis and Bordetella parapertussis. There is a raising concern about specificity of these targets due to sequence similarity with Bordetella holmesii and Bordetella bronchiseptica. The likelihood of false (para)pertussis diagnoses should be correlated with the prevalence of these organisms in the respiratory tract (RT). From October 2010 until September 2011, 2,207 RT samples were submitted to the Belgian reference laboratory for pertussis diagnosis. End-point IS481/IS 1001 PCR and culture were performed for B. pertussis and B. parapertussis. We developed a sensitive culture method followed by screening with matrix-assisted laser desorption/ionisation- time of flight mass spectrometry (MALDI-TOF MS) to look for B. holmesii and B. bronchiseptica in our samples,. Only one B. bronchiseptica and no B. holmesii were detected in RT samples from Belgian patients with pertussis-like symptoms.

  17. Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin.

    PubMed

    Brito, G A; Falcão, J L; Saraiva, S N; Lima, A A; Flores, C A; Ribeiro, R A

    1998-10-01

    In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 +/- 0.19 and Dex = 0.35 +/- 0.13 vs saline (S) = 2.85 +/- 0.59; fMLP: Ptx = 0.43 +/- 0.09 and Dex 0.01 +/- 0.01 vs S = 1.08 +/- 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 +/- 1.4 and Dex = 3.06 +/- 0.76 vs S = 15.94 +/- 3.97; fMLP: Ptx = 3.85 +/- 0.56 and Dex = 0.40 +/- 0.16 vs S = 7.15 +/- 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 microns), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 +/- 0.38 vs S = 4.20 +/- 1.01; fMLP: Dex = 0.25 +/- 0.11 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 +/- 2.25 vs S = 4.20 +/- 1.01; fMLP: Ptx = 4.66 +/- 1.24 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial

  18. Mucosal and systemic antibody responses against an acellular pertussis vaccine in mice after intranasal co-administration with recombinant cholera toxin B subunit as an adjuvant.

    PubMed

    Isaka, Masanori; Yasuda, Yoko; Taniguchi, Tooru; Kozuka, Satoshi; Matano, Keiko; Maeyama, Jun-ichi; Morokuma, Kazunori; Ohkuma, Kunio; Goto, Norihisa; Tochikubo, Kunio

    2003-03-07

    To investigate the possibility of intranasal immunization with an acellular pertussis vaccine, groups of mice were administered intranasally with aluminium-non-adsorbed pertussis toxoid (PTd; 0.5 or 5 microg) and formalin-treated filamentous hemagglutinin (fFHA; 5 microg) with and without recombinant cholera toxin B subunit (rCTB; 10 microg) as a mucosal adjuvant. At a low concentration of PTd, the following things became clear: (1) earlier and higher elevation of serum anti-PTd and anti-FHA IgG antibody titres in the presence of rCTB than in its absence, (2) higher serum anti-PTd and anti-FHA IgG antibody titres than 200 and 100 ELISA units ml(-1) (EU ml(-1)) in all mice, respectively, in the presence of rCTB, which were obtained by calibration against a reference anti-pertussis mouse serum, and (3) in an intranasal challenge experiment with Bordetella pertussis, slightly more rapid elimination of the bacteria from the lungs of mice intranasally immunized in the presence of rCTB, suggesting the effectiveness of rCTB as a mucosal adjuvant. However, irrespective of rCTB and dose of PTd, mice which were immunized four times and sacrificed on day 35 developed high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs; only a slight or no increase of anti-FHA mucosal IgA antibodies was observed in the lung. These facts suggested the immunogenicity and mucosal adjuvanticity of PTd, and therefore, the mucosal adjuvanticity of rCTB seemed to be inconspicuous. Moreover, the addition of rCTB induced higher anti-PTd serum IgE antibody responses than no addition of it depending on dose of PTd. These results show that dose of PTd included in an acellular pertussis vaccine had better be low as possible and the addition of rCTB may not be always necessary in case of this nasal vaccine alone unlike tetanus and diphtheria toxoids and hepatitis B virus vaccine reported before.

  19. Collaborative study for the standardisation of the histamine sensitizing test in mice and the CHO cell-based assay for the residual toxicity testing of acellular pertussis vaccines.

    PubMed

    Xing, D; Maes, A; Behr-Gross, M-E; Costanzo, A; Daas, A; Buchheit, K-H

    2010-04-01

    The European Pharmacopoeia (Ph. Eur.) and the World Health Organisation (WHO) require the performance of extensive quality and safety control testing before the release on the market of vaccine products for human use. Safety testing with regard to residual pertussis toxin (PT) in acellular pertussis combination vaccines is performed through assessment of fatal sensitisation of mice to histamine challenge by the vaccine product under test. Currently, use of different in-house procedures and no requirement for the inclusion of a standard reference in each assay render comparisons of results obtained for identical vaccine batches between different control laboratories very difficult. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM), an international collaborative study was organised for the standardization of the Histamine Sensitizing Test (HIST) in mice and the Chinese Hamster Ovary (CHO)-cell-based assay (performed at the bulk product level) for the residual toxicity testing of acellular pertussis vaccines or acellular pertussis-based combination vaccines. The study was run under the aegis of the Biological Standardisation Programme, jointly supported by the Council of Europe and the European Commission under the project code BSP076. Ten (10) laboratories participated in the study and were requested to perform 3 independent Histamine Sensitizing Tests in mice and to report results of the lethal end-point measurement as prescribed by the Ph. Eur. monographs. Some of them also reported data from an in-house validated CHO-cell-based assay. In addition, some of the laboratories reported concomitantly data obtained by measurement of the drop in temperature induced after the histamine challenge, a method currently under investigation to be added as an alternative end-point for the HIST in the Ph. Eur. monographs for acellular pertussis-based combination vaccines in order to alleviate animal suffering (in application of the 3

  20. Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis.

    PubMed

    Skopova, Karolina; Tomalova, Barbora; Kanchev, Ivan; Rossmann, Pavel; Svedova, Martina; Adkins, Irena; Bibova, Ilona; Tomala, Jakub; Masin, Jiri; Guiso, Nicole; Osicka, Radim; Sedlacek, Radislav; Kovar, Marek; Sebo, Peter

    2017-06-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b(+)) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b(-) cells. The nonhemolytic AC(+) Hly(-) bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC(+) Hly(-) mutant also infected mouse lungs as efficiently as the parental AC(+) Hly(+) strain. Hence, elevation of cAMP in CD11b(-) cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>10(7) CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent. Copyright © 2017 American Society for Microbiology.

  1. Epidermal growth factor (EGF) stimulated Ca/sup 2 +/ mobilization in hepatocytes is abolished by phorbol esters, pertussis toxin and partial hepatectomy

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1986-05-01

    EGF has been demonstrated to increase free intracellular Ca/sup 2 +/ levels in isolated hepatocytes putatively by generation of the second messenger inositol trisphosphate (IP/sub 3/). Pretreatment of cells with phorbol 12-myristate 13-acetate (PMA) inhibited the EGF (66 nM) stimulated Ca/sup 2 +/ response as measured by quin2. Inhibition by PMA was maximal within 3 min and was concentration dependent (IC/sub 50/ = 13.5 nM). Four other active phorbol ester analogues blocked the Ca/sup 2 +/ response while inactive analogues did not. EGF was unable to increase intracellular Ca/sup 2 +/ levels in hepatocytes isolated from rats treated with pertussis toxin for 72 hrs. Neither PMA nor toxin pretreatment was able to inhibit the Ca/sup 2 +/ response to angiotensin II (Ang II). In hepatocytes isolated 24 hrs after partial hepatectomy, the Ca/sup 2 +/ response to EGF (as measured by phosphorylase activity, EC/sub 50/ = 5 nM) was completely abolished and remained attenuated for 7 days post-hepatectomy. The Ca/sup 2 +/ response to Ang II in this model system was also blunted but required 3 days for development of the full effect and within 7 days full activity is nearly restored. The results suggest that fundamental differences exist in the transduction mechanisms used by these two Ca/sup 2 +/-linked hormones to mobilize intracellular Ca/sup 2 +/ (and putatively increase IP/sub 3/ formation).

  2. Unrecognized pertussis infection in adolescents.

    PubMed

    Cromer, B A; Goydos, J; Hackell, J; Mezzatesta, J; Dekker, C; Mortimer, E A

    1993-05-01

    Little information is available regarding the level of immunity to Bordetella pertussis among adolescents. We measured serum antibodies in 156 healthy adolescents to the following pertussis antigens: pertussis toxin, filamentous hemagglutinin, and 69-kd outer membrane protein. In an attempt to identify intercurrent pertussis infections, we also obtained a total of 43 repeated samples during the following 5 years. Using a 50% or greater rise in IgG enzyme-linked immunosorbent assay titers to define seroconversion, we found an annual incidence of 6.1%; by alternative definitions of seropositivity, the predicted annual incidence of infection ranged from 1.2% to 8.2%. These data suggest that infection with B pertussis is common in the adolescent population.

  3. Rapid diagnosis of pertussis in young infants: comparison of culture, PCR, and infant's and mother's serology.

    PubMed Central

    Grimprel, E; Njamkepo, E; Bégué, P; Guiso, N

    1997-01-01

    The contribution of maternal pertussis serology comparing prepartum serum to serum collected during the infant's disease to the diagnosis of pertussis in infants was evaluated for 28 pairs of young infants with pertussis syndrome and their mothers and was compared to those of culture and PCR. Infants had a nasopharyngeal aspiration tested by PCR, and acute and convalescent sera were collected during their disease. Mothers had a first acute serum collected concomitantly with the infant's acute serum, and both acute sera were compared to a prepartum serum. Sera were analyzed by immunoblotting for the detection of anti-pertussis toxin (PT) antibodies. Serological evidence of pertussis in infants was assessed as either an increase in anti-PT antibody levels between the mother's prepartum and acute sera or the presence of antibodies in the infant's acute serum and their absence in both the mother's acute and prepartum sera. Culture and PCR sensitivity were 43 and 89%, respectively. Most infants (18 of 24) had no pertussis antibody detectable in their acute sera, confirming a delayed immune response at this age. A comparison of infant's and mother's serology, using prepartum serum, rapidly confirmed the diagnosis in 57% of the cases. Although less sensitive than PCR, this serological method should be used for a rapid diagnosis of pertussis in young infants when culture and PCR are either not available or negative. PMID:9384297

  4. Unbinding forces of single pertussis toxin-antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance.

    PubMed

    Moreno, Nadège; Chevalier, Michel; Ronzon, Frédéric; Manin, Catherine; Dupuy, Monique; Krell, Tino; Rieu, Jean-Paul

    2011-01-01

    An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant k(a) and the dissociation constant k(d) . A significant reduction in k(a) was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the k(d) value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate k(d) values. The use of DFS as a technique to study ultratight binding is discussed. Copyright © 2011 John Wiley & Sons, Ltd.

  5. Comparative Study of Different Sources of Pertussis Toxin (PT) as Coating Antigens in IgG Anti-PT Enzyme-Linked Immunosorbent Assays

    PubMed Central

    Kapasi, Aditi; Meade, Bruce D.; Plikaytis, Brian; Pawloski, Lucia; Martin, Monte D.; Yoder, Sandra; Rock, Michael T.; Coddens, Séverine; Haezebroeck, Valérie; Fievet-Groyne, Françoise; Bixler, Garvin; Jones, Charles; Hildreth, Stephen; Edwards, Kathryn M.; Messonnier, Nancy E.

    2012-01-01

    In an effort to improve the reliability and reproducibility of serological assays for Bordetella pertussis, a collaborative study was conducted to compare four different sources of pertussis toxin (PT) as coating antigens in the immunoglobulin G (IgG) anti-PT enzyme-linked immunosorbent assay (ELISA). Four sources of PT were used as coating antigens in the IgG anti-PT ELISA in four different testing laboratories (labs A to D) to determine whether the different antigen preparations and different laboratories influenced assay results. A panel of 60 sera consisting of deidentified human specimens from previous vaccination trials of healthy adults and infants and clinical specimens from outbreak settings was tested. In the four laboratories, each sample was tested three times with the four PT antigens according to the standard coating optimization and IgG anti-PT ELISA testing procedures used in that laboratory. Differences among the antigens, as well as intra- and interlaboratory variability, were evaluated. Excellent agreement was observed with the test panel results among the four antigens within each laboratory. Concordance correlation coefficient (rc) measurements among the different antigens ranged from 0.99, 0.99 to 1.00, 1.00, and 0.97 to 1.00 for labs A to D, respectively. The comparisons between pairs of laboratories also indicated a high degree of concordance for each PT preparation, with rc measurements between 0.90 and 0.98, 0.93 and 0.99, 0.92 and 0.98, and 0.93 and 0.99 for antigens 1 to 4, respectively. Relatively minor differences in results were observed among laboratories or among antigens, suggesting that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine evaluation. PMID:22116688

  6. Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin.

    PubMed

    Karst, Johanna C; Barker, Robert; Devi, Usha; Swann, Marcus J; Davi, Marilyne; Roser, Stephen J; Ladant, Daniel; Chenal, Alexandre

    2012-03-16

    The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

  7. Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin*

    PubMed Central

    Karst, Johanna C.; Barker, Robert; Devi, Usha; Swann, Marcus J.; Davi, Marilyne; Roser, Stephen J.; Ladant, Daniel; Chenal, Alexandre

    2012-01-01

    The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1–384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1–489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375–485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1–384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells. PMID:22241477

  8. The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

    SciTech Connect

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. )

    1991-01-01

    In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

  9. Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

    SciTech Connect

    Jones, J.; Schultz, R.M. )

    1990-06-01

    G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

  10. Assignment of functional domains involved in ADP-ribosylation and B-oligomer binding within the carboxyl terminus of the S1 subunit of pertussis toxin.

    PubMed Central

    Krueger, K M; Barbieri, J T

    1994-01-01

    The roles of the carboxyl terminus of the S1 subunit (composed of 235 amino acids) of pertussis toxin in the ADP-ribosylation of transducin (Gt) and in B-oligomer binding were defined by analysis of two carboxyl-terminal deletion mutants of the recombinant S1 (rS1) subunit: C204, which is composed of amino acids 1 through 204 of S1, and C219, which is composed of amino acids 1 through 219 of S1. C204 was expressed in Escherichia coli as a stable, soluble peptide that had an apparent molecular mass of 23.4 kDa. In a linear velocity assay, the specific activity of C180 was 2% and that of C204 was 80% of the activity displayed by rS1 in catalyzing the ADP-ribosylation of Gt. In addition, C204 possessed catalytic efficiencies (kcat/Km) that were 110% at variable Gt concentrations and 40% at variable NAD concentrations of those reported for rS1. These data showed that the catalytic activity of C204 approached the activity of S1. C204 and C219 were unable to associate with the B oligomer under conditions which promoted association of rS1 with the B oligomer. Consistent with these results, mixtures of C204 or C219 with the B oligomer did not elicit a clustering phenotype in CHO cells, whereas rS1 which had associated with the B oligomer was as cytotoxic as native pertussis toxin. These data indicate that residues between 219 and 235 are important in the association of the S1 subunit with the B oligomer. These data allow the assignment of functional regions to the carboxyl terminus of S1. Residues 195 to 204 are required for optimal ADP-ribosyltransferase activity, residues 205 to 219 link the catalytic region of S1 and a B-oligomer-binding region of S1, and residues 220 to 235 are required for association of S1 with the B oligomer. Images PMID:8168972

  11. Survey and Rapid Detection of Bordetella pertussis in Clinical Samples Targeting the BP485 in China.

    PubMed

    Liu, Wei; Xu, Yinghua; Dong, Derong; Li, Huan; Zhao, Xiangna; Li, Lili; Zhang, Ying; Wei, Xiao; Wang, Xuesong; Huang, Simo; Zeng, Ming; Huang, Liuyu; Zhang, Shumin; Yuan, Jing

    2015-01-01

    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 B. 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 groups with different alleles of the virulence-related genes including four alleles of ptxA, six of prn, four of tcfA, two of fim2, and three 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.

  12. Survey and Rapid Detection of Bordetella pertussis in Clinical Samples Targeting the BP485 in China

    PubMed Central

    Liu, Wei; Xu, Yinghua; Dong, Derong; Li, Huan; Zhao, Xiangna; Li, Lili; Zhang, Ying; Wei, Xiao; Wang, Xuesong; Huang, Simo; Zeng, Ming; Huang, Liuyu; Zhang, Shumin; Yuan, Jing

    2015-01-01

    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 B. 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 groups with different alleles of the virulence-related genes including four alleles of ptxA, six of prn, four of tcfA, two of fim2, and three 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. PMID:25798436

  13. Pathogenesis and histopathology of pertussis: implications for immunization.

    PubMed

    Cherry, James D; Paddock, Christopher D

    2014-09-01

    Pertussis is a unique infectious disease in that it can be severe and fatal but occurs without fever and other evidence of an inflammatory illness. The authors with others have studied the histopathology of fatal pertussis and also the unique characteristics of severe pertussis in young infants. Histopathologic observations from approximately 100 years ago, and from recent evaluation, indicate that the histopathologic changes of the upper respiratory tract of patients with fatal pertussis are often relatively normal unless there is a secondary bacterial infection. Bordetella pertussis contains many protein antigens and perhaps a polysaccharide capsule which contribute to the infectious process. However, only two of these antigens contribute to clinical illness. These antigens are pertussis toxin and the yet to be identified 'cough toxin'. The authors speculate as to the nature of the 'cough toxin' and discuss the implications of their observations and concepts for the future control of pertussis.

  14. Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

    SciTech Connect

    Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

    1988-07-12

    The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate (Gpp(NH)p) on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed (/sup 32/P)ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-..cap alpha../sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-..cap alpha../sub i/ or anti-..cap alpha../sub 0/ antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed.

  15. Native and genetically inactivated pertussis toxins induce human dendritic cell maturation and synergize with lipopolysaccharide in promoting T helper type 1 responses.

    PubMed

    Ausiello, Clara M; Fedele, Giorgio; Urbani, Francesca; Lande, Roberto; Di Carlo, Beatrice; Cassone, Antonio

    2002-08-01

    The capacity of pertussis toxin (PT) to induce maturation and functional activities of human monocyte-derived dendritic cells (DCs) was investigated. Both native PT (nPT) and genetically detoxified PT (dPT) efficiently promoted expression on DCs of CD80, CD86, human leukocyte antigen-DR, and CD83 markers, alloreactive antigen presentation, and cytokine production, primarily interferon (IFN)-gamma. Although they did not affect interleukin (IL)-10 production by lipopolysaccharide (LPS)-stimulated DCs, both nPT and dPT strongly synergized with LPS for IL-12 production. PTs plus LPS-stimulated DCs secreted soluble factors fostering IFN-gamma but not IL-4 and IL-5 production by naive T cells. T helper type 1 (Th1) polarization was, as alloreactive antigen presentation, inhibited by anti-IL-12 monoclonal antibody. These findings support the notion that nPT, in addition to inducing specific immune response, is a potent Th1 adjuvant and that dPT fully preserves this adjuvanticity. The synergic interaction between PT and LPS in IL-12 production might be relevant for the mechanisms of vaccine-induced protection.

  16. Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine.

    PubMed

    Isaka, Masanori; Komiya, Takako; Takahashi, Motohide; Yasuda, Yoko; Taniguchi, Tooru; Zhao, Yanqiu; Matano, Keiko; Matsui, Hideyuki; Maeyama, Jun-Ichi; Morokuma, Kazunori; Ohkuma, Kunio; Goto, Norihisa; Tochikubo, Kunio

    2004-08-13

    Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

  17. Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein.

    PubMed

    Gabig, T G; English, D; Akard, L P; Schell, M J

    1987-02-05

    Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.

  18. Inhaled Bordetella pertussis vaccine decreases airway responsiveness in guinea pigs.

    PubMed

    Vargas, M H; Bazán-Perkins, B; Segura, P; Campos, M G; Selman, M; Montaño, L M

    1995-01-01

    Bordetella pertussis (BP) has been used as adjuvant for experimental animal immunization, but its effects on airway responsiveness are uncertain. Three groups of guinea pigs were used: animals with a single exposure to inhaled BP vaccine (strain 134, total dose 1.24 x 10(12) germs), animals submitted to a sensitization procedure through inhalation of ovalbumin plus BP, and healthy control animals. Four weeks after inhalation of BP or after the beginning of sensitization, dose- or concentration-response curves to histamine were constructed in vivo and in vitro (tracheal and parenchymal preparations). We found that BP alone produced lower responses to histamine than control guinea pigs in vivo (insufflation pressure, p = 0.0003) and in tracheal tissues (p = 0.04), but not in parenchymal preparations. Sensitization did not modify the responsiveness compared with their respective controls. These results suggest that some BP component(s), probably pertussis toxin, causes a long lasting airway hyporesponsiveness in guinea pigs.

  19. Sensitive detection of active Shiga toxin using low cost CCD based optical detector

    USDA-ARS?s Scientific Manuscript database

    To reduce the sources and incidence of food-borne illness there is a need to develop inexpensive sensitive devices for detection of active toxin, such as Shiga toxin type 2 (Stx2). This approach increases the availability of foodborne bacterial toxin diagnostics in regions where there are limited r...

  20. Bordetella pertussis.

    PubMed

    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.

  1. [Clinical research of Bordetella pertussis infection in infants with prolonged cough].

    PubMed

    Mi, Rong; Fu, Jin; Wang, Xiao-ying; Kang, Li-min; Li, Li; Xu, Fang-sheng; Cui, Xiao-dai

    2013-06-11

    To explore the prevalence of Bordetella pertussis (B. pertussis) infection in unvaccinated or incomplete vaccinated infants with cough for a prolonged duration. 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 enacted 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. 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. And 40 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. 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

  2. Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

    SciTech Connect

    De Lean, A.; Cantin, M.

    1986-03-05

    The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levels of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with (/sup 32/P) NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production.

  3. Role of CD11b/CD18 in the Process of Intoxication by the Adenylate Cyclase Toxin of Bordetella pertussis

    PubMed Central

    Eby, Joshua C.; Gray, Mary C.; Mangan, Annabelle R.; Donato, Gina M.

    2012-01-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis does not require a receptor to generate intracellular cyclic AMP (cAMP) in a broad range of cell types. To intoxicate cells, ACT binds to the cell surface, translocates its catalytic domain across the cell membrane, and converts intracellular ATP to cAMP. In cells that express the integrin CD11b/CD18 (CR3), ACT is more potent than in CR3-negative cells. We find, however, that the maximum levels of cAMP accumulation inside CR3-positive and -negative cells are comparable. To better understand how CR3 affects the generation of cAMP, we used Chinese hamster ovary and K562 cells transfected to express CR3 and examined the steps in intoxication in the presence and absence of the integrin. The binding of ACT to cells is greater in CR3-expressing cells at all concentrations of ACT, and translocation of the catalytic domain is enhanced by CR3 expression, with ∼80% of ACT molecules translocating their catalytic domain in CR3-positive cells but only 25% in CR3-negative cells. Once in the cytosol, the unregulated catalytic domain converts ATP to cAMP, and at ACT concentrations >1,000 ng/ml, the intracellular ATP concentration is <5% of that in untreated cells, regardless of CR3 expression. This depletion of ATP prevents further production of cAMP, despite the CR3-mediated enhancement of binding and translocation. In addition to characterizing the effects of CR3 on the actions of ACT, these data show that ATP consumption is yet another concentration-dependent activity of ACT that must be considered when studying how ACT affects target cells. PMID:22144488

  4. Guanine nucleotide-dependent, pertussis toxin-insensitive, stimulation of inositol phosphate formation by carbachol in a membrane preparation from astrocytoma cells

    SciTech Connect

    Hepler, J.R.; Harden, T.K.

    1986-03-05

    Formation of the inositol phosphates (InsP), InsP/sub 3/, InsP/sub 2/, and InsP/sub 1/ was increased in a concentration dependent manner (K/sub 0.5/ approx. 5 ..mu..M) by GTP..sigma..S in washed membranes prepared from /sup 3/H-inositol-prelabelled 1321N1 human astrocytoma cells. Both GTP..gamma..S and GppNHp stimulated InsP formation by 2-3 fold over control; GTP and GDP were much less efficacious and GMP had no effect. Although the muscarinic cholinergic receptor agonist carbachol had no effect in the absence of guanine nucleotide, in the presence of 10 ..mu..M GTP..gamma..S, carbachol stimulated (K/sub 0.5/ approx. 10 ..mu.. M) the formation of InsP above the level achieved with GTP..gamma..S alone. The effect of carbachol was completely blocked by atropine. The order of potency for a series of nucleotides for stimulation of InsP formation in the presence of 500 ..mu..M carbachol was GTP..gamma..S > GppNHp > GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate G/sub i/, had no effect on InsP formation in the presence of GTP..gamma..S or GTP..gamma..S plus carbachol. Histamine and bradykinin also stimulated InsP formation in the presence of GTP..gamma..S in washed membranes from 1321N1 cells. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not G/sub i/ is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.

  5. Interaction of the pertussis toxin peptide containing residues 30-42 with DR1 and the T-cell receptors of 12 human T-cell clones.

    PubMed Central

    De Magistris, M T; Di Tommaso, A; Domenighini, M; Censini, S; Tagliabue, A; Oksenberg, J R; Steinman, L; Judd, A K; O'Sullivan, D; Rappuoli, R

    1992-01-01

    The interaction of the immunodominant pertussis toxin peptide containing residues 30-42 (p30-42) with soluble DR1 molecules and the T-cell receptor (TCR) of 12 DR1-restricted human T-cell clones has been analyzed. Peptide analogues of p30-42 containing single alanine substitutions were used in DR1-binding and T-cell proliferation assays to identify the major histocompatibility complex and TCR contact residues. Each T-cell clone was found to recognize p30-42 with a different fine specificity. However, a common core comprising amino acids 33-39 was found to be important for stimulation of all T-cell clones. Within this core two residues, Leu33 and Leu36, interact with the DR1 molecule, whereas Asp34, His35, Thr37, and Arg39 are important for TCR recognition in most of the clones. Computer modeling of the structure of p30-42 showed that an alpha-helical conformation is compatible with the experimental data. The analysis of TCR rearrangement revealed that the peptide was recognized by T-cell clones expressing different variable region alpha (V alpha) and variable region beta (V beta) chains, although a preferential use of V alpha 8-V beta 13 and V alpha 11-V beta 18 combinations was found in clones from the same donor. Understanding the details of the interaction of antigenic peptides with the major histocompatibility complex and TCR molecules should provide the theoretical basis to design T-cell epitopes and obtain more immunogenic vaccines. Images PMID:1313575

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

    PubMed Central

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

    2016-01-01

    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. PMID:27043627

  7. Role of whole-cell pertussis vaccine in severe local reactions to the preschool (fifth) dose of diphtheria-pertussis-tetanus vaccine.

    PubMed Central

    Scheifele, D W; Bjornson, G; Halperin, S H; Mitchell, L; Boraston, S

    1994-01-01

    OBJECTIVE: To estimate the contribution of whole-cell pertussis vaccine to severe local reactions after the preschool (fifth) dose of adsorbed diphtheria toxoid-pertussis vaccine-tetanus toxoid (DPT) vaccine. DESIGN: Double-blind randomized controlled trial. SETTING: Urban community. PARTICIPANTS: Volunteer sample of 200 healthy children 4 to 6 years old who were eligible for the fifth dose of DPT vaccine. INTERVENTIONS: Children received, in both arms, either diphtheria toxoid-tetanus toxoid (DT) and monovalent pertussis vaccines (group A, 99 children) or DPT and meningococcal vaccines (group B, 101 children). All were licensed products from single lots. The children were assessed 24 hours later by a trained observer. Serum samples obtained before vaccination were tested for antibodies to tetanus and diphtheria toxins and five pertussis antigens by means of enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Rates of severe local reactions (an area of redness or swelling or both of 50 mm or greater) 24 hours after vaccination. Relation between serum antibody levels before vaccination and rates of severe local reactions to corresponding vaccines. RESULTS: All of the subjects were followed up 24 hours after vaccination. Severe redness was present in 38% given DPT vaccine, 29% given intramuscular pertussis vaccine and 9% given DT vaccine (p < or = 0.002, three-way comparison). Severe swelling was common after vaccination with all three products. After intramuscular pertussis vaccination a relation was evident between the prevaccination levels of antibody to whole-cell pertussis bacteria and the rates of redness (p < 0.02) but not between the prevaccination subcellular antibody levels and the rates of redness. CONCLUSION: That pertussis vaccine resembled the DPT vaccine in causing severe redness suggests that it is the principal cause of such reactions after DPT vaccination. The DT vaccine was also reactogenic; thus, cumulative sensitization to one or more of

  8. Sensitivity to Alternaria alternata toxin in citrus because of altered mitochondrial RNA processing

    PubMed Central

    Ohtani, Kouhei; Yamamoto, Hiroyuki; Akimitsu, Kazuya

    2002-01-01

    Specificity in the interaction between rough lemon (Citrus jambhiri Lush.) and the fungal pathogen Alternaria alternata rough lemon pathotype is determined by a host-selective toxin, ACR-toxin. Mitochondria from rough lemon are sensitive to ACR-toxin whereas mitochondria from resistant plants, including other citrus species, are resistant. We have identified a C. jambhiri mitochondrial DNA sequence, designated ACRS (ACR-toxin sensitivity gene), that confers toxin sensitivity to Escherichia coli. ACRS is located in the group II intron of the mitochondrial tRNA-Ala and is translated into a SDS-resistant oligomeric protein in C. jambhiri mitochondria but is not translated in the toxin-insensitive mitochondria. ACRS is present in the mitochondrial genome of both toxin-sensitive and -insensitive citrus. However, in mitochondria of toxin-insensitive plants, the transcripts from ACRS are shorter than those in mitochondria of sensitive plants. These results demonstrate that sensitivity to ACR-toxin and hence specificity of the interaction between A. alternata rough lemon pathotype and C. jambhiri is due to differential posttranscriptional processing of a mitochondrial gene. PMID:11842194

  9. Sensitivity to Alternaria alternata toxin in citrus because of altered mitochondrial RNA processing.

    PubMed

    Ohtani, Kouhei; Yamamoto, Hiroyuki; Akimitsu, Kazuya

    2002-02-19

    Specificity in the interaction between rough lemon (Citrus jambhiri Lush.) and the fungal pathogen Alternaria alternata rough lemon pathotype is determined by a host-selective toxin, ACR-toxin. Mitochondria from rough lemon are sensitive to ACR-toxin whereas mitochondria from resistant plants, including other citrus species, are resistant. We have identified a C. jambhiri mitochondrial DNA sequence, designated ACRS (ACR-toxin sensitivity gene), that confers toxin sensitivity to Escherichia coli. ACRS is located in the group II intron of the mitochondrial tRNA-Ala and is translated into a SDS-resistant oligomeric protein in C. jambhiri mitochondria but is not translated in the toxin-insensitive mitochondria. ACRS is present in the mitochondrial genome of both toxin-sensitive and -insensitive citrus. However, in mitochondria of toxin-insensitive plants, the transcripts from ACRS are shorter than those in mitochondria of sensitive plants. These results demonstrate that sensitivity to ACR-toxin and hence specificity of the interaction between A. alternata rough lemon pathotype and C. jambhiri is due to differential posttranscriptional processing of a mitochondrial gene.

