Sample records for francisella tularensis genes

  1. Identifying Francisella tularensis genes required for growth in host cells

    USDA-ARS?s Scientific Manuscript database

    Technical Abstract: Francisella tularensis is a highly virulent Gram negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cel...

  2. Virulent Type A Francisella tularensis actively suppresses cytokine responses in human monocytes

    PubMed Central

    Gillette, Devyn D.; Curry, Heather M.; Cremer, Thomas; Ravneberg, David; Fatehchand, Kavin; Shah, Prexy A.; Wewers, Mark D.; Schlesinger, Larry S.; Butchar, Jonathan P.; Tridandapani, Susheela; Gavrilin, Mikhail A.

    2014-01-01

    Background: Human monocyte inflammatory responses differ between virulent and attenuated Francisella infection. Results: A mixed infection model showed that the virulent F. tularensis Schu S4 can attenuate inflammatory cytokine responses to the less virulent F. novicida in human monocytes. Conclusion: F. tularensis dampens inflammatory response by an active process. Significance: This suppression may contribute to enhanced pathogenicity of F. tularensis. Francisella tularensis is a Gram-negative facultative bacterium that can cause the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. Previous work has shown that monocytes infected with highly virulent F. tularensis subsp. tularensis strain Schu S4 responded with a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes and cytokine production in comparison to those infected with the less virulent related F. novicida. However, it has been unclear whether the virulent Schu S4 was merely evading or actively suppressing monocyte responses. By using mixed infection assays with F. tularensis and F. novicida, we show that F. tularensis actively suppresses monocyte pro-inflammatory responses. Additional experiments show that this suppression occurs in a dose-dependent manner and is dependent upon the viability of F. tularensis. Importantly, F. tularensis was able to suppress pro-inflammatory responses to earlier infections with F. novicida. These results lend support that F. tularensis actively dampens human monocyte responses and this likely contributes to its enhanced pathogenicity. PMID:24783062

  3. Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.

    PubMed

    Le Pihive, E; Blaha, D; Chenavas, S; Thibault, F; Vidal, D; Valade, E

    2009-11-01

    Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.

  4. Francisella tularensis: an arthropod-borne pathogen

    PubMed Central

    Petersen, Jeannine M.; Mead, Paul S.; Schriefer, Martin E.

    2009-01-01

    Arthropod transmission of tularemia occurs throughout the northern hemisphere. Few pathogens show the adaptability of Francisella tularensis to such a wide array of arthropod vectors. Nonetheless, arthropod transmission of F. tularensis was last actively investigated in the first half of the 20th century. This review will focus on arthropod transmission to humans with respect to vector species, modes of transmission, geographic differences and F. tularensis subspecies and clades. PMID:18950590

  5. Systems approach to investigating host-pathogen interactions in infections with the biothreat agent Francisella. Constraints-based model of Francisella tularensis

    PubMed Central

    2010-01-01

    Background Francisella tularensis is a prototypic example of a pathogen for which few experimental datasets exist, but for which copious high-throughout data are becoming available because of its re-emerging significance as biothreat agent. The virulence of Francisella tularensis depends on its growth capabilities within a defined environmental niche of the host cell. Results We reconstructed the metabolism of Francisella as a stoichiometric matrix. This systems biology approach demonstrated that changes in carbohydrate utilization and amino acid metabolism play a pivotal role in growth, acid resistance, and energy homeostasis during infection with Francisella. We also show how varying the expression of certain metabolic genes in different environments efficiently controls the metabolic capacity of F. tularensis. Selective gene-expression analysis showed modulation of sugar catabolism by switching from oxidative metabolism (TCA cycle) in the initial stages of infection to fatty acid oxidation and gluconeogenesis later on. Computational analysis with constraints derived from experimental data revealed a limited set of metabolic genes that are operational during infection. Conclusions This integrated systems approach provides an important tool to understand the pathogenesis of an ill-characterized biothreat agent and to identify potential novel drug targets when rapid target identification is required should such microbes be intentionally released or become epidemic. PMID:20731870

  6. Uncovering the components of the Francisella tularensis virulence stealth strategy

    PubMed Central

    Jones, Bradley D.; Faron, Matthew; Rasmussen, Jed A.; Fletcher, Joshua R.

    2014-01-01

    Over the last decade, studies on the virulence of the highly pathogenic intracellular bacterial pathogen Francisella tularensis have increased dramatically. The organism produces an inert LPS, a capsule, escapes the phagosome to grow in the cytosol (FPI genes mediate phagosomal escape) of a variety of host cell types that include epithelial, endothelial, dendritic, macrophage, and neutrophil. This review focuses on the work that has identified and characterized individual virulence factors of this organism and we hope to highlight how these factors collectively function to produce the pathogenic strategy of this pathogen. In addition, several recent studies have been published characterizing F. tularensis mutants that induce host immune responses not observed in wild type F. tularensis strains that can induce protection against challenge with virulent F. tularensis. As more detailed studies with attenuated strains are performed, it will be possible to see how host models develop acquired immunity to Francisella. Collectively, detailed insights into the mechanisms of virulence of this pathogen are emerging that will allow the design of anti-infective strategies. PMID:24639953

  7. Francisella tularensis Molecular Typing Using Differential Insertion Sequence Amplification

    DTIC Science & Technology

    2011-08-01

    16 May 2011 Tularemia is a potentially fatal disease that is caused by the highly infectious and zoonotic pathogen Francisella tularensis. Despite...and characterizations of tularemia source outbreaks. Francisella tularensis is a facultative intracellular bacterium and the causative agent of the...zoonotic disease tularemia ( 10). This Gram-negative microbe is highly infectious, with as few as 10 organisms being capable of causing disease in

  8. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M [Tracy, CA; Radnedge, Lyndsay [San Mateo, CA; Andersen, Gary L [Berkeley, CA; Ott, Linda L [Livermore, CA; Slezak, Thomas R [Livermore, CA; Kuczmarski, Thomas A [Livermore, CA; Vitalis, Elizabeth A [Livermore, CA

    2007-02-06

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  9. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M [Tracy, CA; Radnedge, Lyndsay [San Mateo, CA; Andersen, Gary L [Berkeley, CA; Ott, Linda L [Livermore, CA; Slezak, Thomas R [Livermore, CA; Kuczmarski, Thomas A [Livermore, CA; Vitalis, Elizabeth A [Livermore, CA

    2009-02-24

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  10. Repression of inflammasome by Francisella tularensis during early stages of infection.

    PubMed

    Dotson, Rachel J; Rabadi, Seham M; Westcott, Elizabeth L; Bradley, Stephen; Catlett, Sally V; Banik, Sukalyani; Harton, Jonathan A; Bakshi, Chandra Shekhar; Malik, Meenakshi

    2013-08-16

    Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1β and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.

  11. Repression of Inflammasome by Francisella tularensis during Early Stages of Infection*

    PubMed Central

    Dotson, Rachel J.; Rabadi, Seham M.; Westcott, Elizabeth L.; Bradley, Stephen; Catlett, Sally V.; Banik, Sukalyani; Harton, Jonathan A.; Bakshi, Chandra Shekhar; Malik, Meenakshi

    2013-01-01

    Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1β and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect. PMID:23821549

  12. Presence of an emerging subclone of Francisella tularensis holarctica in Ixodes ricinus ticks from south-western Germany.

    PubMed

    Gehringer, Heike; Schacht, Erik; Maylaender, Nicole; Zeman, Ella; Kaysser, Philipp; Oehme, Rainer; Pluta, Silvia; Splettstoesser, Wolf D

    2013-02-01

    The zoonotic disease tularaemia is caused by the bacterial pathogen Francisella tularensis. Although the causative agent is known for 100 years, knowledge of its enzootic cycles is still rudimentary. Apart from tabanids and mosquitoes, hard ticks have been described as important vectors and potential reservoirs for F. tularensis. Available data on the incidence of human tularaemia indicate an increase in cases in the federal state of Baden-Wuerttemberg. To determine whether ticks are involved in the reported increase in F. tularensis infections in humans and wildlife in this south-western part of Germany, 916 Ixodes ricinus and 211 adult Dermacentor marginatus and D. reticulatus ticks were collected in two different locations. Screening for the presence of F. tularensis was performed by real-time PCR of the 16S rRNA gene. Of the 95 pools of I. ricinus ticks (representing 916 individual ticks), 8 tick pools (8.4%) were positive in this PCR. 30-bp deletion PCR confirmed that the F. tularensis subspecies holarctica was present. FtM24 VNTR analysis revealed that they belong to the emerging Franco-Iberian subclone group of F. tularensis holarctica. Of the 211 ticks of the genus Dermacentor, 35 randomly chosen DNAs were subjected to 16S rRNA gene screening PCR; 20 of these (57%) gave positive signals. For cluster analysis, the lpnA gene region of all Francisella-positive I. ricinus pools and 6 Dermacentor ticks with a positive reaction in the screening PCR was amplified and sequenced. In the resulting neighbour-joining tree, all Francisella-positive I. ricinus samples clustered with sequences of F. tularensis, whilst all Dermacentor tick samples clustered with FLE (Francisella-like endosymbiont) sequences. This study shows that I. ricinus ticks may serve as vectors and/or reservoirs of F. tularensis in Germany and supports the hypothesis that the state of Baden-Wuerttemberg represents an emerging endemic focus of tularaemia. Copyright © 2012 Elsevier GmbH. All rights

  13. A 52 Kilodalton Protein Vaccine Candidate for Francisella tularensis

    DTIC Science & Technology

    2004-12-01

    du vaccin vivant F. tularensis (LVS). Soixante pourcent (60%) des souris vaccindes ont survdcu la dose ltale multiple alors que toutes les souris non...le lysat des cellules de cultures vivantes du vaccin vivant F. tularensis. Plusieurs composants de Francisella tularensis ont dt6 identifids par cet...antiserum. Le s6rum de souris provenant de souris vaccin6es avec F. tularensis non- vivant n’a pas identifid ces composants. A partir de ces prot6ines

  14. The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis.

    PubMed

    Schmitt, Deanna M; Barnes, Rebecca; Rogerson, Taylor; Haught, Ashley; Mazzella, Leanne K; Ford, Matthew; Gilson, Tricia; Birch, James W-M; Sjöstedt, Anders; Reed, Douglas S; Franks, Jonathan M; Stolz, Donna B; Denvir, James; Fan, Jun; Rekulapally, Swanthana; Primerano, Donald A; Horzempa, Joseph

    2017-01-01

    Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU , or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake ( Pseudechis guttatus ), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.

  15. The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

    PubMed Central

    Schmitt, Deanna M.; Barnes, Rebecca; Rogerson, Taylor; Haught, Ashley; Mazzella, Leanne K.; Ford, Matthew; Gilson, Tricia; Birch, James W.-M.; Sjöstedt, Anders; Reed, Douglas S.; Franks, Jonathan M.; Stolz, Donna B.; Denvir, James; Fan, Jun; Rekulapally, Swanthana; Primerano, Donald A.; Horzempa, Joseph

    2017-01-01

    Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks. PMID:28536678

  16. Identification of Francisella tularensis by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry: fast, reliable, robust, and cost-effective differentiation on species and subspecies levels.

    PubMed

    Seibold, E; Maier, T; Kostrzewa, M; Zeman, E; Splettstoesser, W

    2010-04-01

    Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.

  17. Interleukin-17 protects against the Francisella tularensis live vaccine strain but not against a virulent F. tularensis type A strain.

    PubMed

    Skyberg, Jerod A; Rollins, Maryclare F; Samuel, Joshua W; Sutherland, Marjorie D; Belisle, John T; Pascual, David W

    2013-09-01

    Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17Rα(-/-) mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17Rα(-/-) mice display organ burdens and pulmonary gamma interferon (IFN-γ) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17Rα(-/-) and wild-type mice. While IFN-γ was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-γ(-/-) mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.

  18. Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis

    PubMed Central

    Schwartz, Justin T.; Bandyopadhyay, Sarmistha; Kobayashi, Scott D.; McCracken, Jenna; Whitney, Adeline R.; DeLeo, Frank R.; Allen, Lee-Ann H.

    2013-01-01

    We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24 h time period relative to the uninfected controls. Of ~47,000 genes analyzed, 3,435 were significantly up- or down-regulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes associated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2, and CASP8) and genes that act via the NF B pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2, and RELA, IL1B, CAST, CDK2, GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that X-linked inhibitor of apoptosis (XIAP) protein remained abundant in PMNs over 48 h of LVS infection, whereas BAX mRNA and protein were progressively down-regulated. These data strongly suggest that antiapoptotic and pro-survival mechanisms collaborate to sustain the viability of F. tularensis infected neutrophils. PMID:22986450

  19. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3280...

  20. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3280...

  1. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3280...

  2. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3280...

  3. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3280...

  4. Differential Substrate Usage and Metabolic Fluxes in Francisella tularensis Subspecies holarctica and Francisella novicida

    PubMed Central

    Chen, Fan; Rydzewski, Kerstin; Kutzner, Erika; Häuslein, Ina; Schunder, Eva; Wang, Xinzhe; Meighen-Berger, Kevin; Grunow, Roland; Eisenreich, Wolfgang; Heuner, Klaus

    2017-01-01

    Francisella tularensis is an intracellular pathogen for many animals causing the infectious disease, tularemia. Whereas F. tularensis subsp. holarctica is highly pathogenic for humans, F. novicida is almost avirulent for humans, but virulent for mice. In order to compare metabolic fluxes between these strains, we performed 13C-labeling experiments with F. tularensis subsp. holarctica wild type (beaver isolate), F. tularensis subsp. holarctica strain LVS, or F. novicida strain U112 in complex media containing either [U-13C6]glucose, [1,2-13C2]glucose, [U-13C3]serine, or [U-13C3]glycerol. GC/MS-based isotopolog profiling of amino acids, polysaccharide-derived glucose, free fructose, amino sugars derived from the cell wall, fatty acids, 3-hydroxybutyrate, lactate, succinate and malate revealed uptake and metabolic usage of all tracers under the experimental conditions with glucose being the major carbon source for all strains under study. The labeling patterns of the F. tularensis subsp. holarctica wild type were highly similar to those of the LVS strain, but showed remarkable differences to the labeling profiles of the metabolites from the F. novicida strain. Glucose was directly used for polysaccharide and cell wall biosynthesis with higher rates in F. tularensis subsp. holarctica or metabolized, with higher rates in F. novicida, via glycolysis and the non-oxidative pentose phosphate pathway (PPP). Catabolic turnover of glucose via gluconeogenesis was also observed. In all strains, Ala was mainly synthesized from pyruvate, although no pathway from pyruvate to Ala is annotated in the genomes of F. tularensis and F. novicida. Glycerol efficiently served as a gluconeogenetic substrate in F. novicida, but only less in the F. tularensis subsp. holarctica strains. In any of the studied strains, serine did not serve as a major substrate and was not significantly used for gluconeogenesis under the experimental conditions. Rather, it was only utilized, at low rates, in

  5. Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System.

    PubMed

    Banada, Padmapriya P; Deshpande, Srinidhi; Chakravorty, Soumitesh; Russo, Riccardo; Occi, James; Meister, Gabriel; Jones, Kelly J; Gelhaus, Carl H; Valderas, Michelle W; Jones, Martin; Connell, Nancy; Alland, David

    2017-01-01

    Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive F. tularensis assay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system. F. tularensis DNA in buffer or CFU of F. tularensis was spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected with F. tularensis Schu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/ml F. tularensis in both human and macaque blood. In infected macaques, the assay detected F. tularensis on days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detect F. tularensis in bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection. Copyright © 2016 American Society for Microbiology.

  6. Detection of Francisella tularensis in Alaskan Mosquitoes (Diptera: Culicidae) and Assessment of a Laboratory Model for Transmission

    PubMed Central

    TRIEBENBACH, ALISON N.; VOGL, SIGRID J.; LOTSPEICH-COLE, LEDA; SIKES, DEREK S.; HAPP, GEORGE M.; HUEFFER, KARSTEN

    2013-01-01

    Tularemia is a zoonotic disease caused by the Category A bioterrorism agent Francisella tularensis. In Scandinavia, tularemia transmission by mosquitoes has been widely cited in the literature. We tested >2,500 mosquitoes captured in Alaska and found Francisella DNA in 30% of pooled samples. To examine the potential for transmission of Francisella by mosquitoes, we developed a mosquito model of Francisella infection. Larvae of Anopheles gambiae Giles and Aedes aegypti (L.) readily ingest F. tularensis but do not efficiently transfer infective doses of the bacterium to the pupal or adult stage. After a bloodmeal containing Francisella, adult female An. gambiae and Ae. aegypti retained detectable levels of Francisella DNA for 3 d, but when they took a second bloodmeal, the mammalian host was not infected. This study suggests that although Francisella DNA can be detected in a significant portion of wild-caught mosquitoes, transmission of Francisella is either very inefficient or is species dependent for the Francisella strain or the arthropod vector. PMID:20695280

  7. Francisella tularensis endocarditis: two case reports and a literature review.

    PubMed

    Gaci, Rostane; Alauzet, Corentine; Selton-Suty, Christine; Lozniewski, Alain; Pulcini, Céline; May, Thierry; Goehringer, François

    2017-02-01

    We report the first two cases of infective endocarditis caused by Francisella tularensis in Europe (two cases have previously been reported outside Europe). We suggest clinicians should consider tularemia as a possible diagnosis in endemic regions in cases of culture-negative endocarditis.

  8. Amblyomma americanum as a Bridging Vector for Human Infection with Francisella tularensis

    PubMed Central

    2015-01-01

    The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 104 CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 103 to 108 CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study). In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 101 to 105 CFU/adult at 168 days post-capillary feeding (longest sampling time). For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID50 of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding preference for larger

  9. Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus.

    PubMed

    Genchi, Marco; Prati, Paola; Vicari, Nadia; Manfredini, Andrea; Sacchi, Luciano; Clementi, Emanuela; Bandi, Claudio; Epis, Sara; Fabbi, Massimo

    2015-01-01

    Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results. The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus. Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes. These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view.

  10. Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus

    PubMed Central

    Genchi, Marco; Prati, Paola; Vicari, Nadia; Manfredini, Andrea; Sacchi, Luciano; Clementi, Emanuela; Bandi, Claudio; Epis, Sara; Fabbi, Massimo

    2015-01-01

    Background Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results. Objective The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus. Results Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes. Conclusions These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view. PMID:26244842

  11. Formulation and Stabilization of Francisella tularensis Live Vaccine Strain

    PubMed Central

    OHTAKE, SATOSHI; MARTIN, RUSSELL A.; SAXENA, ATUL; LECHUGA-BALLESTEROS, DAVID; SANTIAGO, ARACELI E; BARRY, EILEEN M.; TRUONG-LE, VU

    2012-01-01

    Francisella tularensis live vaccine strain (F. tularensis LVS), a promising vaccine candidate for protection against F. tularensis exposure, is a particularly thermolabile vaccine and difficult to stabilize sufficiently for storage under refrigerated conditions. Our preliminary data show that F. tularensis LVS can be stabilized in the dried state using foam drying, a modified freeze drying method, with sugar-based formulations. The process was conducted under mild drying conditions, which resulted in a good titer retention following processing. The inclusion of osmolytes in the growth media resulted in an acceleration of growth kinetics, although no change in osmotolerance was observed. The optimized F. tularensis formulation, which contained trehalose, gelatin, and Pluronic F68 demonstrated stability for approximately 1.5 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1 log10 colony forming unit) and for 12 weeks at 25°C. At refrigerator storage condition (4°C), stabilized F. tularensis LVS vaccine exhibited no activity loss for at least 12 weeks. This stabilization method utilizes conventional freeze dryers and pharmaceutically approved stabilizers, and thus can be readily implemented at many manufacturing sites for large-scale production of stabilized vaccines. The improved heat stability of the F. tularensis LVS could mitigate risks of vaccine potency loss during long-term storage, shipping, and distribution. PMID:21491457

  12. Survey of Francisella tularensis in Wild Animals in Japan in Areas Where Tularemia is Endemic.

    PubMed

    Hotta, Akitoyo; Tanabayashi, Kiyoshi; Fujita, Osamu; Shindo, Junji; Park, Chu-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamamoto, Yoshie; Takano, Ai; Kawabata, Hiroki; Sharma, Neekun; Uda, Akihiko; Yamada, Akio; Morikawa, Shigeru

    2016-09-21

    Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.

  13. CpG oligodeoxyribonucleotides protect mice from Burkholderia pseudomallei but not Francisella tularensis Schu S4 aerosols.

    PubMed

    Rozak, David A; Gelhaus, Herbert C; Smith, Mark; Zadeh, Mojgan; Huzella, Louis; Waag, David; Adamovicz, Jeffrey J

    2010-02-05

    Studies have shown that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis live vaccine strain (LVS), when administered before parenteral challenge. Given the potential to develop CpG ODN as a pre-treatment for multiple bacterial biological warfare agents, we examined survival, histopathology, and cytokine data from CpG ODN-treated C57BL/6 mice to determine whether previously-reported protection extended to aerosolized B. pseudomallei 1026b and highly virulent F. tularensis Schu S4 infections. We found that, although CpG ODN protected mice from aerosolized B. pseudomallei challenges, the immunostimulant failed to benefit the animals exposed to F. tularensis Schu S4 aerosols. Our results, which contrast with earlier F. tularensis LVS studies, highlight potential differences in Francisella species pathogenesis and underscore the need to evaluate immunotherapies against human pathogenic species.

  14. Water as Source of Francisella tularensis Infection in Humans, Turkey

    PubMed Central

    Kilic, Selcuk; Birdsell, Dawn N.; Karagöz, Alper; Çelebi, Bekir; Bakkaloglu, Zekiye; Arikan, Muzaffer; Sahl, Jason W.; Mitchell, Cedar; Rivera, Andrew; Maltinsky, Sara; Keim, Paul; Üstek, Duran; Durmaz, Rıza

    2015-01-01

    Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey. PMID:26583383

  15. CpG oligodeoxyribonucleotides protect mice from Burkholderia pseudomallei but not Francisella tularensis Schu S4 aerosols

    PubMed Central

    2010-01-01

    Studies have shown that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis live vaccine strain (LVS), when administered before parenteral challenge. Given the potential to develop CpG ODN as a pre-treatment for multiple bacterial biological warfare agents, we examined survival, histopathology, and cytokine data from CpG ODN-treated C57BL/6 mice to determine whether previously-reported protection extended to aerosolized B. pseudomallei 1026b and highly virulent F. tularensis Schu S4 infections. We found that, although CpG ODN protected mice from aerosolized B. pseudomallei challenges, the immunostimulant failed to benefit the animals exposed to F. tularensis Schu S4 aerosols. Our results, which contrast with earlier F. tularensis LVS studies, highlight potential differences in Francisella species pathogenesis and underscore the need to evaluate immunotherapies against human pathogenic species. PMID:20181102

  16. FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence

    PubMed Central

    Wu, Xiaojun; Ren, Guoping; Gunning, William T.; Weaver, David A.; Kalinoski, Andrea L.; Khuder, Sadik A.; Huntley, Jason F.

    2016-01-01

    Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in

  17. GM-CSF has disparate roles during intranasal and intradermal Francisella tularensis infection.

    PubMed

    Kurtz, Sherry L; Bosio, Catharine M; De Pascalis, Roberto; Elkins, Karen L

    2016-12-01

    Our laboratory has employed in vitro and in vivo mouse models based on Francisella tularensis Live Vaccine Strain (LVS)-induced protection to elucidate immune correlates for intracellular bacteria. Among the effectors found was GM-CSF, a pleiotropic cytokine that is integral to the development and proliferation of myeloid cells, including alveolar macrophages. GM-CSF has roles in resistance to primary murine infection with several intracellular pathogens, but its role during Francisella infection is unknown. Francisella is an intracellular pathogen that infects lungs after inhalation, primarily invading alveolar macrophages. Here we show that GM-CSF has route-dependent roles during primary infection of mice with LVS. GM-CSF deficient (GM-CSF KO) mice were slightly more susceptible than wild type to intradermal infection, but had increased resistance to intranasal infection. Similarly, these mice had increased resistance to pulmonary infection with virulent F. tularensis (SchuS4). LVS-vaccinated GM-CSF KO mice had normal adaptive immune responses, as measured by T cell activities after LVS intradermal or intranasal vaccination, and survived lethal secondary LVS challenge. GM-CSF KO mice also had robust humoral responses, producing elevated levels of serum antibodies following LVS vaccination compared to wild type mice. Taken together, our data demonstrates that the absence of GM-CSF improves resistance to pulmonary, but not intradermal, infection with Francisella. Published by Elsevier Masson SAS.

  18. Photonic Biosensor Assays to Detect and Distinguish Subspecies of Francisella tularensis

    PubMed Central

    Cooper, Kristie L.; Bandara, Aloka B.; Wang, Yunmiao; Wang, Anbo; Inzana, Thomas J.

    2011-01-01

    The application of photonic biosensor assays to diagnose the category-A select agent Francisella tularensis was investigated. Both interferometric and long period fiber grating sensing structures were successfully demonstrated; both these sensors are capable of detecting the optical changes induced by either immunological binding or DNA hybridization. Detection was made possible by the attachment of DNA probes or immunoglobulins (IgG) directly to the fiber surface via layer-by-layer electrostatic self-assembly. An optical fiber biosensor was tested using a standard transmission mode long period fiber grating of length 15 mm and period 260 μm, and coated with the IgG fraction of antiserum to F. tularensis. The IgG was deposited onto the optical fiber surface in a nanostructured film, and the resulting refractive index change was measured using spectroscopic ellipsometry. The presence of F. tularensis was detected from the decrease of peak wavelength caused by binding of specific antigen. Detection and differentiation of F. tularensis subspecies tularensis (type A strain TI0902) and subspecies holarctica (type B strain LVS) was further accomplished using a single-mode multi-cavity fiber Fabry-Perot interferometric sensor. These sensors were prepared by depositing seven polymer bilayers onto the fiber tip followed by attaching one of two DNA probes: (a) a 101-bp probe from the yhhW gene unique to type-A strains, or (b) a 117-bp probe of the lpnA gene, common to both type-A and type-B strains. The yhhW probe was reactive with the type-A, but not the type-B strain. Probe lpnA was reactive with both type-A and type-B strains. Nanogram quantities of the target DNA could be detected, highlighting the sensitivity of this method for DNA detection without the use of PCR. The DNA probe reacted with 100% homologous target DNA, but did not react with sequences containing 2-bp mismatches, indicating the high specificity of the assay. These assays will fill an important void that

  19. Type IV Pili in Francisella tularensis: Roles of pilF and pilT in Fiber Assembly, Host Cell Adherence, and Virulence ▿

    PubMed Central

    Chakraborty, Subhra; Monfett, Michael; Maier, Tamara M.; Benach, Jorge L.; Frank, Dara W.; Thanassi, David G.

    2008-01-01

    Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence. PMID:18426883

  20. Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp novicida

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Varnum, Susan M.; Webb-Robertson, Bobbie-Jo M.; Pounds, Joel G.

    2012-07-06

    Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA); and Pseudomonas aeruginosa. The composition of BALF proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system, however the timing of their induction varied.more » Francisella tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection, however within 24 hours they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response, however this response is dimished by 24 hours. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.« less

  1. Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis

    PubMed Central

    Wilkinson, Rachael C.; Batten, Laura E.; Wells, Neil J.; Oyston, Petra C.F.; Roach, Peter L.

    2015-01-01

    The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the ‘alarmones’ (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5′,3′-dibisphosphate guanosine) with an EC50 of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia. PMID:26450927

  2. Density-Dependent Prevalence of Francisella tularensis in Fluctuating Vole Populations, Northwestern Spain

    PubMed Central

    Rodríguez-Pastor, Ruth; Escudero, Raquel; Vidal, Dolors; Mougeot, François; Arroyo, Beatriz; Lambin, Xavier; Vila-Coro, Ave Maria; Rodríguez-Moreno, Isabel; Anda, Pedro

    2017-01-01

    Tularemia in humans in northwestern Spain is associated with increases in vole populations. Prevalence of infection with Francisella tularensis in common voles increased to 33% during a vole population fluctuation. This finding confirms that voles are spillover agents for zoonotic outbreaks. Ecologic interactions associated with tularemia prevention should be considered. PMID:28726608

  3. Multifaceted effects of Francisella tularensis on human neutrophil function and lifespan.

    PubMed

    Kinkead, Lauren C; Allen, Lee-Ann H

    2016-09-01

    Francisella tularensis in an intracellular bacterial pathogen that causes a potentially lethal disease called tularemia. Studies performed nearly 100 years ago revealed that neutrophil accumulation in infected tissues correlates directly with the extent of necrotic damage during F. tularensis infection. However, the dynamics and details of bacteria-neutrophil interactions have only recently been studied in detail. Herein, we review current understanding regarding the mechanisms that recruit neutrophils to F. tularensis-infected lungs, opsonization and phagocytosis, evasion and inhibition of neutrophil defense mechanisms, as well as the ability of F. tularensis to prolong neutrophil lifespan. In addition, we discuss distinctive features of the bacterium, including its ability to act at a distance to alter overall neutrophil responsiveness to exogenous stimuli, and the evidence which suggests that macrophages and neutrophils play distinct roles in tularemia pathogenesis, such that macrophages are major vehicles for intracellular growth and dissemination, whereas neutrophils drive tissue destruction by dysregulation of the inflammatory response. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Septic pneumonic tularaemia caused by Francisella tularensis subsp. holarctica biovar II.

    PubMed

    Fritzsch, Joerg; Splettstoesser, Wolf D

    2010-09-01

    This case of pneumonic tularaemia elucidates two aspects: it is believed to be the first documented case of bacteraemia caused by Francisella tularensis subsp. holarctica biovar II; furthermore, it illustrates the remission of septic pneumonic tularaemia without appropriate anti-infective therapy. A blood culture from a patient with community-acquired pneumonia was found to be positive for F. tularensis subsp. holarctica biovar II after 10 days of cultivation. Meanwhile, the patient had been treated with ceftriaxone, followed by sultamicillin and clindamycin. The patient continued suffering from fever of up to 40.7 degrees C and rising C-reactive protein (CRP) for 4 days before the fever and CRP declined. The isolated strain was later tested and found to be resistant to the antibiotics used. The present case underlines that F. tularensis subsp. holarctica infections may cause severe symptoms but mostly have a favourable outcome.

  5. The use of Matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis.

    PubMed

    Karatuna, Onur; Celebi, Bekir; Can, Simge; Akyar, Isin; Kilic, Selcuk

    2016-01-15

    Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institute of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica due to RD1 subspecies-specific PCR result. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories.

  6. The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis

    PubMed Central

    Karatuna, Onur; Çelebi, Bekir; Can, Simge; Akyar, Işın; Kiliç, Selçuk

    2016-01-01

    Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institution of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica according to region of difference 1 (RD1) subspecies-specific PCR results. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories. PMID:26773181

  7. Paralogous Outer Membrane Proteins Mediate Uptake of Different Forms of Iron and Synergistically Govern Virulence in Francisella tularensis tularensis*

    PubMed Central

    Ramakrishnan, Girija; Sen, Bhaswati; Johnson, Richard

    2012-01-01

    Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a 55Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host. PMID:22661710

  8. Francisella tularensis detection using magnetic labels and a magnetic biosensor based on frequency mixing

    NASA Astrophysics Data System (ADS)

    Meyer, Martin H. F.; Krause, Hans-Joachim; Hartmann, Markus; Miethe, Peter; Oster, Jürgen; Keusgen, Michael

    2007-04-01

    A biosensor that uses resonant coils with a special frequency-mixing technique and magnetic beads as detectable labels has been established for the detection of Francisella tularensis, the causative agent for tularemia. The detection principle is based on a sandwich immunoassay using an anti-Ft antibody for immunofiltration immobilized to ABICAP ® polyethylene filters, and biotinylated with streptavidin-coated magnetic beads as labels. The linear detection range of this biosensor was found to be 10 4-10 6 cfu F. tularensis lipopolysaccharide (LPS) per ml. Tested sample matrices were physiological PBS buffer and rabbit serum.

  9. Interaction of Francisella tularensis bacterial cells with dynamic speckles

    NASA Astrophysics Data System (ADS)

    Ulianova, Onega V.; Ulyanov, Sergey S.; Sazanova, Elena V.; Zudina, Irina; Zhang, Zhihong; Sibo, Zhou; Luo, Qingming

    2006-08-01

    Influence of low-coherent speckles on the colonies grows is investigated. It has been demonstrated that effects of light on the inhibition of cells (Francisella Tularensis) are caused by speckle dynamics. The regimes of illumination of cell suspension with purpose of devitalization of hazard bacteria, caused very dangerous infections, such as tularemia, are found. Mathematical model of interaction of low-coherent laser radiation with bacteria suspension has been proposed. Computer simulations of the processes of laser-cells interaction have been carried out. Role of coherence of light in the processes of laser-cell interaction is analyzed.

  10. The potential for flower nectar to allow mosquito to mosquito transmission of Francisella tularensis.

    PubMed

    Kenney, Adam; Cusick, Austin; Payne, Jessica; Gaughenbaugh, Anna; Renshaw, Andrea; Wright, Jenna; Seeber, Roger; Barnes, Rebecca; Florjanczyk, Aleksandr; Horzempa, Joseph

    2017-01-01

    Francisella tularensis is disseminated in nature by biting arthropods such as mosquitoes. The relationship between mosquitoes and F. tularensis in nature is highly ambiguous, due in part to the fact that mosquitoes have caused significant tularemia outbreaks despite being classified as a mechanical vector of F. tularensis. One possible explanation for mosquitoes being a prominent, yet mechanical vector is that these insects feed on flower nectar between blood meals, allowing for transmission of F. tularensis between mosquitoes. Here, we aimed to assess whether F. tularensis could survive in flower nectar. Moreover, we examined if mosquitoes could interact with or ingest and transmit F. tularensis from one source of nectar to another. F. tularensis exhibited robust survivability in flower nectar with concentrations of viable bacteria remaining consistent with the rich growth medium. Furthermore, F. tularensis was able to survive (albeit to a lesser extent) in 30% sucrose (a nectar surrogate) over a period of time consistent with that of a typical flower bloom. Although we observed diminished bacterial survival in the nectar surrogate, mosquitoes that fed on this material became colonized with F. tularensis. Finally, colonized mosquitoes were capable of transferring F. tularensis to a sterile nectar surrogate. These data suggest that flower nectar may be capable of serving as a temporary source of F. tularensis that could contribute to the amplification of outbreaks. Mosquitoes that feed on an infected mammalian host and subsequently feed on flower nectar could deposit some F. tularensis bacteria into the nectar in the process. Mosquitoes subsequently feeding on this nectar source could potentially become colonized by F. tularensis. Thus, the possibility exists that flower nectar may allow for vector-vector transmission of F. tularensis.

  11. Identification and characterization of novel and potent transcription promoters of Francisella tularensis.

    PubMed

    Zaide, Galia; Grosfeld, Haim; Ehrlich, Sharon; Zvi, Anat; Cohen, Ofer; Shafferman, Avigdor

    2011-03-01

    Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli σ(70)-related promoter motifs. These motifs include the -10 (TATAAT) and -35 [TTGA(C/T)A] domains and an AT-rich region upstream from -35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.

  12. Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation

    PubMed Central

    Loughman, Kathleen; Hall, Jesse; Knowlton, Samantha; Sindeldecker, Devin; Gilson, Tricia; Schmitt, Deanna M.; Birch, James W.-M.; Gajtka, Tara; Kobe, Brianna N.; Florjanczyk, Aleksandr; Ingram, Jenna; Bakshi, Chandra S.; Horzempa, Joseph

    2016-01-01

    Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia – the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature. PMID:26858709

  13. Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation.

    PubMed

    Loughman, Kathleen; Hall, Jesse; Knowlton, Samantha; Sindeldecker, Devin; Gilson, Tricia; Schmitt, Deanna M; Birch, James W-M; Gajtka, Tara; Kobe, Brianna N; Florjanczyk, Aleksandr; Ingram, Jenna; Bakshi, Chandra S; Horzempa, Joseph

    2016-01-01

    Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia - the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature.