  10. Seroprevalence of pertussis in China

    PubMed Central

    Xu, Ying Hua; Wang, Lichan; Xu, Jin; Wang, Xinjian; Wei, Chen; Luo, Peng; Ma, Xiao; Hou, Qiming; Wang, Junzhi

    2014-01-01

    Pertussis remains an important cause of infant death worldwide and is an ongoing public health concern even in countries with high vaccination coverage. A cross-sectional seroepidemiological study was undertaken to estimate true incidence rates and gain further insight into the epidemiology and burden of pertussis in China. During 2011, a total of 1080 blood samples were obtained from healthy individuals between 0 and 86 y of age in Zhengzhou, Central China. Serum IgG antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) were measured quantitatively using ELISA. The results showed that the geometric mean titers of PT and FHA IgG were 6.48 IU/mL (95% CI: 5.70–7.41 IU/mL) and 11.39 IU/mL (95% CI: 10.22–12.87 IU/mL) among subjects less than 4 y of age, indicating that pertussis antibody levels were low despite high vaccination coverage. Of the 850 subjects ≥4 y of age, 56 (6.6%) had anti-PT IgG titers above 30 IU/mL, and 11 (1.3%) had antibodies titers above 80 IU/mL. The estimated age-specific incidence of infection with B. pertussis revealed a peak incidence in the 31 to 40 y age group, followed by the 41 to 60 y age group. Taken together, these results indicate that pertussis is common in Chinese subjects in Zhengzhou, especially in adults, suggesting that the disease burden is underestimated in China. Therefore, our study stresses the importance of strengthening the diagnostic capacity and improving surveillance system for delineating current epidemiological profiles of pertussis. Most importantly, it may be advisable to re-evaluate the current Chinese pertussis immunization schedule and implement to booster doses for older children, adolescents and adults. PMID:24018405

  11. Survey of pertussis in patients with prolonged cough.

    PubMed

    Hu, Jen Jan; Lu, Chun Yi; Chang, Luan Yin; Huang, Chin Hao; Chou, Chen Cheng; Huang, Fu Yuan; Lee, Chin Yun; Huang, Li Min

    2006-02-01

    Pertussis is an acute respiratory tract illness resulting from Bordetella pertussis. Widespread use of pertussis vaccine over the past 50 years has decreased the incidence of pertussis. The incidence of pertussis in adolescents and adults has increased in many areas of the world. This study aimed to evaluate the etiologic role of B. pertussis in patients with prolonged cough in Taiwan. Patients with cough lasting for more than 1 week were recruited. Nasopharyngeal swabs were taken for culture of B. pertussis and detection of nucleic acid of B. pertussis by polymerase chain reaction. Serum samples were collected in a subset of patients for assay of immunoglobulin G and immunoglobulin A antibodies against pertussis toxin. In total, 111 patients were recruited. Thirty-three patients agreed to have their serum samples taken and tested. Eight patients had evidence of acute infection with B. pertussis; among them, 1 was diagnosed by polymerase chain reaction and 7 by serology. Older subjects were more likely to suffer from pertussis than younger subjects. The incidence of pertussis in patients with prolonged cough was 7.2%. However, the rate could have been as high as 21% in those with serum samples tested. We conclude that pertussis is a prevalent disease in Taiwan, especially in adolescents and adults.

  12. Differentially Expressed Genes in Bordetella pertussis Strains Belonging to a Lineage Which Recently Spread Globally

    PubMed Central

    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

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

    PubMed

    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

  14. Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: comparison with membranous adenylyl cyclase I.

    PubMed

    Schuler, Dominik; Lübker, Carolin; Lushington, Gerald H; Tang, Wei-Jen; Shen, Yuequan; Richter, Mark; Seifert, Roland

    2012-04-01

    The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2'-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Prevalence of Bordetella pertussis and Bordetella parapertussis infections in Tunisian hospitalized infants: results of a 4-year prospective study.

    PubMed

    Zouari, Asma; Smaoui, Hanen; Brun, Delphine; Njamkepo, Elisabeth; Sghaier, Soufien; Zouari, Emna; Félix, Renaud; Menif, Khaled; Ben Jaballah, Najla; Guiso, Nicole; Kechrid, Amel

    2012-04-01

    The prevalence of pertussis in Tunisia remains undetermined essentially because of the unavailability of a basic laboratory diagnostic service. Specific diagnostic tools were applied for the first time in a Tunisian prospective study in order to get a first estimation of the prevalence of Bordetella pertussis/parapertussis infections and to evaluate their use to determine the epidemiologic characteristics of these infections in Tunisian infants. Between 2007 and 2011, a total of 626 samples from 599 infants aged <1 year with and without pertussoid cough were investigated for the presence of B. pertussis/parapertussis using culture and real-time polymerase chain reaction (PCR). The real-time PCR (RT-PCR) targets include IS481 commonly found in B. pertussis, B. bronchiseptica, and B. holmesii; IS1001 specific of B. parapertussis, in combination with the pertussis toxin promoter region gene (ptx) of B. pertussis; and the recA gene specific of B. holmesii. When possible, patients' household contacts provided nasopharyngeal aspirates (NPAs) for RT-PCR detection of B. pertussis/parapertussis or single-serum samples for anti-PT IgG quantification. All except 1 NPAs were negative by conventional culture, whereas PCR gave positive signals for 126 specimens (21%): B. pertussis, B. parapertussis, and Bordetella spp. were detected in 82%, 6%, and 4% of the samples, respectively. The simultaneous presence of B. pertussis and B. parapertussis was noted in 8% of the cases. Pertussis was reported throughout the year with a peak during the summer of the year 2009. The prevalence of Bordetella infection was 20% between 2007 and 2011. Most of these cases corresponded to patients younger than 6 months who received <3 doses of pertussis vaccine. Among the household contacts enrolled in the study, mothers seemed to be the likely source of infection. This study showed that pertussis is still prevalent in Tunisia and that the disease remains a public health problem affecting not only

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

    SciTech Connect

    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.

  17. Antibody Response from Whole-Cell Pertussis Vaccine Immunized Brazilian Children against Different Strains of Bordetella pertussis

    PubMed Central

    Pereira, Alexandre; Pietro Pereira, Aparecida S.; Silva, Célio Lopes; de Melo Rocha, Gutemberg; Lebrun, Ivo; Sant'Anna, Osvaldo A.; Tambourgi, Denise V.

    2010-01-01

    Bordetella pertussis is a gram-negative bacillus that causes the highly contagious disease known as pertussis or whooping cough. Antibody response in children may vary depending on the vaccination schedule and the product used. In this study, we have analyzed the antibody response of cellular pertussis vaccinated children against B. pertussis strains and their virulence factors, such as pertussis toxin, pertactin, and filamentous hemagglutinin. After the completion of the immunization process, according to the Brazilian vaccination program, children serum samples were collected at different periods of time, and tested for the presence of specific antibodies and antigenic cross-reactivity. Results obtained show that children immunized with three doses of the Brazilian whole-cell pertussis vaccine present high levels of serum antibodies capable of recognizing the majority of the components present in vaccinal and non-vaccinal B. pertussis strains and their virulence factors for at least 2 years after the completion of the immunization procedure. PMID:20348518

  18. Pertussis (Whooping Cough) Complications

    MedlinePlus

    ... Controls Search Form Controls Cancel Submit Pertussis (Whooping Cough) Note: Javascript is disabled or is not supported ... Photos of Pertussis Pertussis Vaccination Pregnancy and Whooping Cough Clinicians Disease Specifics Treatment Clinical Features Clinical Complications ...

  19. Pertussis (Whooping Cough) Outbreaks

    MedlinePlus

    ... Controls Search Form Controls Cancel Submit Pertussis (Whooping Cough) Note: Javascript is disabled or is not supported ... Photos of Pertussis Pertussis Vaccination Pregnancy and Whooping Cough Clinicians Disease Specifics Treatment Clinical Features Clinical Complications ...

  20. Pertussis outbreak in university students and evaluation of acellular pertussis vaccine effectiveness in Japan.

    PubMed

    Hara, Megumi; Fukuoka, Mami; Tashiro, Katsuya; Ozaki, Iwata; Ohfuji, Satoko; Okada, Kenji; Nakano, Takashi; Fukushima, Wakaba; Hirota, Yoshio

    2015-02-06

    Recent studies worldwide have reported increasing numbers of adults diagnosed with Bordetella pertussis despite receiving childhood vaccinations. This study describes a pertussis outbreak at a university medical faculty campus and examines the effectiveness of diphtheria, tetanus, and pertussis (DTaP) vaccination completed during infancy in Japan. After the outbreak, self-administered questionnaires and serum samples were collected from students on campus to determine the incidence of pertussis and underlying diseases. Pertussis was diagnosed on the basis of clinical criteria and serum anti-pertussis toxin antibody levels. Using data collected from 248 first and second grade students who had submitted copies of their vaccination records, we evaluated the effectiveness of DTaP vaccination in infancy against adult pertussis. Questionnaire responses were obtained from 636 students (of 671 registered students; 95% response rate). Of 245 students who reported a continuous cough during the outbreak period, 84 (attack rate: 13.2%) were considered "probable" pertussis cases that met clinical criteria. The outbreak occurred mainly in first and second grade students in the Faculty of Medicine. Of 248 students who provided vaccination records, 225 had received 4 DTaP doses (coverage: 90.7%); the relative risk of the complete vaccination series compared to those with fewer than 4 doses or no doses for probable cases was 0.48 (95% confidence interval: 0.24-0.97). Waning protection was suspected due to over time. Booster vaccination for teenagers and development of highly efficacious pertussis vaccines are needed.

  1. Bordetella pertussis: new concepts in pathogenesis and treatment

    PubMed Central

    Carbonetti, Nicholas H.

    2016-01-01

    Purpose of review The purpose of this review is to summarize and discuss recent findings and selected topics of interest in Bordetella pertussis virulence and pathogenesis and treatment of pertussis. It is not intended to cover issues on immune responses to B. pertussis infection or problems with currently used pertussis vaccines. Recent findings Studies on the activities of various B. pertussis virulence factors include the immunomodulatory activities of filamentous hemagglutinin, fimbriae, and adenylate cyclase toxin. Recently emerging B. pertussis strains show evidence of genetic selection for vaccine escape mutants, with changes in vaccine antigen-expressing genes, some of which may have increased the virulence of this pathogen. Severe and fatal pertussis in young infants continues to be a problem, with several studies highlighting predictors of fatality, including the extreme leukocytosis associated with this infection. Treatments for pertussis are extremely limited, though early antibiotic intervention may be beneficial. Neutralizing pertussis toxin activity may be an effective strategy, as well as targeting two host proteins, pendrin and sphingosine-1-phosphate receptors, as novel potential therapeutic interventions. Summary Pertussis is reemerging as a major public health problem and continued basic research is revealing information on bacterial virulence and disease pathogenesis, as well as potential novel strategies for vaccination and targets for therapeutic intervention. PMID:26906206

  2. Evaluation of an in vitro assay system as a potential alternative to current histamine sensitization test for acellular pertussis vaccines.

    PubMed

    Xing, Dorothy; Yuen, Chun-Ting; Asokanathan, Catpagavalli; Rigsby, Peter; Horiuchi, Yoshinobu

    2012-11-01

    The histamine sensitization test (HIST) is a lethal test for batch release of acellular pertussis or its combination vaccines (ACV). Large numbers of animals have been used and it is difficult to standardize. Therefore there is an urgent need to develop an in vitro alternative to HIST. An in vitro test system has been developed as a potential alternative to HIST, to examine both the functional domains of PT based on a combination of enzyme coupled-HPLC (E-HPLC) and carbohydrate binding assays. We describe here an international collaborative study, which involved sixteen laboratories from 9 countries to assess the methodology transferability of the in vitro test system and its suitability for the testing of three different types of ACV products that are currently used worldwide. This study also evaluated further the relationship between the in vivo activity by HIST and the in vitro assay system. The results showed that the methodology of the E-HPLC and carbohydrate binding assays are transferable between laboratories worldwide and is suitable for the three types of ACV products included in the study. Although direct correlation between the in vitro assay system and the in vivo HIST (temperature reduction assay) for each individual vaccine lot cannot be established due to the large variation in the HIST results, the observation that the mean estimates of the in vitro and in vivo activities gave the same rank order of the three vaccine types included in the study is encouraging. The in vitro systems provide reproducible product specific profiles which supports their use as a potential alternative to the HIST.

  3. Your Child's Immunizations: Diphtheria, Tetanus & Pertussis Vaccine (DTaP)

    MedlinePlus

    ... at any age, caused by toxin-producing bacteria contaminating a wound Pertussis (whooping cough) : a respiratory illness ... an earlier DTaP shot: a brain or nervous-system problem, like a seizure the worsening of a ...

  4. Serodiagnosis as adjunct assay for pertussis infection in São Paulo, Brazil.

    PubMed

    Vaz-de-Lima, Lourdes R A; Martin, Monte D; Pawloski, Lucia C; Leite, Daniela; Rocha, Karen C P; de Brito, Cyro A; Vaz, Tânia M I; Martins, Luciano Moura; Alvarenga, Danielly P; Ribeiro, Ana F; Carvalhanas, Telma R M P; Nakasaki, Rosa M D; Oliveira, Silvia S; Waldman, Eliseu A; Tondella, Maria Lucia

    2014-05-01

    Pertussis remains an important public health problem in many countries despite extensive immunization. Cultures and real-time PCR (RT-PCR) assays are the recommended pertussis diagnostic tests, but they lack sensitivity at the later stage of the disease. This study introduces the IgG anti-pertussis toxin enzyme-linked immunosorbent assay (PT ELISA) in our routine diagnosis to improve disease burden estimation. Serum samples and nasopharyngeal swabs (n = 503) were collected at the same time from patients presenting with cough illness suspected of being pertussis and tested by the PT ELISA and culture and/or RT-PCR, respectively. Patients were separated into three age groups: group 1, <1 year (n = 260; mean age, 3 months), group 2, 1 to 6 years (n = 81; mean age, 3 years), and group 3, ≥7 years (n = 162; mean age, 26 years). The times (means) from cough onset to specimen collection were 16, 24, and 26 days, respectively. In group 1, 83 (82.2%) of 101 positive cases were positive for pertussis by culture/RT-PCR, while 40 (39.6%) tested positive by PT ELISA. In group 2, 6 (19.4%) of 31 positive cases were culture/RT-PCR positive, and 29 (93.6%) were seropositive. In group 3, 13 (13.8%) of 94 positive cases were positive by culture/RT-PCR and 91 (96.8%) were positive by serology. Culture/RT-PCR detected more cases of pertussis in infants (P < 0.0001), whereas the PT ELISA detected more cases in adolescents and adults (P < 0.0001). The timing between cough onset and specimen collection or recent vaccination may have partially affected our results. Serology is a suitable, cost-effective, and complementary pertussis diagnostic tool, especially among older children, adolescents, and adults during the later disease phase.

  5. Serodiagnosis as Adjunct Assay for Pertussis Infection in São Paulo, Brazil

    PubMed Central

    Martin, Monte D.; Pawloski, Lucia C.; Leite, Daniela; Rocha, Karen C. P.; de Brito, Cyro A.; Vaz, Tânia M. I.; Martins, Luciano Moura; Alvarenga, Danielly P.; Ribeiro, Ana F.; Carvalhanas, Telma R. M. P.; Nakasaki, Rosa M. D.; Oliveira, Silvia S.; Waldman, Eliseu A.; Tondella, Maria Lucia

    2014-01-01

    Pertussis remains an important public health problem in many countries despite extensive immunization. Cultures and real-time PCR (RT-PCR) assays are the recommended pertussis diagnostic tests, but they lack sensitivity at the later stage of the disease. This study introduces the IgG anti-pertussis toxin enzyme-linked immunosorbent assay (PT ELISA) in our routine diagnosis to improve disease burden estimation. Serum samples and nasopharyngeal swabs (n = 503) were collected at the same time from patients presenting with cough illness suspected of being pertussis and tested by the PT ELISA and culture and/or RT-PCR, respectively. Patients were separated into three age groups: group 1, <1 year (n = 260; mean age, 3 months), group 2, 1 to 6 years (n = 81; mean age, 3 years), and group 3, ≥7 years (n = 162; mean age, 26 years). The times (means) from cough onset to specimen collection were 16, 24, and 26 days, respectively. In group 1, 83 (82.2%) of 101 positive cases were positive for pertussis by culture/RT-PCR, while 40 (39.6%) tested positive by PT ELISA. In group 2, 6 (19.4%) of 31 positive cases were culture/RT-PCR positive, and 29 (93.6%) were seropositive. In group 3, 13 (13.8%) of 94 positive cases were positive by culture/RT-PCR and 91 (96.8%) were positive by serology. Culture/RT-PCR detected more cases of pertussis in infants (P < 0.0001), whereas the PT ELISA detected more cases in adolescents and adults (P < 0.0001). The timing between cough onset and specimen collection or recent vaccination may have partially affected our results. Serology is a suitable, cost-effective, and complementary pertussis diagnostic tool, especially among older children, adolescents, and adults during the later disease phase. PMID:24599531

  6. Seroprevalence of pertussis among Danish patients with cough of unknown etiology.

    PubMed

    Dalby, Tine; Harboe, Zitta B; Krogfelt, Karen Angeliki

    2010-12-01

    The common perception that pertussis is only a childhood disease is not correct. Vaccination or infection with Bordetella pertussis provides only short-lived protection against pertussis-and the majority of the population is consequently at risk of contracting pertussis. We evaluated the seroprevalence of pertussis antibodies (IgG against pertussis toxin) in serum samples from 265 Danish patients, aged 8 years and older, with coughs of unknown etiology. Depending on the cutoff chosen, we found that 2.6% to 10.9% of these patients were seropositive for pertussis. Of 178 patients with a reported duration of cough between 2 weeks and 3 months, 3.4% to 12.4% were seropositive for pertussis, indicating recent infection. Our study indicates that B. pertussis infection may be underdiagnosed among older children and adults with coughs in Denmark.

  7. Frequency of pertussis in children with prolongued cough.

    PubMed

    Yildirim, Inci; Ceyhan, Mehmet; Kalayci, Omer; Cengiz, Ali Bulent; Secmeer, Gulten; Gur, Deniz; Pelton, Steve

    2008-01-01

    To determine the frequency of pertussis in children < or = 16 y who had prolonged cough (> or = 14 d), a prospective study was conducted at an outpatient clinic of a paediatric hospital. Nasopharyngeal swabs were taken for culture and nucleic acid testing by polymerase chain reaction (PCR) for Bordetella pertussis. Immunoglobulin A and immunoglobulin G antibodies against pertussis toxin (PT) were tested by ELISA in paired serum samples. A total of 148 patients were recruited during 1 y. Pertussis was detected in 25 (16.9%) patients with at least 1 of the tests. PCR was positive in 12 patients, and 9 cases was diagnosed serologically. Both PCR and serology were positive in 4 children. Duration of cough was longer in the patients with pertussis (median 33 vs 20, p = 0.03). Seropositivity of pertussis toxin was higher in pertussis negative patients during enrollment (24% vs 65%, p = 0.005). From the results of this study, B. pertussis seems to be common in our population despite high immunization rates with whole cell vaccine. Although the duration of cough is defined as longer than 21 d in some studies for pertussis case definition criteria, it was shorter than this in 3 of our cases.

  8. Plant Compounds Enhance the Assay Sensitivity for Detection of Active Bacillus cereus Toxin

    PubMed Central

    Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

    2015-01-01

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety. PMID:25767986

  9. Plant compounds enhance the assay sensitivity for detection of active Bacillus cereus toxin.

    PubMed

    Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

    2015-03-11

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety.

  10. Thermal sensitivity of Clostridium botulinum type C toxin.

    PubMed Central

    Hubálek, Z.; Halouzka, J.

    1988-01-01

    A sterile suspension containing 950 mouse LD50 per ml of type C botulinum toxin was exposed for various periods to different temperatures. The time required for the 99% (hundred-fold) reduction of toxicity was more than 5 years at -70 degrees C or -20 degrees C, 6 months at +5 degrees C, 3 weeks at +20 degrees C, 2 weeks at +28 degrees C, 2 days at +37 degrees C, 9 h at +42 degrees C, less than 30 min at +56 degrees C, less than 20 min at +60 degrees C, and below 5 min at +80 degrees C. The results suggest that Clostridium botulinum type C toxin, if produced in an ecosystem of the mild climatic zone, might persist there over the winter season and cause the intoxication of vertebrates next early spring in the absence of further microbial toxigenesis. PMID:2972554

  11. 77 FR 52333 - International Workshop on Alternatives to the Murine Histamine Sensitization Test (HIST) for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-29

    ... HUMAN SERVICES International Workshop on Alternatives to the Murine Histamine Sensitization Test (HIST... an ``International Workshop on Alternatives to the Murine Histamine Sensitization Test (HIST) for... histamine sensitization test (HIST) is a key safety test used to monitor residual levels of pertussis toxin...

  12. Sensitivity and specificity of single IgA and IgG antibody concentrations for early diagnosis of pertussis in adults: an evaluation for outbreak management in public health practice

    PubMed Central

    Mertens, Paul LJM; Stals, Frans S; Steyerberg, Ewout W; Richardus, Jan H

    2007-01-01

    Background An accurate, practical laboratory test is needed to confirm clinical diagnosis of pertussis in adults during the first 3 symptomatic weeks, when treatment is effective and transmission can be interrupted. Methods The sensitivity and specificity of single IgA and IgG levels were assessed in a cohort study of a pertussis epidemic in 99 adults in a closed community. Sensitivities were assessed in the sera of 46 laboratory confirmed clinical pertussis cases during the first 3 weeks. Specificities were calculated in sera of 35 asymptomatic controls without clinical symptoms or laboratory confirmed infections from the same community (internal controls). We compared these specificities with the specificities of single IgA and IgG levels in 4275 external controls from a cross-section of the general Dutch population aged 21–79 years who had not coughed for more than 2 weeks in the past year, and without pertussis diagnoses. The study was done in the Netherlands when whole-cell pertussis vaccine was used in the national vaccination programme. Results Levels of 24 U/ml for IgA and 27 U/ml for IgG gave sensitivities of 100% and 75%, respectively, in the first 2 weeks, 100% in the third week, and 97% after the fourth week. The levels were reached within 2 days after onset of increase, and remained above these levels for roughly 7.2 and 5.1 months, respectively. Specificity was 82% for IgA and 89% for IgG in the internal controls and 90% in the external controls, respectively. Conclusion We suggest levels of 24 U/ml for IgA level and 27 U/ml (= 27 International Units (IU)/ml) for IgG as sensitive, specific, and practical for laboratory confirmation of clinical pertussis in adults in the first 3 weeks of outbreak management. PMID:17553132

  13. [Immunogenicity and safety of vaccine preparations based on circulating Bordetella pertussis strains].

    PubMed

    Zaĭtsev, E M; Mertsalova, N Iu; Britsina, M V; Ozeretskovskaia, M N; Bazhanova, I G; Shinkarev, A S; Poddubikov, A V

    2014-01-01

    Study specific activity and safety ofvaccine preparations based on circulating B. pertussis strains with currently predominating allele variants of pertussis toxin (ptxA1) and pertactin (prn2) genes. B. pertussis strains isolated from pertussis patients in Moscow in 2001-2010 were grown in dense and liquid media. The content of separate antigens in B. pertussis strains was determined by EIA. Immunogenicity and safety of the preparations was determined in F1(CBAxC57B16) line mice. All the studied circulating B. pertussis strains expressed pertussis toxin (PT), filamentous hemagglutinin (FHA) and agglutinogens corresponding to the serovar. Whole-cell and acellular pertussis vaccines were prepared based on the circulating strains, and a highly productive recently isolated toxigenic B.pertussis strain that could be used for production ofpertussis vaccines was selected as a result of studies ofimmunogenic, toxic and sensibilizing properties. Vaccine preparations based on a B. pertussis strain adapted to growth in liquid media with pertussis toxin and pertactin ptxAl1 - prn2 gene allele variation characteristic for contemporary population are specifically active and safe.

  14. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    SciTech Connect

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  15. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    PubMed

    Jenko, Kathryn L; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D; Varnum, Susan M

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg mL(-1) (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg mL(-1) (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg mL(-1) (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation <20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little cross-reactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  16. Sensitivity of Hep G2 cells to Bacillus cereus emetic toxin.

    PubMed

    Kamata, Yoichi; Kanno, Shinji; Mizutani, Noriko; Agata, Norio; Kawakami, Hiroshi; Sugiyama, Kei-ichi; Sugita-Konishi, Yoshiko

    2012-11-01

    We herein examined the sensitivity of Hep G2 human hepatoma cells to Bacillus cereus emetic toxin. Hep G2 cells were treated with the emetic toxin, and the cell shape was observed. The same experiments were performed for comparison purposes, using HEp-2 cells, which are currently used by most laboratories for a bioassay of the emetic toxin. Hep G2 cells showed clearer vacuolation in the cytosol within 2 hr and required a shorter incubation period than HEp-2 cells (10 hr). The number of vacuoles in the Hep G2 cells was greater, and the size of the vacuoles was larger than those observed in HEp-2 cells. The minimal concentration of the emetic toxin required to induce the vacuolation of Hep G2 cells was 0.04 ng/ml. The concentration for the HEp-2 cells was 1 ng/ml. These findings indicate that Hep G2 cells show higher sensitivity to the emetic toxin. Hep G2 cells may be superior to the currently used HEp-2 cells for the bioassay of the emetic toxin.

  17. Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells.

    PubMed

    Starost, Laura Julia; Karassek, Sascha; Sano, Yasuteru; Kanda, Takashi; Kim, Kwang Sik; Dobrindt, Ulrich; Rüter, Christian; Schmidt, Marcus Alexander

    2016-10-13

    Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood-brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218's effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB.

  18. Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells

    PubMed Central

    Starost, Laura Julia; Karassek, Sascha; Sano, Yasuteru; Kanda, Takashi; Kim, Kwang Sik; Dobrindt, Ulrich; Rüter, Christian; Schmidt, Marcus Alexander

    2016-01-01

    Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB. PMID:27754355

  19. A novel Love Wave biosensor for rapid and sensitive detection of marine toxins.

    PubMed

    Zhang, Xi; Fang, Jiaru; Zou, Yingchang; Zou, Ling; Hu, Ning; Wang, Ping

    2015-01-01

    Marine toxins are produced by plankton and do a great harm to human through food chain by accumulating in shellfishes and fishes. It is highly required and favorable to develop novel methods for the rapid and efficient detection of marine toxins to avoid the poisoning cases that have occurred frequently in many countries. This study presents a real-time Love Wave biosensor for the rapid detection of okadaic acid (OA), which used HepG2 cell lines as the sensing elements. The results indicate that this cell-based biosensor can provide real-time information of cellular activities induced by okadaic acid and has a higher sensitivity than the conventional cell-based assay. It is suggested that this cell-based biosensor can be used as a convenient and efficient method for marine toxin detection, which has a great potential to contribute to avoid the harmful effects of marine toxins on the human health.

  20. A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

    PubMed Central

    Andersson, M. A.; Mikkola, R.; Helin, J.; Andersson, M. C.; Salkinoja-Salonen, M.

    1998-01-01

    Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen−1. This amount corresponds to 104 to 105 CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 108 CFU ml−1. The detection limit for food was 3 g of rice containing 106 to 107 CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen−1, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa. PMID:9546170

  1. Whooping Cough (Pertussis)

    MedlinePlus

    ... Whooping cough (pertussis) is an infection of the respiratory system caused by the bacterium Bordetella pertussis (or B. ... Immunizations Your Child's Immunizations Coughing Pneumonia Lungs and Respiratory System The Woes of Whooping Cough Contact Us Print ...

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

    PubMed

    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.

  3. Bordetella pertussis transmission

    USDA-ARS?s Scientific Manuscript database

    Bordetella pertussis and Bordetella bronchiseptica are Gram negative bacterial respiratory pathogens. B. pertussis is the causative agent of whooping cough and is considered a human-adapted variant of B. bronchiseptica. B. pertussis and B. bronchiseptica share mechanisms of pathogenesis and are gene...

  4. Preventing Pertussis (Whooping Cough)

    MedlinePlus

    ... Some babies who are less than 3 months old will die from pertussis if they get infected. Most adults do not have severe symptoms of the disease ... pertussis happen in babies less than 1 year old. How can I protect ... babies and children get pertussis from an adult who they are around often, such as a ...

  5. Immunochemical technologies for replacement of rodent bioassays in sensitive detection of toxins in foods

    USDA-ARS?s Scientific Manuscript database

    Rapid sensitive assays for biothreat toxins that can be used to detect intentionally contaminated foods are now typically performed via bioassay in live mice. While bioassay provides essential data on bioavailability, animal models are technically, fiscally, and ethically challenging. Through carefu...

  6. Map-Based Cloning of the Fungal Toxin Sensitivity Gene Tsn1 in Wheat

    USDA-ARS?s Scientific Manuscript database

    The wheat Tsn1 gene on wheat chromosome arm 5BL confers sensitivity to the host-selective proteinaceous toxins Ptr ToxA and SnToxA produced by the pathogenic fungi Pyrenophora tritici-repentis and Stagonospora nodorum, respectively. Compatible Tsn1-ToxA interactions lead to extensive cell death and...

  7. Evaluation of a multitarget real-time PCR assay for detection of Bordetella species during a pertussis outbreak in New Hampshire in 2011.

    PubMed

    Gao, Fengxiang; Mahoney, Jennifer C; Daly, Elizabeth R; Lamothe, Wendy; Tullo, Daniel; Bean, Christine

    2014-01-01

    A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 C(T) of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 C(T) values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 C(T) values of 30 ≤ CT <40.

  8. Evaluation of a Multitarget Real-Time PCR Assay for Detection of Bordetella Species during a Pertussis Outbreak in New Hampshire in 2011

    PubMed Central

    Mahoney, Jennifer C.; Daly, Elizabeth R.; Lamothe, Wendy; Tullo, Daniel; Bean, Christine

    2014-01-01

    A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 CT of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 CT values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 CT values of 30 ≤ CT <40. PMID:24131698

  9. Bordetella pertussis transmission.

    PubMed

    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.

  10. Monitoring Pertussis Infections Using Internet Search Queries.

    PubMed

    Zhang, Yuzhou; Milinovich, Gabriel; Xu, Zhiwei; Bambrick, Hilary; Mengersen, Kerrie; Tong, Shilu; Hu, Wenbiao

    2017-09-05

    This study aims to assess the utility of internet search query analysis in pertussis surveillance. This study uses an empirical time series model based on internet search metrics to detect the pertussis incidence in Australia. Our research demonstrates a clear seasonal pattern of both pertussis infections and Google Trends (GT) with specific search terms in time series seasonal decomposition analysis. The cross-correlation function showed significant correlations between GT and pertussis incidences in Australia and each state at the lag of 0 and 1 months, with the variation of correlations between 0.17 and 0.76 (p < 0.05). A multivariate seasonal autoregressive integrated moving average (SARIMA) model was developed to track pertussis epidemics pattern using GT data. Reflected values for this model were generally consistent with the observed values. The inclusion of GT metrics improved detective performance of the model (β = 0.058, p < 0.001). The validation analysis indicated that the overall agreement was 81% (sensitivity: 77% and specificity: 83%). This study demonstrates the feasibility of using internet search metrics for the detection of pertussis epidemics in real-time, which can be considered as a pre-requisite for constructing early warning systems for pertussis surveillance using internet search metrics.

  11. High heterogeneity in methods used for the laboratory confirmation of pertussis diagnosis among European countries, 2010: integration of epidemiological and laboratory surveillance must include standardisation of methodologies and quality assurance.