  14. Novel Catanionic Surfactant Vesicle Vaccines Protect against Francisella tularensis LVS and Confer Significant Partial Protection against F. tularensis Schu S4 Strain

    PubMed Central

    Richard, Katharina; Mann, Barbara J.; Stocker, Lenea; Barry, Eileen M.; Qin, Aiping; Cole, Leah E.; Hurley, Matthew T.; Ernst, Robert K.; Michalek, Suzanne M.; Stein, Daniel C.; DeShong, Philip

    2014-01-01

    Francisella tularensis is a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensis strains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensis type B live vaccine strain (F. tularensis LVS [LVS-V]) or F. tularensis type A Schu S4 strain (F. tularensis Schu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not express F. tularensis components, partially protected against F. tularensis LVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensis Schu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensis LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensis Schu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensis LVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensis LVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effective F. tularensis subunit vaccine and may be applicable for use with other pathogens. PMID:24351755

  15. The potential for flower nectar to allow mosquito to mosquito transmission of Francisella tularensis

    PubMed Central

    Payne, Jessica; Gaughenbaugh, Anna; Renshaw, Andrea; Wright, Jenna; Seeber, Roger; Barnes, Rebecca; Florjanczyk, Aleksandr; Horzempa, Joseph

    2017-01-01

    Francisella tularensis is disseminated in nature by biting arthropods such as mosquitoes. The relationship between mosquitoes and F. tularensis in nature is highly ambiguous, due in part to the fact that mosquitoes have caused significant tularemia outbreaks despite being classified as a mechanical vector of F. tularensis. One possible explanation for mosquitoes being a prominent, yet mechanical vector is that these insects feed on flower nectar between blood meals, allowing for transmission of F. tularensis between mosquitoes. Here, we aimed to assess whether F. tularensis could survive in flower nectar. Moreover, we examined if mosquitoes could interact with or ingest and transmit F. tularensis from one source of nectar to another. F. tularensis exhibited robust survivability in flower nectar with concentrations of viable bacteria remaining consistent with the rich growth medium. Furthermore, F. tularensis was able to survive (albeit to a lesser extent) in 30% sucrose (a nectar surrogate) over a period of time consistent with that of a typical flower bloom. Although we observed diminished bacterial survival in the nectar surrogate, mosquitoes that fed on this material became colonized with F. tularensis. Finally, colonized mosquitoes were capable of transferring F. tularensis to a sterile nectar surrogate. These data suggest that flower nectar may be capable of serving as a temporary source of F. tularensis that could contribute to the amplification of outbreaks. Mosquitoes that feed on an infected mammalian host and subsequently feed on flower nectar could deposit some F. tularensis bacteria into the nectar in the process. Mosquitoes subsequently feeding on this nectar source could potentially become colonized by F. tularensis. Thus, the possibility exists that flower nectar may allow for vector-vector transmission of F. tularensis. PMID:28486521

  16. Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds

    PubMed Central

    Jayamani, Elamparithi; Tharmalingam, Nagendran; Rajamuthiah, Rajmohan; Kim, Wooseong; Okoli, Ikechukwu; Hernandez, Ana M.; Lee, Kiho; Nau, Gerard J.; Ausubel, Frederick M.

    2017-01-01

    ABSTRACT Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z′ factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro. Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 μg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia. PMID:28652232

  17. Thermal resistance of Francisella tularensis in infant formula and fruit juices.

    PubMed

    Day, J B; Trujillo, S; Hao, Y Y D; Whiting, R C

    2008-11-01

    Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, little information exists on the thermal stability of this organism in food matrices. In the present study, the thermal resistance of the live vaccine strain of F. tularensis in four food products (liquid infant formula, apple juice, mango juice, and orange juice) was investigated. D-values ranged from 12 s (57.5 degrees C) to 580 s (50 degrees C) in infant formula with a z-value of 4.37 degrees C. D-values in apple juice ranged from 8 s (57.5 degrees C) to 59 s (50 degrees C) with a z-value of 9.17 degrees C. The live vaccine strain did not survive at temperatures above 55 degrees C in mango juice and orange juice (>6-log inactivation). D-values at 55 to 47.5 degrees C were 15 to 59 s in mango juice and 16 to 105 s in orange juice with z-values of 9.28 and 12.30 degrees C, respectively. These results indicate that current pasteurization parameters used for destroying common foodborne bacterial pathogens are adequate for eliminating F. tularensis in the four foods tested. This study is the first to determine thermal inactivation of F. tularensis in specific foods and will permit comparisons with the thermal inactivation data of other more traditional foodborne pathogens.

  18. Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors.

    PubMed

    Wallqvist, Anders; Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert; Rajagopala, Seesandra V; Kwon, Keehwan; Yu, Chenggang; Hoover, Timothy A; Reifman, Jaques

    2015-12-29

    Francisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms. We initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response. Direct interactions with specific host proteins and the

  19. Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei Infections.

    PubMed

    Skyberg, Jerod A; Rollins, MaryClare F; Holderness, Jeff S; Marlenee, Nicole L; Schepetkin, Igor A; Goodyear, Andrew; Dow, Steven W; Jutila, Mark A; Pascual, David W

    2012-01-01

    Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens.

  20. An Outbreak of Respiratory Tularemia Caused by Diverse Clones of Francisella tularensis

    PubMed Central

    Johansson, Anders; Lärkeryd, Adrian; Widerström, Micael; Mörtberg, Sara; Myrtännäs, Kerstin; Öhrman, Caroline; Birdsell, Dawn; Keim, Paul; Wagner, David M.; Forsman, Mats; Larsson, Pär

    2014-01-01

    Background. The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens. Methods. A localized outbreak of tularemia in Sweden was investigated. Sixty-seven humans contracted laboratory-verified respiratory tularemia. F. tularensis subspecies holarctica was isolated from the blood or pleural fluid of 10 individuals from July to September 2010. Using whole-genome sequencing and analysis of single-nucleotide polymorphisms (SNPs), outbreak isolates were compared with 110 archived global isolates. Results. There were 757 SNPs among the genomes of the 10 outbreak isolates and the 25 most closely related archival isolates (all from Sweden/Finland). Whole genomes of outbreak isolates were >99.9% similar at the nucleotide level and clustered into 3 distinct genetic clades. Unexpectedly, high-sequence similarity grouped some outbreak and archival isolates that originated from patients from different geographic regions and up to 10 years apart. Outbreak and archival genomes frequently differed by only 1–3 of 1 585 229 examined nucleotides. Conclusions. The outbreak was caused by diverse clones of F. tularensis that occurred concomitantly, were widespread, and apparently persisted in the environment. Multiple independent acquisitions of F. tularensis from the environment over a short time period suggest that natural outbreaks of respiratory tularemia are triggered by environmental cues. The findings additionally caution against interpreting genome sequence identity for this pathogen as proof of a direct epidemiological link. PMID:25097081

  1. NMR Structure of Francisella tularensis Virulence Determinant Reveals Structural Homology to Bet v1 Allergen Proteins.

    PubMed

    Zook, James; Mo, Gina; Sisco, Nicholas J; Craciunescu, Felicia M; Hansen, Debra T; Baravati, Bobby; Cherry, Brian R; Sykes, Kathryn; Wachter, Rebekka; Van Horn, Wade D; Fromme, Petra

    2015-06-02

    Tularemia is a potentially fatal bacterial infection caused by Francisella tularensis, and is endemic to North America and many parts of northern Europe and Asia. The outer membrane lipoprotein, Flpp3, has been identified as a virulence determinant as well as a potential subunit template for vaccine development. Here we present the first structure for the soluble domain of Flpp3 from the highly infectious Type A SCHU S4 strain, derived through high-resolution solution nuclear magnetic resonance (NMR) spectroscopy; the first structure of a lipoprotein from the genus Francisella. The Flpp3 structure demonstrates a globular protein with an electrostatically polarized surface containing an internal cavity-a putative binding site based on the structurally homologous Bet v1 protein family of allergens. NMR-based relaxation studies suggest loop regions that potentially modulate access to the internal cavity. The Flpp3 structure may add to the understanding of F. tularensis virulence and contribute to the development of effective vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Protective immunity against tularemia provided by an adenovirus-vectored vaccine expressing Tul4 of Francisella tularensis.

    PubMed

    Kaur, Ravinder; Chen, Shan; Arévalo, Maria T; Xu, Qingfu; Chen, Yanping; Zeng, Mingtao

    2012-03-01

    Francisella tularensis, a category A bioterrorism agent, is a highly infectious organism that is passed on via skin contact and inhalation routes. A live attenuated vaccine strain (LVS) has been developed, but it has not been licensed for public use by the FDA due to safety concerns. Thus, there exists a need for a safer and improved vaccine. In this study, we have constructed a replication-incompetent adenovirus, Ad/opt-Tul4, carrying a codon-optimized gene for expression of a membrane protein, Tul4, of F. tularensis LVS. Its ability to protect against lethal challenge and its immunogenicity were evaluated in a murine model. An intramuscular injection of a single dose (1 × 10(7) PFU) of Ad/opt-Tul4 elicited a robust Tul4-specific antibody response. Assays suggest a Th1-driven response. A single dose elicited 20% protection against challenge with 100 × 50% lethal dose (LD(50)) F. tularensis LVS; two additional booster shots resulted in 60% protection. In comparison, three doses of 5 μg recombinant Tul4 protein did not elicit significant protection against challenge. Therefore, the Ad/opt-Tul4 vaccine was more effective than the protein vaccine, and protection was dose dependent. Compared to LVS, the protection rate is lower, but an adenovirus-vectored vaccine may be more attractive due to its enhanced safety profile and mucosal route of delivery. Furthermore, simple genetic modification of the vaccine may potentially produce antibodies protective against a fully virulent strain of F. tularensis. Our data support the development and further research of an adenovirus-vectored vaccine against Tul4 of F. tularensis LVS.

  3. Identification of Francisella tularensis lipoproteins that stimulate the toll-like receptor (TLR) 2/TLR1 heterodimer.

    PubMed

    Thakran, Shalini; Li, Hanfen; Lavine, Christy L; Miller, Mark A; Bina, James E; Bina, Xiaowen R; Re, Fabio

    2008-02-15

    The innate immune response to Francisella tularensis is primarily mediated by TLR2, though the bacterial products that stimulate this receptor remain unknown. Here we report the identification of two Francisella lipoproteins, TUL4 and FTT1103, which activate TLR2. We demonstrate that TUL4 and FTT1103 stimulate chemokine production in human and mouse cells in a TLR2-dependent way. Using an assay that relies on chimeric TLR proteins, we show that TUL4 and FTT1103 stimulate exclusively the TLR2/TLR1 heterodimer. Our results also show that yet unidentified Francisella proteins, possibly unlipi-dated, have the ability to stimulate the TLR2/TLR6 heterodimer. Through domain-exchange analysis, we determined that an extended region that comprises LRR 9-17 in the extra-cellular portion of TLR1 mediates response to Francisella lipoproteins and triacylated lipopeptide. Substitution of the corresponding LRR of TLR6 with the LRR derived from TLR1 enables TLR6 to recognize TUL4, FTT1103, and triacylated lipopeptide. This study identifies for the first time specific Fran-cisella products capable of stimulating a proinflammatory response and the cellular receptors they trigger.

  4. Inhibition of AcpA phosphatase activity with ascorbate attenuates Francisella tularensis intramacrophage survival.

    PubMed

    McRae, Steven; Pagliai, Fernando A; Mohapatra, Nrusingh P; Gener, Alejandro; Mahmou, Asma Sayed Abdelgeliel; Gunn, John S; Lorca, Graciela L; Gonzalez, Claudio F

    2010-02-19

    Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (K(i) = 380 +/- 160 microM) and 2-phosphoascorbate (K(i) = 3.2 +/- 0.85 microM) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia.

  5. Inhibition of AcpA Phosphatase Activity with Ascorbate Attenuates Francisella tularensis Intramacrophage Survival

    PubMed Central

    McRae, Steven; Pagliai, Fernando A.; Mohapatra, Nrusingh P.; Gener, Alejandro; Abdelgeliel Mahmou, Asma Sayed; Gunn, John S.; Lorca, Graciela L.; Gonzalez, Claudio F.

    2010-01-01

    Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (Ki = 380 ± 160 μm) and 2-phosphoascorbate (Ki = 3.2 ± 0.85 μm) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia. PMID:20028980

  6. Phylogeographical pattern of Francisella tularensis in a nationwide outbreak of tularaemia in Norway, 2011.

    PubMed

    Afset, J E; Larssen, K W; Bergh, K; Larkeryd, A; Sjodin, A; Johansson, A; Forsman, M

    2015-05-14

    In 2011, a nationwide outbreak of tularaemia occurred in Norway with 180 recorded cases. It was associated with the largest peak in lemming density seen in 40 years. Francisella tularensis was isolated from 18 patients. To study the geographical distribution of F.tularensis genotypes in Norway and correlate genotype with epidemiology and clinical presentation,we performed whole genome sequencing of patient isolates. All 18 genomes from the outbreak carried genetic signatures of F. tularensis subsp. holarctica and were assigned to genetic clades using canonical single nucleotide polymorphisms. Ten isolates were assigned to major genetic clade B.6 (subclade B.7),seven to clade B.12, and one to clade B.4. The B.6 subclade B.7 was most common in southern and central Norway, while clade B.12 was evenly distributed between the southern, central and northern parts of the country. There was no association between genotype and clinical presentation of tularaemia, time of year or specimen type. We found extensive sequence similarity with F. tularensis subsp. holarctica genomes from high-endemic tularaemia areas in Sweden.Finding nearly identical genomes across large geographical distances in Norway and Sweden imply a life cycle of the bacterium without replication between the outbreaks and raise new questions about long-range migration mechanisms.

  7. Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

    PubMed

    Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

    2012-07-01

    Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

  8. Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler.

    PubMed

    Emanuel, Peter A; Bell, Ryan; Dang, Jessica L; McClanahan, Rebecca; David, John C; Burgess, Robert J; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

    2003-02-01

    The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5' hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

  9. Detection of Francisella tularensis within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

    PubMed Central

    Emanuel, Peter A.; Bell, Ryan; Dang, Jessica L.; McClanahan, Rebecca; David, John C.; Burgess, Robert J.; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

    2003-01-01

    The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field. PMID:12574268

  10. An outbreak of respiratory tularemia caused by diverse clones of Francisella tularensis.

    PubMed

    Johansson, Anders; Lärkeryd, Adrian; Widerström, Micael; Mörtberg, Sara; Myrtännäs, Kerstin; Ohrman, Caroline; Birdsell, Dawn; Keim, Paul; Wagner, David M; Forsman, Mats; Larsson, Pär

    2014-12-01

    The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens. A localized outbreak of tularemia in Sweden was investigated. Sixty-seven humans contracted laboratory-verified respiratory tularemia. F. tularensis subspecies holarctica was isolated from the blood or pleural fluid of 10 individuals from July to September 2010. Using whole-genome sequencing and analysis of single-nucleotide polymorphisms (SNPs), outbreak isolates were compared with 110 archived global isolates. There were 757 SNPs among the genomes of the 10 outbreak isolates and the 25 most closely related archival isolates (all from Sweden/Finland). Whole genomes of outbreak isolates were >99.9% similar at the nucleotide level and clustered into 3 distinct genetic clades. Unexpectedly, high-sequence similarity grouped some outbreak and archival isolates that originated from patients from different geographic regions and up to 10 years apart. Outbreak and archival genomes frequently differed by only 1-3 of 1 585 229 examined nucleotides. The outbreak was caused by diverse clones of F. tularensis that occurred concomitantly, were widespread, and apparently persisted in the environment. Multiple independent acquisitions of F. tularensis from the environment over a short time period suggest that natural outbreaks of respiratory tularemia are triggered by environmental cues. The findings additionally caution against interpreting genome sequence identity for this pathogen as proof of a direct epidemiological link. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Utilization of Fc Receptors as a Mucosal Vaccine Strategy against an Intracellular Bacterium, Francisella tularensis1

    PubMed Central

    Rawool, Deepak B.; Bitsaktsis, Constantine; Li, Ying; Gosselin, Diane R.; Lin, Yili; Kurkure, Nitin V.; Metzger, Dennis W.; Gosselin, Edmund J.

    2013-01-01

    Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcγR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen. PMID:18390739

  12. Aged mice display an altered pulmonary host response to Francisella tularensis live vaccine strain (LVS) infections

    PubMed Central

    CA, Mares; SS, Ojeda; Q, Li; EG, Morris; JJ, Coalson; JM, Teale

    2012-01-01

    Aging is a complex phenomenon that has been shown to affect many organ systems including the innate and adaptive immune systems. The current study was designed to examine the potential effect of immunosenescence on the pulmonary immune response using a Francisella tularensis live vaccine strain (LVS) inhalation infection model. F. tularensis is a gram-negative intracellular pathogen that can cause a severe pneumonia.In this study both young (8-12 week old) and aged (20-24 month old) mice were infected intranasally with LVS. Lung tissues from young and aged mice were used to assess pathology, recruitment of immune cell types and cytokine expression levels at various times post infection. Bacterial burdens were also assessed. Interestingly, the lungs of aged animals harbored fewer organisms at early time points of infection (day 1, day 3) compared with their younger counterparts. In addition, only aged animals displayed small perivascular aggregates at these early time points that appeared mostly mononuclear in nature. However, the kinetics of infiltrating polymorphonuclear neutrophils (PMNs) and increased cytokine levels measured in the bronchial alveolar lavage fluid (BALF) were delayed in infected aged animals relative to young infected animals with neutrophils appearing at day 5 post infection (PI) in the aged animals as opposed to day 3 PI in the young infected animals. Also evident were alterations in the ratios of mononuclear to PMNs at distinct post infection times. The above evidence indicates that aged mice elicit an altered immune response in the lung to respiratory Francisella tularensis LVS infections compared to their younger counterparts. PMID:19825409

  13. Characterization of the pathogenicity island protein PdpA and its role in the virulence of Francisella novicida

    PubMed Central

    Schmerk, Crystal L.; Duplantis, Barry N.; Wang, Diana; Burke, Robert D.; Chou, Alicia Y.; Elkins, Karen L.; Ludu, Jagjit S.; Nano, Francis E.

    2009-01-01

    Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice. PMID:19372153

  14. High and novel genetic diversity of Francisella tularensis in Germany and indication of environmental persistence.

    PubMed

    Schulze, C; Heuner, K; Myrtennäs, K; Karlsson, E; Jacob, D; Kutzer, P; GROßE, K; Forsman, M; Grunow, R

    2016-10-01

    In Germany tularemia is a re-emerging zoonotic disease. Therefore, we investigated wild animals and environmental water samples for the presence and phylogenetic diversity of Francisella tularensis in the poorly studied Berlin/Brandenburg region. The phylogenomic analysis of three isolates from wild animals revealed three new subclades within the phylogenetic tree of F. tularensis [B.71 from a raccoon dog (Nyctereutes procyonoides); B.74 from a red fox (Vulpes vulpes), and B.75 from a Eurasian beaver (Castor fiber albicus)]. The results from histological, PCR, and genomic investigations on the dead beaver showed that the animal suffered from a systemic infection. Indications were found that the bacteria were released from the beaver carcass into the surrounding environment. We demonstrated unexpectedly high and novel phylogenetic diversity of F. tularensis in Germany and the fact that the bacteria persist in the environment for at least one climatic season. These findings support a broader host species diversity than previously known regarding Germany. Our data further support the assumption derived from previous serological studies of an underestimated frequency of occurrence of the pathogen in the environment and in wild animals. F. tularensis was isolated from animal species not previously reported as natural hosts in Germany.

  15. About three cases of ulceroglandular tularemia, is this the re-emergence of Francisella tularensis in Belgium?

    PubMed

    Dupont, E; Van Eeckhoudt, S; Thissen, X; Ausselet, N; Fretin, D; Stefanescu, I; Glupczynski, Y; Delaere, B

    2015-10-01

    Tularemia is a zoonosis caused by Francisella tularensis that can be transmitted by several ways to human being and cause different clinical manifestations. We report three clinical cases of tularemia with ulceroglandular presentation in young males acquired during outdoor activities in Southern Belgium. Confirmation of the diagnosis was established by serology. Only three cases of tularemia have been reported in Belgium between 1950 and 2012 by the National Reference Laboratory CODA-CERVA (Ref Lab CODA-CERVA) but re-emergence of tularemia is established in several European countries and F. tularensis is also well known to be present in animal reservoirs and vectors in Belgium. The diagnosis of tularemia has to be considered in case of suggestive clinical presentation associated with epidemiological risk factors.

  16. Detoxified Endotoxin Vaccine (J5dLPS/OMP) Protects Mice Against Lethal Respiratory Challenge with Francisella tularensis SchuS4

    PubMed Central

    Gregory, Stephen H.; Chen, Wilbur H.; Mott, Stephanie; Palardy, John E.; Parejo, Nicholas A.; Heninger, Sara; Anderson, Christine A.; Artenstein, Andrew W.; Opal, Steven M.; Cross, Alan S.

    2010-01-01

    Francisella tularensis is a category A select agent. J5dLPS/OMP is a novel vaccine construct consisting of detoxified, O-polysaccharide side chain-deficient, lipopolysaccharide non-covalently complexed with the outer membrane protein of N. meningitidis group B. Immunization elicits hightiter polyclonal antibodies specific for the highly-conserved epitopes expressed within the glycolipid core that constitutes gram-negative bacteria (e.g., F. tularensis). Mice immunized intranasally with J5dLPS/OMP exhibited protective immunity to intratracheal challenge with the live vaccine strain, as well as the highly-virulent SchuS4 strain, of F. tularensis. The efficacy of J5dLPS/OMP vaccine suggests its potential utility in immunizing the general population against several different gram-negative select agents concurrently. PMID:20170768

  17. Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection of Francisella tularensis.

    PubMed

    Dulay, Samuel B; Gransee, Rainer; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K

    2014-09-15

    Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Rapid Countermeasure Discovery against Francisella tularensis Based on a Metabolic Network Reconstruction

    PubMed Central

    Chaudhury, Sidhartha; Abdulhameed, Mohamed Diwan M.; Singh, Narender; Tawa, Gregory J.; D’haeseleer, Patrik M.; Zemla, Adam T.; Navid, Ali; Zhou, Carol E.; Franklin, Matthew C.; Cheung, Jonah; Rudolph, Michael J.; Love, James; Graf, John F.; Rozak, David A.; Dankmeyer, Jennifer L.; Amemiya, Kei; Daefler, Simon; Wallqvist, Anders

    2013-01-01

    In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 µM, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested

  19. Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays

    PubMed Central

    Nakajima, Rie; Escudero, Raquel; Molina, Douglas M.; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L.; AuCoin, David P.; Anda, Pedro

    2016-01-01

    Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip. PMID:27098957

  20. Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

    PubMed Central

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique

    2014-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  1. Whole genome sequencing of the fish pathogen Francisella noatunensis subsp. orientalis Toba04 gives novel insights into Francisella evolution and pathogenecity

    PubMed Central

    2012-01-01

    Background Francisella is a genus of gram-negative bacterium highly virulent in fishes and human where F. tularensis is causing the serious disease tularaemia in human. Recently Francisella species have been reported to cause mortality in aquaculture species like Atlantic cod and tilapia. We have completed the sequencing and draft assembly of the Francisella noatunensis subsp. orientalisToba04 strain isolated from farmed Tilapia. Compared to other available Francisella genomes, it is most similar to the genome of Francisella philomiragia subsp. philomiragia, a free-living bacterium not virulent to human. Results The genome is rearranged compared to the available Francisella genomes even though we found no IS-elements in the genome. Nearly 16% percent of the predicted ORFs are pseudogenes. Computational pathway analysis indicates that a number of the metabolic pathways are disrupted due to pseudogenes. Comparing the novel genome with other available Francisella genomes, we found around 2.5% of unique genes present in Francisella noatunensis subsp. orientalis Toba04 and a list of genes uniquely present in the human-pathogenic Francisella subspecies. Most of these genes might have transferred from bacterial species through horizontal gene transfer. Comparative analysis between human and fish pathogen also provide insights into genes responsible for pathogenecity. Our analysis of pseudogenes indicates that the evolution of Francisella subspecies’s pseudogenes from Tilapia is old with large number of pseudogenes having more than one inactivating mutation. Conclusions The fish pathogen has lost non-essential genes some time ago. Evolutionary analysis of the Francisella genomes, strongly suggests that human and fish pathogenic Francisella species have evolved independently from free-living metabolically competent Francisella species. These findings will contribute to understanding the evolution of Francisella species and pathogenesis. PMID:23131096

  2. Whole-genome relationships among Francisella bacteria of diverse origins define new species and provide specific regions for detection

    DOE PAGES

    Challacombe, Jean Faust; Petersen, Jeannine M.; Gallegos-Graves, La Verne A.; ...

    2016-11-23

    Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisellamore » strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). Lastly, this study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria.« less

  3. Whole-genome relationships among Francisella bacteria of diverse origins define new species and provide specific regions for detection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Challacombe, Jean Faust; Petersen, Jeannine M.; Gallegos-Graves, La Verne A.

    Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisellamore » strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). Lastly, this study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria.« less

  4. Francisella tularensis type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks post Infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    El-Etr, S H; Margolis, J; Monack, D

    2009-07-28

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown, and the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype (REP)more » is caused by factor(s) secreted by amoebae and/or F. tularensis into the co-culture media. Further, our results indicate that in contrast to LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks post infection and that induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that the interactions between F. tularensis strains and amoeba may play a role in the environmental persistence of F. tularensis.« less

  5. Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA, IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge.

    PubMed

    Jia, Qingmei; Bowen, Richard; Lee, Bai-Yu; Dillon, Barbara Jane; Masleša-Galić, Saša; Horwitz, Marcus A

    2016-09-22

    A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ΔcapB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS

  6. Monophosphoryl Lipid A Enhances Efficacy of a Francisella tularensis LVS-Catanionic Nanoparticle Subunit Vaccine against F. tularensis Schu S4 Challenge by Augmenting both Humoral and Cellular Immunity

    PubMed Central

    Richard, Katharina; Mann, Barbara J.; Qin, Aiping; Barry, Eileen M.; Ernst, Robert K.

    2017-01-01

    ABSTRACT Francisella tularensis, a bacterial biothreat agent, has no approved vaccine in the United States. Previously, we showed that incorporating lysates from partially attenuated F. tularensis LVS or fully virulent F. tularensis Schu S4 strains into catanionic surfactant vesicle (V) nanoparticles (LVS-V and Schu S4-V, respectively) protected fully against F. tularensis LVS intraperitoneal (i.p.) challenge in mice. However, we achieved only partial protection against F. tularensis Schu S4 intranasal (i.n.) challenge, even when employing heterologous prime-boost immunization strategies. We now extend these findings to show that both LVS-V and Schu S4-V immunization (i.p./i.p.) elicited similarly high titers of anti-F. tularensis IgG and that the titers could be further increased by adding monophosphoryl lipid A (MPL), a nontoxic Toll-like receptor 4 (TLR4) adjuvant that is included in several U.S. FDA-approved vaccines. LVS-V+MPL immune sera also detected more F. tularensis antigens than LVS-V immune sera and, after passive transfer to naive mice, significantly delayed the time to death against F. tularensis Schu S4 subcutaneous (s.c.) but not i.n. challenge. Active immunization with LVS-V+MPL (i.p./i.p.) also increased the frequency of gamma interferon (IFN-γ)-secreting activated helper T cells, IFN-γ production, and the ability of splenocytes to control intramacrophage F. tularensis LVS replication ex vivo. Active LVS-V+MPL immunization via heterologous routes (i.p./i.n.) significantly elevated IgA and IgG levels in bronchoalveolar lavage fluid and significantly enhanced protection against i.n. F. tularensis Schu S4 challenge (to ∼60%). These data represent a significant step in the development of a subunit vaccine against the highly virulent type A strains. PMID:28077440

  7. IL-10 restrains IL-17 to limit lung pathology characteristics following pulmonary infection with Francisella tularensis live vaccine strain.

    PubMed

    Slight, Samantha R; Monin, Leticia; Gopal, Radha; Avery, Lyndsay; Davis, Marci; Cleveland, Hillary; Oury, Tim D; Rangel-Moreno, Javier; Khader, Shabaana A

    2013-11-01

    IL-10 production during intracellular bacterial infections is generally thought to be detrimental because of its role in suppressing protective T-helper cell 1 (Th1) responses. Francisella tularensis is a facultative intracellular bacterium that activates both Th1 and Th17 protective immune responses. Herein, we report that IL-10-deficient mice (Il10(-/-)), despite having increased Th1 and Th17 responses, exhibit increased mortality after pulmonary infection with F. tularensis live vaccine strain. We demonstrate that the increased mortality observed in Il10(-/-)-infected mice is due to exacerbated IL-17 production that causes increased neutrophil recruitment and associated lung pathology. Thus, although IL-17 is required for protective immunity against pulmonary infection with F. tularensis live vaccine strain, its production is tightly regulated by IL-10 to generate efficient induction of protective immunity without mediating pathology. These data suggest a critical role for IL-10 in maintaining the delicate balance between host immunity and pathology during pulmonary infection with F. tularensis live vaccine strain. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

    PubMed

    Nakajima, Rie; Escudero, Raquel; Molina, Douglas M; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L; AuCoin, David P; Anda, Pedro; Davies, D Huw

    2016-07-01

    Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    PubMed

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections.

  10. Biochemical responses and oxidative stress in Francisella tularensis infection: a European brown hare model

    PubMed Central

    2011-01-01

    Background The aim of the present study was to investigate biochemical and oxidative stress responses to experimental F. tularensis infection in European brown hares, an important source of human tularemia infections. Methods For these purposes we compared the development of an array of biochemical parameters measured in blood plasma using standard procedures of dry chemistry as well as electrochemical devices following a subcutaneous infection with a wild Francisella tularensis subsp. holarctica strain (a single dose of 2.6 × 109 CFU pro toto). Results Subcutaneous inoculation of a single dose with 2.6 × 109 colony forming units of a wild F. tularensis strain pro toto resulted in the death of two out of five hares. Plasma chemistry profiles were examined on days 2 to 35 post-infection. When compared to controls, the total protein, urea, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were increased, while albumin, glucose and amylase were decreased. Both uric and ascorbic acids and glutathione dropped on day 2 and then increased significantly on days 6 to 12 and 6 to 14 post-inoculation, respectively. There was a two-fold increase in lipid peroxidation on days 4 to 8 post-inoculation. Conclusions Contrary to all expectations, the present study demonstrates that the European brown hare shows relatively low susceptibility to tularemia. Therefore, the circumstances of tularemia in hares under natural conditions should be further studied. PMID:21232117

  11. Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  12. Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers.

    PubMed

    Cer, Regina Z; Herrera-Galeano, J Enrique; Frey, Kenneth G; Schully, Kevin L; Luu, Truong V; Pesce, John; Mokashi, Vishwesh P; Keane-Myers, Andrea M; Bishop-Lilly, Kimberly A

    2017-01-01

    Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5 α . RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis -exposed hPBMCs.

  13. Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers

    PubMed Central

    Herrera-Galeano, J. Enrique; Frey, Kenneth G.; Schully, Kevin L.; Luu, Truong V.; Pesce, John; Mokashi, Vishwesh P.; Keane-Myers, Andrea M.; Bishop-Lilly, Kimberly A.

    2017-01-01

    Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5α. RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis-exposed hPBMCs. PMID:28791299

  14. Genome Sequencing of 18 Francisella Strains To Aid in Assay Development and Testing

    DOE PAGES

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.; ...

    2015-04-30

    Francisella tularensis is a highly infectious bacterium that has the potential of causing high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

  15. Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

    PubMed

    Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed; Miller, Barbara A; Melendez, J Andrés

    2016-02-19

    As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Subburaman, P.; Austin, B.P.; Shaw, G.X.

    2010-11-03

    Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.

  17. Long-range dispersal moved Francisella tularensis into Western Europe from the East

    PubMed Central

    Dwibedi, Chinmay; Birdsell, Dawn; Lärkeryd, Adrian; Myrtennäs, Kerstin; Öhrman, Caroline; Nilsson, Elin; Karlsson, Edvin; Hochhalter, Christian; Rivera, Andrew; Maltinsky, Sara; Bayer, Brittany; Keim, Paul; Scholz, Holger C.; Tomaso, Herbert; Wittwer, Matthias; Beuret, Christian; Schuerch, Nadia; Pilo, Paola; Hernández Pérez, Marta; Rodriguez-Lazaro, David; Escudero, Raquel; Anda, Pedro; Forsman, Mats; Wagner, David M.; Larsson, Pär

    2016-01-01

    For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species. PMID:28348839

  18. Long-range dispersal moved Francisella tularensis into Western Europe from the East.

    PubMed

    Dwibedi, Chinmay; Birdsell, Dawn; Lärkeryd, Adrian; Myrtennäs, Kerstin; Öhrman, Caroline; Nilsson, Elin; Karlsson, Edvin; Hochhalter, Christian; Rivera, Andrew; Maltinsky, Sara; Bayer, Brittany; Keim, Paul; Scholz, Holger C; Tomaso, Herbert; Wittwer, Matthias; Beuret, Christian; Schuerch, Nadia; Pilo, Paola; Hernández Pérez, Marta; Rodriguez-Lazaro, David; Escudero, Raquel; Anda, Pedro; Forsman, Mats; Wagner, David M; Larsson, Pär; Johansson, Anders

    2016-12-01

    For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis , the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains ( n =205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains ( n =195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.

  19. Modeling early events in Francisella tularensis pathogenesis.

    PubMed

    Gillard, Joseph J; Laws, Thomas R; Lythe, Grant; Molina-París, Carmen

    2014-01-01

    Computational models can provide valuable insights into the mechanisms of infection and be used as investigative tools to support development of medical treatments. We develop a stochastic, within-host, computational model of the infection process in the BALB/c mouse, following inhalational exposure to Francisella tularensis SCHU S4. The model is mechanistic and governed by a small number of experimentally verifiable parameters. Given an initial dose, the model generates bacterial load profiles corresponding to those produced experimentally, with a doubling time of approximately 5 h during the first 48 h of infection. Analytical approximations for the mean number of bacteria in phagosomes and cytosols for the first 24 h post-infection are derived and used to verify the stochastic model. In our description of the dynamics of macrophage infection, the number of bacteria released per rupturing macrophage is a geometrically-distributed random variable. When combined with doubling time, this provides a distribution for the time taken for infected macrophages to rupture and release their intracellular bacteria. The mean and variance of these distributions are determined by model parameters with a precise biological interpretation, providing new mechanistic insights into the determinants of immune and bacterial kinetics. Insights into the dynamics of macrophage suppression and activation gained by the model can be used to explore the potential benefits of interventions that stimulate macrophage activation.

  20. Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis.

    PubMed Central

    Waag, D M; Galloway, A; Sandstrom, G; Bolt, C R; England, M J; Nelson, G O; Williams, J C

    1992-01-01

    Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. We evaluated a new lot of live F. tularensis vaccine for its immunogenicity in human volunteers. Scarification vaccination induced humoral and cell-mediated immune responses. Indications of a positive immune response after vaccination included an increase in specific antibody levels, which were measured by enzyme-linked immunosorbent and immunoblot assays, and the ability of peripheral blood lymphocytes to respond to whole F. tularensis bacteria as recall antigens. Vaccination caused a significant rise (P less than 0.05) in immunoglobulin A (IgA), IgG, and IgM titers. Lymphocyte stimulation indices were significantly increased (P less than 0.01) in vaccinees 14 days after vaccination. These data verify that this new lot of live F. tularensis vaccine is immunogenic. Images PMID:1400988

  1. Insights to Genetic Characterization Tools for Epidemiological Tracking of Francisella tularensis in Sweden

    PubMed Central

    Wahab, Tara; Birdsell, Dawn N.; Hjertqvist, Marika; Mitchell, Cedar L.; Wagner, David M.; Keim, Paul S.; Hedenström, Ingela; Löfdahl, Sven

    2014-01-01

    Tularaemia, caused by the bacterium Francisella tularensis, is endemic in Sweden and is poorly understood. The aim of this study was to evaluate the effectiveness of three different genetic typing systems to link a genetic type to the source and place of tularemia infection in Sweden. Canonical single nucleotide polymorphisms (canSNPs), MLVA including five variable number of tandem repeat loci and PmeI-PFGE were tested on 127 F. tularensis positive specimens collected from Swedish case-patients. All three typing methods identified two major genetic groups with near-perfect agreement. Higher genetic resolution was obtained with canSNP and MLVA compared to PFGE; F. tularensis samples were first assigned into ten phylogroups based on canSNPs followed by 33 unique MLVA types. Phylogroups were geographically analysed to reveal complex phylogeographic patterns in Sweden. The extensive phylogenetic diversity found within individual counties posed a challenge to linking specific genetic types with specific geographic locations. Despite this, a single phylogroup (B.22), defined by a SNP marker specific to a lone Swedish sequenced strain, did link genetic type with a likely geographic place. This result suggests that SNP markers, highly specific to a particular reference genome, may be found most frequently among samples recovered from the same location where the reference genome originated. This insight compels us to consider whole-genome sequencing (WGS) as the appropriate tool for effectively linking specific genetic type to geography. Comparing the WGS of an unknown sample to WGS databases of archived Swedish strains maximizes the likelihood of revealing those rare geographically informative SNPs. PMID:25401326

  2. In vitro and in vivo evaluation of fluoroquinolone resistance associated with DNA gyrase mutations in Francisella tularensis, including in tularaemia patients with treatment failure.