    PubMed

    He, Q; Barkoff, A M; Mertsola, J; Glismann, S; Bacci, S

    2012-08-09

    Despite extensive childhood immunisation, pertussis remains one of the world’s leading causes of vaccine preventable deaths. The current methods used for laboratory diagnosis of pertussis include bacterial culture, polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) serology. We conducted a questionnaire survey to identify variations in the laboratory methods and protocols used among participating countries included in the European surveillance network for vaccine-preventable diseases(EUVAC.NET). In February 2010, we performed the survey using a web-based questionnaire and sent it to the country experts of 25 European Union countries,and two European Economic Area (EEA) countries,Norway and Iceland. The questionnaire consisted of 37 questions which covered both general information on surveillance methods and detailed laboratory methods used. A descriptive analysis was performed.Questionnaires were answered by all 27 contacted countries. Nineteen countries had pertussis reference laboratories at the national level; their functions varied from performing diagnosis to providing technical advice for routine microbiology laboratories. Culture,PCR and serology were used in 17, 18 and 20 countries,respectively. For PCR, nine laboratories used insertion sequence IS481 as the target gene, which is present in multiple copies in the Bordetella pertussis genome and thus has a greater sensitivity over single copy targets, but has been proved not to be specific for B.pertussis. Antibodies directed against pertussis toxin(PT) are specific for B. pertussis infections. For ELISA serology, only 13 countries’ laboratories used purified PT as coating antigen and 10 included World Health Organization (WHO) or Food and Drug Administration (FDA) reference sera in their tests. This present survey shows that methods used for laboratory confirmation of pertussis differ widely among European countries and that there is a great heterogeneity of the reference

  12. Bordetella pertussis modulates human macrophage defense gene expression.

    PubMed

    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. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Cercospora beticola Toxin Inhibits Vanadate-Sensitive H+ Transport in Corn Root Membrane Vesicles

    PubMed Central

    Blein, Jean-Pierre; Bourdil, Isabelle; Rossignol, Michel; Scalla, René

    1988-01-01

    The effect of Cercospora beticola toxin on the transport of protons by vanadate-sensitive ATPase was studied with corn (Zea mays) root microsomal vesicles prepared by differential centrifugation, sedimentation through a sucrose cushion, and washing with Triton X-100 plus KBr. In these preparations, addition of ATP induced intravesicular H+-accumulation as evidenced by a rapid quenching of the fluorescence of 9-amino-6-chloro-2-methoxy acridine. This quenching was relatively unaffected by inhibitors of mitochondrial and tonoplast-type ATPases, but was strongly reduced by inhibitors of plasma membrane H+-ATPase. C. beticola toxin markedly inhibited ATP dependent H+-transport, and this effect increased with the length of preincubation with the toxin. The same observations were made concerning ATPase activity. Inhibition of H+-transport was greater at pH 7.3 than at pH 5.7. Lineweaver-Burk plot analysis showed that inhibition kinetics were competitive with respect to ATP. These data suggest a direct effect of C. beticola toxin on vanadate-sensitive ATPase presumed to be associated with the plasma membrane. PMID:16666321

  14. Cercospora beticola Toxin Inhibits Vanadate-Sensitive H Transport in Corn Root Membrane Vesicles.

    PubMed

    Blein, J P; Bourdil, I; Rossignol, M; Scalla, R

    1988-10-01

    The effect of Cercospora beticola toxin on the transport of protons by vanadate-sensitive ATPase was studied with corn (Zea mays) root microsomal vesicles prepared by differential centrifugation, sedimentation through a sucrose cushion, and washing with Triton X-100 plus KBr. In these preparations, addition of ATP induced intravesicular H(+)-accumulation as evidenced by a rapid quenching of the fluorescence of 9-amino-6-chloro-2-methoxy acridine. This quenching was relatively unaffected by inhibitors of mitochondrial and tonoplast-type ATPases, but was strongly reduced by inhibitors of plasma membrane H(+)-ATPase. C. beticola toxin markedly inhibited ATP dependent H(+)-transport, and this effect increased with the length of preincubation with the toxin. The same observations were made concerning ATPase activity. Inhibition of H(+)-transport was greater at pH 7.3 than at pH 5.7. Lineweaver-Burk plot analysis showed that inhibition kinetics were competitive with respect to ATP. These data suggest a direct effect of C. beticola toxin on vanadate-sensitive ATPase presumed to be associated with the plasma membrane.

  15. Effect of Prepregnancy Pertussis Vaccination in Young Infants.

    PubMed

    Maertens, Kirsten; Tran, Thao Mai Phuong; Hens, Niel; Van Damme, Pierre; Leuridan, Elke

    2017-06-15

    Maternal antibodies to pertussis can hamper infant immune responses to pertussis vaccines. The effect a maternal tetanus, diphtheria, acellular pertussis (Tdap) vaccine booster between 2 consecutive pregnancies is investigated. A prospective study was conducted in Belgium during 2008-2014 on the kinetics of maternal pertussis antibodies in unvaccinated women and their infants (group A; 86 mother-infant pairs) and in siblings born after the women received Tdap vaccine (group B; 58 mother-infant pairs). Levels of antibody to pertussis toxin, antibody to filamentous hemagglutinin, and antibody to pertactin were measured in maternal blood before and after vaccination and at both deliveries, in cord blood from both siblings, and in infants before and after they received a priming series of acellular pertussis containing vaccines. Levels of pertussis antibodies in all group B siblings at birth were significantly higher than those in their siblings at birth, even as the interval since maternal vaccination increased. Blunting of the infant pertussis vaccine response was detected in group B siblings. We estimated the maximum interval between repeat Tdap vaccine doses in adult women that would yield a beneficial effect for the consecutive infant. Prepregnancy Tdap vaccination significantly increases maternal antibody concentrations in consecutive infants. However, similar to the effect of Tdap vaccination during pregnancy, immune responses of later-born infants born to mothers who received a prepregnancy immunization, are blunted.

  16. Involvement of a Botulinum Toxin-Sensitive 22-kDa G Protein in Stimulated Exocytosis of Human Neutrophils

    DTIC Science & Technology

    1994-01-01

    FUNDING NUMBERS ]involvement -of -a Botulinun toxin -sesftUv&2_2At6-d -- Protein in stinulated exocytosis of humnan neutrophi1s. WICD I. AUTHOR(S) Nath...Botulinum Toxin -Sensitive 22-kDa G Protein in Stimulated Exocytosis of Human Neutrophils jaymaree Nath,’ Annette Powledge, and Daniel G. Wright2...observed. Although both peslussis toxin and ST-O Inhibited excocytosis In FlMvLP-stimvulated PMNs, the inhibitory effects o( the two toxins were found to be

  17. Pertussis immunization: an update

    PubMed Central

    Morgan, Lon G

    1997-01-01

    A segment of chiropractic has historically opposed the practice of immunization. This opposition has been based on historical and philosophical precedent, but with little support from the scientific literature. Pertussis immunization has successfully controlled a disease with a prior history of high childhood morbidity. An evaluation of the literature fails to find supporting evidence that whole-cell pertussis vaccine causes SIDS, asthma, or encephalopathy. Countries who discontinued pertussis immunization experienced a return of the disease, and in every case pertussis immunization has been reinstated. The recent successful clinical trials and subsequent approval of an acellular pertussis vaccine should reduce the local reactions and discomfort sometimes experienced with the whole-cell product. In view of the considerable scientific evidence for the desirability and efficacy of pertussis immunization, chiropractic should encourage patient participation in this worthwhile public health service.

  18. Sensitization of spinal cord nociceptive neurons with a conjugate of substance P and cholera toxin

    PubMed Central

    Caudle, Robert M; Mannes, Andrew J; Keller, Jason; Perez, Federico M; Suckow, Shelby K; Neubert, John K

    2007-01-01

    Background Several investigators have coupled toxins to neuropeptides for the purpose of lesioning specific neurons in the central nervous system. By producing deficits in function these toxin conjugates have yielded valuable information about the role of these cells. In an effort to specifically stimulate cells rather than kill them we have conjugated the neuropeptide substance P to the catalytic subunit of cholera toxin (SP-CTA). This conjugate should be taken up selectively by neurokinin receptor expressing neurons resulting in enhanced adenylate cyclase activity and neuronal firing. Results The conjugate SP-CTA stimulates adenylate cyclase in cultured cells that are transfected with either the NK1 or NK2 receptor, but not the NK3 receptor. We further demonstrate that intrathecal injection of SP-CTA in rats induces the phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) and also enhances the expression of the immediate early gene c-Fos. Behaviorally, low doses of SP-CTA (1 μg) injected intrathecally produce thermal hyperalgesia. At higher doses (10 μg) peripheral sensitivity is suppressed suggesting that descending inhibitory pathways may be activated by the SP-CTA induced sensitization of spinal cord neurons. Conclusion The finding that stimulation of adenylate cyclase in neurokinin receptor expressing neurons in the spinal cord produces thermal hyperalgesia is consistent with the known actions of these neurons. These data demonstrate that cholera toxin can be targeted to specific cell types by coupling the catalytic subunit to a peptide agonist for a g-protein coupled receptor. Furthermore, these results demonstrate that SP-CTA can be used as a tool to study sensitization of central neurons in vivo in the absence of an injury. PMID:17493276

  19. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    PubMed

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

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

    PubMed

    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 <16% and homologous 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.

  1. Seroprevalence of Pertussis in Senegal: A Prospective Study

    PubMed Central

    Gaayeb, Lobna; Sarr, Jean Biram; Ndiath, Mamadou O.; Hanon, Jean-Baptiste; Debrie, Anne-Sophie; Seck, Modou; Schacht, Anne-Marie; Remoué, Franck; Hermann, Emmanuel; Riveau, Gilles

    2012-01-01

    Background Pertussis, also known as whooping cough, is a vaccine-preventable respiratory disease caused by Bordetella pertussis infection, against which Senegalese children are immunized with the diphtheria-tetanus-whole cell pertussis vaccine (DTwP). Seroepidemiology of pertussis has been widely described in industrialized countries, but rare are the studies referring to it in developing countries. Methods We conducted a longitudinal survey in Northern Senegal to investigate the epidemiology of B. pertussis by evaluating the IgG antibody (Ab) response against pertussis toxin (PT). A cohort of 410 children aged 1 to 9 from five villages in the Middle Senegal River Valley were followed-up for 18 months. During that period, five visits were made to assess the immunological status of the children. Principal Findings PT-specific IgG responses were significantly different according to age. Until the age of 3, there was a decrease in the Ab response, which then increased in the older groups. Assessment of IgG antibodies to PT (IgG-PT) suggested evidence of recent exposures to the pathogen. Surprisingly, in one of the five villages the average Ab response to PT was very low at all ages during the first 6 months of the study. At the third visit, IgG-PT concentrations peaked to very high levels, to slightly decline at the end of the survey. This indicates an outbreak of B. pertussis, whereas in the other villages a pertussis endemic profile could be observed. Conclusions Pertussis is endemic in Northern Senegal despite the introduction of vaccination. The circulation of the bacteria seems to differ between geographic locations and over time. A more complete understanding of the epidemiology of pertussis and its environmental determinants could provide information to adapt vaccination programs. PMID:23119090

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

    PubMed

    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. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

  3. An integrated microfluidic platform for sensitive and rapid detection of biological toxins.

    PubMed

    Meagher, Robert J; Hatch, Anson V; Renzi, Ronald F; Singh, Anup K

    2008-12-01

    Towards designing a portable diagnostic device for detecting biological toxins in bodily fluids, we have developed microfluidic chip-based immunoassays that are rapid (< 20 minutes), require minimal sample volume (<10 microL) and have appreciable sensitivity and dynamic range (microM-pM). The microfluidic chip is being integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is intended for rapid, point-of-care (and, in future, point-of-incident) testing in case of an accidental or intentional exposure/intoxication to biotoxins. Detection of toxins and potential host-response markers is performed using microfluidic electrophoretic immunoassays integrated with sample preconcentration and mixing of analytes with fluorescently labeled antibodies. Preconcentration is enabled by photopolymerizing a thin, nanoporous membrane with a MW cut-off of approximately 10 kDa in the sample loading region of the chip. Polymeric gels with larger pores are located adjacent to the size exclusion membrane to perform electrophoretic separation of antibody-analyte complex and excess antibody. Measurement of the ratio of bound and unbound immune-complex using sensitive laser-induced fluorescence detection provides quantitation of analyte in the sample. We have demonstrated electrophoretic immunoassays for the biotoxins ricin, Shiga toxin I, and Staphylococcal enterotoxin B (SEB). With off-chip mixing and no sample preconcentration, the limits of detection (LOD) were 300 pM for SEB, 500 pM for Shiga toxin I, and 20 nM for ricin. With a 10 min on-chip preconcentration, the LOD for SEB is <10 pM. The portable device being developed is readily applicable to detection of proteinaceous biomarkers of many other diseases and is intended to represent the next-generation diagnostic devices capable of rapid and quantitative measurements of multiple analytes simultaneously.

  4. Pertussis in north-central and northwestern regions of Algeria.

    PubMed

    Benamrouche, Nabila; Tali Maamar, Hassiba; Lazri, Malika; Hasnaoui, Sonia; Radoui, Abdelkarim; Lafer, Ourida; Boukari, Rachida; Kaddache, Chawki; Arrada, Zakia; Rahal, Kheira

    2016-11-24

    Pertussis outbreaks continue to occur in many countries despite high vaccination coverage. Under-diagnosed cases in adolescents and adults may result in increased transmission to infants, who are at risk of severe pertussis. Additional measures to protect both groups should be considered. Nasopharyngeal samples and sera were collected from patients and household contacts with clinically suspected pertussis. Diagnoses were confirmed by culture, real-time polymerase chain reaction (PCR), and serology. Bordetella pertussis isolates were characterized by antimicrobial sensitivity and fimbrial serotyping. Of 392 participants, 134/248 patients (54%) and 66/144 contacts (45.8%) had confirmed pertussis infections. B. parapertussis was not detected. All B. pertussis isolates were sensitive to the antibiotics tested, and all expressed the Fim3, not the Fim2, fimbrial serotype. Most patients (81.2%) were <6 months (51.8% of whom were <3 months) of age; 77.6% were unvaccinated, and most positive contacts were mothers 20-40 years of age. Despite high vaccination coverage, pertussis is circulating in Algeria. Most infections occur in unvaccinated infants <6 months of age, with mothers as the main source of infection. An adolescent/adult booster should be considered. Adoption of sensitive and specific laboratory tests would improve pertussis diagnosis and surveillance.

  5. Open-sandwich immunoassay for sensitive and broad-range detection of a shellfish toxin gonyautoxin.

    PubMed

    Hara, Yuko; Dong, Jinhua; Ueda, Hiroshi

    2013-09-02

    At present, the analytical method for paralytic shellfish poisoning (PSP) toxins in shellfish is the mouse bioassay (MBA), which is an official method of the Association of Analytical Communities (AOAC [8]). However, the low sensitivity and concerns over the number of live animals required for testing have been cited as the major reason for seeking its replacement. In this report, we employed an open-sandwich immunoassay (OS-IA) to detect gonyautoxin (GTX2/3), a kind of PSP toxins. OS-IA, which utilizes the antigen-induced enhancement of antibody VH/VL interaction, can measure a small molecule antigen in a noncompetitive format. Hence it has a wider working range and shorter measurement time. We isolated anti-GTX2/3 antibody gene from a hybridoma GT-13A by screening a Fab-displaying phage library. Then the vectors for OS-IA were constructed, and examined for antigen concentration-dependency of the VH/VL interaction by OS-ELISA. As a result, in each case, signal intensity increases notably in a wide concentration range (0.1 to >1000 ng mL(-1)) of free GTX2/3, which was enough to cover its regulation value (80 μg 100 g(-1)) in many countries. So OS-IA will be widely applicable to detect PSP toxins in shellfish meats and in drinking water.

  6. Sensitization of human umbilical vein endothelial cells to Shiga toxin: involvement of protein kinase C and NF-kappaB.

    PubMed Central

    Louise, C B; Tran, M C; Obrig, T G

    1997-01-01

    Infection of humans with Shiga toxin-producing Escherichia coli O157:H7 and Shigella dysenteriae 1 is strongly associated with vascular endothelial cell damage and the development of hemolytic-uremic syndrome. The cytotoxic effect of Shiga toxins on vascular endothelial cells in vitro is enhanced by prior exposure to bacterial lipopolysaccharide (LPS) or either of the host cytokines tumor necrosis factor alpha (TNF) and interleukin-1beta (IL-1). The purpose of this study was to examine individual signal transduction components involved in the sensitization of human umbilical vein endothelial cells (HUVEC) to Shiga toxin 1. The results demonstrate that class I and II protein kinase C (PKC) isozymes are required for sensitization of HUVEC to Shiga toxin by phorbol myristate acetate (PMA) or LPS but not by TNF or IL-1. Thus, the specific competitive inhibitor of class I/II PKC, 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG), prevented only the action of PMA and LPS on HUVEC. Additional data obtained with ATP binding site inhibitors which affect all PKCs (i.e., classes I, II, and III) suggest that TNF may utilize class III PKC isozymes in the Shiga toxin sensitization of HUVEC. Transcriptional activator NF-kappaB did not appear to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA. Thus, the specific serine protease inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) did not inhibit the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA despite its ability to inhibit NF-kappaB activation and the induction of the NF-kappaB-dependent tissue factor gene by these agents. Finally, all-trans retinoic acid partially inhibited the sensitization of HUVEC to Shiga toxin, by unknown mechanisms which also appeared to be independent of NF-kappaB activation. These results indicate that PKC plays a role in the sensitization of HUVEC to Shiga toxin in response to some, but not all, sensitizing agents. In contrast, NF

  7. Physiopathological roles of spontaneously released outer membrane vesicles of Bordetella pertussis.

    PubMed

    Gasperini, Gianmarco; Arato, Vanessa; Pizza, Mariagrazia; Aricò, Beatrice; Leuzzi, Rosanna

    2017-10-05

    Bordetella pertussis has been shown to release outer membrane vesicles (OMV) both in vitro and in vivo but little is known about their biological role during the initial phases of B. pertussis infection of the airways. We have demonstrated that OMV are released by B. pertussis in a human ciliated-airway cell model and purified vesicles can interact with host cells. Binding and uptake are strictly Bvg-regulated and OMV-associated. Pertussis toxin contributes to host-cell intoxication. Furthermore, we have shown that OMV act as iron-delivery systems complementing the B. pertussis growth defect in iron-limiting conditions. We have proved that OMV play different roles in B. pertussis physiopathology and we opened new perspectives to be further investigated.

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

    PubMed

    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.

  9. Usefulness of laboratory methods in diagnosis of pertussis in adult with paroxysmal cough.

    PubMed

    Piekarska, Katarzyna; Rzeczkowska, Magdalena; Rastawicki, Waldemar; Dąbrowska-Iwanicka, Anna; Paradowska-Stankiewicz, Iwona

    2014-01-01

    Pertussis is an acute, highly contagious bacterial infection of respiratory system caused by Bordetella pertussis. Principally, disease affects young children, however, recently it is also reported in adolescents and adults. Symptoms of pertussis in adults are non-specific, i.e. dry, paroxysmal and protracted cough. Thus, it is rarely diagnosed in this group. This paper aimed at evaluating the usefulness of the laboratory methods in diagnosis of pertussis in adults based on a case presenting with dry, paroxysmal and chronic cough. Sputum (collected on 25th January 2013) and paired serum samples (collected on 13th February and 19 April 2013) were tested. Pertussis diagnostics involved culture, in-house PCR, real-time PCR and ELISA. Sputum culture, using commercial medium Bordetella Selective Medium by Oxoid did not reveal the presence of B. pertussis. Real-time PCR and PCR, however, confirmed the presence of insertion sequence IS481 and pertussis toxin promoter sequence ptx-Pr, markers indicative of B. pertussis infection. Serological testing revealed the high titres of IgA, IgG and IgM antibodies to B. pertussis in the first sample. In the second sample, collected 2 months following the first one, a significant decrease in IgA antibodies was reported. These data suggest a high usefulness of the laboratory methods in the diagnosis of pertussis in adults with chronic cough. Application of such methods ensures adequate diagnosis of disease, quick introduction of proper treatment and implementation of procedures preventing the spread of infection.

  10. How to fight pertussis?

    PubMed Central

    2013-01-01

    Universal pertussis vaccination has successfully decreased pertussis mortality and morbidity in childhood. However, despite intensive vaccination of young children, pertussis remains a major public health problem in both developing and industrialized regions. Recent epidemics in California and Australia demonstrated that the agent of the disease is still circulating. They also revealed several aspects that must not be neglected concerning vaccine-preventable diseases. Indeed, pertussis is one of the oldest vaccine-preventable bacterial diseases, so can provide a good illustration of all of the aspects associated with the need for surveillance after the introduction of vaccination. (i) The type of vaccine: two types of pertussis vaccine, whole cell and acellular, inducing different types of immunity are now used around the world. (ii) The vaccine strategy, the vaccine coverage and the duration of vaccine immunity: pertussis epidemics provide evidence that 90% of the infants must be vaccinated, vaccination must be sufficiently early and both vaccine-induced immunity and natural infection-induced immunity to pertussis wane with time indicating that pertussis is not only a pediatric disease. (iii) The agents of the disease, Bordetella pertussis and Bordetella parapertussis: the intensive vaccination of young infants modified the herd immunity, controlled bacteria similar to the vaccine strains but not all, revealing polymorphism of the agents of the disease evidencing the importance of continuing their isolation and their surveillance as well as monitoring their antibiotic resistance. (iv) The diagnosis of the disease: the epidemics showed the importance of specific diagnostic techniques that are easy to use by medical laboratories and the availability of the reagents required. (v) Communication with the public, the health authorities and the health providers: any changes of vaccine type, vaccine strategy, characteristics of the disease, and biological diagnosis must

  11. Different IgG-subclass distributions after whole-cell and acellular pertussis infant primary vaccinations in healthy and pertussis infected children.

    PubMed

    Hendrikx, Lotte H; Schure, Rose-Minke; Oztürk, Kemal; de Rond, Lia G H; de Greeff, S C; Sanders, Elisabeth A M; Berbers, Guy A M; Buisman, Anne-Marie

    2011-09-16

    The distribution of IgG-subclasses provides insight in the immunological mechanisms of protection against whooping cough. We investigated the effect of Dutch whole-cell pertussis and acellular pertussis vaccines administered in infancy on the IgG-subclass distributions in healthy children aged 12 months, 4 years and 9 years as well as in children who have been infected with Bordetella pertussis. A fluorescent bead-based multiplex immunoassay was used for the measurement of IgG1, IgG2, IgG3 and IgG4 responses against pertussis toxin, filamentous heamagglutinin and pertactin. Although IgG1 was the predominant subclass for all pertussis antigens in both healthy and infected children, elevated IgG4 levels were only present in children who had received repeated number of acellular pertussis vaccinations. IgG2 and IgG3 antibodies did not contribute to the IgG response. No differences in IgG-subclasses between healthy vaccinated or infected children were found. The pertussis vaccine used for priming seems to determine the IgG-subclass composition elicited after a secondary antibody response either induced by pertussis vaccination or infection. The pronounced anti-pertussis IgG4 response might reflect the Th2-skewing of the immune response after aP vaccination.

  12. Rapid, Sensitive Detection of Botulinum Toxin on a Flexible Microfluidics Platform

    SciTech Connect

    Warner, Marvin G.; Dockendorff, Brian P.; Feldhaus, Michael J.; Anheier, Norman C.; Marks, James D.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2004-10-30

    In this paper we will describe how high affinity reagents and a sensor configuration enabling rapid mass transport can be combined for rapid, sensitive biodetection. The system that we have developed includes a renewable surface immunoassay, which involves on-column detection of a fluorescently labeled secondary antibody in a sandwich immunoassay. Yeast display and directed molecular evolution were used to create high affinity antibodies to the botulinum toxin heavy chain receptor binding domain, AR1 and 3D12. A rotating rod renewable surface microcolumn was used to form a microliter-sized column containing beads functionalized with the capture antibody (AR1). The column was perfused with sample, wash solutions, and a fluorescently labeled secondary antibody (3D12) while the on-column fluorescence was monitored. Detection was accomplished in less than 5 minutes, with a total processing time of about 10 minutes. On-column detection of botulinum toxin was more sensitive and much faster than flow cytometry analysis on microbeads using the same reagents.

  13. [Antigenic variability of Bordetella pertussis strains isolated in 1967-2010 in the Czech Republic--possible explanation for the rise in cases of pertussis?].

    PubMed

    Zavadilová, J; Lžičařová, D; Musílek, M; Křížová, P; Fabiánová, K

    2015-09-01

    Comparison of antigenic structures of Bordetella pertussis (B. pertussis) strains isolated from 1967 to 2010 in the Czech Republic. Seventy strains of B. pertussis were referred to the National Reference Laboratory (NRL) for Pertussis and Diphtheria within the surveillance of pertussis from all over the Czech Republic (CR) between 1967 and 2010. To study the strains, the analysis was performed of the genome sequences encoding the surface immunogenic structures--the pertussis toxin S1 subunit gene (ptxA), pertactin gene region 1 (prnA), type 3 fimbriae gene (fim3)--and pertussis toxin promoter (ptxP) responsible for the regulation of the production of pertussis toxin. For the study set of B. pertussis strains, the sequencing analysis revealed changes in all genomic regions studied. The isolates from three periods differ in the allelic profile. In period I (19671978) with the use of whole cell pertussis vaccine (wP), the following two profiles were the most common: ptxP(1), ptxA(2), prnA(1), fim3(1) and ptxP(1), ptxA(1), prnA(3), fim3(1). In period 2 (19902007) with the switch to acellular pertussis vaccine (aP), the most common profile was: ptxP(3), ptxA(1), prnA(2), fim3(2). Period 3 (20082010) with the use of aP was characterized by the predominance of the following two profiles which had never been found in period 1: ptxP(3), ptxA(1), prnA(2), fim3(2) and ptxP(3) ptxA(1), prnA(2), fim3(1). Sequencing of the genomic regions ptxP, ptxA, prnA, and fim3 of B. pertussis strains isolated in the CR between 1967 and 2010 confirmed changes in the allelic variants of these regions. The incidence of strains carrying the new allelic variants was increasing after 1995 at the expense of those carrying the original variants. The study results can be interpreted as a partial genetic escape of pathogenic strains of B. pertussis beyond the reach of the pertussis vaccines.

  14. Travelers' Health: Pertussis

    MedlinePlus

    ... 6–21 days) after exposure, followed by a cough that becomes paroxysmal. Coughing paroxysms can vary in ... minimal. The clinical case definition for pertussis includes cough for ≥2 weeks with paroxysms, whoop, or posttussive ...

  15. Pertussis Signs & Symptoms

    MedlinePlus

    ... Dis . 2014;58(11):1523–9. Stehr K, Cherry JD, Heininger U, et al. A comparative effectiveness ... Pediatrics . 2003;112(5):1069–75. Ward JI, Cherry JD, Swei-Ju C, et al. Bordetella pertussis ...

  16. Seroprevalence study of B. pertussis infection in health care workers in Catalonia, Spain.

    PubMed

    Urbiztondo, Luis; Broner, Sonia; Costa, Josep; Rocamora, Laura; Bayas, José M; Campins, Magda; Esteve, Maria; Borras, Eva; Domínguez, Angela; For The Study Of The Immune Status In Health Care, The Working Group

    2015-01-01

    Pertussis is a re-emerging infection in countries with high infant immunization coverage. Healthcare workers (HCW) are exposed and can transmit the infection to especially-vulnerable patients. Therefore, pertussis vaccination of HCW is recommended. Between June 2008 and December 2010, 460 HCW from hospital and primary healthcare centers were recruited to determine susceptibility to pertussis. IgG antibodies against pertussis (anti-pertussis ab) were measured, using a routine technique that detects antibodies against pertussis including pertussis toxin (PT) and filamentous hemagglutinin (FHA). Positive results were confirmed with a more-specific technique that only assesses anti-PT IgG antibodies. The median age was 42 years (range, 21-65), 77.3% were female. 172 were nurses, 133 physicians, 60 other clinical workers and 95 non-clinical workers. None had received pertussis vaccination since childhood. The overall prevalence of anti-pertussis antibodies was 51.7%, (95% CI 47.1-56.4). Anti-PT antibodies were determined in the 220 HCW with positive anti-pertussis antibodies: 4 (1.8%) were negative and 33 (15%) had a high titer (≥ 45 IU/mL). No significant differences between the prevalence of anti-pertussis antibodies or anti-TP antibodies were found according to age, type of occupation or type of center. Our study confirms the need for vaccination of HCW because at least half are susceptible to pertussis. High anti-PT titers found in 15% of seropositive HCW showed that they had had recent contact with B. pertussis.

  17. Seroprevalence study of B. pertussis infection in health care workers in Catalonia, Spain

    PubMed Central

    Urbiztondo, Luis; Broner, Sonia; Costa, Josep; Rocamora, Laura; Bayas, José M; Campins, Magda; Esteve, Maria; Borras, Eva; Domínguez, Angela; for the Study of the Immune Status in Health Care, the Working Group

    2014-01-01

    Pertussis is a re-emerging infection in countries with high infant immunization coverage. Healthcare workers (HCW) are exposed and can transmit the infection to especially-vulnerable patients. Therefore, pertussis vaccination of HCW is recommended. Between June 2008 and December 2010, 460 HCW from hospital and primary healthcare centers were recruited to determine susceptibility to pertussis. IgG antibodies against pertussis (anti-pertussis ab) were measured, using a routine technique that detects antibodies against pertussis including pertussis toxin (PT) and filamentous hemagglutinin (FHA). Positive results were confirmed with a more-specific technique that only assesses anti-PT IgG antibodies. The median age was 42 years (range, 21–65), 77.3% were female. 172 were nurses, 133 physicians, 60 other clinical workers and 95 non-clinical workers. None had received pertussis vaccination since childhood. The overall prevalence of anti-pertussis antibodies was 51.7%, (95% CI 47.1–56.4). Anti-PT antibodies were determined in the 220 HCW with positive anti-pertussis antibodies: 4 (1.8%) were negative and 33 (15%) had a high titer (≥ 45 IU/mL). No significant differences between the prevalence of anti-pertussis antibodies or anti-TP antibodies were found according to age, type of occupation or type of center. Our study confirms the need for vaccination of HCW because at least half are susceptible to pertussis. High anti-PT titers found in 15% of seropositive HCW showed that they had had recent contact with B. pertussis. PMID:25483549

  18. Airborne transmission of Bordetella pertussis.

    PubMed

    Warfel, Jason M; Beren, Joel; Merkel, Tod J

    2012-09-15

    Pertussis is a contagious, acute respiratory illness caused by the bacterial pathogen Bordetella pertussis. Although it is widely believed that transmission of B. pertussis occurs via aerosolized respiratory droplets, no controlled study has ever documented airborne transmission of pertussis. We set out to determine if airborne transmission occurs between infected and naive animals, utilizing the baboon model of pertussis. Our results showed that 100% of exposed naive animals became infected even when physical contact was prevented, demonstrating that pertussis transmission occurs via aerosolized respiratory droplets.

  19. Genetic Variation of Bordetella pertussis in Austria.

    PubMed

    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.

  20. Genetic Variation of Bordetella pertussis in Austria

    PubMed Central

    Wagner, Birgit; Melzer, Helen; Freymüller, Georg; Stumvoll, Sabine; Rendi-Wagner, Pamela; Paulke-Korinek, Maria; Repa, Andreas; Mooi, Frits R.; Kollaritsch, Herwig; 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. PMID

  1. Review of the neutrophil response to Bordetella pertussis infection

    PubMed Central

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

    2015-01-01

    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. PMID:26432818

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

    PubMed

    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. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Sensitive voltammetric and amperometric responses of respiratory toxins at hemin-adsorbed carbon-felt.

    PubMed

    Hasebe, Yasushi; Wang, Yue

    2013-06-01

    A hemin [iron-Fe(III) protoporphyrin IX chloride] was adsorbed onto a carbon-felt (CF), which is a microelectrode ensemble of micro carbon fiber (ca. 7 microm diameter). The resulting hemin-adsorbed-CF (hemin-CF) showed a well-defined redox wave based on the hemin-Fe(III)/Fe(II) redox process with the formal potential of -0.225 V vs. Ag/AgCl in deoxygenated phosphate/citrate buffer solution (0.1 mol/L, pH 5.0). The apparent heterogenous electron transfer rate constant was estimated to be 8.6 sec(-1). In air-saturated electrolyte solution, the hemin-CF exhibited an excellent electrocatalytic activity for the reduction of dioxygen (O2). This activity was reversibly inhibited by respiratory toxins such as cyanide and azide, which bind sixth coordination position of iron active center of hemin. The electrocatalytic 02 reduction current at the hemin-CF was modulated by the toxins in a concentration-depending manner. Based on the relationship between the %inhibition and the toxin concentration, apparent inhibition constants of cyanide and azide were evaluated to be 4.52 and 1.98 micromol/L, respectively. When the hemin-CF was used as a working electrode unit of the CF-based electrochemical flow-through detector with air-saturated carrier, the injection of the azide induced peak-shape current responses, which allowed rapid and continuous flow-amperometric determination of azide with high sensitivity.

  4. Role of nuclear factor-kappa B in the regulation of intercellular adhesion molecule 1 after infection of human bronchial epithelial cells by Bordetella pertussis.

    PubMed

    Ishibashi, Yoshio; Nishikawa, Akemi

    2003-10-01

    Previous work has demonstrated that infection of human bronchial epithelial cells by Bordetella pertussis up-regulates intercellular adhesion molecule-1 (ICAM-1) gene and protein expression. It has also been shown that interaction of the Arg-Gly-Asp (RGD) site of filamentous hemagglutinin (FHA) with host cell very late antigen (VLA)-5 (alpha 5 beta 1 integrin) is required for the up-regulation of epithelial ICAM-1 expression, and that pertussis toxin (PT) impairs this response. We therefore examined the molecular mechanisms leading to B. pertussis-induced ICAM-1 up-regulation in BEAS-2B human bronchial epithelial cells. A colorimetric nuclear factor kappa B (NF-kappa B) activation assay demonstrated that NF-kappa B was activated in response to infection of these cells with B. pertussis. This activation occurred in an FHA(RGD)-dependent manner, and was blocked by an antibody against VLA-5, implying that binding of the RGD to VLA-5 integrin is involved in NF-kappa B activation. Western blot analysis revealed that the activation of NF-kappa B by B. pertussis was preceded by degradation of I kappa B alpha, a major cytoplasmic inhibitor of NF-kappa B. Pretreatment of the BEAS-2B cells with the NF-kappa B inhibitors pyrrolidine dithiocarbamate (PDTC), MG-132, and SN50 resulted in a marked decrease in B. pertussis-induced ICAM-1 expression, implying the involvement of NF-kappa B in ICAM-1 expression. Purified PT abrogated both NF-kappa B activation and I kappa B alpha degradation. These results suggest that ligation of VLA-5 integrin by FHA induces RGD-dependent NF-kappa B activation, thus leading to the up-regulation of epithelial ICAM-1 expression, and that a PT-sensitive G protein may be involved in this signaling pathway.

  5. Crystallization of pertussigen from Bordetella pertussis.

    PubMed Central

    Arai, H; Munoz, J J

    1981-01-01

    A method is described for crystallizing pertussigen from Bordetella pertussis. The crystalline material induced histamine hypersensitivity in mice at a dose of 0.5 ng of protein and leukocytosis at a dose of 100 ng and was toxic at a dose of 429 microgram. The histamine-sensitizing activity and the toxicity were as high as ever reported. Images PMID:6260667

  6. Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

    PubMed Central

    Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R.; Dorner, Brigitte G.; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

    2017-01-01

    Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test. PMID:28700661

  7. Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices.

    PubMed

    Féraudet-Tarisse, Cécile; Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R; Dorner, Brigitte G; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

    2017-01-01

    Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

  8. A novel sensitive cell-based Love Wave biosensor for marine toxin detection.

    PubMed

    Zhang, Xi; Fang, Jiaru; Zou, Ling; Zou, Yingchang; Lang, Lang; Gao, Fan; Hu, Ning; Wang, Ping

    2016-03-15

    A novel HepG2 cell-based biosensor using Love Wave sensor was developed to implement the real-time and sensitive detection of a diarrheic shellfish poisoning (DSP) toxin, Okadaic acid (OA). Detachable Love Wave sensor unit and miniaturized 8-channel recording instrument were designed for the convenient experimental preparation and sensor response signal measurement. The Love Wave sensor, whose synchronous frequency is around 160 MHz, was fabricated with ST-cut quartz substrate. To establish a cell-based biosensor, HepG2 cells as sensing elements were cultured onto the Love Wave sensor surface, and the cell attachment process was recorded by this biosensor. Results showed this sensor could monitor the cell attachment process in real time and response signals were related to the initial cell seeding densities. Furthermore, cell-based Love Wave sensor was treated with OA toxin. This biosensor presented a good performance to various OA concentrations, with a wide linear detection range (10-100 μg/L). Based on the ultrasensitive acoustic wave platform, this cell-based biosensor will be a promising tool for real-time and convenient OA screening.

  9. Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin.

    PubMed

    Wang, Pingyan; Li, Guanghui; Yan, Junrong; Hu, Yonghong; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun

    2014-12-15

    The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin.

  10. Increase in pertussis cases along with high prevalence of two emerging genotypes of Bordetella pertussis in Perú, 2012.

    PubMed

    Bailon, H; León-Janampa, N; Padilla, C; Hozbor, D

    2016-08-17

    As has occurred in many regions worldwide, in 2012 the incidence of pertussis increased in Perú. This epidemiologic situation has been associated with a waning vaccine-induced immunity and the adaptation of Bordetella pertussis to vaccine-induced immunity along with improved diagnostic methods. The study comprised a total of 840 pertussis-suspected cases reported in Perú during 2012. We summarize here the distribution of pertussis cases according to age and immunization status along with the immunization-coverage rate. Laboratory diagnosis was performed by culture test and real-time polymerase-chain reaction (PCR). B. pertussis bacteria recovered from infected patients were characterized by pulsed-field gel electrophoresis (PFGE), and the DNA sequencing of the pertussis-toxin (promoter and subunit A), pertactin, and fimbriae (fim2 and fim3) genes. From the total pertussis-suspected cases, 191 (22.7 %) infections were confirmed by real-time PCR and 18 through cultivation of B. pertussis (2.1 %), while one infection of B. parapertussis (0.11 %) was also detected by culture. Pertussis was significantly higher in patients that had had 0-3 vaccine doses (pentavalent vaccine alone) than in those who had had 4-5 vaccine doses (pentavalent plus DwPT boosters) at 94.3 vs. 5.7 %, respectively (p < 0.00001). The relative risk (RR) for patients with 4-5 doses compared to those with fewer than 4 doses or no dose was 0.23 (95 % Confidence Interval: 0.11-0.44), while the vaccine effectiveness was 77 % and coverage 50.5 %. Genetic analysis of B. pertussis isolates from different Peruvian regions detected two clonal groups as identified by PFGE. Those two groups corresponded to the B. pertussis genotypes emerging worldwide ptxP3-ptxA1-prn2 or 9-fim3-1 and ptxP3-ptxA1-prn2 or 9-fim3-2. Two emerging B. pertussis genotypes similar to isolates involved in worldwide epidemics were detected in Perú. Low vaccine coverage (<50 %) and genetic divergence between the vaccine

  11. Role of neutrophils in response to Bordetella pertussis infection in mice.

    PubMed

    Andreasen, Charlotte; Carbonetti, Nicholas H

    2009-03-01

    Pertussis is an acute respiratory disease caused by the bacterium Bordetella pertussis, for which humans are the only known reservoir. During infection, B. pertussis releases several toxins, including pertussis toxin (PT) and adenylate cyclase toxin (ACT), which have both been shown to play roles in promoting bacterial growth during early infection in a mouse model. Furthermore, in vitro and in vivo studies suggest that PT and ACT affect neutrophil chemotaxis and/or function, thereby altering the innate immune response. In this study we depleted animals of neutrophils to investigate whether neutrophils play a protective role during B. pertussis infection in mice. In addition, by infection with toxin-deficient strains, we investigated whether neutrophils are the main targets for PT and/or ACT activity in promoting bacterial growth. Surprisingly, we found no role for neutrophils during B. pertussis infection in naïve mice. However, in previously infected (immune) mice or in mice receiving immune serum, we observed a significant role for neutrophils during infection. Furthermore, in this immune mouse model our evidence indicates that neutrophils appear to be the main target cells for ACT, but not for PT.

  12. Seroprevalence of pertussis in China: need to improve vaccination strategies.

    PubMed

    Xu, Yinghua; Wang, Lichan; Xu, Jin; Wang, Xinjian; Wei, Chen; Luo, Peng; Ma, Xiao; Hou, Qiming; Wang, Junzhi

    2014-01-01

    Pertussis remains an important cause of infant death worldwide and is an ongoing public health concern even in countries with high vaccination coverage. A cross-sectional seroepidemiological study was undertaken to estimate true incidence rates and gain further insight into the epidemiology and burden of pertussis in China. During 2011, a total of 1080 blood samples were obtained from healthy individuals between 0 and 86 y of age in Zhengzhou, Central China. Serum IgG antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) were measured quantitatively using ELISA. The results showed that the geometric mean titers of PT and FHA IgG were 6.48 IU/mL (95% CI: 5.70-7.41 IU/mL) and 11.39 IU/mL (95% CI: 10.22-12.87 IU/mL) among subjects less than 4 y of age, indicating that pertussis antibody levels were low despite high vaccination coverage. Of the 850 subjects≥4 y of age, 56 (6.6%) had anti-PT IgG titers above 30 IU/mL, and 11 (1.3%) had antibodies titers above 80 IU/mL. The estimated age-specific incidence of infection with B. pertussis revealed a peak incidence in the 31 to 40 y age group, followed by the 41 to 60 y age group. Taken together, these results indicate that pertussis is common in Chinese subjects in Zhengzhou, especially in adults, suggesting that the disease burden is underestimated in China. Therefore, our study stresses the importance of strengthening the diagnostic capacity and improving surveillance system for delineating current epidemiological profiles of pertussis. Most importantly, it may be advisable to re-evaluate the current Chinese pertussis immunization schedule and implement to booster doses for older children, adolescents and adults.

  13. Cutting Edge: Resistance to Bacillus anthracis Infection Mediated by a Lethal Toxin Sensitive Allele of Nalp1b/Nlrp1b

    DTIC Science & Technology

    2010-01-01

    Nalp1b/Nlrp1b Lethal Toxin Sensitive Allele of Infection Mediated by aanthracis BacillusCutting Edge: Resistance to http://www.jimmunol.org/cgi/content...COVERED - 4. TITLE AND SUBTITLE Cutting edge: resistance to Bacillus anthracis infection mediated by a lethal toxin -sensitive allele of Nalp1b...Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase

  14. Does tetanus-diphtheria-acellular pertussis vaccination interfere with serodiagnosis of pertussis infection?

    PubMed

    Pawloski, Lucia C; Kirkland, Kathryn B; Baughman, Andrew L; Martin, Monte D; Talbot, Elizabeth A; Messonnier, Nancy E; Tondella, Maria Lucia

    2012-06-01

    An anti-pertussis toxin (PT) IgG enzyme-linked immunosorbent assay (ELISA) was analytically validated for the diagnosis of pertussis at a cutoff of 94 ELISA units (EU)/ml. Little was known about the performance of this ELISA in the diagnosis of adults recently vaccinated with tetanus-diphtheria-acellular pertussis (Tdap) vaccine, which contains PT. The goal of this study was to determine when the assay can be used following Tdap vaccination. A cohort of 102 asymptomatic health care personnel (HCP) vaccinated with Tdap (Adacel; Sanofi Pasteur) were aged 19 to 79 years (median, 47 years) at vaccination. For each HCP, specimens were available for evaluation at 2 to 10 time points (prevaccination to 24 months postvaccination), and geometric mean concentrations (GMC) for the cohort were calculated at each time point. Among 97 HCP who responded to vaccination, a mixed-model analysis with prediction and tolerance intervals was performed to estimate the time at which serodiagnosis can be used following vaccination. The GMCs were 8, 21, and 9 EU/ml at prevaccination and 4 and 12 months postvaccination, respectively. Eight (8%) of the 102 HCP reached antibody titers of ≥94 EU/ml during their peak response, but none had these titers by 6 months postvaccination. The calculated prediction and tolerance intervals were <94 EU/ml by 45 and 75 days postvaccination, respectively. Tdap vaccination 6 months prior to testing did not confound result interpretation. This seroassay remains a valuable diagnostic tool for adult pertussis.

  15. An improved sensitive assay for the detection of PSP toxins with neuroblastoma cell-based impedance biosensor.

    PubMed

    Zou, Ling; Wu, Chunsheng; Wang, Qin; Zhou, Jie; Su, Kaiqi; Li, Hongbo; Hu, Ning; Wang, Ping

    2015-05-15

    Paralytic shellfish poisoning (PSP) toxins are well-known sodium channel-blocking marine toxins, which block the conduction of nerve impulses and lead to a series of neurological disorders symptoms. However, PSP toxins can inhibit the cytotoxicity effect of compounds (e.g., ouabain and veratridine). Under the treatment of ouabain and veratridine, neuroblastoma cell will swell and die gradually, since veratridine causes the persistent inflow of Na(+) and ouabain inhibits the activity of Na(+)/K(+)-ATPases. Therefore, PSP toxins with antagonism effect can raise the chance of cell survival by blocking inflow of Na(+). Based on the antagonism effect of PSP toxins, we designed an improved cell-based assay to detect PSP toxins using a neuroblastoma cell-based impedance biosensor. The results demonstrated that this biosensor showed high sensitivity and good specificity for saxitoxins detection. The detection limit of this biosensor was as low as 0.03 ng/ml, which was lower than previous reported cell-based assays and mouse bioassays. With the improvement of biosensor performance, the neuroblastoma cell-based impedance biosensor has great potential to be a universal PSP screening method.

  16. Investigation in a model system of the effects of combinations of anthrax and pertussis vaccines administered to service personnel in the 1991 Gulf War.

    PubMed

    Rijpkema, Sjoerd G; Adams, Trudy; Rigsby, Peter; Xing, Dorothy K; Corbel, Michael J

    2005-01-01

    The toxicity and immunogenicity of the anthrax and pertussis vaccine combinations used in the 1991 Gulf War was assessed in NIH, A/J and Balb/c mice. Inoculation of pertussis vaccines, vaccine combinations, or aluminium salt caused illness, splenomegaly and significant weight loss. Although some animals recovered eventually, a lethal form of ascites developed in some NIH mice and body weights of A/J and Balb/c mice remained below normal levels. Inoculation of anthrax vaccine produced little effect. Exposure to diluted vaccine combinations produced less serious side effects of shorter duration. Single vaccinations induced specific IgG1 antibodies whereas a mixture of IgG1 and IgG2a was produced after multiple injections. Antigen stimulation of spleen cells from mice exposed to pertussis vaccines induced high levels of NO and IL-6, whereas stimulated spleen cells from mice exposed to anthrax vaccine produced only low levels of IL-6. In mice, pertussis vaccines act as an adjuvant for anthrax vaccine, but these vaccines are also the major cause of toxicity of the vaccine combination. The relatively high vaccine dose used, together with the low sensitivity of mice to anthrax toxin, emphasises that caution should be exercised in applying these results to human recipients of these vaccines.

  17. Sensitive-cell-based fish chromatophore biosensor

    NASA Astrophysics Data System (ADS)

    Plant, Thomas K.; Chaplen, Frank W.; Jovanovic, Goran; Kolodziej, Wojtek; Trempy, Janine E.; Willard, Corwin; Liburdy, James A.; Pence, Deborah V.; Paul, Brian K.

    2004-07-01

    A sensitive biosensor (cytosensor) has been developed based on color changes in the toxin-sensitive colored living cells of fish. These chromatophores are highly sensitive to the presence of many known and unknown toxins produced by microbial pathogens and undergo visible color changes in a dose-dependent manner. The chromatophores are immobilized and maintained in a viable state while potential pathogens multiply and fish cell-microbe interactions are monitored. Low power LED lighting is used to illuminate the chromatophores which are magnified using standard optical lenses and imaged onto a CCD array. Reaction to toxins is detected by observing changes is the total area of color in the cells. These fish chromatophores are quite sensitive to cholera toxin, Staphococcus alpha toxin, and Bordatella pertussis toxin. Numerous other toxic chemical and biological agents besides bacterial toxins also cause readily detectable color effects in chromatophores. The ability of the chromatophore cell-based biosensor to distinguish between different bacterial pathogens was examined. Toxin producing strains of Salmonella enteritis, Vibrio parahaemolyticus, and Bacillus cereus induced movement of pigmented organelles in the chromatophore cells and this movement was measured by changes in the optical density over time. Each bacterial pathogen elicited this measurable response in a distinctive and signature fashion. These results suggest a chromatophore cell-based biosensor assay may be applicable for the detection and identification of virulence activities associated with certain air-, food-, and water-borne bacterial pathogens.

  18. Immunogenicity and safety of a monovalent, multicomponent acellular pertussis vaccine in 15 month-6-year-old German children. Monovalent Acellular Pertussis Vaccine Study Group.

    PubMed

    Stehr, K; Heininger, U; Uhlenbusch, R; Angersbach, P; Hackell, J; Eckhardt, T

    1995-03-01

    Immunization against pertussis has been re-recommended for healthy children in Germany in 1991. In addition the former restriction of immunizing only in the first 2 years of life was abolished. In children born before 1991 immunization rates against pertussis were 15% or less. With the new recommendations physicians are now faced with an increasing demand of parents for catch-up vaccinations in these children. Since they were immunized against diphtheria and tetanus previously monovalent pertussis vaccines are needed for this indication. Therefore a monovalent, multicomponent acellular pertussis vaccine was studied in 249 German children 15 months to 6 years of age. Three doses were administered at 6-10 week intervals. Reactogenicity and antibody responses against the vaccine antigens pertussis toxin (PT), filamentous haemagglutinin (FHA), 69-kd antigen (pertactin) and fimbriae-2 (agglutinogen) were investigated. Local and systemic reactions were minimal in frequency and severity. Antibody responses against all vaccine antigens were pronounced with 93%-100% of vaccinees demonstrating at least four fold titre rises above pre-immunization after the third dose. These findings indicate that this monovalent, multicomponent acellular pertussis vaccine with excellent immunogenicity and low reactogenicity is an appropriate candidate for closing immunization gaps in older children in countries with previously low vaccination rates against pertussis. Based on the results of this study the monovalent acellular pertussis vaccine was licensed in Germany in January 1994.

  19. [Multicenter clinical investigation of pertussis in children and adolescents with persistent cough].

    PubMed

    2010-10-01

    Pertussis is an acute respiratory tract infection caused by Bordetella pertussis. This disease, though preventable by vaccination, the resurgence or persistence was reported frequently in developed countries. The current clinical epidemiology of pertussis in China is scarcely known. In this multicenter clinical study, we investigated the prevalence of pertussis in children and adolescents with persistent cough in Shanghai, Chongqing, Kunming and Yinchuan. The nasopharyngeal swabs and serum (single) were collected from subjects aged ≥ 6 years with cough for more than 2 weeks during November 2008 to October 2009. The culture and multiplex PCR assays were used to identify the B. pertussis, and ELISA assay (Viron/serio, Germany) was used to test the antibody to pertussis toxin (PTIgG). The laboratory confirmed B. pertussis were culture positive and or PCR positive, and or PTIgG > 80 FDA-U/ml (single serum). Totally 1001 subjects were enrolled; 93.5% of subjects were immunized with diphtheria-pertussis-tetanus vaccine (DPT). The subjects were divided into group 1 aged from 6 to 8 years, group 2 (age -10 years), group 3 (age -12 years) and group 4 (age -18 years). The numbers of subjects of the four groups were 307, 249, 212, and 233; 113 subjects were B. pertussis positive (11.3%), 888 subjects were B. pertussis negative. The infection rates of the four groups were 7.2% (22, 307), 11.2% (28, 249), 10.4% (22, 212) and 17.6% (41, 233) respectively (χ(2) = 2915.2, P < 0.01). The total infection rate in Shanghai was 16.7% (65, 402), which was higher than that in the other regions (χ(2) = 3003.0, P < 0.01). Although the immunization rates of DPT is high, B. pertussis infection is still an important etiological factor associated with persistent cough in children and adolescent. Doctors and the laboratory staff should pay attention to diagnosis of pertussis.

  20. Adult pertussis is unrecognized public health problem in Thailand.

    PubMed

    Siriyakorn, Nirada; Leethong, Pornvimol; Tantawichien, Terapong; Sripakdee, Saowalak; Kerdsin, Anusak; Dejsirilert, Surang; Paitoonpong, Leilani

    2016-01-25

    Although pertussis has been considered a disease of childhood, it is also recognized as an important respiratory tract infection in adolescents and adults. However, in countries with routine vaccination against pertussis with high coverage, pertussis is not usually taken into consideration for the etiology of prolonged cough in adults. Previous studies in a variety of populations in developed countries have documented that pertussis is quite common, ranging from 2.9 to 32% of adolescents and adults with prolonged cough. The anticipation and early recognition of this change in the epidemiology is important because the affected adolescents and adults act as reservoirs of the disease and source of infection to the vulnerable population of infants, for whom the disease can be life threatening. We conducted a prospective study to determine the prevalence of pertussis in Thai adults with prolonged cough. Seventy-six adult patients with a cough lasting for more than 2 weeks (range, 14-180 days) were included in the present study. The data regarding medical history and physical examination were carefully analyzed. Nasopharyngeal swabs from all patients were obtained for the detection of deoxyribonucleic acid of Bordetella pertussis by the polymerase chain reaction (PCR) method. Paired serum samples were collected and tested for IgG antibody against pertussis toxin by using an ELISA method. Of 76 adult patients, 14 patients (18.4%) with the mean age of 59 (range, 28-85) years and the mean duration of cough of 34 (range, 14-120) days had laboratory evidence of acute pertussis infection. One patient was diagnosed by the PCR method, while the rest had serological diagnosis. Whooping cough is a significantly associated symptom of patients with chronic cough who had laboratory evidence of pertussis. (p < .05, odds ratio 3.75, 95% confidence interval: 1.00, 14.06) CONCLUSION: Pertussis is being increasingly recognized as a cause of prolonged, distressing cough among adults in

  1. Characterization of two Achromobacter xylosoxidans isolates from patients with pertussis-like symptoms.

    PubMed

    Orellana-Peralta, Fiorella; Jacinto, Michelle; Pons, Maria J; Gomes, Cláudia; Bada, Carlos; Reyes, Isabel; Del Valle Mendoza, Juana; Ruiz, Joaquim

    2015-06-01

    To characterize two Achromobacter xylosoxidans recovered from 2 patients diagnosed with pertussis during a Bordetella pertussis surveillance program. Nasopharyngeal swabs from 2 children under 1 year of age with clinical suspicion of pertussis were analyzed by culture and PCR. 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. Although more detailed studies are needed, the present data highlight the possibility that Achromobacter xylosoxidans, closely related Bordetella pertusssis 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. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

  2. Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine-sensitive Ca2+ channels

    PubMed Central

    Shahbazzadeh, Delavar; Srairi-Abid, Najet; Feng, Wei; Ram, Narendra; Borchani, Lamia; Ronjat, Michel; Akbari, Abolfazl; Pessah, Isaac N.; De Waard, Michel; El Ayeb, Mohamed

    2007-01-01

    In the present work, we purified and characterized a novel toxin named hemicalcin from the venom of the Iranian chactoid scorpion Hemiscorpius lepturus where it represents 0.6% of the total protein content. It is a 33-mer basic peptide reticulated by three disulfide bridges, and that shares between 85 and 91% sequence identity with four other toxins, all known or supposed to be active on ryanodine-sensitive calcium channels. Hemicalcin differs from these other toxins by seven amino acids at positions 9 (leucine/arginine), 12 (alanine/glutamic acid), 13 (aspartic acid/asparagine), 14 (lysine/asparagine), 18 (serine/glycine), 26 (threonine/alanine) and 28 (proline/isoleucine/alanine). In spite of these differences, hemicalcin remains active on ryanodine-sensitive Ca2+ channels, since it increases [3H]ryanodine binding on RyR1 (ryanodine receptor type 1) and triggers Ca2+ release from sarcoplasmic vesicles. Bilayer lipid membrane experiments, in which the RyR1 channel is reconstituted and its gating properties are analysed, indicate that hemicalcin promotes an increase in the opening probability at intermediate concentration and induces a long-lasting subconductance level of 38% of the original amplitude at higher concentrations. Mice intracerebroventricular inoculation of 300 ng of hemicalcin induces neurotoxic symptoms in vivo, followed by death. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on the ryanodine-sensitive channel. PMID:17291197

  3. Biotyping of Candida albicans and other fungi by yeast killer toxins sensitivity.

    PubMed

    Polonelli, Luciano; Conti, Stefania

    2009-01-01

    Intraspecific differentiation of pathogenic microorganisms is a major need in epidemiological studies concerning the source and spread of infections. This requirement is paramount for those etiologic agents of infectious diseases, which are mainly grouped into one species within the genus, such as Candida albicans. Ideally, laboratory methods for biotyping purposes should be sensitive, reproducible, easy, and economical to perform. In addition, the methods should be flexible in their application to taxonomically unrelated pathogens. We have shown that the toxins produced by a selected panel of killer yeasts, each characterized by a wide spectrum of antimicrobial activity, may be used to discriminate strains belonging to the species of the genus Candida and to other species of eukaryotic and prokaryotic pathogenic microorganisms. The "yeast killer system," which may be sharply increased in sensitivity by addition of further standardized killer yeasts, has proven to be of value in the resolution of many cases of clinical and nosocomial fungal infections. Owing to its reliability, economy, and versatility, this phenotypic system can be used as an alternative biotyping method in laboratories lacking the financial and training resources necessary to perform more sophisticated and expensive molecular approaches.

  4. Engineered bakers yeast as a sensitive bioassay indicator organism for the trichothecene toxin deoxynivalenol.

    PubMed

    Abolmaali, Shamsozoha; Mitterbauer, Rudolf; Spadiut, Oliver; Peruci, Michaela; Weindorfer, Hanna; Lucyshyn, Doris; Ellersdorfer, Günther; Lemmens, Marc; Moll, Wulf-Dieter; Adam, Gerhard

    2008-03-01

    The aim of this study was to increase the sensitivity of Saccharomyces cerevisiae towards trichothecene toxins, in particular to deoxynivalenol (DON), in order to improve the utility of this yeast as a bioassay indicator organism. We report the construction of a strain with inactivated genes (PDR5, PDR10, PDR15) encoding ABC transporter proteins with specificity for the trichothecene deoxynivalenol, with inactivated AYT1 (encoding a trichothecene-3-O-acetyltransferase), and inactivated UBI4 and UBP6 genes. Inactivation of the stress inducible polyubiquitin gene UBI4 or the ubiquitin protease UBP6 increased DON sensitivity, the inactivation of both genes had a synergistic effect. The resulting pdr5 pdr10 pdr15 ayt1 ubp6 ubi4 mutant strain showed 50% growth inhibition at a DON concentration of 5 mg/l under optimal conditions. The development of a simple two step assay for microbial DON degradation in 96 well microtiter format and its testing with the DON detoxifying bacterium BBSH 797 is reported.

  5. The RNA Chaperone Hfq Is Required for Virulence of Bordetella pertussis

    PubMed Central

    Bibova, Ilona; Skopova, Karolina; Masin, Jiri; Cerny, Ondrej; Hot, David; Sebo, Peter

    2013-01-01

    Bordetella pertussis is a Gram-negative pathogen causing the human respiratory disease called pertussis or whooping cough. Here we examined the role of the RNA chaperone Hfq in B. pertussis virulence. Hfq mediates interactions between small regulatory RNAs and their mRNA targets and thus plays an important role in posttranscriptional regulation of many cellular processes in bacteria, including production of virulence factors. We characterized an hfq deletion mutant (Δhfq) of B. pertussis 18323 and show that the Δhfq strain produces decreased amounts of the adenylate cyclase toxin that plays a central role in B. pertussis virulence. Production of pertussis toxin and filamentous hemagglutinin was affected to a lesser extent. In vitro, the ability of the Δhfq strain to survive within macrophages was significantly reduced compared to that of the wild-type (wt) strain. The virulence of the Δhfq strain in the mouse respiratory model of infection was attenuated, with its capacity to colonize mouse lungs being strongly reduced and its 50% lethal dose value being increased by one order of magnitude over that of the wt strain. In mixed-infection experiments, the Δhfq strain was then clearly outcompeted by the wt strain. This requirement for Hfq suggests involvement of small noncoding RNA regulation in B. pertussis virulence. PMID:23980112

  6. Purification of binding protein for Tityus gamma toxin identified with the gating component of the voltage-sensitive Na+ channel.

    PubMed Central

    Norman, R I; Schmid, A; Lombet, A; Barhanin, J; Lazdunski, M

    1983-01-01

    The gating component associated with the voltage-sensitive Na+ channel from electroplax membranes of Electrophorus electricus has been purified by using toxin gamma from the venom of the scorpion Tityus serrulatus serrulatus. The toxin-binding site was efficiently solubilized with Lubrol PX, resulting in an extract of high initial specific activity. Purification was achieved by adsorption of the toxin-binding component to DEAE-Sephadex A-25 followed by desorption at high ionic strength and chromatography on either wheat germ agglutinin-Ultrogel or Sepharose 6B. Maximal final specific activities were at least 42% of the specific activity expected for a pure toxin-binding component. The purified material exhibited a Stokes radius of 85 A, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated a single polypeptide component of Mr 270,000. Furthermore, tetrodotoxin binding activity and Tityus gamma toxin binding activity copurified, suggesting that the selectivity filter and the gating component of the Na+ channel are carried by the same polypeptide chain. Images PMID:6306665

  7. Pertussis: a reemerging infection.

    PubMed

    Kline, Jonathan M; Lewis, William D; Smith, Eleanor A; Tracy, Lloyd R; Moerschel, Sarah K

    2013-10-15

    Pertussis, also known as whooping cough, is an acute respiratory tract infection that has increased in incidence in recent years. The initial catarrhal stage presents with nonspecific symptoms of malaise, rhinorrhea, sneezing, lacrimation, and mild cough. During the paroxysmal stage, severe outbreaks of coughing often lead to the classic high-pitched whooping sound patients make when gasping for breath. The paroxysmal stage is followed by the convalescent stage and resolution of symptoms. Complications vary by age, with infants more likely to experience severe complications such as apnea, pneumonia, seizures, or death. In adolescents and adults, complications are the result of chronic cough. The diagnosis depends on clinical signs and laboratory testing. Both culture and polymerase chain reaction testing can be used to confirm the diagnosis; serologic testing is not standardized or routinely recommended. Although antibiotics have not shown clear effectiveness in the treatment of pertussis, they eradicate nasal bacterial carriage and may reduce transmission rates. Macrolide antibiotics such as azithromycin are first-line treatments to prevent transmission; trimethoprim/sulfamethoxazole is an alternative in cases of allergy or intolerance to macrolides. Immunization against pertussis is essential for disease prevention. Current recommendations in the United States consist of administering five doses of the diphtheria and tetanus toxoids and acellular pertussis (DTaP) vaccine to children before seven years of age, and administering a tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) booster between 11 and 18 years of age. Recent efforts have focused on the vaccination of adolescents and adults, with new recommendations for a single dose of the Tdap booster if it has not been previously administered.

  8. Whooping Cough and the Pertussis Vaccine

    MedlinePlus

    ... Gynecology Medical Conditions Nutrition & Fitness Emotional Health Whooping Cough and the Pertussis Vaccine Posted under Health Guides . ... Content Key Facts Pertussis is also called Whooping Cough. The best way to protect yourself from Pertussis ...

  9. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules.

    PubMed

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells.

  10. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules

    PubMed Central

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S. Y.

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This “shock” approach is then followed by “kill” of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  11. Antibody Responses to Individual Bordetella pertussis Fimbrial Antigen Fim2 or Fim3 following Immunization with the Five-Component Acellular Pertussis Vaccine or to Pertussis Disease

    PubMed Central

    Alexander, Frances; Matheson, Mary; Fry, Norman K.; Labram, Briony

    2012-01-01

    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. PMID:22956654

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

    PubMed

    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.

  13. Characterization of Post-Translational Modifications and Cytotoxic Properties of the Adenylate-Cyclase Hemolysin Produced by Various Bordetella pertussis and Bordetella parapertussis Isolates.

    PubMed

    Bouchez, Valérie; Douché, Thibaut; Dazas, Mélody; Delaplane, Sophie; Matondo, Mariette; Chamot-Rooke, Julia; Guiso, Nicole

    2017-09-26

    Bordetella pertussis and Bordetella parapertussis are the causal agents of whooping cough in humans. They produce diverse virulence factors, including adenylate cyclase-hemolysin (AC-Hly), a secreted toxin of the repeat in toxins (RTX) family with cyclase, pore-forming, and hemolytic activities. Post-translational modifications (PTMs) are essential for the biological activities of the toxin produced by B. pertussis. In this study, we compared AC-Hly toxins from various clinical isolates of B. pertussis and B. parapertussis, focusing on (i) the genomic sequences of cyaA genes, (ii) the PTMs of partially purified AC-Hly, and (iii) the cytotoxic activity of the various AC-Hly toxins. The genes encoding the AC-Hly toxins of B. pertussis and B. parapertussis displayed very limited polymorphism in each species. Most of the sequence differences between the two species were found in the C-terminal part of the protein. Both toxins harbored PTMs, mostly corresponding to palmitoylations of the lysine 860 residue and palmoylations and myristoylations of lysine 983 for B. pertussis and AC-Hly and palmitoylations of lysine 894 and myristoylations of lysine 1017 for B. parapertussis AC-Hly. Purified AC-Hly from B. pertussis was cytotoxic to macrophages, whereas that from B. parapertussis was not.

  14. Role of PCR in the diagnosis of pertussis infection in infants: 5 years' experience of provision of a same-day real-time PCR service in England and Wales from 2002 to 2007.