    PubMed

    Sutera, V; Hoarau, G; Renesto, P; Caspar, Y; Maurin, M

    2017-09-01

    Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis, but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone-resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and gyrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ-susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  3. The phylogeographic pattern of Francisella tularensis in Sweden indicates a Scandinavian origin of Eurosiberian tularaemia.

    PubMed

    Karlsson, Edvin; Svensson, Kerstin; Lindgren, Petter; Byström, Mona; Sjödin, Andreas; Forsman, Mats; Johansson, Anders

    2013-02-01

    Previous studies of the causative agent of tularaemia, Francisella tularensis have identified phylogeographic patterns suggestive of environmental maintenance reservoirs. To investigate the phylogeography of tularaemia in Sweden, we selected 163 clinical isolates obtained during 1995-2009 in 10 counties and sequenced one isolate's genome to identify new genetic markers. An improved typing scheme based on two indels and nine SNPs was developed using hydrolysis or TaqMan MGB probe assays. The results showed that much of the known global genetic diversity of F. tularensis subsp. holarctica is present in Sweden. Thirteen of the 163 isolates belonged to a new genetic group that is basal to all other known members of the major genetic clade B.I, which is spread across the Eurosiberian region. One hundred and twenty-five of the 163 Swedish isolates belonged to B.I, but individual clades' frequencies differed from county to county (P < 0.001). Subsequent analyses revealed a correlation between genotype variation over time and recurrent outbreaks at specific places, supporting the 'maintenance reservoir' environmental maintenance hypothesis. Most importantly, the findings reveal the presence of diverse source populations of F. tularensis subsp. holarctica in Sweden and suggest a historical spread of the disease from Scandinavia to other parts of Eurosiberia. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  4. Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

    PubMed Central

    Lindgren, Helena

    2015-01-01

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo. PMID:26503658

  5. Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis.

    PubMed

    Lindgren, Helena; Sjöstedt, Anders

    2016-01-01

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

    PubMed

    Parthasarathy, N; Saksena, R; Kováč, P; Deshazer, D; Peacock, S J; Wuthiekanun, V; Heine, H S; Friedlander, A M; Cote, C K; Welkos, S L; Adamovicz, J J; Bavari, S; Waag, D M

    2008-11-03

    We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

  7. Lipids Derived from Virulent Francisella tularensis Broadly Inhibit Pulmonary Inflammation via Toll-Like Receptor 2 and Peroxisome Proliferator-Activated Receptor α

    PubMed Central

    Crane, Deborah D.; Ireland, Robin; Alinger, Joshua B.; Small, Pamela

    2013-01-01

    Francisella tularensis is a Gram-negative facultative intracellular pathogen that causes an acute lethal respiratory disease in humans. The heightened virulence of the pathogen is linked to its unique ability to inhibit Toll-like receptor (TLR)-mediated inflammatory responses. The bacterial component and mechanism of this inhibition are unknown. Here we show that lipids isolated from virulent but not attenuated strains of F. tularensis are not detected by host cells, inhibit production of proinflammatory cytokines by primary macrophages in response to known TLR ligands, and suppress neutrophil recruitment in vivo. We further show that lipid-mediated inhibition of inflammation is dependent on TLR2, MyD88, and the nuclear hormone and fatty acid receptor peroxisome proliferator-activated receptor α (PPARα). Pathogen lipid-mediated interference with inflammatory responses through the engagement of TLR2 and PPARα represents a novel manipulation of host signaling pathways consistent with the ability of highly virulent F. tularensis to efficiently evade host immune responses. PMID:23925884

  8. Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages

    PubMed Central

    Woolard, Matthew D.; Barrigan, Lydia M.; Fuller, James R.; Buntzman, Adam S.; Bryan, Joshua; Manoil, Colin; Kawula, Thomas H.; Frelinger, Jeffrey A.

    2013-01-01

    Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E2 (PGE2). Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial) that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain F. tularensis subspecies novicida U112 (U112) two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI). Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used in the host for PGE2

  9. RipA, a Cytoplasmic Membrane Protein Conserved among Francisella Species, Is Required for Intracellular Survival▿

    PubMed Central

    Fuller, James R.; Craven, Robin R.; Hall, Joshua D.; Kijek, Todd M.; Taft-Benz, Sharon; Kawula, Thomas H.

    2008-01-01

    Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ΔripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ΔripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ΔripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence. PMID:18765722

  10. Seroprevalence for Coxiella burnetii, Francisella tularensis, Brucella abortus and Brucella melitensis in Austrian adults: a cross-sectional survey among military personnel and civilians.

    PubMed

    Tobudic, Selma; Nedomansky, Klara; Poeppl, Wolfgang; Müller, Maria; Faas, Angelus; Mooseder, Gerhard; Allerberger, Franz; Stanek, Gerold; Burgmann, Heinz

    2014-04-01

    The prevalence of Coxiella burnetii, Francisella tularensis, Brucella abortus, and Brucella melitensis infections in Austria and the exposure risk of military personnel were assessed in an exploratory nationwide cross-sectional seroprevalence survey in 526 healthy adult individuals, 222 of which were soldiers and 304 were civilians. Screening for IgA/IgG antibodies to C. burnetii (Phase I) and IgG/IgM antibodies to C. burnetii (Phase II), and to F. tularensis was done with commercial enzyme-linked immunosorbent assays. To detect antibodies against B. abortus and B. melitensis, an in-house complement fixation test was used. Overall, 11 individuals (2.0%) showed antibodies to C. burnetii, 3 individuals (0.5%) were seropositive for F. tularensis, and one (0.3%) individual was borderline positive. All individuals positive or borderline for F. tularensis tested negative for antibodies against C. burnetii. All individuals tested negative for antibodies against B. melitensis/B. abortus. There were no significant differences between the seroprevalence of C. burnetii and F. tularensis among military personnel and civilians. Our data demonstrate serological evidence of a low rate of exposure to C. burnetii and F. tularensis among the Austrian adult population and military personnel. Copyright © 2014 Elsevier GmbH. All rights reserved.

  11. Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen1

    PubMed Central

    Clay, Corey D.; Soni, Shilpa; Gunn, John S.; Schlesinger, Larry S.

    2009-01-01

    The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag. PMID:18832715

  12. Temporal manipulation of mitochondrial function by virulent Francisella tularensis to limit inflammation and control cell death.

    PubMed

    Jessop, Forrest; Schwarz, Benjamin; Heitmann, Emily; Buntyn, Robert; Wehrly, Tara; Bosio, Catharine M

    2018-05-14

    Francisella tularensis ssp tularensis (Ftt) is a highly pathogenic intracellular bacterium that suppresses host inflammation by impairing the metabolic shift from oxidative phosphorylation to glycolysis. Decreased mitochondrial metabolism is central to initiating a metabolic shift to glycolysis and regulating inflammation, but Ftt manipulation of host mitochondrial function has not been explored. We demonstrate using extracellular flux analysis that Ftt infection initially improves host macrophage mitochondrial bioenergetics in a capsule dependent manner. Enhancement of mitochondrial function by Ftt allowed for modest replication and inhibition of apoptosis early after infection. However, using live cell imaging we found that Ftt facilitated the loss of mitochondrial function at later time points during infection in a capsule independent fashion. This loss of function was paired with oncosis and rapid bacterial replication. Inhibition of oncosis reduced intracellular bacteria numbers, underscoring the requirement for this process during Ftt infection. These findings establish that temporal mitochondrial manipulation by Ftt is critical for maintenance of a non-inflammatory environment and subsequently aids in optimal replication and dissemination of this pathogenic organism. Copyright © 2018 American Society for Microbiology.

  13. B-Cell Epitopes in GroEL of Francisella tularensis

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Madico, Guillermo; Li, Sheng; Yang, Chiou-Ying; Perkins, Hillary M.; Sompuram, Seshi R.; Kodela, Vani; Liu, Tong; Morris, Timothy; Wang, Daphne; Roche, Marly I.; Seaton, Barbara A.; Sharon, Jacqueline

    2014-01-01

    The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL. PMID:24968190

  14. Five cases of vector-borne Francisella tularensis holarctica infections in south-western Germany and genetic diversity.

    PubMed

    Borde, Johannes P; Zange, Sabine; Antwerpen, Markus H; Georgi, Enrico; von Buttlar, Heiner; Kern, Winfried V; Rieg, Siegbert

    2017-08-01

    Tularemia is a rare zoonotic disease in Germany. Francisella tularensis has been isolated previously from ticks in southern Germany underscoring the importance of ticks (Ixodes ricinus) in tularemia transmission, but there have been only few reports from this region with single cases or small case series of tick-borne transmissions of tularemia. We report five cases of non-game animal associated tularemia diagnosed from 2010 to 2016 in southwestern Germany - Baden-Wuerttemberg. Our case series and molecular typing (MLVA) results add published clinical experience to this underdiagnosed disease and consolidate previous findings regarding tick-borne transmission of tularemia and phylogenetic diversity in Germany. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E.

    Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. In the bacterial FASII pathway we found it a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here, we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promisingmore » low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, the improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.« less

  16. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

    DOE PAGES

    Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E.; ...

    2015-01-29

    Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. In the bacterial FASII pathway we found it a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here, we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promisingmore » low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, the improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.« less

  17. Mouse models of aerosol-acquired tularemia caused by Francisella tularensis types A and B.

    PubMed

    Fritz, David L; England, Marilyn J; Miller, Lynda; Waag, David M

    2014-10-01

    After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.

  18. A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia

    PubMed Central

    Chance, Taylor; Toothman, Ronald G.; Nuss, Jonathan E.; Raymond, Jo Lynne; Biot, Fabrice V.; Demons, Samandra; Miller, Lynda; Halasohoris, Stephanie; Mou, Sherry; Koroleva, Galina; Lovett, Sean; Palacios, Gustavo; Vietri, Nicholas J.; Worsham, Patricia L.; Cote, Christopher K.; Kijek, Todd M.; Bozue, Joel A.

    2017-01-01

    Francisella tularensis, a gram–negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures. PMID:28328947

  19. Comparison of virulence of Francisella tularensis ssp. holarctica genotypes B.12 and B.FTNF002-00.

    PubMed

    Kreizinger, Zsuzsa; Erdélyi, Károly; Felde, Orsolya; Fabbi, Massimo; Sulyok, Kinga M; Magyar, Tibor; Gyuranecz, Miklós

    2017-02-10

    Two main genetic groups (B.12 and B.FTNF002-00) of Francisella tularensis ssp. holarctica are endemic in Europe. The B.FTNF002-00 group proved to be dominant in Western European countries, while strains of the B.12 group were isolated mainly in Northern, Central and Eastern Europe. The clinical course of tularemia in the European brown hare (Lepus europaeus) also shows distinct patterns according to the geographical area. Acute course of the disease is observed in hares in Western European countries, while signs of sub-acute or chronic infection are more frequently detected in the eastern part of the continent. The aim of the present study was to examine whether there is any difference in the virulence of the strains belonging to the B.FTNF002-00 and B.12 genetic clades. Experimental infection of Fischer 344 rats was performed by intra-peritoneal injection of three dilutions of a Hungarian (B.12 genotype) and an Italian (B.FTNF002-00 genotype) F. tularensis ssp. holarctica strain. Moderate difference was observed in the virulence of the two genotypes. Significant differences were observed in total weight loss values and scores of clinical signs between the two genotypes with more rats succumbing to tularemia in groups infected with the B.FTNF002-00 genotype. Results of the experimental infection are consistent with previous clinical observations and pathological studies suggesting that F. tularensis ssp. holarctica genotype B.FTNF002-00 has higher pathogenic potential than the B.12 genotype.

  20. Historical distribution and host-vector diversity of Francisella tularensis, the causative agent of tularemia, in Ukraine.

    PubMed

    Hightower, Jake; Kracalik, Ian T; Vydayko, Nataliya; Goodin, Douglas; Glass, Gregory; Blackburn, Jason K

    2014-10-16

    Francisella tularensis, the causative agent of tularemia, is a zoonotic agent that remains across much of the northern hemisphere, where it exists in enzootic cycles. In Ukraine, tularemia has a long history that suggests a need for sustained surveillance in natural foci. To better characterize the host-vector diversity and spatial distribution of tularemia, we analyzed historical data from field collections carried out from 1941 to 2008. We analyzed the spatial-temporal distribution of bacterial isolates collected from field samples. Isolates were characterized by source and dominant land cover type. To identify environmental persistence and spatial variation in the source of isolation, we used the space-time permutation and multinomial models in SaTScan. A total of 3,086 positive isolates were taken from 1,084 geographic locations. Isolation of F. tularensis was more frequent among arthropods [n = 2,045 (66.3%)] followed by mammals [n = 619 (20.1%)], water [n = 393 (12.7%)], and farm produce [n = 29 (0.94%)], respectively. Four areas of persistent bacterial isolation were identified. Water and farm produce as sources of bacterial isolation were clustered. Our findings confirm the presence of long-standing natural foci of F. tularensis in Ukraine. Given the history of tularemia as well as its environmental persistence there exists a possibility of (re)emergence in human populations. Heterogeneity in the distribution of tularemia isolate recovery related to land cover type supports the theory of natural nidality and clusters identify areas to target potential sources of the pathogen and improve surveillance.

  1. A Molecular Survey for Francisella tularensis and Rickettsia spp. in Haemaphysalis leporispalustris (Acari: Ixodidae) in Northern California.

    PubMed

    Roth, Tara; Lane, Robert S; Foley, Janet

    2017-03-01

    Francisella tularensis and Rickettsia spp. have been cultured from Haemaphysalis leporispalustris Packard, but their prevalence in this tick has not been determined using modern molecular methods. We collected H. leporispalustris by flagging vegetation and leaf litter and from lagomorphs (Lepus californicus Gray and Sylvilagus bachmani (Waterhouse)) in northern California. Francisella tularensis DNA was not detected in any of 1,030 ticks tested by polymerase chain reaction (PCR), whereas 0.4% of larvae tested in pools, 0 of 117 individual nymphs, and 2.3% of 164 adult ticks were PCR-positive for Rickettsia spp. Positive sites were Laurel Canyon Trail in Tilden Regional Park in Alameda Contra Costa County, with a Rickettsia spp. prevalence of 0.6% in 2009, and Hopland Research and Extension Center in Mendocino County, with a prevalence of 4.2% in 1988. DNA sequencing revealed R. felis, the agent of cat-flea typhus, in two larval pools from shaded California bay and live oak leaf litter in Contra Costa County and one adult tick from a L. californicus in chaparral in Mendocino County. The R. felis in unfed, questing larvae demonstrates that H. leporispalustris can transmit this rickettsia transovarially. Although R. felis is increasingly found in diverse arthropods and geographical regions, prior literature suggests a typical epidemiological cycle involving mesocarnivores and the cat flea, Ctenocephalides felis. To our knowledge, this is the first report of R. felis in H. leporispalustris. Natural infection and transovarial transmission of this pathogen in the tick indicate the existence of a previously undocumented wild-lands transmission cycle that may intersect mesocarnivore-reservoired cycles and collectively affect human health risk. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Characterization of Francisella tularensis Schu S4 mutants identified from a transposon library screened for O-antigen and capsule deficiencies

    PubMed Central

    Rasmussen, Jed A.; Fletcher, Joshua R.; Long, Matthew E.; Allen, Lee-Ann H.; Jones, Bradley D.

    2015-01-01

    The lipopolysaccharide (LPS) and O-antigen polysaccharide capsule structures of Francisella tularensis play significant roles in helping these highly virulent bacteria avoid detection within a host. We previously created pools of F. tularensis mutants that we screened to identify strains that were not reactive to a monoclonal antibody to the O-antigen capsule. To follow up previously published work, we characterize further seven of the F. tularensis Schu S4 mutant strains identified by our screen. These F. tularensis strains carry the following transposon mutations: FTT0846::Tn5, hemH::Tn5, wbtA::Tn5, wzy::Tn5, FTT0673p/prsA::Tn5, manB::Tn5, or dnaJ::Tn5. Each of these strains displayed sensitivity to human serum, to varying degrees, when compared to wild-type F. tularensis Schu S4. By Western blot, only FTT0846::Tn5, wbtA::Tn5, wzy::Tn5, and manB::Tn5 strains did not react to the capsule and LPS O-antigen antibody 11B7, although the wzy::Tn5 strain did have a single O-antigen reactive band that was detected by the FB11 monoclonal antibody. Of these strains, manB::Tn5 and FTT0846 appear to have LPS core truncations, whereas wbtA::Tn5 and wzy::Tn5 had LPS core structures that are similar to the parent F. tularensis Schu S4. These strains were also shown to have poor growth within human monocyte derived macrophages (MDMs) and bone marrow derived macrophages (BMDMs). We examined the virulence of these strains in mice, following intranasal challenge, and found that each was attenuated compared to wild type Schu S4. Our results provide additional strong evidence that LPS and/or capsule are F. tularensis virulence factors that most likely function by providing a stealth shield that prevents the host immune system from detecting this potent pathogen. PMID:25999917

  3. Isolation of Francisella tularensis and Yersinia pestis from Blood Cultures by Plasma Purification and Immunomagnetic Separation Accelerates Antibiotic Susceptibility Determination

    PubMed Central

    Aloni-Grinstein, Ronit; Schuster, Ofir; Yitzhaki, Shmuel; Aftalion, Moshe; Maoz, Sharon; Steinberger-Levy, Ida; Ber, Raphael

    2017-01-01

    The early symptoms of tularemia and plague, which are caused by Francisella tularensis and Yersinia pestis infection, respectively, are common to other illnesses, resulting in a low index of suspicion among clinicians. Moreover, because these diseases can be treated only with antibiotics, rapid isolation of the bacteria and antibiotic susceptibility testing (AST) are preferable. Blood cultures of patients may serve as a source for bacteria isolation. However, due to the slow growth rates of F. tularensis and Y. pestis on solid media, isolation by plating blood culture samples on proper agar plates may require several days. Thus, improving the isolation procedure prior to antibiotic susceptibility determination is a major clinically relevant need. In this study, we developed a rapid, selective procedure for the isolation of F. tularensis and Y. pestis from blood cultures. We examined drop-plating and plasma purification followed by immunomagnetic separation (IMS) as alternative isolation methods. We determined that replacing the classical isolation method with drop-plating is advantageous with respect to time at the expense of specificity. Hence, we also examined isolation by IMS. Sub-localization of F. tularensis within blood cultures of infected mice has revealed that the majority of the bacteria are located within the extracellular fraction, in the plasma. Y. pestis also resides within the plasma. Therefore, the plasma fraction was isolated from blood cultures and subjected to an IMS procedure using polyclonal anti-F. tularensis live vaccine strain (LVS) or anti-Y. pestis antibodies conjugated to 50-nm nano-beads. The time required to reach an inoculum of sufficient bacteria for AST was shortest when using the plasma and IMSs for both bacteria, saving up to 2 days of incubation for F. tularensis and 1 day for Y. pestis. Our isolation procedure provides a proof of concept for the clinical relevance of rapid isolation for AST from F. tularensis- and Y. pestis

  4. Exposure of Laboratory Workers to Francisella tularensis despite a Bioterrorism Procedure

    PubMed Central

    Shapiro, Daniel S.; Schwartz, Donald R.