    PubMed

    Fry, Norman K; Duncan, John; Wagner, Karen; Tzivra, Oceanis; Doshi, Nita; Litt, David J; Crowcroft, Natasha; Miller, Elizabeth; George, Robert C; Harrison, Timothy G

    2009-08-01

    As part of an enhanced surveillance programme for pertussis in England and Wales, a real-time PCR service for the detection of Bordetella pertussis was introduced for infants aged pertussis. Two real-time fluorescent resonance energy transfer hybridization probe LightCycler (Roche Diagnostics) PCR assays were used. One (designed in-house) targeted the pertussis toxin S1 promoter (ptxA-pr), and included an internal process control to test for sample inhibition and reagent performance. The other (already published) targeted the insertion element IS481. The analytical sensitivities of the assays were 100 and 10 fg per reaction for the ptxA-pr and IS481 PCRs, respectively. The ptxA-pr assay was specific for B. pertussis, whilst the IS481 PCR also showed some cross-reactivity with Bordetella holmesii and the type strain of Bordetella parapertussis. From April 2002 to March 2007, 848 samples were received from 774 patients and DNA was extracted. Of 824 samples that were suitable for testing, 183 (22.2 %) had evidence of Bordetella infection (18.9 % ptxA-pr and IS481; 3.3 % IS481 only), 621 (75.4 %) were negative and 20 (2.4 %) were inhibitory for the PCR. Within the targeted age group of pertussis where results can affect patient and contact management.

  15. NQO1-Knockout Mice Are Highly Sensitive to Clostridium Difficile Toxin A-Induced Enteritis.

    PubMed

    Nam, Seung Taek; Hwang, Jung Hwan; Kim, Dae Hong; Lu, Li Fang; Hong, Ji; Zhang, Peng; Yoon, I Na; Hwang, Jae Sam; Chung, Hyo Kyun; Shong, Minho; Lee, Chul-Ho; Kim, Ho

    2016-08-28

    Clostridium difficile toxin A causes acute gut inflammation in animals and humans. It is known to downregulate the tight junctions between colonic epithelial cells, allowing luminal contents to access body tissues and trigger acute immune responses. However, it is not yet known whether this loss of the barrier function is a critical factor in the progression of toxin A-induced pseudomembranous colitis. We previously showed that NADH:quinone oxidoreductase 1 (NQO1) KO (knockout) mice spontaneously display weak gut inflammation and a marked loss of colonic epithelial tight junctions. Moreover, NQO1 KO mice exhibited highly increased inflammatory responses compared with NQO1 WT (wild-type) control mice when subjected to DSS-induced experimental colitis. Here, we tested whether toxin A could also trigger more severe inflammatory responses in NQO1 KO mice compared with NQO1 WT mice. Indeed, our results show that C. difficile toxin A-mediated enteritis is significantly enhanced in NQO1 KO mice compared with NQO1 WT mice. The levels of fluid secretion, villus disruption, and epithelial cell apoptosis were also higher in toxin A-treated NQO1 KO mice compared with WT mice. The previous and present results collectively show that NQO1 is involved in the formation of tight junctions in the small intestine, and that defects in NQO1 enhance C. difficile toxin A-induced acute inflammatory responses, presumably via the loss of epithelial cell tight junctions.

  16. Loss of multi-epitope specificity in memory CD4(+) T cell responses to B. pertussis with age.

    PubMed

    Han, Wanda G H; van Twillert, Inonge; Poelen, Martien C M; Helm, Kina; van de Kassteele, Jan; Verheij, Theo J M; Versteegh, Florens G A; Boog, Claire J P; van Els, Cécile A C M

    2013-01-01

    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.

  17. Differential growth inhibition as a tool to increase the discriminating power of killer toxin sensitivity in fingerprinting of yeasts.

    PubMed

    Buzzini, P; Martini, A

    2000-12-01

    A panel of 27 cell-free crude killer toxin preparations were used in fingerprinting 45 Saccharomyces cerevisiae and 11 Saccharomyces exiguus strains. The differential sensitivity to different mycocins was evaluated both as binary data matrix (presence-absence of killing effect), and by considering the growth inhibition areas (measured by agar diffusion well bioassay). The first approach gave an individual fingerprinting of 68% of sensitive strains, whereas the second gave a total and reproducible (P<0.01) discrimination of all tested strains.

  18. Characterisation of the Bordetella pertussis secretome under different media.

    PubMed

    Luu, Laurence Don Wai; Octavia, Sophie; Zhong, Ling; Raftery, Mark; Sintchenko, Vitali; Lan, Ruiting

    2017-03-31

    Our understanding of the Bordetella pertussis secretome remains limited including the role of different growth conditions in the secretome. In this study the secretome of L1423, a clinical isolate from the 2008-2012 Australian epidemic, cultured on Stainer-Scholte (SS) and Thalen-IJssel (THIJS) media for 12h was characterised using liquid chromatography-mass spectrometry (LC-MS/MS). In the supernatant, LC-MS/MS identified 260 proteins with 143 bioinformatically predicted to be secreted. Eighty percent of proteins were identified in both media. Proteins secreted were functionally associated with cell surface (41%), pathogenicity (16%) and transport (17%). The most abundant proteins identified were pathogenic proteins including toxins (PtxA and CyaA), adhesins (TcfA) and type III secretion (T3SS) proteins. There were 46 proteins found uniquely in THIJS including 8 virulence associated proteins. These included T3SS proteins, adhesins (FhaL and FhaS) and a putative toxin (BP1251). Nine proteins were found uniquely in SS and these were metabolic and transport-related proteins. None of the unique proteins detected in SS were known to be virulence associated. This study found that THIJS promotes secretion of virulence factors based on the number of unique virulence proteins found and may be a growth media of choice for the study of B. pertussis virulence and vaccine development. Over the past two decades, the number of B. pertussis notifications has risen despite vaccination. There is a greater need to understand the biology behind B. pertussis infections. The secretome of B. pertussis in two different media was characterised using LC-MS/MS. The results showed that THIJS promotes secretion of importance virulence factors which may be important for the development of vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Treatment failure of nosocomial pertussis infection in a very-low-birth-weight neonate.

    PubMed

    Bonacorsi, Stéphane; Farnoux, Caroline; Bidet, Philippe; Caro, Valérie; Aizenfisz, Sophie; Benhayoun, Mounir; Aujard, Yannick; Guiso, Nicole; Bingen, Edouard

    2006-10-01

    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.

  20. Treatment Failure of Nosocomial Pertussis Infection in a Very-Low-Birth-Weight Neonate

    PubMed Central

    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

  1. High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures.

    PubMed

    Boyer, Anne E; Gallegos-Candela, Maribel; Quinn, Conrad P; Woolfitt, Adrian R; Brumlow, Judith O; Isbell, Katherine; Hoffmaster, Alex R; Lins, Renato C; Barr, John R

    2015-04-01

    Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3-9.9% coefficient of variation), and accurate (0.1-12.7%error; n ≥ 25). Diagnostic sensitivity was 100% (n = 27 animal/clinical cases). Diagnostic specificity was 100% (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.

  2. Prevalence, diagnosis, and disease course of pertussis in adults with acute cough: a prospective, observational study in primary care

    PubMed Central

    Teepe, Jolien; Broekhuizen, Berna DL; Ieven, Margareta; Loens, Katherine; Huygen, Kris; Kretzschmar, Mirjam; de Melker, Hester; Butler, Chris C; Little, Paul; Stuart, Beth; Coenen, Samuel; Goossens, Herman; Verheij, Theo JM

    2015-01-01

    Background Most cases of adult pertussis probably remain undiagnosed. Aim To explore the prevalence, diagnosis, and disease course of acute pertussis infection in adult patients presenting with acute cough. Design and setting Prospective observational study between 2007 and 2010 in primary care in 12 European countries. Method Adults presenting with acute cough (duration of ≤28 days) were included. Bordetella pertussis infection was determined by polymerase chain reaction (from nasopharyngeal flocked swabs and sputa) and by measurement of immunoglobulin G antibodies to pertussis toxin (PT) in venous blood at day 28. An antibody titre to PT of ≥125 IU/ml or PCR positive result in a respiratory sample defined recent infection. Patients completed a symptom diary for 28 days. Results Serum and/or respiratory samples were obtained in 3074 patients. Three per cent (93/3074) had recent B. pertussis infection. Prior cough duration >2 weeks discriminated to some extent between those with and without pertussis (adjusted odds ratio 1.89, 95% confidence interval = 1.17 to 3.07; P = 0.010). Median cough duration after presentation was 17 and 12 days in patients with and without pertussis, respectively (P = 0.008). Patients with pertussis had longer duration of phlegm production (P = 0.010), shortness of breath (P = 0.037), disturbed sleep (P = 0.013) and interference with normal activities or work (P = 0.033) after presentation. Conclusion Pertussis infection plays a limited role among adults presenting with acute cough in primary care, but GPs should acknowledge the possibility of pertussis in uncomplicated lower respiratory tract infection. As in children, pertussis also causes prolonged symptoms in adults. However, pertussis is difficult to discern from other acute cough syndromes in adults at first presentation. PMID:26412843

  3. Prevalence, diagnosis, and disease course of pertussis in adults with acute cough: a prospective, observational study in primary care.

    PubMed

    Teepe, Jolien; Broekhuizen, Berna D L; Ieven, Margareta; Loens, Katherine; Huygen, Kris; Kretzschmar, Mirjam; de Melker, Hester; Butler, Chris C; Little, Paul; Stuart, Beth; Coenen, Samuel; Goossens, Herman; Verheij, Theo J M

    2015-10-01

    Most cases of adult pertussis probably remain undiagnosed. To explore the prevalence, diagnosis, and disease course of acute pertussis infection in adult patients presenting with acute cough. Prospective observational study between 2007 and 2010 in primary care in 12 European countries. Adults presenting with acute cough (duration of ≤28 days) were included. Bordetella pertussis infection was determined by polymerase chain reaction (from nasopharyngeal flocked swabs and sputa) and by measurement of immunoglobulin G antibodies to pertussis toxin (PT) in venous blood at day 28. An antibody titre to PT of ≥125 IU/ml or PCR positive result in a respiratory sample defined recent infection. Patients completed a symptom diary for 28 days. Serum and/or respiratory samples were obtained in 3074 patients. Three per cent (93/3074) had recent B. pertussis infection. Prior cough duration >2 weeks discriminated to some extent between those with and without pertussis (adjusted odds ratio 1.89, 95% confidence interval = 1.17 to 3.07; P = 0.010). Median cough duration after presentation was 17 and 12 days in patients with and without pertussis, respectively (P = 0.008). Patients with pertussis had longer duration of phlegm production (P = 0.010), shortness of breath (P = 0.037), disturbed sleep (P = 0.013) and interference with normal activities or work (P = 0.033) after presentation. Pertussis infection plays a limited role among adults presenting with acute cough in primary care, but GPs should acknowledge the possibility of pertussis in uncomplicated lower respiratory tract infection. As in children, pertussis also causes prolonged symptoms in adults. However, pertussis is difficult to discern from other acute cough syndromes in adults at first presentation. © British Journal of General Practice 2015.

  4. Investigation in a murine model of possible mechanisms of enhanced local reactions to post-primary diphtheria-tetanus toxoid boosters in recipients of acellular pertussis-diphtheria-tetanus vaccine

    PubMed Central

    Ochiai, Masaki; Horiuchi, Yoshinobu; Yuen, Chun-Ting; Asokanathan, Catpagavalli; Yamamoto, Akihiko; Okada, Kenji; Kataoka, Michiyo; Markey, Kevin; Corbel, Michael; Xing, Dorothy

    2014-01-01

    In recipients primed with acellular pertussis diphtheria-tetanus combined vaccine (DTaP) an increased incidence of severe local reactions with extensive redness/swelling has been reported for each subsequent dose of diphtheria-tetanus based combination vaccine given as a booster. This has been attributed to residual active pertussis toxin (PT) in the primary vaccine. In this study, we investigated the possible contribution of the A-subunit enzymatic activity and the B-oligomer carbohydrate binding activity of residual PT in DTaP to local reactions in a murine model using Japanese DTaP batches produced before and after the introduction of a test for reversion of pertussis toxoid to toxin. Residual PT activity was correlated with the B-oligomer carbohydrate binding activity. The in vivo mouse footpad swelling model assay indicated that the B-oligomer carbohydrate binding activity and possibly other factors were associated with intensified sensitization to local reaction following diphtheria toxoid booster. PMID:25424818

  5. Qualitative assessment of pertussis diagnostics in United States laboratories.

    PubMed

    Tatti, Kathleen M; Martin, Stacey W; Boney, Kathryn O; Brown, Kristin; Clark, Thomas A; Tondella, Maria Lucia

    2013-09-01

    United States national surveillance data show that the use of culture for pertussis diagnostics has sharply declined, whereas polymerase chain reaction (PCR) is now the most common testing method. PCR testing for pertussis is rapid and sensitive, but the lack of standardization and variable specificity is concerning. A web-based survey containing 12 questions was sent to public health, commercial and hospital-based US laboratories performing clinical diagnostics to determine the pertussis diagnostics used. An extensive real-time PCR (RT-PCR) questionnaire accompanied a proficiency panel assessing the types of extraction methods, RT-PCR methods and current quality control in place at the laboratories. The proficiency panel of 12 specimens containing Bordetella pertussis at various concentrations and negative controls was created to detect cross-contamination and assess the lower limit of detection. One hundred twenty-three (35%) of 355 respondents from the web-based survey performed diagnostic tests for the presence of B. pertussis. Eighty-three (71%) labs reported performing culture, whereas 67 (54%) labs used PCR. All 41 laboratories that consented to participate in the proficiency exercise used the IS481 RT-PCR target; however, a variety of extraction and RT-PCR methods were employed. The laboratories correctly identified 92% of the B. pertussis specimens, and 5% of the laboratories (1.8% of the panel specimens) reported at least 1 false-positive. The small percentage of false-positives suggests that adequate procedures are in place to prevent cross-contamination. Differing extraction and PCR methods as well as variable analytic sensitivity emphasize the necessity for an external well-defined quality control program and interlaboratory pertussis PCR harmonization.

  6. New Data on Vaccine Antigen Deficient Bordetella pertussis Isolates

    PubMed Central

    Bouchez, Valérie; Hegerle, Nicolas; Strati, Francesco; Njamkepo, Elisabeth; Guiso, Nicole

    2015-01-01

    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. PMID:26389958

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

    PubMed Central

    Heikkinen, Eriikka; Xing, Dorothy K; Ölander, Rose-Marie; Hytönen, Jukka; Viljanen, Matti K; Mertsola, Jussi; He, Qiushui

    2008-01-01

    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 coincided with nationwide

  8. Bordetella pertussis Infection of Primary Human Monocytes Alters HLA-DR Expression

    PubMed Central

    Shumilla, Jennifer A.; Lacaille, Vashti; Hornell, Tara M. C.; Huang, Jennifer; Narasimhan, Supraja; Relman, David A.; Mellins, Elizabeth D.

    2004-01-01

    Bordetella pertussis is the causative agent of whooping cough, a potentially lethal respiratory disease in children. In immunocompetent individuals, B. pertussis infection elicits an effective adaptive immune response driven by activated CD4+ T cells. However, live B. pertussis persists in the host for 3 to 4 weeks prior to clearance. Thus, B. pertussis appears to have evolved short-term mechanisms for immune system evasion. We investigated the effects of B. pertussis wild-type strain BP338 on antigen presentation in primary human monocytes. BP338 infection reduced cell surface expression of HLA-DR and CD86 but not that of major histocompatibility complex class I proteins. This change in cell surface HLA-DR expression reflected intracellular redistribution of HLA-DR. The proportion of peptide-loaded molecules was unchanged in infected cells, suggesting that intracellular retention occurred after peptide loading. Although B. pertussis infection of monocytes induced rapid and robust expression of interleukin-10 (IL-10), HLA-DR redistribution did not appear to be explained by increased IL-10 levels. BP338-infected monocytes exhibited reduced synthesis of HLA-DR dimers. Interestingly, those HLA-DR proteins that were generated appeared to be longer-lived than HLA-DR in uninfected monocytes. BP338 infection also prevented gamma interferon (IFN-γ) induction of HLA-DR protein synthesis. Using mutant strains of B. pertussis, we found that reduction in HLA-DR surface expression was due in part to the presence of pertussis toxin whereas the inhibition of IFN-γ induction of HLA-DR could not be linked to any of the virulence factors tested. These data demonstrate that B. pertussis utilizes several mechanisms to modulate HLA-DR expression. PMID:14977950

  9. Bordetella pertussis infection of primary human monocytes alters HLA-DR expression.

    PubMed

    Shumilla, Jennifer A; Lacaille, Vashti; Hornell, Tara M C; Huang, Jennifer; Narasimhan, Supraja; Relman, David A; Mellins, Elizabeth D

    2004-03-01

    Bordetella pertussis is the causative agent of whooping cough, a potentially lethal respiratory disease in children. In immunocompetent individuals, B. pertussis infection elicits an effective adaptive immune response driven by activated CD4(+) T cells. However, live B. pertussis persists in the host for 3 to 4 weeks prior to clearance. Thus, B. pertussis appears to have evolved short-term mechanisms for immune system evasion. We investigated the effects of B. pertussis wild-type strain BP338 on antigen presentation in primary human monocytes. BP338 infection reduced cell surface expression of HLA-DR and CD86 but not that of major histocompatibility complex class I proteins. This change in cell surface HLA-DR expression reflected intracellular redistribution of HLA-DR. The proportion of peptide-loaded molecules was unchanged in infected cells, suggesting that intracellular retention occurred after peptide loading. Although B. pertussis infection of monocytes induced rapid and robust expression of interleukin-10 (IL-10), HLA-DR redistribution did not appear to be explained by increased IL-10 levels. BP338-infected monocytes exhibited reduced synthesis of HLA-DR dimers. Interestingly, those HLA-DR proteins that were generated appeared to be longer-lived than HLA-DR in uninfected monocytes. BP338 infection also prevented gamma interferon (IFN-gamma) induction of HLA-DR protein synthesis. Using mutant strains of B. pertussis, we found that reduction in HLA-DR surface expression was due in part to the presence of pertussis toxin whereas the inhibition of IFN-gamma induction of HLA-DR could not be linked to any of the virulence factors tested. These data demonstrate that B. pertussis utilizes several mechanisms to modulate HLA-DR expression.

  10. Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification.

    PubMed

    Yamazaki, Wataru; Seto, Kazuko; Taguchi, Masumi; Ishibashi, Masanori; Inoue, Kiyoshi

    2008-06-12

    Vibrio cholerae is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Cholera toxin (CT) is a major virulence determinant of V. cholerae. Detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of cholera toxin (CT)-producing Vibrio cholerae. The assay provided markedly more sensitive and rapid detection of CT-producing V. cholerae strains than conventional biochemical and PCR assays. The assay correctly identified 34 CT-producing V. cholerae strains, but did not detect 13 CT non-producing V. cholerae and 53 non-V. cholerae strains. Sensitivity of the LAMP assay for direct detection of CT-producing V. cholerae in spiked human feces was 7.8 x 102 CFU per g (1.4 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay for detection of CT-producing V. cholerae required less than 35 min with a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 70 min with human feces from the beginning of DNA extraction to final determination. The LAMP assay is a sensitive, rapid and simple tool for the detection of CT-producing V. cholerae and will be useful in facilitating the early diagnosis of human V. cholerae infection.

  11. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  12. Probing the genome-scale metabolic landscape of Bordetella pertussis, the causative agent of whooping cough.

    PubMed

    Branco Dos Santos, Filipe; Olivier, Brett G; Boele, Joost; Smessaert, Vincent; De Rop, Philippe; Krumpochova, Petra; Klau, Gunnar W; Giera, Martin; Dehottay, Philippe; Teusink, Bas; Goffin, Philippe

    2017-08-25

    Whooping cough is a highly-contagious respiratory disease caused by Bordetella pertussis. Despite vaccination, its incidence has been rising alarmingly, and yet, the physiology of B. pertussis remains poorly understood. We combined genome-scale metabolic reconstruction, a novel optimization algorithm and experimental data to probe the full metabolic potential of this pathogen, using strain Tohama I as a reference. Experimental validation showed that B. pertussis secretes a significant proportion of nitrogen as arginine and purine nucleosides, which may contribute to modulation of the host response. We also found that B. pertussis can be unexpectedly versatile, being able to metabolize many compounds while displaying minimal nutrient requirements. It can grow without cysteine - using inorganic sulfur sources such as thiosulfate - and it can grow on organic acids such as citrate or lactate as sole carbon sources, providing in vivo demonstration that its TCA cycle is functional. Although the metabolic reconstruction of eight additional strains indicates that the structural genes underlying this metabolic flexibility are widespread, experimental validation suggests a role of strain-specific regulatory mechanisms in shaping metabolic capabilities. Among five alternative strains tested, three were shown to grow on substrate combinations requiring a functional TCA cycle, but only one could use thiosulfate. Finally, the metabolic model was used to rationally design growth media with over two-fold improvements in pertussis toxin production. This study thus provides novel insights into B. pertussis physiology, and highlights the potential, but also limitations of models solely based on metabolic gene content.IMPORTANCE The metabolic capabilities of Bordetella pertussis - the causative agent of whooping cough - were investigated from a systems-level perspective. We constructed a comprehensive genome-scale metabolic model for B. pertussis, and challenged its predictions

  13. Tetanus, Diphtheria, Pertussis (Tdap) Vaccine

    MedlinePlus

    ... the throat. It can lead to breathing problems, paralysis, heart failure, and death.PERTUSSIS (Whooping Cough) causes severe coughing spells, which can cause difficulty breathing, vomiting and disturbed sleep. It can also lead to weight loss, incontinence, ...

  14. Development of improved pertussis vaccine

    PubMed Central

    Rumbo, Martin; Hozbor, Daniela

    2014-01-01

    Rates of infection with Bordetella pertussis, the gram-negative bacterium that causes the respiratory disease called whooping cough or pertussis, have not abated and 16 million cases with almost 200,000 deaths are estimated by the WHO to have occurred worldwide in 2008. Despite relatively high vaccination rates, the disease has come back in recent years to afflict people in numbers not seen since the pre-vaccine days. Indeed, pertussis is now recognized as a frequent infection not only in newborn and infants but also in adults. The disease symptoms also can be induced by the non-vaccine-preventable infection with the close species B. parapertussis for which an increasing number of cases have been reported. The epidemiologic situation and current knowledge of the limitations of pertussis vaccine point out the need to design improved vaccines. Several alternative approaches and their challenges are summarized. PMID:25424954

  15. Substitutions in the Domain III Voltage-sensing Module Enhance the Sensitivity of an Insect Sodium Channel to a Scorpion β-Toxin*

    PubMed Central

    Song, Weizhong; Du, Yuzhe; Liu, Zhiqi; Luo, Ningguang; Turkov, Michael; Gordon, Dalia; Gurevitz, Michael; Goldin, Alan L.; Dong, Ke

    2011-01-01

    Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT3, from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNav). Like other scorpion β-toxins, Lqh-dprIT3 shifts the voltage dependence of activation of BgNav channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNav splice variants tested for their sensitivity to the toxin, only BgNav1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT3. Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation. PMID:21454658

  16. Detection of Protein Toxins

    USDA-ARS?s Scientific Manuscript database

    We have focused on ricin, shiga-like toxin, botulinum neurotoxin (BoNT), and staphylococcal enterotoxin A (SEA), developing sensitive test methods for toxins and marker compounds in food matrices. Although animal models provide the best means for risk assessment, especially for crude toxins in compl...

  17. Pertussis Serodiagnosis in Belgium from 1990 to 2009 ▿

    PubMed Central

    Vincent, Muriel; Rodeghiero, Caroline; Eylenbosch, Romain; Mans, Yvan; Swalus-Steenhouwer, Jeannine; Piérard, Denis; Huygen, Kris; Vanhoof, Raymond

    2011-01-01

    Diagnosis of pertussis by culture and PCR is most sensitive when performed on nasopharyngeal specimens collected <2 weeks and <3 weeks, respectively, after the onset of clinical disease. Conversely, serological testing allows the diagnosis of patients (mostly adults) with less typical whooping cough symptoms, for whom clinical samples are often collected at later time points. Here, we report on a 20-year serodiagnostic survey of pertussis in Belgium from 1990 to 2009. In total, 13,163 patients were analyzed for Bordetella pertussis-specific antibodies by agglutination, complement fixation, immunofluorescence, and ELISA. The number of positive pertussis cases detected by serodiagnosis ranged between 50 and 150 annually. The mean age of positive cases increased from 9.9 years in 1990 to 33.9 years in 2009. Whereas from 1990 to 2003, children and young adolescents made up the majority of cases, from 2004 onwards, cases were detected in all age groups and the distribution became bimodal, with a first peak at the age of 10 to 20 years and a second at the age of 35 to 50 years. In contrast, patients diagnosed since 2001 by PCR and/or culture were mostly children younger than 1 year of age. Despite extensive childhood vaccination campaigns, whooping cough is still present in Belgium. Our findings confirm the potential role of adults in the continued transmission of pertussis and strongly warrant booster or cocoon vaccinations in older age groups. PMID:21346057

  18. Whooping cough in school age children presenting with persistent cough in UK primary care after introduction of the preschool pertussis booster vaccination: prospective cohort study.

    PubMed

    Wang, Kay; Fry, Norman K; Campbell, Helen; Amirthalingam, Gayatri; Harrison, Timothy G; Mant, David; Harnden, Anthony

    2014-06-24

    To estimate the prevalence and clinical severity of whooping cough (pertussis) in school age children presenting with persistent cough in primary care since the introduction and implementation of the preschool pertussis booster vaccination. Prospective cohort study (November 2010 to December 2012). General practices in Thames Valley, UK. 279 children aged 5 to 15 years who presented in primary care with a persistent cough of two to eight weeks' duration. Exclusion criteria were cough likely to be caused by a serious underlying medical condition, known immunodeficiency or immunocompromise, participation in another clinical research study, and preschool pertussis booster vaccination received less than one year previously. Evidence of recent pertussis infection based on an oral fluid anti-pertussis toxin IgG titre of at least 70 arbitrary units. Cough frequency was measured in six children with laboratory confirmed pertussis. 56 (20%, 95% confidence interval 16% to 25%) children had evidence of recent pertussis infection, including 39 (18%, 13% to 24%) of 215 children who had been fully vaccinated. The risk of pertussis was more than three times higher (21/53; 40%, 26% to 54%) in children who had received the preschool pertussis booster vaccination seven years or more previously than in those who had received it less than seven years previously (20/171; 12%, 7% to 17%). The risk of pertussis was similar between children who received five and three component preschool pertussis booster vaccines (risk ratio for five component vaccine 1.14, 0.64 to 2.03). Four of six children in whom cough frequency was measured coughed more than 400 times in 24 hours. Pertussis can still be found in a fifth of school age children who present in primary care with persistent cough and can cause clinically significant cough in fully vaccinated children. These findings will help to inform consideration of the need for an adolescent pertussis booster vaccination in the United Kingdom. UK

  19. Licensed pertussis vaccines in the United States

    PubMed Central

    Klein, Nicola P

    2014-01-01

    The United States switched from whole cell to acellular pertussis vaccines in the 1990s following global concerns with the safety of the whole cell vaccines. Despite high levels of acellular pertussis vaccine coverage, the United States and other countries are experiencing large pertussis outbreaks. The aim of this article is to describe the historical context which led to acellular pertussis vaccine development, focusing on vaccines currently licensed in the US, and to review evidence that waning protection following licensed acellular pertussis vaccines have been significant factors in the widespread reappearance of pertussis. PMID:25483496

  20. Economic evaluation of a new acellular vaccine for pertussis in Canada.

    PubMed

    Iskedjian, M; Einarson, T R; O'Brien, B J; De Serres, J G; Gold, R; Gemmill, I M; Milkovich, N; Rosner, A

    2001-01-01

    Pertussis is a highly contagious infection affecting mainly children. Acellular pertussis vaccines were recently introduced in Canada based on evidence of improved safety and efficacy over whole cell vaccines, the current standard of care. The following study reports the economic impact of replacing the whole cell vaccine (wP) by a new acellular vaccine (aP) in the Ontario pertussis immunisation programme. For a hypothetical cohort of 100,000 children from birth to the age of 8 years, the costs and consequences of pertussis vaccination with either aP or wP were compared. A decision analytical model was constructed for vaccine delivery, treatment of pertussis cases and vaccine adverse events, with analyses from the viewpoints of the Ontario Ministry of Health and society. The main outcomes were expected number of pertussis cases, hospitalisations, and workdays lost by parents. Data on vaccine effectiveness, pertussis incidence, and other parameters used in the model were from published literature. Costs were discounted at 5%, and extensive sensitivity analyses were undertaken. Over 8 years, in a cohort of 100,000 children, the introduction of aP would prevent 10,500 cases of pertussis, avoiding 504 hospital admissions and 73,500 days of work absence. For Ontario, healthcare cost savings over the same period would amount to 275,585 Canadian dollars ($Can), and societal savings to $Can9,752,864

  1. Immune response to Bordetella pertussis is associated with season and undernutrition in Senegalese children.

    PubMed

    Gaayeb, Lobna; Pinçon, Claire; Cames, Cécile; Sarr, Jean-Biram; Seck, Modou; Schacht, Anne-Marie; Remoué, Franck; Hermann, Emmanuel; Riveau, Gilles

    2014-06-05

    While vaccines elicit a protective response in most recipients, studies suggest that environmental and nutritional factors can influence the strength of the individual response to immunization and to subsequent natural infectious challenges. We conducted a longitudinal survey in Senegal to assess the individual response to B. pertussis, a respiratory disease against which Senegalese children are vaccinated before the age of one (Clinicaltrials.gov ID: NCT01545115). A cohort of 203 children aged 1-9 from four villages of the Senegal River Valley was followed-up for 14 months (October 2008-January 2010). During that period, four visits have been made to the villages to assess the immunological and nutritional status of these children and to determine risk factors involved in the modulation of their humoral immune response to B. pertussis toxin. A multivariate model has demonstrated that birth season and nutritional status appeared to modulate humoral response to pertussis toxin. Moreover, response to B. pertussis was dependent on age, village and time of visit. These results are consistent with the hypothesis that environmental and nutritional factors modulate children's response to pertussis following natural infection or vaccination. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. An evaluation of pertussis vaccine.

    PubMed

    Mortimer, E A; Jones, P K

    1979-01-01

    Infant mortality from pertussis in the United States was 4.5 deaths/1,000 in 1900 but decreased to 0.003 deaths/1,000 by 1974. The attribution of this decrease in mortality to the widespread use of pertussis vaccine, which began in the 1940s, has been questioned because death rates from pertussis in infants steadily declined by 70% between 1900-1904 and 1935-1939. Thes doubts are compounded by the uncertain frequency and significance of untoward reactions to the vaccine. An attempt was made to clarify this issue by statistical analysis. Because most deaths from pertussis occur in the young, death rates were determined for consecutive five-year periods from 1900 through 1974 among children younger than one year of age (infants) and among those from one to four years of age. there was an accelerated decline in mortality beginning in 1940, especially among infants (P < 0.01 vs. mortality in 1930-1939). On the basis of the rate of decline before 1940, 4,000-8,000 deaths from pertussis would be expected to hve occurred in 1970-1974; however, only 52 such deaths occurred. It is unlikely that factors other than pertussis vaccine caused this decline in mortality. Therefore, the vaccine's benefit-risk ratio probably is high.

  3. Pertussis epidemic--California, 2014.

    PubMed

    Winter, Kathleen; Glaser, Carol; Watt, James; Harriman, Kathleen

    2014-12-05

    On June 13, 2014, the California Department of Public Health (CDPH) declared that a pertussis epidemic was occurring in the state when reported incidence was more than five times greater than baseline levels. The incidence of pertussis in the United States is cyclical, with peaks every 3-5 years, as the number of susceptible persons in the population increases. The last pertussis epidemic in California occurred in 2010, when approximately 9,000 cases were reported, including 808 hospitalizations and 10 infant deaths, for a statewide incidence of 24.6 cases per 100,000 population. During January 1-November 26, 2014, a total of 9,935 cases of pertussis with onset in 2014 were reported to CDPH, for a statewide incidence of 26.0 cases per 100,000. CDPH is working closely with local health departments to prioritize public health activities, with the primary goal of preventing severe cases of pertussis, which typically occurs in infants. All prenatal care providers are being encouraged to provide tetanus, diphtheria, and acellular pertussis vaccine (Tdap) to pregnant women during each pregnancy, ideally at 27-36 weeks' gestation, as is recommended by the Advisory Committee on Immunization Practices (ACIP), or refer patients to an alternative provider, such as a pharmacy or local public health department, to receive Tdap.

  4. Estimating the Duration of Pertussis Immunity Using Epidemiological Signatures

    PubMed Central

    Wearing, Helen J.; Rohani, Pejman

    2009-01-01

    Case notifications of pertussis have shown an increase in a number of countries with high rates of routine pediatric immunization. This has led to significant public health concerns over a possible pertussis re-emergence. A leading proposed explanation for the observed increase in incidence is the loss of immunity to pertussis, which is known to occur after both natural infection and vaccination. Little is known, however, about the typical duration of immunity and its epidemiological implications. Here, we analyze a simple mathematical model, exploring specifically the inter-epidemic period and fade-out frequency. These predictions are then contrasted with detailed incidence data for England and Wales. We find model output to be most sensitive to assumptions concerning naturally acquired immunity, which allows us to estimate the average duration of immunity. Our results support a period of natural immunity that is, on average, long-lasting (at least 30 years) but inherently variable. PMID:19876392

  5. The Effect of Maternal Pertussis Immunization on Infant Vaccine Responses to a Booster Pertussis-Containing Vaccine in Vietnam

    PubMed Central

    Maertens, Kirsten; Hoang, Thi Thu Ha; Nguyen, Trung Dac; Caboré, Raïssa Nadège; Duong, Thi Hong; Huygen, Kris; Hens, Niel; Van Damme, Pierre; Dang, Duc Anh; Leuridan, Elke

    2016-01-01

    Background. Maternal vaccination with an acellular pertussis (aP)–containing vaccine is a recommended strategy in a growing number of industrialized countries, to protect young infants from disease. Little is known on the effect of this strategy in low- and middle-income countries. Following a previous report on the effect of adding a pertussis and diphtheria component to the tetanus vaccination program in pregnant women in Vietnam, we report on infant immune responses to a booster aP vaccine dose in this randomized controlled clinical trial. Methods. Thirty infants of Tdap (tetanus, diphtheria, and acellular pertussis)–vaccinated pregnant women and 37 infants of women vaccinated with a tetanus-only vaccine received a fourth aP-containing vaccine dose in the second year of life. Blood was taken 1 month after the fourth infant dose. Immunoglobulin G (IgG) antibodies against pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (Prn), tetanus toxoid (TT), and diphtheria toxoid (DT) were measured using commercially available enzyme-linked immunosorbent assays (ELISA). Results. One month after the booster dose, significantly lower antibody titers were measured in the Tdap group for anti-TT IgG (P < .001) only. Anti-DT IgG, anti-PT IgG, anti-Prn IgG, and anti-FHA IgG antibody titers were comparable for both groups. A rise in antibody concentrations was elicited for all (except DT) antigens after boosting. Conclusions. The present results indicate that the blunting of infant pertussis responses induced by maternal immunization, measured after a primary series of aP vaccines, was resolved with the booster aP vaccine dose. These results add to the evidence for national and international decision makers on maternal immunization as a vaccination strategy for protection of young infants against infectious diseases. PMID:27838673

  6. Estimated and reported incidence of pertussis in Estonian adults: A seroepidemiological study.

    PubMed

    Jõgi, Piia; Oona, Marje; Toompere, Karolin; Lutsar, Irja

    2015-09-11

    Rates of pertussis immunisation among children in Estonia are high (∼95%), but pertussis is still the most common vaccine preventable childhood disease. Adults are suspected to be sources of pertussis in children. We aimed to measure pertussis toxin (PT) IgG in adults to estimate pertussis infection activity and compare estimated and reported pertussis incidences. In a cross-sectional serosurvey, consecutive leftover blood sera (n=3327) from subjects aged 20-99 years old were collected at Quattromed HTI laboratories between the 7th January and 27th February 2013. Anti-PT IgG concentration was measured by ELISA (Euroimmun, Lübeck, Germany). Estimated annual pertussis incidence was calculated for 10-year age classes using de Melker et al. (2006. J Infect. 53(2):106-13) formula. The mean number of samples in each 10-year age class was 466 (SD 20.5), except for 90-99 year olds which contained 65 samples. More than half of all subjects (58.1%) had anti-PT IgG <5.0IU/mL, 2.7% had 62.5 to <125IU/mL and 0.6% ≥125IU/mL; no differences occurred between 10-year age classes. Estimated incidence of pertussis infection was 5.8% (95% CI 4.8-7.0) in 2012, with peaks observed in 20-29 year olds (11.0%; 95% CI 7.4-15.6) and 90-99 year olds (10.8%; 95% CI 3.0-26.2). Estimated pertussis incidence rate was 915 times higher than reported. Of 80 subjects with anti-PT IgG ≥62.5IU/mL, 25 (31.3%) had complained of coughing to their GP during the previous six months. The frequency of pertussis infection was similar for all ages, suggesting similar Bordetella pertussis activity in adults and children. The wide gap between reported and estimated incidence indicates poor recognition of pertussis, likely owing to it being an asymptomatic or mild disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Identification of Microcystis aeruginosa Peptides Responsible for Allergic Sensitization and Characterization of Functional Interactions between Cyanobacterial Toxins and Immunogenic Peptides

    PubMed Central

    Geh, Esmond N.; Ghosh, Debajyoti; McKell, Melanie; de la Cruz, Armah A.; Stelma, Gerard

    2015-01-01

    Background The cyanobacterium species Microcystis aeruginosa produces microcystin and an array of diverse metabolites believed responsible for their toxicity and/or immunogenicity. Previously, chronic rhinitis patients were demonstrated to elicit a specific IgE response to nontoxic strains of M. aeruginosa by skin-prick testing, indicating that cyanobacteria allergenicity resides in a non-toxin–producing component of the organism. Objectives We sought to identify and characterize M. aeruginosa peptide(s) responsible for allergic sensitization in susceptible individuals, and we investigated the functional interactions between cyanobacterial toxins and their coexpressed immunogenic peptides. Methods Sera from patients and extracts from M. aeruginosa toxic [MC(+)] and nontoxic [MC(–)] strains were used to test IgE-specific reactivity by direct and indirect ELISAs; 2D gel electrophoresis, followed by immunoblots and mass spectrometry (MS), was performed to identify the relevant sensitizing peptides. Cytotoxicity and mediator release assays were performed using the MC(+) and MC(–) lysates. Results We found specific IgE to be increased more in response to the MC(–) strain than the MC(+) strain. This response was inhibited by preincubation of MC(–) lysate with increasing concentrations of microcystin. MS revealed that phycocyanin and the core-membrane linker peptide are the responsible allergens, and MC(–) extracts containing these proteins induced β-hexosaminidase release in rat basophil leukemia cells. Conclusions Phycobiliprotein complexes in M. aeruginosa have been identified as the relevant sensitizing proteins. Our finding that allergenicity is inhibited in a dose-dependent manner by microcystin toxin suggests that further investigation is warranted to understand the interplay between immunogenicity and toxicity of cyanobacteria under diverse environmental conditions. Citation Geh EN, Ghosh D, McKell M, de la Cruz AA, Stelma G, Bernstein JA. 2015

  8. Discrimination between Candida albicans and Other Pathogenic Species of the Genus Candida by Their Differential Sensitivities to Toxins of a Panel of Killer Yeasts

    PubMed Central

    Buzzini, P.; Martini, A.

    2001-01-01

    The differential sensitivities to toxins produced by a short panel of four killer yeasts allowed discrimination between 91 strains of the yeast Candida albicans and 223 non-C. albicans Candida strains. One hundred percent of C. albicans isolates exhibited negative results to the toxin panel, while 100% of non-C. albicans cultures gave well-defined and reproducible positive results to at least one of the four killer toxins. Among C. albicans strains only 96 and 87% gave germ tube (GT)- and chlamydospore-positive results, respectively. In addition a few GT-false-positive strains were detected among non-C. albicans isolates. Susceptibility to the toxin panel is apparently expressed more consistently than either GT or chlamydospore production and may constitute a promising basis for a new simple and easy-to-use procedure for routine discrimination between the species C. albicans and other species of the genus Candida. PMID:11526179

  9. Prior exposure to Bordetella species as an exclusion criterion in the baboon model of pertussis

    PubMed Central

    NGUYEN, Annalee W.; WAGNER, Ellen K.; POSADA, Luciano; LIU, Xinlei; CONNELLY, Sheila; PAPIN, James F.; WOLF, Roman F.; KALEKO, Michael; MAYNARD, Jennifer A.

    2016-01-01

    The baboon model of Bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date. However, among the 15 experimentally infected baboons in this study, a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count. Moreover, cultures of nasopharyngeal wash samples from several baboons suggested B. bronchiseptica coinfection. Analysis of serum antibodies recognizing filamentous hemagglutinin, pertussis toxin and B. pertussis lipo-oligosaccharide indicated that several baboons had likely been previously exposed to Bordetella species and that prior exposure correlated with partial protection from B. pertussis infection. Notably, all animals with a baseline Fha titer of 5 IU/ml or below exhibited symptoms typical of the model, suggesting this value can be used as inclusion criteria for animals prior to study enrollment. While B. pertussis infection is endemic to human populations and B. bronchiseptica is common in wild small mammals, this study illustrates that baboons can readily harbor both organisms. Awareness of Bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis. PMID:27666464

  10. Epidemiological features of pertussis resurgence based on community populations with high vaccination coverage in China.

    PubMed

    Huang, H; Zhu, T; Gao, C; Gao, Z; Liu, Y; Ding, Y; Sun, J; Guo, L; Liu, P; Chen, D; Wang, L; Wu, S; Zhang, Y

    2015-07-01

    Active symptom surveillance was applied to three selected communities ( 160,147 persons) in Tianjin from 2010 to 2012. We examined 1089 individuals showing pertussis-like symptoms, of which 1022 nasopharyngeal specimens were tested for pertussis by polymerase chain reaction and 802 sera for anti-pertussis toxin antibodies. Of the total cases tested, 113 were confirmed, and their demographic, clinical, and vaccination-related data were collected. The annual incidence was 23.52 cases/100,000 persons among communities, which was 16.22 times that obtained via hospital reports for the same period (P < 0.001). The actual incidence in the 15-69 years age group was most significantly underestimated by hospitals, given that it was 43.08 times that of the reported hospital rate. Among the cases aged <15 years, 84.5% were individuals who had been fully vaccinated. The misdiagnosis rate was as high as 94.69%, and only 5.31% of the confirmed pertussis cases were properly diagnosed as pertussis at their first medical visit. Pertussis incidence in China has been severely underestimated and this was in part due to a high misdiagnosis rate. Adolescents and adults have become new high-risk populations. Future work should focus on reinforcing immunization programmes, especially among adolescents and adults.

  11. A proteoliposome formulation derived from Bordetella pertussis induces protection in two murine challenge models.

    PubMed

    Fernández, Sonsire; Fajardo, Esther M; Mandiarote, Aleida; Año, Gemma; Padrón, Maria A; Acosta, Michel; Cabrera, Rubén A; Riverón, Luis A; Álvarez, Maydelis; Blaín, Kirenia; Fariñas, Mildrey; Cardoso, Daniel; García, Luis G; Campa, Concepción; Pérez, José L

    2013-01-01

    Whooping cough remains a health problem despite high vaccination coverage. It has been recommended that development of new strategies provide long-lasting immunity. The aim of this work was to evaluate the potential of proteoliposomes (PL) extracted from Bordetella pertussis as a vaccine candidate against whooping cough. The size of the B. pertussis PL was estimated to be 96.7 ± 50.9 nm by Scanning Correlation Spectroscopy and the polydispersity index was 0.268. Western blots using monoclonal antibodies revealed the presence of pertussis toxin, pertactin, and fimbriae 3. The Limulus Amebocyte Lisate (LAL) assay showed endotoxin levels lower than those reported for whole cell pertussis licensed vaccines, while the Pyrogen Test indicated 75 ng/mL/Kg. The PL showed high protection capacity in mouse challenge models. There was 89.7% survival in the intracerebral challenge and total reduction of the number of CFU in the intranasal challenge. No significant differences (p > 0.05) were observed between mice immunized with B. pertussis PL and the Cuban DTwP vaccine, whichever challenge model used. These results encouraged us to continue the development of the B. pertussis PL as a component of a new combined vaccine formulated with tetanus and diphtheria toxoids or as a booster dose for adolescents and adults.

  12. High incidence of Pertussis among schoolchildren with prolonged cough in Turkey.

    PubMed

    Aksakal, Fatma Nur; Cöplü, Nilay; Ceyhan, Mustafa N; Sönmez, Cemile; Ozkan, Seçil; Esen, Berrin; Ilhan, Mustafa N; Aygün, Remzi

    2007-04-01

    Older children, adolescents and adults with prolonged cough should be screened for pertussis, because they can be a major source of transmission for infants who are not yet fully immunized. This study aimed to estimate the incidence of pertussis among schoolchildren of 6-14 years old with prolonged cough attending two government primary schools in central Ankara. A questionnaire and an informed consent form were sent to the parents of 1,859 schoolchildren. The former comprised questions regarding sociodemographics, and the presence and duration of cough. Parents of 1,698 (91.3%) schoolchildren returned the questionnaire. Three hundred and eighty three (22.6%) of the schoolchildren had cough for more than two weeks, and 307 (80.2%) of them, whose parents gave consent, were included in the study. Their blood samples were collected twice, with a two-week interval. Anti-pertussis toxin IgG levels were measured by enzyme-linked immunosorbent assay (ELISA). Fifty-one children (16.6%) had evidence of recent pertussis infection documented by either a titer > or = 100 ELISA Unit (EU)/ml in one of two serum samples or seroconversion in paired sera. None of the schoolchildren who had attended health care facilities for cough had been diagnosed as pertussis by a physician. In conclusion, pertussis is evident among older children and adolescents in Turkey, and in order to control the disease, priority should be given to strengthen primary immunization services along with the implementation of booster vaccinations beyond childhood.

  13. Pertussis Antibody Concentrations in Infants Born Prematurely to Mothers Vaccinated in Pregnancy.

    PubMed

    Kent, Alison; Ladhani, Shamez N; Andrews, Nick J; Matheson, Mary; England, Anna; Miller, Elizabeth; Heath, Paul T

    2016-07-01

    Maternal antenatal pertussis-containing vaccination is recommended for the prevention of neonatal pertussis, but the ability of maternal vaccination to protect premature infants is unknown. We hypothesized that that infants born prematurely to antenatally vaccinated women would have higher pertussis antibody concentrations than those born to unvaccinated women. Mothers had been offered a combined tetanus, diphtheria, 5-component acellular pertussis, inactivated polio vaccine from 28 weeks' gestation as part of their routine antenatal care. Premature infants of vaccinated and unvaccinated mothers enrolled in a randomized controlled trial of pneumococcal conjugate vaccine schedules had antibody concentrations (pertussis toxin, filamentous hemoagglutinin [FHA], and fimbriae 2 and 3) measured at 2 months (before primary vaccination), 5 months (1 month after primary vaccination), and 12 months of age. Mothers of 31 (19%) of 160 premature infants had received combined tetanus, diphtheria, 5-component acellular pertussis, inactivated polio vaccine in pregnancy. Compared with infants of unvaccinated mothers, those born to vaccinated mothers had significantly higher antibody concentrations at 2 months for all measured vaccine antigens (P < .001). The number of days between maternal vaccination and delivery and immunoglobulin G concentration at 2 months of age was positively correlated for pertussis toxin (P = .011) and FHA (P = .001). After primary immunization, infants of vaccinated mothers had significantly lower antibody concentrations for FHA (P = .003) compared with infants of unvaccinated mothers; these differences had resolved by 12 months of age. Maternal vaccination administered early in the third trimester may provide protection for infants born prematurely. Copyright © 2016 by the American Academy of Pediatrics.

  14. Fast, antigen-saving multiplex immunoassay to determine levels and avidity of mouse serum antibodies to pertussis, diphtheria, and tetanus antigens.

    PubMed

    Stenger, Rachel M; Smits, Mieke; Kuipers, Betsy; Kessen, Sabine F M; Boog, Claire J P; van Els, Cécile A C M

    2011-04-01

    To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra- and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.

  15. Botulinum toxin type A selectivity for certain types of pain is associated with capsaicin-sensitive neurons.

    PubMed

    Matak, Ivica; Rossetto, Ornella; Lacković, Zdravko

    2014-08-01

    Unlike most classical analgesics, botulinum toxin type A (BoNT/A) does not alter acute nociceptive thresholds, and shows selectivity primarily for allodynic and hyperalgesic responses in certain pain conditions. We hypothesized that this phenomenon might be explained by characterizing the sensory neurons targeted by BoNT/A in the central nervous system after its axonal transport. BoNT/A's central antinociceptive activity following its application into the rat whisker pad was examined in trigeminal nucleus caudalis (TNC) and higher-level nociceptive brain areas using BoNT/A-cleaved synaptosomal-associated protein 25 (SNAP-25) and c-Fos immunohistochemistry. Occurrence of cleaved SNAP-25 in TNC was examined after nonselective ganglion ablation with formalin or selective denervation of capsaicin-sensitive (vanilloid receptor-1 or TRPV1-expressing) neurons, and in relation to different cellular and neuronal markers. Regional c-Fos activation and effect of TRPV1-expressing afferent denervation on toxin's antinociceptive action were studied in formalin-induced orofacial pain. BoNT/A-cleaved SNAP-25 was observed in TNC, but not in higher-level nociceptive nuclei. Cleaved SNAP-25 in TNC disappeared after formalin-induced trigeminal ganglion ablation or capsaicin-induced sensory denervation. Occurrence of cleaved SNAP-25 in TNC and BoNT/A antinociceptive activity in formalin-induced orofacial pain were prevented by denervation with capsaicin. Cleaved SNAP-25 localization demonstrated toxin's presynaptic activity in TRPV1-expressing neurons. BoNT/A reduced the c-Fos activation in TNC, locus coeruleus, and periaqueductal gray. Present experiments suggest that BoNT/A alters the nociceptive transmission at the central synapse of primary afferents. Targeting of TRPV1-expressing neurons might be associated with observed selectivity of BoNT/A action only in certain types of pain.

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

    PubMed

    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

  17. A Rapid and Sensitive Method to Measure the Functional Activity of Shiga Toxins in Human Serum

    PubMed Central

    Arfilli, Valentina; Carnicelli, Domenica; Ardissino, Gianluigi; Torresani, Erminio; Scavia, Gaia; Brigotti, Maurizio

    2015-01-01

    Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins). PMID:26556372

  18. Characterization of the key antigenic components of pertussis vaccine based on outer membrane vesicles.

    PubMed

    Ormazábal, Maximiliano; Bartel, Erika; Gaillard, María Emilia; Bottero, Daniela; Errea, Agustina; Zurita, M Eugenia; Moreno, Griselda; Rumbo, Martin; Castuma, Celina; Flores, Dario; Martín, María Julia; Hozbor, Daniela

    2014-10-21

    Pertussis has resurged during the last two decades in different countries. In particular in the 2010-2013 period large outbreaks were detected in US, Australia, UK and The Netherlands with significant mortality in infants. The epidemiological situation of pertussis points out the need to develop new vaccines and in this regard we previously developed a new vaccine based on outer membrane vesicles (OMVs) which have been shown to be safe and to induce protection in mice. Here we have further investigated the properties of OMVs vaccines; in particular we studied the contribution of pertussis toxin (PTx) and pertactin (Prn) in OMVs-mediated protection against pertussis. PTx-deficient OMVs and Prn-deficient OMVs were obtained from defective Bordetella pertussis mutants. The absence of PTx or Prn did compromise the protective capacity of the OMVs formulated as Tdap vaccine. Whereas the protective efficacy of the PTx-deficient OMVs in mice was comparable to Prn-deficient OMVs, the protective capacity of both of them was significantly impaired when it was compared with the wild type OMVs. Interestingly, using OMVs obtained from a B. pertussis strain which does not express any of the virulence factors but expresses the avirulent phenotype; we observed that the protective ability of such OMVs was lower than that of OMVs obtained from virulent B. pertussis phase. However, it was surprising that although the protective capacity of avirulent OMVs was lower, they were still protective in the used mice model. These results allow us to hypothesize that OMVs from avirulent phase shares protective components with all OMVs assayed. Using an immune proteomic strategy we identified some common components that could play an important role in protection against pertussis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Epidemiology of pertussis in Casablanca (Morocco): contribution of conventional and molecular diagnosis tools.

    PubMed

    Katfy, Khalid; Guiso, Nicole; Diawara, Idrissa; Zerouali, Khalid; Slaoui, Bouchra; Jouhadi, Zineb; Zineddine, Abdelhadi; Belabbes, Houria; Elmdaghri, Naima

    2017-05-16

    highlights the circulation of B. pertussis but also of B. holmesii in Casablanca-Morocco with a high proportion of co-infections B. holmesii/B. pertussis in infants and their mothers, indicate that infection of non-vaccinated infants could be more associated with young parents. Moreover, the RT- PCR provides a sensitive and specific diagnosis of B. pertussis infections and distinguishes it from other Bordetella species, and is therefore suitable for implementation in the diagnostic laboratory.

  20. IgG responses after booster vaccination with different pertussis vaccines in Dutch children 4 years of age: effect of vaccine antigen content.

    PubMed

    Hendrikx, Lotte H; Berbers, Guy A M; Veenhoven, Reinier H; Sanders, Elisabeth A M; Buisman, Anne-Marie

    2009-11-05

    Since whooping cough is reemerging in the Netherlands from 1996 onwards, several changes in the national immunization program have been implemented regarding the pertussis vaccinations. The aim of this study is to investigate IgG responses in whole cell (wP) and acellular (aP) pertussis vaccine primed children following revaccination with different pertussis booster vaccines at 4 years of age. IgG levels to pertussis toxin (Pt), filamentous heamagglutin (FHA), pertactin (Prn) and fimbriae type 2 and 3 (Fim2/3) and avidities of Pt and Prn antibodies were measured using a multiplex immunoassay. Before and after the booster we found significantly higher IgG levels to Pt, FHA and Prn in aP compared to wP primed children. In all children a booster vaccination with a pertussis vaccine containing a high antigen dose (Infanrix) induced higher IgG responses compared to a low antigen dose containing vaccine (Triaxis). Avidities of Pt- and Prn-antibodies before and after booster vaccination were significantly higher in aP than in wP primed children. This study shows that a booster vaccine with high pertussis antigen concentrations induces higher antibody levels than a low antigen containing vaccine. In children primed with the Dutch DTwP-IPV-Hib vaccine we suggest to administer a booster vaccine containing high pertussis antigens to optimize IgG responses. The pertussis vaccination history has to be taken into account in decisions on changes in pertussis vaccination policy.

  1. Bordetella pertussis diagnosis in children under five years of age in the Regional Hospital of Cajamarca, Northern Peru.

    PubMed

    Del Valle-Mendoza, Juana; Casabona-Oré, Veronica; Petrozzi-Helasvuo, Veronica; Cornejo-Tapia, Angela; Weilg, Pablo; Pons, Maria J; Cieza-Mora, Erico; Bazán-Mayra, Jorge; Cornejo-Pacherres, Hernan; Ruiz, Joaquin

    2015-11-30

    Bordetella pertussis is an important human pathogen that causes whooping cough (pertussis), an endemic illness responsible of significant morbidity and mortality, especially in infants and children. Worldwide, there are an estimated of 16 million cases of pertussis, resulting in about 195,000 child deaths per year. In Peru, pertussis is a major health problem that has been on the increase despite immunization efforts. The objective of this study was to determine the prevalence of B. pertussis among children under five years of age suspected to have whopping cough in Cajamarca, Peru. Children diagnosed with whooping cough admitted to the Hospital Regional de Cajamarca from August 2010 to July 2013 were included. Nasopharyngeal samples were obtained for B. pertussis culture and polymerase chain reaction (PCR) detection. In 133 children, the pertussis toxin and IS481 gene were detected in 38.35% (51/133) of the cases by PCR, while only 9.02% (12/133) of the Bordetella cultures were positive. The most frequent symptoms in patients with positive B. pertussis were paroxysm of coughing 68.63% (35/51), cyanosis 56.86% (29/51), respiratory distress 43.14% (22/51), and fever 39.22% (20/51). Pneumonia and acute bronchial obstructive syndrome were present in 17.65% (9/51) and 13.72% (7/51) of the cases, respectively. B. pertussis is responsible for an important proportion of whooping cough in hospitalized children in Cajamarca. Epidemiologic surveillance programs for B. pertussis are essential in Peru, especially in children who could most benefit from the vaccine.

  2. Identifying long-term memory B-cells in vaccinated children despite waning antibody levels specific for Bordetella pertussis proteins.

    PubMed

    Hendrikx, Lotte H; Oztürk, Kemal; de Rond, Lia G H; Veenhoven, Reinier H; Sanders, Elisabeth A M; Berbers, Guy A M; Buisman, Anne-Marie

    2011-02-04

    Whooping cough is a respiratory disease caused by Bordetella pertussis. Since the 1950s in developed countries pertussis vaccinations are included in the national immunization program. However, antibody levels rapidly wane after both whole cell and acellular pertussis vaccination. Therefore protection against pertussis may depend largely on long-term B- and T-cell immunities. We investigated long-term pertussis-specific memory B-cell responses in children who were primed at infant age with the Dutch wP-vaccine (ISRCTN65428640). Purified B-cells were characterized by FACS-analysis and after polyclonal stimulation memory B-cells were detected by ELISPOT-assays specific for pertussis toxin, filamentous haemagglutinin, pertactin and tetanus. In addition, plasma IgG levels directed to the same antigens were measured by a fluorescent bead-based multiplex immunoassay. Two and 3 years after wP priming as well as 2 and 5 years after the aP booster at the age of 4, low plasma IgG levels to the pertussis proteins were found. At the same time, however pertussis protein-specific memory B-cells could be detected and their number increased with age. The number of tetanus-specific memory B-cells was similar in all age groups, whereas IgG-tetanus levels were high 2 years after tetanus booster compared to pre- and 5 years post-booster levels. This study shows the presence of long-term pertussis protein-specific memory B-cells in children despite waning antibody levels after vaccination, which suggests that memory B-cells in addition to antibodies may contribute to protection against pertussis.

  3. Sensitization of radio-resistant prostate cancer cells with a unique cytolethal distending toxin

    PubMed Central

    Lai, Chih-Ho; Chang, Chia-Shuo; Liu, Hsin-Ho; Tsai, Yuh-Shyan; Hsu, Feng-Ming; Yu, Yung-Luen; Lai, Cheng-Kuo; Gandee, Leah; Pong, Rey-Chen; Hsu, Heng-Wei; Yu, Lan; Saha, Debabrata; Hsieh, Jer-Tsong

    2014-01-01

    Cytolethal distending toxin (CDT) produced by Campylobacter jejuni is a genotoxin that induces cell-cycle arrest and apoptosis in mammalian cells. Recent studies have demonstrated that prostate cancer (PCa) cells can acquire radio-resistance when DOC-2/DAB2 interactive protein (DAB2IP) is downregulated. In this study, we showed that CDT could induce cell death in DAB2IP-deficient PCa cells. A combination of CDT and radiotherapy significantly elicited cell death in DAB2IP-deficient PCa cells by inhibiting the repair of ionizing radiation (IR)-induced DNA double-strand break (DSB) during G2/M arrest, which is triggered by ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses. We also found that CDT administration significantly increased the efficacy of radiotherapy in a xenograft mouse model. These results indicate that CDT can be a potent therapeutic agent for radio-resistant PCa. PMID:25015118

  4. Simple, rapid, sensitive, and versatile SWNT-paper sensor for environmental toxin detection competitive with ELISA.

    PubMed

    Wang, Libing; Chen, Wei; Xu, Dinghua; Shim, Bong Sup; Zhu, Yingyue; Sun, Fengxia; Liu, Liqiang; Peng, Chifang; Jin, Zhengyu; Xu, Chuanlai; Kotov, Nicholas A

    2009-12-01

    Safety of water was for a long time and still is one of the most pressing needs for many countries and different communities. Despite the fact that there are potentially many methods to evaluate water safety, finding a simple, rapid, versatile, and inexpensive method for detection of toxins in everyday items is still a great challenge. In this study, we extend the concept of composites, impregnated porous fibrous materials, such as fabrics and papers, by single-walled carbon nanotubes (SWNTs), toward very simple but high-performance biosensors. They utilize the strong dependence of electrical conductivity through nanotubes percolation network on the width of nanotube-nanotube tunneling gap and can potentially satisfy all the requirements outlined above for the routine toxin monitoring. An antibody to the microcystin-LR (MC-LR), one of the common culprits in mass poisonings, was dispersed together with SWNTs. This dispersion was used to dip-coat the paper rendering it conductive. The change in conductivity of the paper was used to sense the MC-LR in the water rapidly and accurately. The method has the linear detection range up to 10 nmol/L and nonlinear detection up to 40 nmol/L. The limit of detection was found to be 0.6 nmol/L (0.6 ng/mL), which satisfies the strictest World Health Organization standard for MC-LR content in drinking water (1 ng/mL) and is comparable to the detection limit of the traditional ELISA method of MC-LR detection, while drastically reducing the time of analysis by more than an order of magnitude, which is one of the major hurdles in practical applications. Similar technology of sensor preparation can also be used for a variety of other rapid environmental sensors.

  5. Safe, rapid, and sensitive method of quantitating and distinguishing among Shiga toxins in complex media and human serum

    USDA-ARS?s Scientific Manuscript database

    Shiga toxins are primarily responsible for the virulence associated with Shiga toxin producing Escherichia coli (STEC) infections. The expression of the Shiga toxins is controlled by a phage that infects the host. More than one phage can infect a host and the host can inactivate infecting phages. T...

  6. [Antibody levels for diphtheria, tetanus and pertussis in young adult females immunized with whole cell pertussis-diphtheria-tetanus toxoid vaccine in infancy].

    PubMed

    Meno, Y; Okada, K; Yamaguchi, Y; Morokuma, K; Okuma, K; Ueda, K

    2000-02-01

    Antibody levels for diphtheria, tetanus and pertussis in 84 young adult females were measured. They had been immunized with whole cell pertussis-diphtheria-tetanus toxoid (DTwP) vaccine as a routine immunization in their infancy. Their history of DTwP vaccination were confirmed in their Maternal and Child Health Handbook, which includes their immunization record. Among the 84 cases, 4 cases (4.7%) had been immunized with the first dose of DTwP, 5 cases (6.0%) with the second dose, 23 cases (27.4%) with the third dose and 52 cases (61.9%) with the fourth dose. Of the 84 cases, 89.3% had received DTwP vaccine more than the third dose. In the 15-19 years after the last DTwP vaccination, the antibody positive rate for diphtheria and tetanus (> or = 0.01 IU/ml) were 86.9% and 94.0%, respectively. On the other hand, antibody positive rate for anti-pertussis toxin (anti-PT) and anti-filamentous hemaggulutinin (anti-FHA) (> or = 10 EU/ml) were 35.7% and 55.9%, respectively. The positive rate for pertussis compared with those for diphtheria and tetanus were lower. These findings suggested that DTwP vaccination in infancy does not provide sufficient immunity for young adults against pertussis, but DTwP vaccination provides adequate immunity against diphtheria and tetanus.

  7. Pertussis: Microbiology, Disease, Treatment, and Prevention

    PubMed Central

    Salim, Abdulbaset M.; Zervos, Marcus J.; Schmitt, Heinz-Josef

    2016-01-01

    SUMMARY Pertussis is a severe respiratory infection caused by Bordetella pertussis, and in 2008, pertussis was associated with an estimated 16 million cases and 195,000 deaths globally. Sizeable outbreaks of pertussis have been reported over the past 5 years, and disease reemergence has been the focus of international attention to develop a deeper understanding of pathogen virulence and genetic evolution of B. pertussis strains. During the past 20 years, the scientific community has recognized pertussis among adults as well as infants and children. Increased recognition that older children and adolescents are at risk for disease and may transmit B. pertussis to younger siblings has underscored the need to better understand the role of innate, humoral, and cell-mediated immunity, including the role of waning immunity. Although recognition of adult pertussis has increased in tandem with a better understanding of B. pertussis pathogenesis, pertussis in neonates and adults can manifest with atypical clinical presentations. Such disease patterns make pertussis recognition difficult and lead to delays in treatment. Ongoing research using newer tools for molecular analysis holds promise for improved understanding of pertussis epidemiology, bacterial pathogenesis, bioinformatics, and immunology. Together, these advances provide a foundation for the development of new-generation diagnostics, therapeutics, and vaccines. PMID:27029594

  8. [Pertussis infection and fatal pulmonary hypertension].