    2002-01-01

    A rapidly fatal case of pulmonary tularemia in a 43-year-old man who was transferred to a tertiary care facility is presented. The microbiology laboratory and autopsy services were not notified of the clinical suspicion of tularemia by the service caring for the patient. Despite having a laboratory bioterrorism procedure in place and adhering to established laboratory protocol, 12 microbiology laboratory employees were exposed to Francisella tularensis and the identification of the organism was delayed due to lack of notification of the laboratory of the clinical suspicion of tularemia. A total of 11 microbiology employees and two persons involved in performing the patient's autopsy received prophylactic doxycycline due to concerns of transmission. None of them developed signs or symptoms of tularemia. One microbiology laboratory employee was pregnant and declined prophylactic antibiotics. As a result of this event, the microbiology laboratory has incorporated flow charts directly into the bench procedures for several highly infectious agents that may be agents of bioterrorism. This should permit more rapid recognition of an isolate for referral to a Level B laboratory for definitive identification and should improve laboratory safety. PMID:12037110

  5. Review of Processing and Analytical Methods for Francisella ...

    EPA Pesticide Factsheets

    Journal Article The etiological agent of tularemia, Francisella tularensis, is a resilient organism within the environment and can be acquired many ways (infectious aerosols and dust, contaminated food and water, infected carcasses, and arthropod bites). However, isolating F. tularensis from environmental samples can be challenging due to its nutritionally fastidious and slow-growing nature. In order to determine the current state of the science regarding available processing and analytical methods for detection and recovery of F. tularensis from water and soil matrices, a review of the literature was conducted. During the review, analysis via culture, immunoassays, and genomic identification were the most commonly found methods for F. tularensis detection within environmental samples. Other methods included combined culture and genomic analysis for rapid quantification of viable microorganisms and use of one assay to identify multiple pathogens from a single sample. Gaps in the literature that were identified during this review suggest that further work to integrate culture and genomic identification would advance our ability to detect and to assess the viability of Francisella spp. The optimization of DNA extraction, whole genome amplification with inhibition-resistant polymerases, and multiagent microarray detection would also advance biothreat detection.

  6. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis.

    PubMed

    Hua, Fei; Zhang, Pingping; Zhang, Fuli; Zhao, Yong; Li, Chunfeng; Sun, Chongyun; Wang, Xiaochen; Yang, Ruifu; Wang, Chengbin; Yu, Ailian; Zhou, Lei

    2015-11-26

    Francisella tularensis is a potential biowarfare/bioterrorism agent and zoonotic pathogen that causes tularemia; thus, surveillance of F. tularensis and first-level emergency response using point-of-care testing (POCT) are essential. The UPT-LF POCT assay was established to quantitatively detect F. tularensis within 15 min, and the sensitivity of the assay was 10(4) CFU · mL(-1) (100 CFU/test). The linear quantitative range covered five orders of magnitude, and the coefficients of variation were less than 10%. Except Shigella dysenteriae, UPT-LF showed excellent specificity to four strains that are also potential biowarfare/bioterrorism agents and 13 food-borne pathogenic strains. Samples with pH 2-13, high ion strengths (≥ 2 mol · L(-1) solution of KCl and NaCl), high viscosities (≤ 50 mg · mL(-1) PEG20000 or ≥ 20% glycerol), and high concentrations of biomacromolecules (≥ 400 mg · mL(-1) bovine serum albumin or ≥ 80 mg · mL(-1) casein) showed little influence on the assay. For practical utilization, the tolerance limits for seven powders and eight viscera were determined, and operation errors of liquid measurement demonstrated a minor influence on the strip. Ftu-UPT-LF is a candidate POCT method because of its excellent sensitivity, specificity, and stability in complex samples, as well as low operation error.

  7. Geographic Differentiation of Francisella Tularensis using Molecular Methods

    DTIC Science & Technology

    2006-05-01

    most often through direct exposure to infected animals or by bites from infected arthropod vectors. Recently, terrestrial and aquatic life cycles have...virulent in humans. F. tularensis species have been isolated throughout the Northern Hemisphere, but subsp. tularensis isolates appear restricted to North...John Wise for administering Pathogene-server access. From Northern Airzona University, I am grateful to Dr. Paul Keim, Miles Stanley, Julia Rhodes, Dr

  8. Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species

    PubMed Central

    Tlapák, Hana; Köppen, Kristin; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus

    2018-01-01

    We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species. PMID:29594068

  9. Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.

    PubMed

    Tlapák, Hana; Köppen, Kristin; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus

    2018-01-01

    We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 ( F. tularensis subsp. novicida -like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val ( Francisella Integration Vector-tRNA Val -specific), using the attL/R- sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli . The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp . where FIV-Val stably integrated site specifically into the tRNA Val gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

  10. Establishment of Three Francisella Infections in Zebrafish Embryos at Different Temperatures

    PubMed Central

    Brudal, Espen; Ulanova, Lilia S.; O. Lampe, Elisabeth; Rishovd, Anne-Lise; Winther-Larsen, Hanne C.

    2014-01-01

    Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells. PMID:24614659

  11. The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis Subspecies tularensis Strain SCHU S4.

    PubMed

    Hutt, Julie A; Lovchik, Julie A; Dekonenko, Alexander; Hahn, Andrew C; Wu, Terry H

    2017-02-01

    The inbred Fischer 344 rat is being evaluated for testing novel vaccines and therapeutics against pneumonic tularemia. Although primary pneumonic tularemia in humans typically occurs by inhalation of aerosolized bacteria, the rat model has relied on intratracheal inoculation of organisms because of safety and equipment issues. We now report the natural history of pneumonic tularemia in female Fischer 344 rats after nose-only inhalational exposure to lethal doses of aerosolized Francisella tularensis subspecies tularensis, strain SCHU S4. Our results are consistent with initial uptake of aerosolized SCHU S4 from the nasal cavity, lungs, and possibly the gastrointestinal tract. Bacteremia with hematogenous dissemination was first detected 2 days after exposure. Shortly thereafter, the infected rats exhibited fever, tachypnea, and hypertension that persisted for 24 to 36 hours and then rapidly decreased as animals succumbed to infection between days 5 and 8 after exposure. Tachycardia was observed briefly, but only after the core body temperature and blood pressure began to decrease as the animals were near death. Initial neutrophilic and histiocytic inflammation in affected tissues became progressively more fibrinous and necrotizing over time. At death, as many as 10 10 colony-forming units were found in the lungs, spleen, and liver. Death was attributed to sepsis and disseminated intravascular coagulation. Overall, the pathogenesis of pneumonic tularemia in the female F344 rat model appears to replicate the disease in humans. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  12. AR-13, a Celecoxib Derivative, Directly Kills Francisella In Vitro and Aids Clearance and Mouse Survival In Vivo

    PubMed Central

    Hoang, Ky V.; Adcox, Haley E.; Fitch, James R.; Gordon, David M.; Curry, Heather M.; Schlesinger, Larry S.; White, Peter; Gunn, John S.

    2017-01-01

    Francisella tularensis (F. tularensis) is the causative agent of tularemia and is classified as a Tier 1 select agent. No licensed vaccine is currently available in the United States and treatment of tularemia is confined to few antibiotics. In this study, we demonstrate that AR-13, a derivative of the cyclooxygenase-2 inhibitor celecoxib, exhibits direct in vitro bactericidal killing activity against Francisella including a type A strain of F. tularensis (SchuS4) and the live vaccine strain (LVS), as well as toward the intracellular proliferation of LVS in macrophages, without causing significant host cell toxicity. Identification of an AR-13-resistant isolate indicates that this compound has an intracellular target(s) and that efflux pumps can mediate AR-13 resistance. In the mouse model of tularemia, AR-13 treatment protected 50% of the mice from lethal LVS infection and prolonged survival time from a lethal dose of F. tularensis SchuS4. Combination of AR-13 with a sub-optimal dose of gentamicin protected 60% of F. tularensis SchuS4-infected mice from death. Taken together, these data support the translational potential of AR-13 as a lead compound for the further development of new anti-Francisella agents. PMID:28955308

  13. AR-13, a Celecoxib Derivative, Directly Kills Francisella In Vitro and Aids Clearance and Mouse Survival In Vivo.

    PubMed

    Hoang, Ky V; Adcox, Haley E; Fitch, James R; Gordon, David M; Curry, Heather M; Schlesinger, Larry S; White, Peter; Gunn, John S

    2017-01-01

    Francisella tularensis ( F. tularensis ) is the causative agent of tularemia and is classified as a Tier 1 select agent. No licensed vaccine is currently available in the United States and treatment of tularemia is confined to few antibiotics. In this study, we demonstrate that AR-13, a derivative of the cyclooxygenase-2 inhibitor celecoxib, exhibits direct in vitro bactericidal killing activity against Francisella including a type A strain of F. tularensis (SchuS4) and the live vaccine strain (LVS), as well as toward the intracellular proliferation of LVS in macrophages, without causing significant host cell toxicity. Identification of an AR-13-resistant isolate indicates that this compound has an intracellular target(s) and that efflux pumps can mediate AR-13 resistance. In the mouse model of tularemia, AR-13 treatment protected 50% of the mice from lethal LVS infection and prolonged survival time from a lethal dose of F. tularensis SchuS4. Combination of AR-13 with a sub-optimal dose of gentamicin protected 60% of F. tularensis SchuS4-infected mice from death. Taken together, these data support the translational potential of AR-13 as a lead compound for the further development of new anti- Francisella agents.

  14. Francisella tularensis SchuS4 and SchuS4 lipids inhibit IL-12p40 in primary human dendritic cells by inhibition of IRF1 and IRF8*

    PubMed Central

    Ireland, Robin; Wang, Rong; Alinger, Joshua B.; Small, Pamela; Bosio, Catharine M.

    2013-01-01

    Induction of innate immunity is essential for host survival of infection. Evasion and inhibition of innate immunity is a strategy used by pathogens, such as the highly virulent bacterium Francisella tularensis, to ensure their replication and transmission. The mechanism and bacterial components responsible for this suppression of innate immunity by F. tularensis are not defined. Here, we demonstrate that lipids enriched from virulent F. tularensis strain SchuS4, but not attenuated Live Vaccine Strain (LVS), inhibit inflammatory responses in vitro and in vivo. Suppression of inflammatory responses is associated with IκBα independent inhibition of NF-κBp65 activation and selective inhibition of activation of Interferon Regulatory Factors (IRFs). Interference with NF-κBp65 and IRFs is also observed following infection with viable SchuS4. Together these data provide novel insight as to how highly virulent bacteria selectively modulate the host to interfere innate immune responses required for survival of infection. PMID:23817430

  15. Benzimidazole-Based Antibacterial Agents Against F. tularensis

    PubMed Central

    Kumar, Kunal; Awasthi, Divya; Lee, Seung-Yub; Cummings, Jason E.; Knudson, Susan E.; Slayden, Richard A.; Ojima, Iwao

    2013-01-01

    Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles were screened against F. tularensis LVS strain. In a preliminary screening assay, remarkably, 23 of 2,5,6- and 2,5,7-trisubstituted benzimidazoles showed excellent activity exhibiting greater than 90 % growth inhibition at 1 µg/mL. Among those hits, 21 compounds showed MIC90 values in the range of 0.35–48.6 µg/mL after accurate MIC determination. In ex-vivo efficacy assays, four of these compounds exhibited 2–3 Log reduction in colony forming units (CFU) per mL at concentrations of 10 and 50 µg/mL. PMID:23623254

  16. Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition

    PubMed Central

    Kinkead, Lauren C.; Whitmore, Laura C.; McCracken, Jenna M.; Fletcher, Joshua R.; Ketelsen, Brandi B.; Kaufman, Justin W.; Jones, Bradley D.; Weiss, David S.; Barker, Jason H.

    2017-01-01

    Abstract Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14C] acetate‐labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose‐dependent neutrophil apoptosis inhibition via a TLR2‐dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function. PMID:29063667

  17. Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production

    PubMed Central

    Dean, Scott N.; Chung, Myung-Chul

    2015-01-01

    In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation. PMID:26231649

  18. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Seiner, Derrick R.; Colburn, Heather A.; Baird, Cheryl L.

    2013-04-29

    To evaluate the sensitivity and specificity of the Idaho Technologies FilmArray® Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft), and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results: DNA samples from Ba, Ft and Yp strains and near-neighbors, and live Ba spores were analyzed using the Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA suggest a limit of detection of 250 genome equivalents (GEs) per sample. Furthermore, the correct call of Ft, Yp or Bacillus species was made in 63 of 72 samplesmore » tested at 25 GE or less. With samples containing 25 Ba Sterne spores, at least one of the two possible Ba markers were identified in all samples tested. We observed no cross-reactivity with near-neighbor DNAs.« less

  19. Phylogeography of Francisella tularensis from Tibet, China: Evidence for an asian origin and radiation of holarctica-type Tularemia.

    PubMed

    Lu, Yongfeng; Yu, Yonghui; Feng, Le; Li, Yanwei; He, Jun; Zhu, Hong; Duan, Qing; Song, Lihua

    2016-07-01

    The geographical origin and radiation of holarctica-type tularemia, which has spread across the northern hemisphere, is open to scientific debate. Here, through phylogenetics, we show that five Tibetan Francisella tularensis isolates subsp. holarctica cluster between basal-positioned Japanese isolates and all other subspecies strains in the world, providing evidence for a previously unknown intermediate lineage next to the Japanese isolates. Importantly, identification of this new intermediate lineage complements current knowledge of tularemia epidemiology, supporting a geographical origin and radiation of the subsp. holarctica in Asia. In addition, thirteen Tibetan isolates belonging to a clade previously found only in North America and Scandinavia, further increases the diversity of holarctica strains in Asia. In summary, this study provides evidence for an Asian origin and radiation of holarctica-type tularemia. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Antibodies to Both Terminal and Internal B-Cell Epitopes of Francisella tularensis O-Polysaccharide Produced by Patients with Tularemia

    PubMed Central

    Lu, Zhaohua; Perkins, Hillary M.

    2014-01-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals. PMID:24351753

  1. Environmental Monitoring and Surveillance of Rodents and Vectors for Francisella tularensis Following Outbreaks of Human Tularemia in Georgia.

    PubMed

    Elashvili, Eka; Kracalik, Ian; Burjanadze, Irma; Datukishvili, Sophio; Chanturia, Gvantsa; Tsertsvadze, Nikoloz; Beridze, Levan; Shavishvili, Merab; Dzneladze, Archil; Grdzelidze, Marina; Imnadze, Paata; Pearson, Andrew; Blackburn, Jason K

    2015-10-01

    Tularemia is a re-emerging bacterial zoonosis, broadly distributed across the northern hemisphere. In Georgia, there is a history of human tularemia outbreaks dating back to the 1940s. In response to outbreaks, health officials initiated long-term field surveillance and environmental monitoring. The objective of our study was to obtain information from 57 years of field surveys to identify species that play a role in the occurrence Francisella tularensis subsp. holarctica in the environment in Georgia. We collected historical data on human outbreaks, field collections, population dynamics of the common vole (Microtus arvalis), and conducted surveys on small mammals and vectors from five regions in Georgia during 1956-2012. Bacterial isolation was conducted using standard culturing techniques, and isolation rates for species were obtained for a subset of years. We used a Spearman rank correlation to test for associations between the density of the common vole and isolation rates. From 1956 through 2012, there were four recorded outbreaks of human tularemia (362 cases). A total of 465 bacterial isolates of F. tularensis subsp. holarctica were obtained from 27 species and environmental samples. The number of isolations was highest in the common vole (M. arvalis; 149 isolates; 32%) and Dermacentor marginatus ticks (132 isolates; 28%); isolation rates ranged between 0-0.91% and 0-0.47%, respectively. Population dynamics of the common vole were not correlated with the isolation rate. Given the history of tularemia re-emergence in Georgia, continued field surveys and environmental monitoring may provide an early indication of outbreak risk in humans. In conclusion, our findings provide evidence of long-standing foci of F. tularensis subsp. holarctica that are likely maintained by the common vole-tick cycle.

  2. Growth conditions and environmental factors impact aerosolization but not virulence of Francisella tularensis infection in mice

    PubMed Central

    Faith, Seth A.; Smith, Le'Kneitah P.; Swatland, Angela S.; Reed, Douglas S.