    PubMed

    Menif, K; Bouziri, A; Khaldi, A; Hamdi, A; Belhadj, S; Benjaballah, N

    2010-11-01

    Pertussis is ranked among the leading causes of childhood mortality. The most catastrophic clinical complication of pertussis in infants, intractable pulmonary hypertension with shock, is not very well known. We describe the clinical course of a fatal case of severe pertussis complicated by refractory pulmonary hypertension and shock in a 2-month-old infant. Copyright © 2010. Published by Elsevier SAS.

  9. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Clostridium difficile Toxin B and Sensitive Detection in Human Fecal Matter

    PubMed Central

    Maher, Eamonn; Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Toxin B is one of the major virulence factors of Clostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence of C. difficile induced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity (Kd = 47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin, Staphylococcus aureus alpha toxin, Pseudomonas aeruginosa exotoxin A, and cholera toxin of Vibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations. PMID:25734010

  10. Using a Bayesian Latent Class Model to Evaluate the Utility of Investigating Persons with Negative Polymerase Chain Reaction Results for Pertussis

    PubMed Central

    Tarr, Gillian A. M.; Eickhoff, Jens C.; Koepke, Ruth; Hopfensperger, Daniel J.; Davis, Jeffrey P.; Conway, James H.

    2013-01-01

    Pertussis remains difficult to control. Imperfect sensitivity of diagnostic tests and lack of specific guidance regarding interpretation of negative test results among patients with compatible symptoms may contribute to its spread. In this study, we examined whether additional pertussis cases could be identified if persons with negative pertussis test results were routinely investigated. We conducted interviews among 250 subjects aged ≤18 years with pertussis polymerase chain reaction (PCR) results reported from 2 reference laboratories in Wisconsin during July–September 2010 to determine whether their illnesses met the Centers for Disease Control and Prevention's clinical case definition (CCD) for pertussis. PCR validity measures were calculated using the CCD as the standard for pertussis disease. Two Bayesian latent class models were used to adjust the validity measures for pertussis detectable by 1) culture alone and 2) culture and/or more sensitive measures such as serology. Among 190 PCR-negative subjects, 54 (28%) had illnesses meeting the CCD. In adjusted analyses, PCR sensitivity and the negative predictive value were 1) 94% and 99% and 2) 43% and 87% in the 2 types of models, respectively. The models suggested that public health follow-up of reported pertussis patients with PCR-negative results leads to the detection of more true pertussis cases than follow-up of PCR-positive persons alone. The results also suggest a need for a more specific pertussis CCD. PMID:23735308

  11. Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library.

    PubMed

    Xu, Chongxin; Liu, Xiaoqin; Zhang, Cunzheng; Zhang, Xiao; Zhong, Jianfeng; Liu, Yuan; Hu, Xiaodan; Lin, Manman; Liu, Xianjin

    2017-02-01

    Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL(-1) and the medium inhibition of control (IC50) was 0.73 ng mL(-1). It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%-96.6% and with a coefficient of variation (CV) among 2.0%-8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.

  12. Pertussis toxin nullifies the depolarization of the membrane potential and the stimulation of the rapid phase of Ca entry through L-type calcium channels that are produced by follicle stimulating hormone in 10- to 12-day-old rat Sertoli cells.

    PubMed

    Jacobus, Ana Paula; Loss, Eloísa Silveira; Wassermann, Guillermo Federico

    2010-01-01

    The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca(2+) into cells and to the Ca(2+)-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of (45)Ca(2+) uptake and [(14)C]-methyl aminoisobutyric acid ([(14)C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca(2+) channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of (45)Ca(2+) entry and [(14)C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca(2+) channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I.

  13. Pertussis Toxin Nullifies the Depolarization of the Membrane Potential and the Stimulation of the Rapid Phase of 45Ca2+ Entry Through L-type Calcium Channels that are Produced by Follicle Stimulating Hormone in 10- to 12-Day-Old Rat Sertoli Cells

    PubMed Central

    Jacobus, Ana Paula; Loss, Eloísa Silveira; Wassermann, Guillermo Federico

    2010-01-01

    The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca2+ into cells and to the Ca2+-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of 45Ca2+ uptake and [14C]-methyl aminoisobutyric acid ([14C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca2+ channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of 45Ca2+ entry and [14C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca2+ channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I. PMID:21423378

  14. Under-recognized pertussis in adults from Asian countries: a cross-sectional seroprevalence study in Malaysia, Taiwan and Thailand.

    PubMed

    Koh, M T; Liu, C-S; Chiu, C-H; Boonsawat, W; Watanaveeradej, V; Abdullah, N; Zhang, Xh; Devadiga, R; Chen, J

    2016-04-01

    Surveillance data on the burden of pertussis in Asian adults are limited. This cross-sectional study evaluated the prevalence of serologically confirmed pertussis in adults with prolonged cough in Malaysia, Taiwan and Thailand. Adults (⩾19 years) with cough lasting for ⩾14 days without other known underlying cause were enrolled from outpatient clinics of seven public and/or private hospitals. Single blood samples for anti-pertussis toxin antibodies (anti-PT IgG) were analysed and economic impact and health-related quality of life (EQ-5D) questionnaires assessed. Sixteen (5·13%) of the 312 chronically coughing adults had serological evidence of pertussis infection within the previous 12 months (anti-PT IgG titre ⩾62·5 IU/ml). Three of them were teachers. Longer duration of cough, paroxysms (75% seroconfirmed, 48% non-seroconfirmed) and breathlessness/chest pain (63% seroconfirmed, 36% non-seroconfirmed) were associated with pertussis (P < 0·04). Of the seroconfirmed patients, the median total direct medical cost per pertussis episode in public hospitals (including physician consultations and/or emergency room visits) was US$13 in Malaysia, US$83 in Taiwan (n = 1) and US$26 in Thailand. The overall median EQ-5D index score of cases was 0·72 (range 0·42-1·00). Pertussis should be considered in the aetiology of adults with a prolonged or paroxysmal cough, and vaccination programmes considered.

  15. Tracking Pertussis and Evaluating Control Measures through Enhanced Pertussis Surveillance, Emerging Infections Program, United States.

    PubMed

    Skoff, Tami H; Baumbach, Joan; Cieslak, Paul R

    2015-09-01

    Despite high coverage with pertussis-containing vaccines, pertussis remains endemic to the United States. There have been increases in reported cases in recent years, punctuated by striking epidemics and shifting epidemiology, both of which raise questions about current policies regarding its prevention and control. Limited data on pertussis reported through the National Notifiable Disease Surveillance System have proved insufficient to answer these questions. To address shortcomings of national pertussis data, the Emerging Infections Program at the US Centers for Disease Control and Prevention launched Enhanced Pertussis Surveillance (EPS), which is characterized by systematic case ascertainment, augmented data collection, and collection of Bordetella pertussis isolates. Data collected through EPS have been instrumental in understanding the rapidly evolving epidemiology and molecular epidemiology of pertussis and have contributed essential information regarding pertussis vaccines. EPS also serves as a platform for conducting critical and timely evaluations of pertussis prevention and control strategies, including targeting of vaccinations and antimicrobial prophylaxis.

  16. Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme.

    PubMed Central

    Rogel, A; Schultz, J E; Brownlie, R M; Coote, J G; Parton, R; Hanski, E

    1989-01-01

    Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties. Images PMID:2555185

  17. A specific real-time PCR assay for the detection of Bordetella pertussis.

    PubMed

    Vincart, Benoit; De Mendonça, Ricardo; Rottiers, Sylvianne; Vermeulen, Françoise; Struelens, Marc J; Denis, Olivier

    2007-07-01

    A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

  18. SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

    PubMed Central

    Bak, Nicola; Rajagopal, Shalini; Stickings, Paul; Sesardic, Dorothea

    2017-01-01

    Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay. PMID:28726719

  19. SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E.

    PubMed

    Bak, Nicola; Rajagopal, Shalini; Stickings, Paul; Sesardic, Dorothea

    2017-07-20

    Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

  20. Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE.

    PubMed

    Diago-Navarro, Elizabeth; Mora, Liliana; Buckingham, Richard H; Díaz-Orejas, Ramón; Lemonnier, Marc

    2009-01-01

    Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.

  1. Shiga Toxin 1-Induced Inflammatory Response in Lipopolysaccharide-Sensitized Astrocytes Is Mediated by Endogenous Tumor Necrosis Factor Alpha▿

    PubMed Central

    Landoni, Verónica I.; de Campos-Nebel, Marcelo; Schierloh, Pablo; Calatayud, Cecilia; Fernandez, Gabriela C.; Ramos, M. Victoria; Rearte, Bárbara; Palermo, Marina S.; Isturiz, Martín A.

    2010-01-01

    Hemolytic-uremic syndrome (HUS) is generally caused by Shiga toxin (Stx)-producing Escherichia coli. Endothelial dysfunction mediated by Stx is a central aspect in HUS development. However, inflammatory mediators such as bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) contribute to HUS pathophysiology by potentiating Stx effects. Acute renal failure is the main feature of HUS, but in severe cases, patients can develop neurological complications, which are usually associated with death. Although the mechanisms of neurological damage remain uncertain, alterations of the blood-brain barrier associated with brain endothelial injury is clear. Astrocytes (ASTs) are the most abundant inflammatory cells of the brain that modulate the normal function of brain endothelium and neurons. The aim of this study was to evaluate the effects of Stx type 1 (Stx1) alone or in combination with LPS in ASTs. Although Stx1 induced a weak inflammatory response, pretreatment with LPS sensitized ASTs to Stx1-mediated effects. Moreover, LPS increased the level of expression of the Stx receptor and its internalization. An early inflammatory response, characterized by the release of tumor necrosis factor alpha (TNF-α) and nitric oxide and PMN-chemoattractant activity, was induced by Stx1 in LPS-sensitized ASTs, whereas activation, evidenced by higher levels of glial fibrillary acid protein and cell death, was induced later. Furthermore, increased adhesion and PMN-mediated cytotoxicity were observed after Stx1 treatment in LPS-sensitized ASTs. These effects were dependent on NF-κB activation or AST-derived TNF-α. Our results suggest that TNF-α is a pivotal effector molecule that amplifies Stx1 effects on LPS-sensitized ASTs, contributing to brain inflammation and leading to endothelial and neuronal injury. PMID:20008539

  2. Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using loop-mediated isothermal amplification.

    PubMed

    Yamazaki, Wataru

    2011-01-01

    Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. The LAMP assay requires 12-18 min for amplification with a single colony on selective agar from cholera toxin (CT)-producing Vibrio cholerae strains and less than 60 min with human feces and seafood samples. The assay requires less than 35 and 80 min for the detection of CT-producing V. cholerae with a colony on selective agar and with human feces and seafood samples from the beginning of DNA extraction to final determination. The LAMP amplification can be judged by both turbidimetric analysis and visual assessment with the unaided eye. The sensitivity of the LAMP assay is tenfold higher than that of the PCR assay. The LAMP assay is a powerful tool for rapid, simple, and sensitive detection of CT-producing V. cholerae which may facilitate the investigation of V. cholerae contamination in seafood, as well as the early diagnosis of cholera in humans.

  3. Cells transformed by PLC-gamma 1 overexpression are highly sensitive to clostridium difficile toxin A-induced apoptosis and mitotic inhibition.

    PubMed

    Nam, Hyo Jung; Kang, Jin Ku; Chang, Jong Soo; Lee, Min Soo; Nam, Seung Taek; Jung, Hyun Woo; Kim, Sung-Kuk; Ha, Eun-Mi; Seok, Heon; Son, Seung Woo; Park, Young Joo; Kim, Ho

    2012-01-01

    Phospholipase C-γl (PLC-γl) expression is associated with cellular transformation. Notably, PLC-gamma is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-γl overexpression. We found that PLC-γl-transformed cells, but not vectortransformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-γl-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-γl-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-γl is highly up-regulated.

  4. Immunization status of Iranian military recruits against Bordetella pertussis infection (whooping cough).

    PubMed

    Izadi, Morteza; Afsharpaiman, Shahla; Jonaidi Jafari, Nematollah; Ranjbar, Reza; Gooya, Mohammad Mahdi; Robat Sarpooshi, Javad; Esfahani, Ali Akbar; Soheylipoor, Hamid

    2011-03-21

    Military recruits are susceptible to respiratory pathogens because of increased antibiotic resistance and the lack of an effective vaccine. The goal of the current study was to determine the immunological status of the Bordetella pertussis among conscripts in Iranian military garrisons. The study population consisted of 424 conscripts aged 18 to 21 years who enrolled for military service. They were selected using cluster stratified sampling from all military garrisons in Tehra, Iran. To determine the seroprevalence of infection, blood specimens from all recruits were collected and stored at - 20 °C until assayed. All serum samples were screened for immunoglobulin G (IgG) antibodies against Bordetella pertussis toxin (PT) and by using enzyme-linked immunosorbent assay (ELISA). The overall prevalence of B. pertussis seropositivity in military recruits was 60.6. Only 55.0% of the recruits had low awareness about the record of vaccination against B. pertussis during childhood. Among 424 studied individuals, 48 recruits (11.3%) had a positive history of whooping cough; prevalence of seropositivity in these recruits was 70.0%. Among these subjects, 61.7% were referred to a physician for treatment and only 39.6% of them were administered anti-pertussis therapy. Our study showed that military conscripts in Tehran garrisons were not serologically immune to pertussis and also confirmed the low awareness about vaccination and medical history related to pertussis infection in this high-risk subgroup of the Iranian population. Routine acellular booster vaccination, particularly before 18 years of age, is recommended.

  5. Molecular epidemiology of Bordetella pertussis in the Philippines in 2012-2014.

    PubMed

    Galit, Salvacion Rosario L; Otsuka, Nao; Furuse, Yuki; Almonia, Daryl Joy V; Sombrero, Lydia T; Capeding, Rosario Z; Lupisan, Socorro P; Saito, Mariko; Oshitani, Hitoshi; Hiramatsu, Yukihiro; Shibayama, Keigo; Kamachi, Kazunari

    2015-06-01

    The present study was designed to determine the genotypes of circulating Bordetella pertussis in the Philippines by direct molecular typing of clinical specimens. Nasopharyngeal swabs (NPSs) were collected from 50 children hospitalized with pertussis in three hospitals during 2012-2014. Multilocus variable-number tandem repeat analysis (MLVA) was performed on the DNA extracts from NPSs. B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter, ptxP, were also investigated by DNA sequence-based typing. Twenty-six DNA extracts yielded a complete MLVA profile, which were sorted into 10 MLVA types. MLVA type 34 (MT34), which is rare in Australia, Europe, Japan, and the USA, was the predominant strain (50%). Seven MTs (MT29, MT32, MT33, and MT283-286, total 42%) were single-locus variants of MT34, while two (MT141 and MT287, total 8%) were double-locus variants of MT34. All MTs had the combination of virulence-associated allelic genes, ptxP1-ptxA1-prn1-fim3A. The B. pertussis population in the Philippines comprises genetically related strains. These strains are markedly different from those found in patients from other countries where acellular pertussis vaccines are used. The differences in vaccine types between these other countries and the Philippines, where the whole-cell vaccine is still used, may select for distinct populations of B. pertussis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Seroprevalence of antibodies to diphtheria, tetanus and pertussis among healthy adolescents and adults in Iran.

    PubMed

    Pourakbari, Babak; Moradi, Behnaz; Mirzaee, Farin; Mahmoudi, Shima; Teymuri, Mostafa; Mamishi, Setareh

    2013-01-01

    Serologic data on diseases that are preventable by vaccine are useful to evaluate the success of immunization programs. In this study we evaluated the serologic levels of antibodies to diphtheria, tetanus, and pertussis. In a cross sectional study, a total of 360 people aged 10-25 years were randomly selected and classified by sex and age (10-14, 15-20, 21-25 years). Overall, 78.8% of people aged 10-25 years had fully protected levels of diphtheria antibody (> or = 0.1 IU/ML), and 89.7% had fully protected levels of tetanus antibody (> or = 0.1 IU/ML), 94.3% of women aged 15-25 years had anti tetanus antibody sufficient to protect against neonatal tetanus (> or = 0.1 IU/ML). Antibodies to Pertussis toxin (PT) were found in 44.2% samples but only 1.4% had fully protective levels. Antibodies to PT increased with age, ranging from 33.5% in aged 10-14 years to 54.6 % in aged 21-25 years. No differences were found between male and female, except for diphtheria in age group 21-25 years. Results of this study reveal that diphtheria and tetanus (dT) are efficient between booster doses. About pertussis, most people are susceptible to pertussis and increased PT antibodies with age suggest acquired asymptomatic Bordeella pertussis infection. Also B. pertussis infections in adolescents and adults are of concern, as they are the most important source of transmission of pertussis to young, unprotected infants. So one booster dose in adolescents and adults (as CDC recommended), to reduce mortality and morbidity in infants, is therefore suggested.

  7. Global Population Structure and Evolution of Bordetella pertussis and Their Relationship with Vaccination

    PubMed Central

    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; Mooi, Frits R.

    2014-01-01

    ABSTRACT 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. PMID:24757216

  8. Plant compounds enhance assay sensitivity for detection of active bacillus cereus toxin

    USDA-ARS?s Scientific Manuscript database

    Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. It has been estimated that there are 84,000 cases of B. cereus food poisoning in the US each year, with an annual cost of USD 36 million. The ability to sensitively trace and...

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

    PubMed

    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

  10. Identification of pertussis-specific effector memory T cells in preschool children.

    PubMed

    de Rond, Lia; Schure, Rose-Minke; Öztürk, Kemal; Berbers, Guy; Sanders, Elisabeth; van Twillert, Inonge; Carollo, Maria; Mascart, Françoise; Ausiello, Clara M; van Els, Cecile A C M; Smits, Kaat; Buisman, Anne-Marie

    2015-05-01

    Whooping cough remains a problem despite vaccination, and worldwide resurgence of pertussis is evident. Since cellular immunity plays a role in long-term protection against pertussis, we studied pertussis-specific T-cell responses. Around the time of the preschool acellular pertussis (aP) booster dose at 4 years of age, T-cell memory responses were compared in children who were primed during infancy with either a whole-cell pertussis (wP) or an aP vaccine. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with pertussis vaccine antigens for 5 days. T cells were characterized by flow-based analysis of carboxyfluorescein succinimidyl ester (CFSE) dilution and CD4, CD3, CD45RA, CCR7, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) expression. Before the aP preschool booster vaccination, both the proliferated pertussis toxin (PT)-specific CD4(+) and CD8(+) T-cell fractions (CFSE(dim)) were higher in aP- than in wP-primed children. Post-booster vaccination, more pertussis-specific CD4(+) effector memory cells (CD45RA(-) CCR7(-)) were induced in aP-primed children than in those primed with wP. The booster vaccination did not appear to significantly affect the T-cell memory subsets and functionality in aP-primed or wP-primed children. Although the percentages of Th1 cytokine-producing cells were alike in aP- and wP-primed children pre-booster vaccination, aP-primed children produced more Th1 cytokines due to higher numbers of proliferated pertussis-specific effector memory cells. At present, infant vaccinations with four aP vaccines in the first year of life result in pertussis-specific CD4(+) and CD8(+) effector memory T-cell responses that persist in children until 4 years of age and are higher than those in wP-primed children. The booster at 4 years of age is therefore questionable; this may be postponed to 6 years of age. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Identification of Pertussis-Specific Effector Memory T Cells in Preschool Children

    PubMed Central

    Schure, Rose-Minke; Öztürk, Kemal; Berbers, Guy; Sanders, Elisabeth; van Twillert, Inonge; Carollo, Maria; Mascart, Françoise; Ausiello, Clara M.; van Els, Cecile A. C. M.; Smits, Kaat; Buisman, Anne-Marie

    2015-01-01

    Whooping cough remains a problem despite vaccination, and worldwide resurgence of pertussis is evident. Since cellular immunity plays a role in long-term protection against pertussis, we studied pertussis-specific T-cell responses. Around the time of the preschool acellular pertussis (aP) booster dose at 4 years of age, T-cell memory responses were compared in children who were primed during infancy with either a whole-cell pertussis (wP) or an aP vaccine. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with pertussis vaccine antigens for 5 days. T cells were characterized by flow-based analysis of carboxyfluorescein succinimidyl ester (CFSE) dilution and CD4, CD3, CD45RA, CCR7, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) expression. Before the aP preschool booster vaccination, both the proliferated pertussis toxin (PT)-specific CD4+ and CD8+ T-cell fractions (CFSEdim) were higher in aP- than in wP-primed children. Post-booster vaccination, more pertussis-specific CD4+ effector memory cells (CD45RA− CCR7−) were induced in aP-primed children than in those primed with wP. The booster vaccination did not appear to significantly affect the T-cell memory subsets and functionality in aP-primed or wP-primed children. Although the percentages of Th1 cytokine-producing cells were alike in aP- and wP-primed children pre-booster vaccination, aP-primed children produced more Th1 cytokines due to higher numbers of proliferated pertussis-specific effector memory cells. At present, infant vaccinations with four aP vaccines in the first year of life result in pertussis-specific CD4+ and CD8+ effector memory T-cell responses that persist in children until 4 years of age and are higher than those in wP-primed children. The booster at 4 years of age is therefore questionable; this may be postponed to 6 years of age. PMID:25787136

  12. Antibiotics for whooping cough (pertussis).

    PubMed

    Altunaiji, S; Kukuruzovic, R; Curtis, N; Massie, J

    2007-07-18

    Whooping cough is a highly contagious disease. Infants are at highest risk of severe disease and death. Erythromycin for 14 days is currently recommended for treatment and contact prophylaxis, but is of uncertain benefit. To study the benefits and risks of antibiotic treatment of and contact prophylaxis against whooping cough. We searched the Cochrane Central Register of Controlled Trials (CENTRAL), the Database of Abstracts of Reviews of Effects (DARE) (The Cochrane Library Issue 1, 2007); MEDLINE (January 1966 to March 2007); EMBASE (January 1974 to March 2007). All randomised and quasi-randomised controlled trials of antibiotics for treatment of, and contact prophylaxis against, whooping cough. Three to four review authors independently extracted data and assessed the quality of each trial. Thirteen trials with 2197 participants met the inclusion criteria: 11 trials investigated treatment regimens; 2 investigated prophylaxis regimens. The quality of the trials was variable.Short-term antibiotics (azithromycin for three to five days, or clarithromycin or erythromycin for seven days) were as effective as long-term (erythromycin for 10 to 14 days) in eradicating Bordetella pertussis (B. pertussis) from the nasopharynx (relative risk (RR) 1.02, 95% confidence interval (CI) 0.98 to 1.05), but had fewer side effects (RR 0.66, 95% CI 0.52 to 0.83). Trimethoprim/sulfamethoxazole for seven days was also effective. Nor were there differences in clinical outcomes or microbiological relapse between short and long-term antibiotics. Contact prophylaxis of contacts older than six months of age with antibiotics did not significantly improve clinical symptoms or the number of cases developing culture-positive B. pertussis. Although antibiotics were effective in eliminating B. pertussis, they did not alter the subsequent clinical course of the illness. There is insufficient evidence to determine the benefit of prophylactic treatment of pertussis contacts.

  13. UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W, P22, P27 and P32.

    PubMed

    Bloch, Sylwia; Nejman-Faleńczyk, Bożena; Topka, Gracja; Dydecka, Aleksandra; Licznerska, Katarzyna; Narajczyk, Magdalena; Necel, Agnieszka; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-09-21

    Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m², their infectivity dropped by 1-3 log10, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells.

  14. UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W, P22, P27 and P32

    PubMed Central

    Bloch, Sylwia; Nejman-Faleńczyk, Bożena; Topka, Gracja; Dydecka, Aleksandra; Licznerska, Katarzyna; Narajczyk, Magdalena; Necel, Agnieszka; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-01-01

    Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m2, their infectivity dropped by 1–3 log10, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells. PMID:26402701

  15. Retrograde trafficking of AB₅ toxins: mechanisms to therapeutics.

    PubMed

    Mukhopadhyay, Somshuvra; Linstedt, Adam D

    2013-10-01

    Bacterial AB5 toxins are a clinically relevant class of exotoxins that include several well-known members such as Shiga, cholera, and pertussis toxins. Infections with toxin-producing bacteria cause devastating human diseases that affect millions of individuals each year and have no definitive medical treatment. The molecular targets of AB5 toxins reside in the cytosol of infected cells, and the toxins reach the cytosol by trafficking through the retrograde membrane transport pathway that avoids degradative late endosomes and lysosomes. Focusing on Shiga toxin as the archetype member, we review recent advances in understanding the molecular mechanisms involved in the retrograde trafficking of AB5 toxins and highlight how these basic science advances are leading to the development of a promising new therapeutic approach based on inhibiting toxin transport.

  16. Aptamer biosensor for sensitive detection of toxin A of Clostridium difficile using gold nanoparticles synthesized by Bacillus stearothermophilus.

    PubMed

    Luo, Peng; Liu, Yi; Xia, Yun; Xu, Huajian; Xie, Guoming

    2014-04-15

    A sensitive electrochemical biosensor was developed to detect toxin A (TOA) of Clostridium difficile based on an aptamer selected by the systematic evolution of ligands using exponential enrichment and gold nanoparticles (GNPS) synthesized by Bacillus stearothermophilus. The thiolated single-stranded DNA used as the capture probe (CP) was first self-assembled on a Nafion-thionine-GNPS-modified screen-printed electrode (SPE) through an Au-thiol interaction. The horseradish peroxidase (HRP)-labeled aptamer probe (AP) was then hybridized to the complementary oligonucleotide of CP to form an aptamer-DNA duplex. In the absence of TOA, the aptamer-DNA duplex modified the electrode surface with HRP, so that an amperometric response was induced based on the electrocatalytic properties of thionine. This was mediated by the electrons that were generated in the enzymatic reaction of hydrogen peroxide under HRP catalysis. After the specific recognition of TOA, an aptamer-TOA complex was produced rather than the aptamer-DNA duplex, forcing the HRP-labeled AP to dissociate from the electrode surface, which reduced the catalytic capacity of HRP and reduced the response current. The reduction in the response current correlated linearly with the concentration of TOA in the range of 0-200 ng/mL. The detection limit was shown to be 1 nM for TOA. This biosensor was applied to the analysis of TOA and showed good selectivity, reproducibility, stability, and accuracy.

  17. Toward a new vaccine for pertussis

    PubMed Central

    Robbins, John B.; Schneerson, Rachel; Kubler-Kielb, Joanna; Keith, Jerry M.; Trollfors, Birger; Vinogradov, Evgeny; Shiloach, Joseph

    2014-01-01

    To overcome the limitations of the current pertussis vaccines, those of limited duration of action and failure to induce direct killing of Bordetella pertussis, a synthetic scheme was devised for preparing a conjugate vaccine composed of the Bordetella bronchiseptica core oligosaccharide with one terminal trisaccharide to aminooxylated BSA via their terminal ketodeoxyoctanate residues. Conjugate-induced antibodies, by a fraction of an estimated human dose injected into young outbred mice as a saline solution, were bactericidal against B. pertussis, and their titers correlated with their ELISA values. The carrier protein is planned to be genetically altered pertussis toxoid. Such conjugates are easy to prepare, stable, and should add both to the level and duration of immunity induced by current vaccine-induced pertussis antibodies and reduce the circulation of B. pertussis. PMID:24556987

  18. Economic impact of the introduction of an acellular pertussis vaccine in Canada: a 6-year analysis.

    PubMed

    Iskedjian, Michael; De Serres, Gaston; Einarson, Thomas R; Walker, John H

    2010-01-08

    Between July 1997 and April 1998, Canadian public health agencies switched from the whole cell vaccine to the acellular vaccine for pertussis immunization. The acellular vaccine provided better efficacy and fewer adverse events than the whole cell vaccine did. To determine the economic impact of replacing the whole cell vaccine with an acellular vaccine in Canada. A decision analytic model was developed comparing costs and outcomes of pertussis vaccination for Canadian children born in the years 1991-2004. Effectiveness was measured as number of avoided pertussis cases as well as the number of avoided hospital admissions. Incremental costs per avoided pertussis case and per avoided hospital admission were calculated for Ministry of Health (MoH) and societal (SOC) perspectives. Various one-way sensitivity analyses as well as a Monte Carlo simulation were performed by varying key model parameters. The switch in immunization programs resulted in an incremental cost to the MoH of CAD $108 per pertussis case avoided (CAD $0.96 per child-year). From the SOC perspective, there was a savings of CAD $184 per pertussis case avoided (CAD $0.13 per child-year). The one-way sensitivity analyses provided incremental cost-effective ratios (ICERs) ranging from an incremental cost of CAD $1034 per avoided pertussis case from the MoH perspective to a saving of CAD $1583 per avoided case from the SOC perspective. The Monte Carlo simulation confirmed the robustness of these results. Pertussis vaccination with AcE was cost-saving from the societal perspective and cost-effective from the Ministry of Health perspective.

  19. Modelling the return on investment of preventively vaccinating healthcare workers against pertussis.

    PubMed

    Tariq, Luqman; Mangen, Marie-Josée J; Hövels, Anke; Frijstein, Gerard; de Boer, Hero

    2015-02-19

    Healthcare workers (HCWs) are at particular risk of acquiring pertussis and transmitting the infection to high-risk susceptible patients and colleagues. In this paper, the return on investment (ROI) of preventively vaccinating HCWs against pertussis to prevent nosocomial pertussis outbreaks is estimated using a hospital ward perspective, presuming an outbreak occurs once in 10 years. Data on the pertussis outbreak on the neonatology ward in 2004 in the Academic Medical Center Amsterdam (The Netherlands) was used to calculate control costs and other outbreak related costs. The study population was: neonatology ward staff members (n = 133), parents (n = 40), neonates (n = 20), and newborns transferred to other hospitals (n = 23). ROI is presented as the amount of Euros saved in averting outbreaks by investing one Euro in preventively vaccinating HCWs. Sensitivity analysis was performed to study the robustness of the ROI. Results are presented at 2012 price level. Total nosocomial pertussis outbreak costs were €48,682. Direct control costs (i.e. antibiotic therapy, laboratory investigation and outbreak management control) were €11,464. Other outbreak related costs (i.e. sick leave of HCWs; restrictions on the neonatology ward, savings due to reduced working force required) accounted for €37,218. Vaccination costs were estimated at €12,208. The ROI of preventively vaccinating HCWs against pertussis was 1:4, meaning 4 Euros could be saved by every Euro invested in vaccinating HCWs to avert outbreaks. ROI was sensitive to a lower vaccine price, considering direct control costs only, average length of stay of neonates on the neonatology ward, length of patient uptake restrictions, assuming no reduced work force due to ward closer and presuming more than one outbreak to occur in 10 years' time. From a hospital ward perspective, preventive vaccination of HCWs against pertussis to prevent nosocomial pertussis outbreaks results in a positive ROI, presuming an outbreak

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

    SciTech Connect

    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.

  1. Phylogenetic changes in sensitivity to Anemonia sulcata toxin (ATX II), and impact of first interaction with the toxin (imprinting) on later response to it.

    PubMed

    Csaba, G; Dobozy, O; Darvas, Z; László, V; Beress, L

    1984-01-01

    The Anemonia sulcata toxin ATX II is cardiotoxic and neurotoxic, and--at a high dose level--even lethal for the mouse, neurotoxic, but non-lethal for the frog, and has no adverse influence whatever on the Planaria and Tetrahymena; it even stimulates the growth of the Tetrahymena at a low dose level. It also induces imprinting in the Tetrahymena, as judged from the altered response of the latter to ATX II on re-exposure. No similar imprinting effect was demonstrable in mice.

  2. Diphtheria, Tetanus, and Pertussis (DTaP) Vaccine

    MedlinePlus

    Certiva® (as a combination product containing Diphtheria, Tetanus Toxoids, Acellular Pertussis Vaccine) ... Daptacel® (as a combination product containing Diphtheria, Tetanus Toxoids, Acellular Pertussis Vaccine)

  3. What Pertussis Mortality Rates Make Maternal Acellular Pertussis Immunization Cost-Effective in Low- and Middle-Income Countries? A Decision Analysis

    PubMed Central

    Russell, Louise B.; Pentakota, Sri Ram; Toscano, Cristiana Maria; Cosgriff, Ben; Sinha, Anushua

    2016-01-01

    Background. Despite longstanding infant vaccination programs in low- and middle-income countries (LMICs), pertussis continues to cause deaths in the youngest infants. A maternal monovalent acellular pertussis (aP) vaccine, in development, could prevent many of these deaths. We estimated infant pertussis mortality rates at which maternal vaccination would be a cost-effective use of public health resources in LMICs. Methods. We developed a decision model to evaluate the cost-effectiveness of maternal aP immunization plus routine infant vaccination vs routine infant vaccination alone in Bangladesh, Nigeria, and Brazil. For a range of maternal aP vaccine prices, one-way sensitivity analyses identified the infant pertussis mortality rates required to make maternal immunization cost-effective by alternative benchmarks ($100, 0.5 gross domestic product [GDP] per capita, and GDP per capita per disability-adjusted life-year [DALY]). Probabilistic sensitivity analysis provided uncertainty intervals for these mortality rates. Results. Infant pertussis mortality rates necessary to make maternal aP immunization cost-effective exceed the rates suggested by current evidence except at low vaccine prices and/or cost-effectiveness benchmarks at the high end of those considered in this report. For example, at a vaccine price of $0.50/dose, pertussis mortality would need to be 0.051 per 1000 infants in Bangladesh, and 0.018 per 1000 in Nigeria, to cost 0.5 per capita GDP per DALY. In Brazil, a middle-income country, at a vaccine price of $4/dose, infant pertussis mortality would need to be 0.043 per 1000 to cost 0.5 per capita GDP per DALY. Conclusions. For commonly used cost-effectiveness benchmarks, maternal aP immunization would be cost-effective in many LMICs only if the vaccine were offered at less than $1–$2/dose. PMID:27838677

  4. Differential Expression of Alpha 4 Integrins on Effector Memory T Helper Cells during Bordetella Infections. Delayed Responses in Bordetella pertussis

    PubMed Central

    Ferguson, Ryan; Tarlton, Nicole; Wu, Victoria; Sequeira, Christopher S.; Bremer, Martina; Abramson, Tzvia

    2012-01-01

    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. PMID:23300813

  5. [Pertussis--not only a childhood disease].

    PubMed

    Stock, Ingo

    2006-06-01

    Whouping cough (pertussis) is a highly contagious respiratory illness of humans that is caused predominantly by the gram-negative bacterial pathogen Bordetella pertussis. The disease shows a variety of symptoms, dependent on the patients' age and immunity. During the last years, the pertussis incidence has increased in several parts of the world. There has been a shift in the epidemiology of disease, with more cases reported in older age groups, specifically adolescents and adults. Macrolides are regarded to be the treatment of choice for the management of B. pertussis infections. Active immunization with whole cell and in particular acellular vaccines is the mainstay for prevention of disease.

  6. Bordetella pertussis pathogenesis: current and future challenges

    PubMed Central

    Melvin, Jeffrey A.; Scheller, Erich V.; Miller, Jeff F.; Cotter, Peggy A.

    2014-01-01

    Pertussis, or whooping cough, has recently reemerged as a major public health threat despite high levels of vaccination against the etiological agent, Bordetella pertussis. In this Review, we describe the pathogenesis of this disease, with a focus on recent mechanistic insights into virulence factor function. We also discuss the changing epidemiology of pertussis and the challenges of vaccine development. Despite decades of research, many aspects of B. pertussis physiology and pathogenesis remain poorly understood. We highlight knowledge gaps that must be addressed to develop improved vaccines and therapeutic strategies. PMID:24608338

  7. Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE

    PubMed Central

    Diago-Navarro, Elizabeth; Mora, Liliana; Buckingham, Richard H; Díaz-Orejas, Ramón; Lemonnier, Marc

    2008-01-01

    Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin–antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a ∼10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1. PMID:19019162

  8. In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

    PubMed Central

    Hong, Ka L.; Battistella, Luisa; Salva, Alysia D.; Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved. PMID:25633102

  9. Structure of Bordetella pertussis peptidoglycan

    SciTech Connect

    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.

  10. Zygocin, a secreted antifungal toxin of the yeast Zygosaccharomyces bailii, and its effect on sensitive fungal cells.

    PubMed

    Weiler, Frank; Schmitt, Manfred J

    2003-03-01

    Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.

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

    PubMed

    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.

  12. Anti-pertussis antibody kinetics following DTaP-IPV booster vaccination in Norwegian children 7-8 years of age.

    PubMed

    Aase, Audun; Herstad, Tove Karin; Jørgensen, Silje Bakken; Leegaard, Truls Michael; Berbers, Guy; Steinbakk, Martin; Aaberge, Ingeborg

    2014-10-14

    At the age of 7-8 years a booster of diphtheria, tetanus, acellular pertussis and polio vaccine is recommended for children in Norway. In this cross-sectional study we have analysed the antibody levels against pertussis vaccine antigens in sera from 498 children aged 6-12 years. The purposes of this study were to investigate the duration of the booster response against the pertussis vaccine antigens pertussis toxin (PT) and filamentous haemagglutinin (FHA); to determine the presence of high levels of pertussis antibodies in absence of recent vaccination; and to analyse how booster immunisation may interfere with the serological pertussis diagnostics. Prior to the booster the IgG antibody levels against PT revealed a geometric mean of 7.3IU/ml. After the booster the geometric mean peak anti-PT IgG response reached to 45.6IU/ml, followed by a steady decline in antibody levels over the next few years. The IgG anti-FHA levels followed the anti-PT IgG profiles. Three years after the booster the geometric mean IgG levels were only slightly above pre-booster levels. Prior to the booster 44% of the sera contained ≤5IU/ml of anti-PT IgG compared to18% 3 years after and 30% 4 years after the booster. When recently vaccinated children were excluded, 6.2% of the children had anti-PT IgG levels above 50IU/ml which may indicate pertussis infection within the last 2 years. This study indicates that the currently used acellular pertussis vaccines induce moderate immune responses to the pertussis antigens and that the antibodies wane within few years after the booster. This lack of sustained immune response may partly be responsible for the increased number of pertussis cases observed in this age group during the last years.

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

    PubMed

    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.

  14. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

    PubMed Central

    Martín, César; Etxaniz, Asier; Uribe, Kepa B.; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M.; Aréchaga, Juan; Ostolaza, Helena

    2015-01-01

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of “toxin-coated bacteria” proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or “free” in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. PMID:26346097

  15. Experience with monocomponent acellular pertussis combination vaccines for infants, children, adolescents and adults--a review of safety, immunogenicity, efficacy and effectiveness studies and 15 years of field experience.

    PubMed

    Thierry-Carstensen, Birgit; Dalby, Tine; Stevner, Michael A; Robbins, John B; Schneerson, Rachel; Trollfors, Birger

    2013-10-25

    Combination vaccines containing a monocomponent acellular pertussis (aP) vaccine, manufactured at Statens Serum Institut (SSI), Denmark, have successfully controlled Bordetella pertussis infections in Denmark since 1997. The efficacy of this aP vaccine was 71% in a double-blind, randomised and controlled clinical trial. Its safety and immunogenicity have been demonstrated in infants, children, adolescents and adults. In approximately 500,000 children it was effective against pertussis requiring hospitalisation (VE: 93% after 3 doses) and against pertussis not requiring hospitalisation (VE: 78% after 3 doses). IgG antibodies against pertussis toxin (IgG anti-PT) response rates after booster vaccination of adults with tetanus, diphtheria and aP combination vaccine (TdaP) were considerably higher for this monocomponent aP vaccine containing 20μg pertussis toxoid, inactivated by hydrogen peroxide (92.0%), than for two multicomponent aP vaccines inactivated by formaldehyde and/or glutaraldehyde: 3-component aP with 8μg pertussis toxoid (77.2%) and 5-component aP with 2.5μg pertussis toxoid (47.1%), without compromising the safety profile. In Denmark where this monocomponent aP vaccine has been the only pertussis vaccine in use for 15 years, there has been no pertussis epidemic since 2002 (population incidence 36 per 100,000), in contrast to neighbouring countries, where epidemics have occurred. This monocomponent aP vaccine can be used in combination vaccines for primary and booster vaccination against pertussis in all age groups and is an important tool for successful pertussis control. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Tdap (tetanus, diphtheria, pertussis) Vaccine and Pregnancy

    MedlinePlus

    ... Canada, and many other countries. Pertussis (also called whooping cough) is a bacterial illness that usually begins with symptoms like those of the common cold. Severe coughing can develop over several weeks. Fast, heavy ... sound when breathing in. Pertussis is most serious ...

  17. Estimating Pertussis Susceptibility Among 0-23-Month-Old Children in the United States: Using National Immunization Survey (NIS) 2013.

    PubMed

    Childs, Lana; Bednarczyk, Robert A

    2017-08-01

    Despite high pertussis-containing vaccine coverage in the United States, children who are unvaccinated or not fully vaccinated remain susceptible to pertussis. Over multiple birth cohorts of incomplete vaccination, the number of children not immune to pertussis will accumulate because of factors such as age-specific vaccination status and dose-specific vaccine effectiveness. The total number of pertussis-susceptible children 0-23 months of age in the United States is unknown. Using data on age-specific pertussis-containing vaccine receipt among children evaluated in the 2013 National Immunization Survey (born between February 2011 and June 2012) and accounting for vaccine effectiveness and maternal transfer of antipertussis antibodies, we estimated the cumulative number of pertussis-susceptible children 0-23 months of age. Of an estimated 7,905,672 children 0-23 months of age in the United States, we estimated that approximately 22% (1,716,429) are susceptible to pertussis. Age was a large factor in susceptibility, with 89% of children less than 2 months of age not immune to pertussis compared with 7% of children 21-23 months of age. In sensitivity analysis, increasing maternal pertussis vaccination coverage from 10% to 42% decreased susceptibility in children less than 2 months of age to 68%. When considering waning immunity after the fourth dose of vaccine, the herd protection threshold was no longer reached. These estimates underscore the need to monitor age-specific pertussis vaccine coverage, to increase childhood and maternal pertussis vaccine coverage, to maintain population-level immunity and to prevent the spread of pertussis among young children.

  18. Monoclonal antibodies and toxins--a perspective on function and isotype.

    PubMed

    Chow, Siu-Kei; Casadevall, Arturo

    2012-06-01

    Antibody therapy remains the only effective treatment for toxin-mediated diseases. The development of hybridoma technology has allowed the isolation of monoclonal antibodies (mAbs) with high specificity and defined properties, and numerous mAbs have been purified and characterized for their protective efficacy against different toxins. This review summarizes the mAb studies for 6 toxins--Shiga toxin, pertussis toxin, anthrax toxin, ricin toxin, botulinum toxin, and Staphylococcal enterotoxin B (SEB)--and analyzes the prevalence of mAb functions and their isotypes. Here we show that most toxin-binding mAbs resulted from immunization are non-protective and that mAbs with potential therapeutic use are preferably characterized. Various common practices and caveats of protection studies are discussed, with the goal of providing insights for the design of future research on antibody-toxin interactions.

  19. Is Pertussis Infection Neglected in China? Evidence from a Seroepidemiology Survey in Zhejiang, an Eastern Province of China.

    PubMed

    He, Hanqing; Yao, Pingping; Zhou, Yang; Deng, Xuan; Pan, Jinren

    2016-01-01

    The resurgence of pertussis has occurred in many countries. However, the epidemiological profiles of pertussis cannot be well understood by the current surveillance system in China. This study was designed to investigate the age specific serologic evidence of antibodies against pertussis, and to offer information regarding the existence of pertussis infection in Zhejiang Province, China. A cross-sectional serosurvey was carried out in 6 counties of Zhejiang Province during September and October of 2014. The immunoglobulin G-pertussis toxin (IgG-PT) levels were measured quantitatively with a commercially available enzyme-linked immunosorbent assay (ELISA). The antibody activities were expressed in the Food and Drug Administration (FDA)-U/ml and a level ≥30 FDA-U/ml was considered seropositive. An IgG-PT >80 FDA-U/ml indicated recent pertussis infection if the patient had not received immunization with the pertussis vaccine within the last year. The mean IgG-PT seropositivity rate among the 2107 subjects was 33.32% with a geometric mean concentration of 17.73 (95% confidence interval: 16.90-18.60) FDA-U/ml. The difference in the seropositivity rates reached significant means among the different age groups (waldχ2 = 198.41, P<0.0005), and children aged 3 years had the highest percentage (63.24%) of undetectable IgG-PT level. Of the 1707 subjects ≥3 years of age, 169 (9.90%) had evidence of a recent infection. The highest proportion of IgG-PT levels ≥80 FDA-U/ml was found in ≥60 years age group followed by 11-15 and 16-25 years age groups. This study indicates the rather lower IgG-PT level sustained 1 year after the acellular pertussis vaccine booster dose, and substantial proportion of population susceptibility to pertussis in Zhejiang Province, China. Moreover, pertussis infection is not uncommon; it was estimated that 10% of subjects were recently infected approximately within the last 100 days. We highly suggest that the surveillance capacity should be

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

    PubMed

    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).

  1. Immunization of teenagers with a fifth dose of reduced DTaP-IPV induces high levels of pertussis antibodies with a significant increase in opsonophagocytic activity.

    PubMed

    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 < 0.001). The prebooster sera showed a geometric mean OPA titer of 65.1 and IgG binding to live bacteria at a geometric mean concentration of 164.9 arbitrary units (AU)/ml. Following the fifth dose, the OPA increased to a titer of 360.4, and the IgG concentration against live bacteria increased to 833.4 AU/ml (P < 0.001 for both). The correlation analyses between the different assays suggest that antibodies against FHA and PRN contribute the most to the OPA and IgG binding.

  2. [Pertussis (Whooping cough)--an update].

    PubMed

    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.

  3. Sensitive and specific in-situ myoneural junction model to study the pharmacological mechanisms of agents and toxins

    SciTech Connect

    Von Bredow, J.; Lowensohn, H.; Adams, N.

    1993-05-13

    Many biological warfare agents simultaneously intoxicate vital central and peripheral organ systems leading to death of the exposed victim from multiple unexplainable causes. The lethal effect of exposure to biological toxins is most often associated with (but not exclusively limited to) neuromuscular paralysis which contributes to death due to respiratory cessation. The systemic administration of the toxin often involves many different systems simultaneously leading to a confusing evaluation of the effect of the toxin and an erroneous assessment of the effectiveness of pretreatments or antidotes. In order to correct for these ambiguous results an in-situ animal model has been modified to more accurately characterize the toxic effect of biological agents on a physiologically functioning gastrocnemius muscle preparation. In the improved animal model both sciatic nerves of phenobarbital anesthetized rabbits are isolated for electrical stimulation of the corresponding gastrocnemius muscles. Toxin and drug solutions are administered directly to the gastrocnemius muscle mass of the experimental leg by intraarterial injections to the femoral artery through a catheterized arterial branch of the femoral artery. The administration of minute quantities of drug or toxin solutions directly to the gastrocnemius muscle mass without contaminating the whole animal (systemic circulation or the central nervous system) assures a normal circulation and no interference from other physiological systems.

  4. Experimental evolution of defense against a competitive mold confers reduced sensitivity to fungal toxins but no increased resistance in Drosophila larvae.

    PubMed

    Trienens, Monika; Rohlfs, Marko

    2011-07-14

    Fungal secondary metabolites have been suggested to function as chemical defenses against insect antagonists, i.e. predators and competitors. Because insects and fungi often compete for dead organic material, insects may achieve protection against fungi by reducing sensitivity to fungal chemicals. This, in turn, may lead to increased resistance allowing insects better to suppress the spread of antagonistic but non-pathogenic microbes in their habitat. However, it remains controversial whether fungal toxins serve as a chemical shield that selects for insects that are less sensitive to toxins, and hence favors the evolution of insect resistance against microbial competitors. To examine the relationship between the ability to survive competition with toxic fungi, sensitivity to fungal toxins and resistance, we created fungal-selected (FS) replicated insect lines by exposing Drosophila melanogaster larvae to the fungal competitor Aspergillus nidulans over 26 insect generations. Compared to unselected control lines (UC), larvae from the FS lines had higher survival rates in the presence of A. nidulans indicating selection for increased protection against the fungal antagonist. In line with our expectation, FS lines were less susceptible to the A. nidulans mycotoxin Sterigmatocystin. Of particular interest is that evolved protection against A. nidulans and Sterigmatocytin was not correlated with increased insect survival in the presence of other fungi and mycotoxins. We found no evidence that FS lines were better at suppressing the expansion of fungal colonies but observed a trend towards a less detrimental effect of FS larvae on fungal growth. Antagonistic but non-pathogenic fungi favor insect variants better protected against the fungal chemical arsenal. This highlights the often proposed but experimentally underexplored importance of secondary metabolites in driving animal-fungus interactions. Instead of enhanced resistance, insect larvae tend to have evolved

  5. [Features of Bordetella pertussis, Bordetella spp. infection and whopping cough in Córdoba province, Argentina].

    PubMed

    Giayetto, Víctor O; Blanco, Sebastián; Mangeaud, Arnaldo; Barbás, María G; Cudolá, Analía; Gallego, Sandra V

    2017-04-01

    Whooping cough is a re-emerging infection in the world and Latin America. It was considered relevant to investigate the clinical and epidemiological profile of Bordetella spp. and Bordetella pertussis infection in Córdoba province, Argentina; evaluating, at the same time, the co-infection with virus producing respiratory infections that may be confused with whooping cough. All whooping cough suspected cases were studied by Polimerase Chain Reaction, amplifying the repeated insertion sequence (IS) 481 and the promoter gene encoding pertussis toxin, between 2011 and 2013. The data were obtained from the clinical and epidemiological records. From 2,588 whooping cough suspected cases, 11.59% was infected by Bordetella spp. and 9.16% was confirmed as Bordetella pertussis infection. The rate of infection was 7.22 and 1.84 per 100,000 for 2011 and 2012, respectively. The infection presented a seasonal tendency and it was mainly found on the group of children between 13 and 24 months old. The co-infection with virus producing respiratory infections, were uncommon. Paroxysmal cough, cyanosis and/or vomiting were predictors of the infection for Bordetella pertussis. To deal with the re-emergence of whooping cough is important the knowledge of the regional epidemiological situation. This paper shows the situation of these infections in the regional clinical and epidemiological context, and makes the information available for health decision-making.

  6. Investigation of cellular and humoral immune responses to whole cell and acellular pertussis vaccines.

    PubMed

    Canthaboo, C; Williams, L; Xing, D K; Corbel, M J

    2000-11-08

    New generation acellular pertussis vaccines were compared with the established whole cell pertussis vaccine for the induction of humoral and cellular immune-responses in mice. At the same time, the in vivo protective effect of these two types of vaccine was also compared in both intracerebral (ic) and aerosol challenge models. In general, whole cell vaccine induced lower antibody titres to pertussis toxin, filamentous haemagglutinin and pertactin than the acellular vaccine. Nitric oxide concentration in macrophage cultures was used as a marker for macrophage activation. The nitric oxide concentrations in the macrophage cultures from mice following immunisation with the whole cell vaccine were higher than those from mice immunised with the acellular vaccine, which indicated that the whole cell vaccine was more effective than the acellular vaccine in activating macrophages. This was associated with better protection in vivo after challenge. After ic challenge of mice following immunisation with whole cell or acellular vaccine, 90% of the whole cell vaccine group survived compared with 40% of the acellular vaccine group at the vaccine dose selected. Following aerosol challenge, mice in the whole cell vaccine group showed faster clearance of bacteria from the lungs than those in the acellular vaccine group. Our findings suggest that the different types of pertussis vaccines may achieve protection in different ways and that CMI may play an important role in eliminating bacteria which escape humoral defence mechanisms.

  7. A novel method for evaluating natural and vaccine induced serological responses to Bordetella pertussis antigens.

    PubMed

    Berbers, G A M; van de Wetering, M S E; van Gageldonk, P G M; Schellekens, J F P; Versteegh, F G A; Teunis, P F M

    2013-08-12

    We studied the time course of serum IgG antibodies against 3 different pertussis vaccine antigens: PT (pertussis toxin), FHA (filamentous hemagglutinin), Prn (pertactin) in sera from individuals vaccinated with four different pertussis vaccines at 4 years of age: (N=44, 44, 23 and 23, respectively,) and compared the responses to/after natural infection with Bordetella pertussis (N=44, age 1-8 years). These longitudinal data were analyzed with a novel method, using a mathematical model to describe the observed responses, and their variation among subjects. This allowed us to estimate biologically meaningful characteristics of the serum antibody response, like peak level and decay rate, and to compare these among natural infections and vaccine responses. Compared to natural infection, responses to PT after vaccination with the tested vaccines are smaller in magnitude and tend to decay slightly faster. When present in vaccines, FHA and Prn tend to produce high peak levels, higher than those in naturally infected patients, but these decay faster. As expected, the Dutch whole cell vaccine produced lower antibody responses than the acellular vaccines. This model allows a better comparison of the kinetics of vaccine induced antibody responses and after natural infection over a long follow up period.

  8. Seroprevalence of antibodies to pertussis and diphtheria among healthy adults in China.

    PubMed

    Zhang, Qi; Han, Fulian; Nie, Qing; Ren, Hongyu; Zhang, Baoqiang; Liu, Qiyong; He, Qiushui; Shao, Zhujun

    2011-12-01

    Despite extensive childhood immunization, pertussis remains one of the world's leading causes of vaccine-preventable deaths. Incidence of pertussis in adolescents and adults has increased in many countries despite high vaccination coverage. In China, booster vaccinations against diphtheria, tetanus and pertussis are not used in adults, and little is known about pertussis incidence in the age group. The aim of this study was to determine seroprevalence of IgG antibodies to pertussis toxin (PT) and diphtheria among adults in China. Blood samples were obtained from 210 healthy adults aged 18-50 years in Weifang city, China during the period of May and June 2010. Serum IgG antibodies against PT (anti-PT IgG) and diphtheria were determined by the commercial ELISA kits, respectively. According to the kit, concentration of anti-PT IgG higher than 30 IU/mL was considered positive. An antibody concentration of ≥ 0.1 IU/mL was defined as evidence of seroprotection against diphtheria. The mean concentration of anti-PT IgG antibodies was 9.95 IU/mL (95% confidence interval (CI) 8.45-11.44). Eleven (5.24%) of the studied subjects were proved to be seropositive to pertussis. Of the 210 subjects, 161 (76.6%) had anti-diphtheria antibody concentration ≥ 0.1 IU/mL and 49 (23.3%) had the antibody concentration between 0.01 and 0.099 IU/mL. Our study indicated that about 5% of adults aged 18-50 years had positive anti-PT IgG antibodies, suggesting that adult pertussis is not uncommon in China. Although a high proportion of studied subjects had a protective level of immunity against diphtheria, the antibody level decreased with the increasing age of adults. Booster vaccinations against pertussis should be considered in adults in China. Copyright © 2011 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  9. Improved specificity of a multiplex immunoassay for quantitation of anti-diphtheria toxin antibodies with the use of diphtheria toxoid.

    PubMed

    van Gageldonk, Pieter G M; von Hunolstein, Christina; van der Klis, Fiona R M; Berbers, Guy A M

    2011-07-01

    A nonspecific binding of antibodies to diphtheria toxin, especially in adult serum samples, was observed in our diphtheria-tetanus-pertussis multiplex immunoassay (DTaP4 MIA). This can be significantly reduced by the use of diphtheria toxoid, achieving a good correlation with the Vero cell neutralization test and the toxin binding inhibition assay.

  10. Characterization of Human Bactericidal Antibodies to Bordetella pertussis

    PubMed Central

    Weiss, Alison A.; Mobberley, Paula S.; Fernandez, Rachel C.; Mink, ChrisAnna M.

    1999-01-01

    The Bordetella pertussis BrkA protein protects against the bactericidal activity of complement and antibody; however, some individuals mount an immune response that overcomes this bacterial defense. To further characterize this process, the bactericidal activities of sera from 13 adults with different modes of exposure to B. pertussis (infected as adults, occupational exposure, immunized with an acellular vaccine, or no identified exposure) against a wild-type strain and a BrkA complement-sensitive mutant were evaluated. All of the sera killed the BrkA mutant, suggesting past exposure to B. pertussis or cross-reactive organisms. Several samples had no or minimal activity against the wild type. All of the sera collected from the infected and occupationally exposed individuals but not all of the sera from vaccinated individuals had bactericidal activity against the wild-type strain, suggesting that some types of exposure can induce an immune response that can overcome the BrkA resistance mechanism. Adsorbing serum with the wild-type strain removed the bactericidal antibodies; however, adsorbing the serum with a lipopolysaccharide (LPS) mutant or an avirulent (bvg mutant) strain did not always result in loss of bactericidal activity, suggesting that antibodies to either LPS or bvg-regulated proteins could be bactericidal. All the samples, including those that lacked bactericidal activity, contained antibodies that recognized the LPS of B. pertussis. Bactericidal activity correlated best with the presence of the immunoglobulin G3 (IgG3) antibodies to LPS, the IgG subtype that is most effective at fixing complement. PMID:10024590

  11. Development of monoclonal antibodies and immunoassays for sensitive and specific detection of Shiga Toxin Stx2f

    USDA-ARS?s Scientific Manuscript database

    Background: Shiga-like toxin 2 (Stx2) is a major virulence factor in gastrointestinal diseases caused by Eschericia coli. Although Stx2a (prototypical Stx2) is well-studied, all seven subtypes of Stx2 have been associated with disease in mammals. Several subtypes of Stx2, including Stx2f, are diffi...

  12. How well do ICD-9 physician claim diagnostic codes identify confirmed pertussis cases in Alberta, Canada? A Canadian Immunization Research Network (CIRN) Study.

    PubMed

    Fathima, Sumana; Simmonds, Kimberley A; Drews, Steven J; Svenson, Lawrence W; Kwong, Jeffrey C; Mahmud, Salaheddin M; Quach, Susan; Johnson, Caitlin; Schwartz, Kevin L; Crowcroft, Natasha S; Russell, Margaret L

    2017-07-12

    Rates of Bordetella pertussis have been increasing in Alberta, Canada despite vaccination programs. Waning immunity from existing acellular component vaccines may be contributing to this. Vaccine effectiveness can be estimated using a variety of data sources including diagnostic codes from physician billing claims, public health records, reportable disease and laboratory databases. We sought to determine if diagnostic codes from billing claims (administrative data) are adequately sensitive and specific to identify pertussis cases among patients who had undergone disease-specific laboratory testing. Data were extracted for 2004-2014 from a public health communicable disease database that contained data on patients under investigation for B. pertussis (both those who had laboratory tests and those who were epidemiologically linked to laboratory-confirmed cases) in Alberta, Canada. These were deterministically linked using a unique lifetime person identifier to the provincial billing claims database, which contains International Classification of Disease version 9 (ICD-9) diagnostic codes for physician visits. We examined visits within 90 days of laboratory testing. ICD-9 codes 033 (whooping cough), 033.0 (Bordetella pertussis), 033.1 (B. parapertussis), 033.8 (whooping cough, other specified organism), and 033.9 (whooping cough, other unspecified organism) in any of the three diagnostic fields for a claim were classified as being pertussis-specific codes. We calculated sensitivity, specificity, positive (PPV) and negative (NPV) predictive values. We identified 22,883 unique patients under investigation for B. pertussis. Of these, 22,095 underwent laboratory testing. Among those who had a laboratory test, 2360 tested positive for pertussis. The sensitivity of a pertussis-specific ICD-9 code for identifying a laboratory-confirmed case was 38.6%, specificity was 76.9%, PPV was 16.0%, and NPV was 91.6%. ICD-9 codes from physician billing claims data have low sensitivity

  13. Whooping cough in school age children presenting with persistent cough in UK primary care after introduction of the preschool pertussis booster vaccination: prospective cohort study

    PubMed Central

    Fry, Norman K; Campbell, Helen; Amirthalingam, Gayatri; Harrison, Timothy G; Mant, David; Harnden, Anthony

    2014-01-01

    Objective To estimate the prevalence and clinical severity of whooping cough (pertussis) in school age children presenting with persistent cough in primary care since the introduction and implementation of the preschool pertussis booster vaccination. Design Prospective cohort study (November 2010 to December 2012). Setting General practices in Thames Valley, UK. Participants 279 children aged 5 to 15 years who presented in primary care with a persistent cough of two to eight weeks’ duration. Exclusion criteria were cough likely to be caused by a serious underlying medical condition, known immunodeficiency or immunocompromise, participation in another clinical research study, and preschool pertussis booster vaccination received less than one year previously. Main outcome measures Evidence of recent pertussis infection based on an oral fluid anti-pertussis toxin IgG titre of at least 70 arbitrary units. Cough frequency was measured in six children with laboratory confirmed pertussis. Results 56 (20%, 95% confidence interval 16% to 25%) children had evidence of recent pertussis infection, including 39 (18%, 13% to 24%) of 215 children who had been fully vaccinated. The risk of pertussis was more than three times higher (21/53; 40%, 26% to 54%) in children who had received the preschool pertussis booster vaccination seven years or more previously than in those who had received it less than seven years previously (20/171; 12%, 7% to 17%). The risk of pertussis was similar between children who received five and three component preschool pertussis booster vaccines (risk ratio for five component vaccine 1.14, 0.64 to 2.03). Four of six children in whom cough frequency was measured coughed more than 400 times in 24 hours. Conclusions Pertussis can still be found in a fifth of school age children who present in primary care with persistent cough and can cause clinically significant cough in fully vaccinated children. These findings will help to inform consideration of the

  14. Excitability parameters and sensitivity to anemone toxin ATX-II in rat small diameter primary sensory neurones discriminated by Griffonia simplicifolia isolectin IB4.

    PubMed

    Snape, Alistair; Pittaway, James F; Baker, Mark D

    2010-01-01

    Sensory neurone subtypes (< or = 25 microm apparent diameter) express a variety of Na(+) channels, where expression is linked to action potential duration, and associated with differential IB4-lectin binding. We hypothesized that sensitivity to ATX-II might also discriminate neurones and report that 1 microm has negligible or small effects on action potentials in IB4 +ve, but dramatically increased action potential duration in IB4 ve, neurones. The toxin did not act on tetrodotoxin-resistant (TTX-r) Na(V)1.8 currents; discrimination was based on tetrodotoxin-sensitive (TTX-s) Na(+) channel expression. We also explored the effects of varying the holding potential on current threshold, and the effect of repetitive activation on action currents in IB4 +ve and ve neurones. IB4 +ve neurones became more excitable with depolarization over the range 100 to 20 mV, but IB4 ve neurones exhibited peak excitability near 55 mV, and were inexcitable at 20 mV. Eliciting action potentials at 2 Hz, we found that peak inward action current in IB4 +ve neurones was reduced, whereas changes in the current amplitude were negligible in most IB4 ve neurones. Our findings are consistent with relatively toxin-insensitive channels including Na(V)1.7 being expressed in IB4 +ve neurones, whereas toxin sensitivity indicates that IB4 ve neurones may express Na(V)1.1 or Na(V)1.2, or both. The retention of excitability at low membrane potentials, and the responses to repetitive stimulation are explained by the known preferential expression of Na(V)1.8 in IB4 +ve neurones, and the reduction in action current in IB4 +ve neurones with repetitive stimulation supports a novel hypothesis explaining the slowing of conduction velocity in C-fibres by the build-up of Na(+) channel inactivation.

  15. Influence of maternal vaccination against diphtheria, tetanus, and pertussis on the avidity of infant antibody responses to a pertussis containing vaccine in Belgium.

    PubMed

    Caboré, Raïssa Nadège; Maertens, Kirsten; Dobly, Alexandre; Leuridan, Elke; Van Damme, Pierre; Huygen, Kris

    2017-02-22

    Maternal antibodies induced by vaccination during pregnancy cross the placental barrier and can close the susceptibility gap to pertussis in young infants up to the start of primary immunization. As not only the quantity but also the quality of circulating antibodies is important for protection, we assessed whether maternal immunization affects the avidity of infant vaccine-induced IgG antibodies, in the frame of a prospective clinical trial on pregnancy vaccination in Belgium. Infants born from Tdap (Boostrix®) vaccinated (N = 55) and unvaccinated (N = 26) mothers were immunized with a hexavalent pertussis containing vaccine (Infanrix Hexa®) at 8, 12 and 16 weeks, followed by a f