    2012-01-01

    In refining methodology to develop a mouse model for inhalation of Francisella tularensis, it was noted that both relative humidity and growth media impacted the aerosol concentration of the live vaccine strain (LVS) of F. tularensis. A relative humidity of less than 55% had a negative impact on the spray factor, the ratio between the concentration of LVS in the aerosol and the nebulizer. The spray factor was significantly higher for LVS grown in brain heart infusion (BHI) broth than LVS grown in Mueller–Hinton broth (MHb) or Chamberlain's chemically defined medium (CCDM). The variability between aerosol exposures was also considerably less with BHI. LVS grown in BHI survived desiccation far longer than MHb-grown or CCDM-grown LVS (~70% at 20 min for BHI compared to <50% for MHb and CCDM). Removal of the capsule by hypertonic treatment impacted the spray factor for CCDM-grown LVS or MHb-grown LVS but not BHI-grown LVS, suggesting the choice of culture media altered the adherence of the capsule to the cell membrane. The choice of growth media did not impact the LD50 of LVS but the LD99 of BHI-grown LVS was 1 log lower than that for MHb-grown LVS or CCDM-grown LVS. Splenomegaly was prominent in mice that succumbed to MHb- and BHI-grown LVS but not CCDM-grown LVS. Environmental factors and growth conditions should be evaluated when developing new animal models for aerosol infection, particularly for vegetative bacterial pathogens. PMID:23087911

  3. Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

    PubMed

    del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

    2014-04-15

    Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. © 2013 Elsevier B.V. All rights reserved.

  4. Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

    PubMed Central

    2010-01-01

    Background Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories. Results The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes. Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. Conclusion We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples. PMID:20205957

  5. Differential expression of microRNAs in Francisella tularensis-infected human macrophages: miR-155-dependent downregulation of MyD88 inhibits the inflammatory response.

    PubMed

    Bandyopadhyay, Sarmistha; Long, Matthew E; Allen, Lee-Ann H

    2014-01-01

    Francisella tularensis is a Gram-negative, facultative intracellular pathogen that replicates in the cytosol of macrophages and is the causative agent of the potentially fatal disease tularemia. A characteristic feature of F. tularensis is its limited proinflammatory capacity, but the mechanisms that underlie the diminished host response to this organism are only partially defined. Recently, microRNAs have emerged as important regulators of immunity and inflammation. In the present study we investigated the microRNA response of primary human monocyte-derived macrophages (MDMs) to F. tularensis and identified 10 microRNAs that were significantly differentially expressed after infection with the live vaccine strain (LVS), as judged by Taqman Low Density Array profiling. Among the microRNAs identified, miR-155 is of particular interest as its established direct targets include components of the Toll-like receptor (TLR) pathway, which is essential for innate defense and proinflammatory cytokine production. Additional studies demonstrated that miR-155 acted by translational repression to downregulate the TLR adapter protein MyD88 and the inositol 5'-phosphatase SHIP-1 in MDMs infected with F. tularensis LVS or the fully virulent strain Schu S4. Kinetic analyses indicated that miR-155 increased progressively 3-18 hours after infection with LVS or Schu S4, and target proteins disappeared after 12-18 hours. Dynamic modulation of MyD88 and SHIP-1 was confirmed using specific pre-miRs and anti-miRs to increase and decrease miR-155 levels, respectively. Of note, miR-155 did not contribute to the attenuated cytokine response triggered by F. tularensis phagocytosis. Instead, this microRNA was required for the ability of LVS-infected cells to inhibit endotoxin-stimulated TNFα secretion 18-24 hours after infection. Thus, our data are consistent with the ability of miR-155 to act as a global negative regulator of the inflammatory response in F. tularensis-infected human

  6. The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Francisella tularensis SCHU S4 Infection in BALB/c Mice and Cynomolgus Macaques

    PubMed Central

    Anderson, Michael S.; Christ, David; Gooldy, Melanie; Henning, Lisa N.; Heine, Henry S.; Kindt, M. Victoria; Lin, Winston; Siefkas-Patterson, Kaylyn; Radcliff, Anne K.; Tam, Vincent H.; Sutcliffe, Joyce A.

    2017-01-01

    ABSTRACT TP-271 is a novel, fully synthetic fluorocycline in development for complicated bacterial respiratory infections. TP-271 was active in vitro against a panel of 29 Francisella tularensis isolates, showing MICs against 50% and 90% of isolates of 0.25 and 0.5 μg/ml, respectively. In a mouse model of inhalational tularemia, animals were exposed by aerosol to 91 to 283 50% lethal doses (LD50)/mouse of F. tularensis SCHU S4. Following 21 days of once-daily intraperitoneal dosing with TP-271 at 3, 6, 12, and 18 mg/kg of body weight/day, initiating at 24 h postchallenge, survival was 80%, 100%, 100%, and 100%, respectively. When treatment was initiated at 72 h postchallenge, survival was 89%, 100%, 100%, and 100% in the 3-, 6-, 12-, and 18-mg/kg/day TP-271 groups, respectively. No mice treated with the vehicle control survived. Surviving mice treated with TP-271 showed little to no relapse during 14 days posttreatment. In a nonhuman primate model of inhalational tularemia, cynomolgus macaques received an average aerosol exposure of 1,144 CFU of F. tularensis SCHU S4. Once-daily intravenous infusion with 1 or 3 mg/kg TP-271, or vehicle control, for 21 days was initiated within 6 h of confirmed fever. All animals treated with TP-271 survived to the end of the study, with no relapse during 14 days after the last treatment, whereas no vehicle control-treated animals survived. The protection and low relapse afforded by TP-271 treatment in these studies support continued investigation of TP-271 for use in the event of aerosolized exposure to F. tularensis. PMID:28559261

  7. A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1

    PubMed Central

    Schmerk, Crystal L.; Duplantis, Barry N.; Howard, Perry L.; Nano, Francis E.

    2009-01-01

    Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The ΔpdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the ΔpdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the ΔpdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands. PMID:19372155

  8. The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Francisella tularensis SCHU S4 Infection in BALB/c Mice and Cynomolgus Macaques.

    PubMed

    Grossman, Trudy H; Anderson, Michael S; Christ, David; Gooldy, Melanie; Henning, Lisa N; Heine, Henry S; Kindt, M Victoria; Lin, Winston; Siefkas-Patterson, Kaylyn; Radcliff, Anne K; Tam, Vincent H; Sutcliffe, Joyce A

    2017-08-01

    TP-271 is a novel, fully synthetic fluorocycline in development for complicated bacterial respiratory infections. TP-271 was active in vitro against a panel of 29 Francisella tularensis isolates, showing MICs against 50% and 90% of isolates of 0.25 and 0.5 μg/ml, respectively. In a mouse model of inhalational tularemia, animals were exposed by aerosol to 91 to 283 50% lethal doses (LD 50 )/mouse of F. tularensis SCHU S4. Following 21 days of once-daily intraperitoneal dosing with TP-271 at 3, 6, 12, and 18 mg/kg of body weight/day, initiating at 24 h postchallenge, survival was 80%, 100%, 100%, and 100%, respectively. When treatment was initiated at 72 h postchallenge, survival was 89%, 100%, 100%, and 100% in the 3-, 6-, 12-, and 18-mg/kg/day TP-271 groups, respectively. No mice treated with the vehicle control survived. Surviving mice treated with TP-271 showed little to no relapse during 14 days posttreatment. In a nonhuman primate model of inhalational tularemia, cynomolgus macaques received an average aerosol exposure of 1,144 CFU of F. tularensis SCHU S4. Once-daily intravenous infusion with 1 or 3 mg/kg TP-271, or vehicle control, for 21 days was initiated within 6 h of confirmed fever. All animals treated with TP-271 survived to the end of the study, with no relapse during 14 days after the last treatment, whereas no vehicle control-treated animals survived. The protection and low relapse afforded by TP-271 treatment in these studies support continued investigation of TP-271 for use in the event of aerosolized exposure to F. tularensis . Copyright © 2017 American Society for Microbiology.

  9. Respiratory Francisella tularensis live vaccine strain infection induces Th17 cells and prostaglandin E2, which inhibits generation of gamma interferon-positive T cells.

    PubMed

    Woolard, Matthew D; Hensley, Lucinda L; Kawula, Thomas H; Frelinger, Jeffrey A

    2008-06-01

    Two key routes of Francisella tularensis infection are through the skin and airway. We wished to understand how the route of inoculation influenced the primary acute adaptive immune response. We show that an intranasal inoculation of the F. tularensis live vaccine strain (LVS) with a 1,000-fold-smaller dose than an intradermal dose results in similar growth kinetics and peak bacterial burdens. In spite of similar bacterial burdens, we demonstrate a difference in the quality, magnitude, and kinetics of the primary acute T-cell response depending on the route of inoculation. Further, we show that prostaglandin E(2) secretion in the lung is responsible for the difference in the gamma interferon (IFN-gamma) response. Intradermal inoculation led to a large number of IFN-gamma(+) T cells 7 days after infection in both the spleen and the lung. In contrast, intranasal inoculation induced a lower number of IFN-gamma(+) T cells in the spleen and lung but an increased number of Th17 cells in the lung. Intranasal infection also led to a significant increase of prostaglandin E(2) (PGE(2)) in the bronchoalveolar lavage fluid. Inhibition of PGE(2) production with indomethacin treatment resulted in increased numbers of IFN-gamma(+) T cells and decreased bacteremia in the lungs of intranasally inoculated mice. This research illuminates critical differences in acute adaptive immune responses between inhalational and dermal infection with F. tularensis LVS mediated by the innate immune system and PGE(2).

  10. Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase*

    PubMed Central

    Arulanandam, Bernard P.; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J.; Chambers, James P.

    2012-01-01

    Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella. PMID:22923614

  11. Francisella DnaK inhibits tissue-nonspecific alkaline phosphatase.

    PubMed

    Arulanandam, Bernard P; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J; Chambers, James P

    2012-10-26

    Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

  12. Low Dose Vaccination with Attenuated Francisella tularensis Strain SchuS4 Mutants Protects against Tularemia Independent of the Route of Vaccination

    PubMed Central

    Rockx-Brouwer, Dedeke; Chong, Audrey; Wehrly, Tara D.; Child, Robert; Crane, Deborah D.

    2012-01-01

    Tularemia, caused by the Gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia. PMID:22662210

  13. Human Dose-Response Data for Francisella tularensis and a Dose- and Time-Dependent Mathematical Model of Early-Phase Fever Associated with Tularemia After Inhalation Exposure.

    PubMed

    McClellan, Gene; Coleman, Margaret; Crary, David; Thurman, Alec; Thran, Brandolyn

    2018-04-25

    Military health risk assessors, medical planners, operational planners, and defense system developers require knowledge of human responses to doses of biothreat agents to support force health protection and chemical, biological, radiological, nuclear (CBRN) defense missions. This article reviews extensive data from 118 human volunteers administered aerosols of the bacterial agent Francisella tularensis, strain Schu S4, which causes tularemia. The data set includes incidence of early-phase febrile illness following administration of well-characterized inhaled doses of F. tularensis. Supplemental data on human body temperature profiles over time available from de-identified case reports is also presented. A unified, logically consistent model of early-phase febrile illness is described as a lognormal dose-response function for febrile illness linked with a stochastic time profile of fever. Three parameters are estimated from the human data to describe the time profile: incubation period or onset time for fever; rise time of fever; and near-maximum body temperature. Inhaled dose-dependence and variability are characterized for each of the three parameters. These parameters enable a stochastic model for the response of an exposed population through incorporation of individual-by-individual variability by drawing random samples from the statistical distributions of these three parameters for each individual. This model provides risk assessors and medical decisionmakers reliable representations of the predicted health impacts of early-phase febrile illness for as long as one week after aerosol exposures of human populations to F. tularensis. © 2018 Society for Risk Analysis.

  14. Resistance of Francisella Novicida to Fosmidomycin Associated with Mutations in the Glycerol-3-Phosphate Transporter

    PubMed Central

    Mackie, Ryan S.; McKenney, Elizabeth S.; van Hoek, Monique L.

    2012-01-01

    The methylerythritol phosphate (MEP) pathway is essential in most prokaryotes and some lower eukaryotes but absent from human cells, and is a validated target for antimicrobial drug development. The formation of MEP is catalyzed by 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR). MEP pathway genes have been identified in many category A and B biothreat agents, including Francisella tularensis, which causes the zoonosis tularemia. Fosmidomycin (Fos) inhibits purified Francisella DXR. This compound also inhibits the growth of F. tularensis NIH B38, F. novicida and F. tularensis subsp. holarctica LVS bacteria. Related compounds such as FR900098 and the lipophilic prodrug of FR900098 (compound 1) have been developed to improve the bioavailability of these DXR inhibitors. In performing disk-inhibition assays with these compounds, we observed breakthrough colonies of F. novicida in the presence of Fos, suggesting spontaneous development of Fos resistance (FosR). FosR bacteria had decreased sensitivity to both Fos and FR900098. The two most likely targets for the development of mutants would be the DXR enzyme itself or the glycerol-3-phosphate transporter (GlpT) that allows entry of Fos into the bacteria. Sensitivity of FosR F. novicida bacteria to compound 1 was not abated suggesting that spontaneous resistance is not due to mutation of DXR. We thus predicted that the glpT transporter may be mutated leading to this resistant phenotype. Supporting this, transposon insertion mutants at the glpT locus were also found to be resistant to Fos. DNA sequencing of four different spontaneous FosR colonies demonstrated a variety of deletions in the glpT coding region. The overall frequency of FosR mutations in F. novicida was determined to be 6.3 × 10−8. Thus we conclude that one mechanism of resistance of F. novicida to Fos is caused by mutations in GlpT. This is the first description of spontaneous mutations in Francisella leading to FosR. PMID:22905031

  15. The FupA/B protein uniquely facilitates transport of ferrous iron and siderophore-associated ferric iron across the outer membrane of Francisella tularensis live vaccine strain

    PubMed Central

    Sen, Bhaswati

    2014-01-01

    Francisella tularensis is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of F. tularensis is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore–ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene fupA/B, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used 55Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. 55Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore–ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation. PMID:24307666

  16. Medical Countermeasure Models. Volume 4. Francisella tularensis

    DTIC Science & Technology

    2013-04-12

    34 Medicine. 64(4). 1985. 187 Flax, L. " TYPHOIDAL TULAREMIA." Maryland state medical journal. 12(601). 1963. 188 Ford-Jones Let al."" Muskrat fever ...Work Lost A-37 Period of Fever A-37 Work Lost in an Individual Who Recovers A-38 Medical Countermeasure Models Volume 4: Francisella...symptom onset, period of fever , day of death, days of work lost due to MCM adverse effects, days of work lost due to illness, day of relapse, and

  17. Seroprevalence of West Nile virus, Crimean-Congo hemorrhagic fever virus, Francisella tularensis and Borrelia burgdorferi in rural population of Manisa, western Turkey.

    PubMed

    Gazi, Hörü; Özkütük, Nuri; Ecemis, Özkütük; Atasoylu, Gonca; Köroglu, Galip; Kurutepe, Semra; Horasan, Gönül Dinç

    2016-01-01

    Zoonotic diseases are well recognised threat to public health globally. The information of regional prevalence and associated risk factors allow the national programmes to determine and frame better strategies for their control, as they also provide the actual status of zoonosis in the region. The aim of this study was to determine the seroprevalence of West Nile virus (WNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Francisella tularensis and Borrelia burgdorferi among the rural residents of Manisa region, Turkey and to identify the associated risk factors. Cross sectional study was conducted in rural parts of Manisa, Aegean region of western Turkey in 2012. Blood samples from 324 randomly selected subjects were screened for the presence of IgG antibodies to WNV, CCHFV, F. tularensis and B. burgdorferi with commercially available kits. The demographic structure of the rural residents and risk factors related to lifestyle such as outdoor agriculture activities, animal husbandry, hunting and history of tick bite were questioned and their relationships with positive results were analyzed statistically. It was observed that 49 subjects (15%) had IgG antibodies to at least one of the zoonotic agents studied. The seroprevalence of F. tularensis was highest with a percentage of 7.1% (n = 23). Distribution of the positive results for WNV, CCHFV and B. burgdorferi were 4.3% (n = 14), 3.7% (n = 12) and 0.9% (n = 3), respectively. Older age and uncompleted secondary education were the statistically significant risk factors for seropositivity to at least one zoonotic agent investigated. Logistic regression analyses confirmed that older age (over 50) increased the risk of WNV and CCHFV seropositivity. Seropositivity rates were not found to be higher than the expected rates. Further, studies are needed to evaluate the threat of vector borne zoonoses and associated risk factors in the study area.

  18. Gallium Disrupts Iron Uptake by Intracellular and Extracellular Francisella Strains and Exhibits Therapeutic Efficacy in a Murine Pulmonary Infection Model ▿

    PubMed Central

    Olakanmi, Oyebode; Gunn, John S.; Su, Shengchang; Soni, Shilpa; Hassett, Daniel J.; Britigan, Bradley E.

    2010-01-01

    Francisella tularensis requires iron (Fe) for growth, but the biologic sources of Fe for this organism are largely unknown. We found that Francisella sp. growing in broth culture or within human macrophages can acquire Fe from the two major host Fe-binding proteins, lactoferrin (Lf) and transferrin (Tf). Fe acquisition is a potential target for novel therapies. Gallium (Ga) is a transition metal that interferes with cellular Fe metabolism by competing with Fe for uptake/utilization. Growth of either F. tularensis live vaccine strain (LVS) or Francisella novicida was inhibited by ≥2 μM Ga chelated to Tf or Lf, with GaLf being somewhat more potent. Francisella spp. express two Fe-containing antioxidant enzymes, catalase (KatG) and Fe cofactored superoxide dismutase (FeSOD). Growth of LVS with 10 μM GaTf or GaLf led to a dramatic decrease in bacterial catalase activity and in FeSOD activity that was associated with an increased susceptibility to H2O2. Ga also protected mice from intranasal challenge with F. novicida. Whereas 100% of the F. novicida-infected mice died by day 9, 75% of the mice receiving Ga continued to survive to at least day 15. Thus, a single intranasal dose of Ga followed by daily intraperitoneal Ga at a dose tolerated by the animals resulted in prolonged survival. These data support the potential utility of Ga as a therapy for F. tularensis infection of the lung. PMID:19917753

  19. Bioavailability and Efficacy of Levofloxacin against Francisella tularensis in the Common Marmoset (Callithrix jacchus)▿

    PubMed Central

    Nelson, Michelle; Lever, Mark S.; Dean, Rachel E.; Pearce, Peter C.; Stevens, Daniel J.; Simpson, Andrew J. H.

    2010-01-01

    Pharmacokinetic and efficacy studies with levofloxacin were performed in the common marmoset (Callithrix jacchus) model of inhalational tularemia. Plasma levofloxacin pharmacokinetics were determined in six animals in separate single-dose and multidose studies. Plasma drug concentrations were analyzed using liquid chromatography-tandem mass spectrometry-electrospray ionization. On day 7 of a twice-daily dosing regimen of 40 mg/kg, the levofloxacin half-life, maximum concentration, and area under the curve in marmoset plasma were 2.3 h, 20.9 μg/ml, and 81.4 μg/liter/h, respectively. An efficacy study was undertaken using eight treated and two untreated control animals. Marmosets were challenged with a mean of 1.5 × 102 CFU of Francisella tularensis by the airborne route. Treated animals were administered 16.5 mg/kg levofloxacin by mouth twice daily, based on the pharmacokinetic parameters, beginning 24 h after challenge. Control animals had a raised core body temperature by 57 h postchallenge and died from infection by day 5. All of the other animals survived, remained afebrile, and lacked overt clinical signs. No bacteria were recovered from the organs of these animals at postmortem after culling at day 24 postchallenge. In conclusion, postexposure prophylaxis with orally administered levofloxacin was efficacious against acute inhalational tularemia in the common marmoset. The marmoset appears to be an appropriate animal model for the evaluation of postexposure therapies. PMID:20625157

  20. Live Attenuated Tularemia Vaccines for Protection Against Respiratory Challenge With Virulent F. tularensis subsp. tularensis

    PubMed Central

    Jia, Qingmei; Horwitz, Marcus A.

    2018-01-01

    Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed “Foshay” vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals—especially mice—but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated—but not killed or subunit—vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development—safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the

  1. Inactivation of F.tularensis Utah-112 on food and food contact surfaces by ultraviolet light

    USDA-ARS?s Scientific Manuscript database

    Francisella tularensis is the causative agent of tularemia, a plague-like illness that affects animals and humans, and has caused large illness pandemics in the last century. It has also been used as a biological warfare agent, and tularemia can be contracted through consumption of contaminated food...

  2. Respiratory infection with Francisella novicida induces rapid dystrophic cardiac calcinosis (DCC).

    PubMed

    Roth, Kimberly M; Oghumu, Steve; Satoskar, Anjali A; Gunn, John S; van Rooijen, Nico; Satoskar, Abhay R

    2008-06-01

    Francisella tularensis causes pulmonary tularemia and death in humans when left untreated. Here, using a novel aerosol infection model, we show that acute pulmonary Francisella novicida infection not only causes pneumonia and liver damage, but also induces dystrophic cardiac calcinosis (DCC) in BALB/c mice. C57BL/6 mice also develop pneumonia and hepatic damage, but fail to develop DCC. Development of DCC in BALB/c mice is associated with significant induction of RANKL but not osteopontin in their organs. Depletion of lung macrophages prior to infection markedly reduces pericarditis and calcification in BALB/c mice but does not increase their susceptibility to infection.

  3. Aerosol delivery of liposome-encapsulated ciprofloxacin: aerosol characterization and efficacy against Francisella tularensis infection in mice.

    PubMed

    Conley, J; Yang, H; Wilson, T; Blasetti, K; Di Ninno, V; Schnell, G; Wong, J P

    1997-06-01

    The aerosol delivery of liposome-encapsulated ciprofloxacin by using 12 commercially available jet nebulizers was evaluated in this study. Aerosol particles containing liposome-encapsulated ciprofloxacin generated by the nebulizers were analyzed with a laser aerodynamic particle sizer. Mean mass aerodynamic diameters (MMADs) and geometric standard deviations (GSDs) were determined, and the drug contents of the sampling filters from each run onto which aerosolized liposome-encapsulated ciprofloxacin had been deposited were analyzed spectrophotometrically. The aerosol particles of liposome-encapsulated ciprofloxacin generated by these nebulizers ranged from 1.94 to 3.5 microm, with GSDs ranging from 1.51 to 1.84 microm. The drug contents of the sampling filters exposed for 1 min to aerosolized liposome-encapsulated ciprofloxacin range from 12.7 to 40.5 microg/ml (0.06 to 0.2 mg/filter). By using the nebulizer selected on the basis of most desirable MMADs, particle counts, and drug deposition, aerosolized liposome-encapsulated ciprofloxacin was used for the treatment of mice infected with 10 times the 50% lethal dose of Francisella tularensis. All mice treated with aerosolized liposome-encapsulated ciprofloxacin survived the infection, while all ciprofloxacin-treated or untreated control mice succumbed to the infection (P < 0.001). These results suggest that aerosol delivery of liposome-encapsulated ciprofloxacin to the lower respiratory tract is feasible and that it may provide an effective therapy for the treatment of respiratory tract infections.

  4. Aerosol delivery of liposome-encapsulated ciprofloxacin: aerosol characterization and efficacy against Francisella tularensis infection in mice.

    PubMed Central

    Conley, J; Yang, H; Wilson, T; Blasetti, K; Di Ninno, V; Schnell, G; Wong, J P

    1997-01-01

    The aerosol delivery of liposome-encapsulated ciprofloxacin by using 12 commercially available jet nebulizers was evaluated in this study. Aerosol particles containing liposome-encapsulated ciprofloxacin generated by the nebulizers were analyzed with a laser aerodynamic particle sizer. Mean mass aerodynamic diameters (MMADs) and geometric standard deviations (GSDs) were determined, and the drug contents of the sampling filters from each run onto which aerosolized liposome-encapsulated ciprofloxacin had been deposited were analyzed spectrophotometrically. The aerosol particles of liposome-encapsulated ciprofloxacin generated by these nebulizers ranged from 1.94 to 3.5 microm, with GSDs ranging from 1.51 to 1.84 microm. The drug contents of the sampling filters exposed for 1 min to aerosolized liposome-encapsulated ciprofloxacin range from 12.7 to 40.5 microg/ml (0.06 to 0.2 mg/filter). By using the nebulizer selected on the basis of most desirable MMADs, particle counts, and drug deposition, aerosolized liposome-encapsulated ciprofloxacin was used for the treatment of mice infected with 10 times the 50% lethal dose of Francisella tularensis. All mice treated with aerosolized liposome-encapsulated ciprofloxacin survived the infection, while all ciprofloxacin-treated or untreated control mice succumbed to the infection (P < 0.001). These results suggest that aerosol delivery of liposome-encapsulated ciprofloxacin to the lower respiratory tract is feasible and that it may provide an effective therapy for the treatment of respiratory tract infections. PMID:9174185

  5. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.

    Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, providemore » better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.« less

  6. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

    DOE PAGES

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; ...

    2016-09-26

    Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, providemore » better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.« less

  7. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella Tularensis: Residence Time and In Vivo Activity

    PubMed Central

    Lu, Hao; England, Kathleen; Ende, Christopher am; Truglio, James J.; Luckner, Sylvia; Reddy, B. Gopal; Marlenee, Nikki; Knudson, Susan E.; Knudson, Dennis L.; Bowen, Richard A.; Kisker, Caroline; Slayden, Richard A.; Tonge, Peter J.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 μg/ml. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency. PMID:19206187

  8. Population Genomics of Francisella tularensis subsp. holarctica and its Implication on the Eco-Epidemiology of Tularemia in Switzerland

    PubMed Central

    Wittwer, Matthias; Altpeter, Ekkehard; Pilo, Paola; Gygli, Sebastian M.; Beuret, Christian; Foucault, Frederic; Ackermann-Gäumann, Rahel; Karrer, Urs; Jacob, Daniela; Grunow, Roland; Schürch, Nadia

    2018-01-01

    Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of Francisella tularensis subsp. holarctica, the causative agent of tularemia in Switzerland. A total of 59 Fth isolates were obtained from castor bean ticks (Ixodes ricinus), animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the Fth population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting Fth to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks. PMID:29623260

  9. Francisella philomiragia Infection and Lethality in Mammalian Tissue Culture Cell Models, Galleria mellonella, and BALB/c Mice

    PubMed Central

    Propst, Crystal N.; Pylypko, Stephanie L.; Blower, Ryan J.; Ahmad, Saira; Mansoor, Mohammad; van Hoek, Monique L.

    2016-01-01

    Francisella (F.) philomiragia is a Gram-negative bacterium with a preference for brackish environments that has been implicated in causing bacterial infections in near-drowning victims. The purpose of this study was to characterize the ability of F. philomiragia to infect cultured mammalian cells, a commonly used invertebrate model, and, finally, to characterize the ability of F. philomiragia to infect BALB/c mice via the pulmonary (intranasal) route of infection. This study shows that F. philomiragia infects J774A.1 murine macrophage cells, HepG2 cells and A549 human Type II alveolar epithelial cells. However, replication rates vary depending on strain at 24 h. F. philomiragia infection after 24 h was found to be cytotoxic in human U937 macrophage-like cells and J774A.1 cells. This is in contrast to the findings that F. philomiragia was non-cytotoxic to human hepatocellular carcinoma cells, HepG2 cells and A549 cells. Differential cytotoxicity is a point for further study. Here, it was demonstrated that F. philomiragia grown in host-adapted conditions (BHI, pH 6.8) is sensitive to levofloxacin but shows increased resistance to the human cathelicidin LL-37 and murine cathelicidin mCRAMP when compared to related the Francisella species, F. tularensis subsp. novicida and F. tularensis subsp. LVS. Previous findings that LL-37 is strongly upregulated in A549 cells following F. tularensis subsp. novicida infection suggest that the level of antimicrobial peptide expression is not sufficient in cells to eradicate the intracellular bacteria. Finally, this study demonstrates that F. philomiragia is lethal in two in vivo models; Galleria mellonella via hemocoel injection, with a LD50 of 1.8 × 103, and BALB/c mice by intranasal infection, with a LD50 of 3.45 × 103. In conclusion, F. philomiragia may be a useful model organism to study the genus Francisella, particularly for those researchers with interest in studying microbial ecology or environmental strains of Francisella

  10. Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Freudenberger Catanzaro, Kelly C.; Champion, Anna E.; Mohapatra, Nrusingh

    Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. But, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locusmore » in F. tularensis. Following daily subculture of F. novicida in Chamberlain’s defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ11212–1218. The subcultured mutant F. novicida Δ11212–1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Δ11212–1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10–14 days post-challenge. Mice immunized intranasally with F. novicida Δ11212–1218 were partially protected against challenge with the parent

  11. Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in Mice

    DOE PAGES

    Freudenberger Catanzaro, Kelly C.; Champion, Anna E.; Mohapatra, Nrusingh; ...

    2017-05-30

    Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. But, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locusmore » in F. tularensis. Following daily subculture of F. novicida in Chamberlain’s defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Δ11212–1218. The subcultured mutant F. novicida Δ11212–1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Δ11212–1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10–14 days post-challenge. Mice immunized intranasally with F. novicida Δ11212–1218 were partially protected against challenge with the parent

  12. Macrophage Pro-Inflammatory Response to Francisella novicida Infection Is Regulated by SHIP

    PubMed Central

    Parsa, Kishore V. L; Ganesan, Latha P; Rajaram, Murugesan V. S; Gavrilin, Mikhail A; Balagopal, Ashwin; Mohapatra, Nrusingh P; Wewers, Mark D; Schlesinger, Larry S; Gunn, John S; Tridandapani, Susheela

    2006-01-01

    Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida–induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida–induced cytokine production through the inhibition of NFκB. Consistently, macrophages lacking SHIP displayed enhanced NFκB-driven gene transcription, whereas overexpression of SHIP led to decreased NFκB activation. Thus, we propose that SHIP negatively regulates F. novicida–induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFκB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia. PMID:16848641

  13. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lu, H.; England, K; Ende, C

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzymemore » with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.« less

  14. Francisella tularensis Susceptibility to Antibiotics: A Comprehensive Review of the Data Obtained In vitro and in Animal Models

    PubMed Central

    Caspar, Yvan; Maurin, Max

    2017-01-01

    The antibiotic classes that are recommended for tularaemia treatment are the aminoglycosides, the fluoroquinolones and the tetracyclines. However, cure rates vary between 60 and 100% depending on the antibiotic used, the time to appropriate antibiotic therapy setup and its duration, and the presence of complications, such as lymph node suppuration. Thus, antibiotic susceptibility testing (AST) of F. tularensis strains remains of primary importance for detection of the emergence of antibiotic resistances to first-line drugs, and to test new therapeutic alternatives. However, the AST methods reported in the literature were poorly standardized between studies and AST data have not been previously evaluated in a comprehensive and comparative way. The aim of the present review was to summarize experimental data on antibiotic susceptibilities of F. tularensis obtained in acellular media, cell models and animal models since the introduction of fluoroquinolones in the treatment of tularaemia in 1989. We compiled MIC data of 33 antibiotics (including aminoglycosides, fluoroquinolones, tetracyclines, macrolides, β-lactams, chloramphenicol, rifampicin, and linezolid) against 900 F. tularensis strains (504 human strains), including 107 subsp. tularensis (type A), 789 subsp. holarctica (type B) and four subsp. mediasiatica strains, using various AST methods. Specific culture media were identified or confirmed as unsuitable for AST of F. tularensis. Overall, MICs were the lowest for ciprofloxacin (≤ 0.002–0.125 mg/L) and levofloxacin, and ranged from ≤ 0.016 to 2 mg/L for gentamicin, and 0.064 to 4 mg/L for doxycycline. No resistant strain to any of these antibiotics was reported. Fluoroquinolones also exhibited a bactericidal activity against intracellular F. tularensis and lower relapse rates in animal models when compared with the bacteriostatic compound doxycycline. As expected, lower MIC values were found for macrolides against type A and biovar I type B strains

  15. Efficient Inactivation of Burkholderia pseudomallei or Francisella tularensis in infected Cells for Safe Removal from Biosafety Level 3 Containment Laboratories

    PubMed Central

    Emery, Felicia D.; Stabenow, Jennifer M.; Miller, Mark A.

    2014-01-01

    Working with infectious agents that require BSL-3 level containment agents offers many challenges for researchers. BSL-3 containment laboratories are usually not equipped with expensive specialty equipment that is needed for studies such as flow cytometric analysis, microscopy, and proteomic analyses. Therefore, for most researchers that are working with BSL-3 level infectious agents, removal of samples from BSL-3 labs for these types of studies is necessary, and methods for complete and dependable inactivation of the samples are required. In this report we have done a thorough characterization of the effectiveness of paraformaldehyde fixation for inactivation of cell samples infected with the intracellular bacterial agents Burkholderia pseudomallei (Bp) and Francisella tularensis (Ft), both of which are Tier 1 select agent pathogens that require BSL-3 containment. We have demonstrated that cells infected with these pathogens are completely inactivated via 5-minute treatment with 4% paraformaldehyde. Moreover, a 15-minute treatment with 2% paraformaldehyde completely sterilized both Bp- and Ft-infected cells. These studies also revealed that Bp is significantly more sensitive to paraformaldehyde treatment than Ft. Our findings have clearly demonstrated that a 15-minute treatment of Bp- or Ft-infected cells with 4% paraformaldehyde solution will allow for safe removal of the cell samples from BSL-3 labs for downstream studies. PMID:24449562

  16. Evaluation of an Immunochromatographic Test for Rapid and Reliable Serodiagnosis of Human Tularemia and Detection of Francisella tularensis-Specific Antibodies in Sera from Different Mammalian Species ▿

    PubMed Central

    Splettstoesser, W.; Guglielmo-Viret, V.; Seibold, E.; Thullier, P.

    2010-01-01

    Tularemia is a highly contagious infectious zoonosis caused by the bacterial agent Francisella tularensis. Serology is still considered to be a cornerstone in tularemia diagnosis due to the low sensitivity of bacterial culture and the lack of standardization in PCR methodology for the direct identification of the pathogen. We developed a novel immunochromatographic test (ICT) to efficiently detect F. tularensis-specific antibodies in sera from humans and other mammalian species (nonhuman primate, pig, and rabbit). This new tool requires none or minimal laboratory equipment, and the results are obtained within 15 min. When compared to the method of microagglutination, which was shown to be more specific than the enzyme-linked immunosorbent assay, the ICT had a sensitivity of 98.3% (58 positive sera were tested) and a specificity of 96.5% (58 negative sera were tested) on human sera. On animal sera, the overall sensitivity was 100% (22 positive sera were tested) and specificity was also 100% (70 negative sera were tested). This rapid test preferentially detects IgG antibodies that may occur early in the course of human tularemia, but further evaluation with human sera is important to prove that the ICT can be a valuable field test to support a presumptive diagnosis of tularemia. The ICT can also be a useful tool to monitor successful vaccination with subunit vaccines or live vaccine strains containing lipopolysaccharide (e.g., LVS) and to detect seropositive individuals or animals in outbreak situations or in the context of epidemiologic surveillance programs in areas of endemicity as recently recommended by the World Health Organization. PMID:20220165

  17. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

    DOE PAGES

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; ...

    2016-01-28

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we havemore » discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

  18. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellularmore » signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.« less

  19. Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via CR1 and CR3 in nonimmune serum

    PubMed Central

    Schwartz, Justin T.; Barker, Jason H.; Long, Matthew E.; Kaufman, Justin; McCracken, Jenna; Allen, Lee-Ann H.

    2012-01-01

    A fundamental step in the life cycle of F. tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum, nor the receptors that mediate infection of neutrophils has been defined. Herein human neutrophil uptake of GFP-expressing F. tularensis strains LVS and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis whereas C5 was not. Second, we used purification and immuno-depletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-antigen and capsule as prominent targets of these antibodies on the bacterial surface. Finally, we demonstrate using receptor-blocking antibodies that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-antigen polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3-opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner. PMID:22888138

  20. Manipulation of host membranes by the bacterial pathogens Listeria, Francisella, Shigella and Yersinia.

    PubMed

    Pizarro-Cerdá, Javier; Charbit, Alain; Enninga, Jost; Lafont, Frank; Cossart, Pascale

    2016-12-01

    Bacterial pathogens display an impressive arsenal of molecular mechanisms that allow survival in diverse host niches. Subversion of plasma membrane and cytoskeletal functions are common themes associated to infection by both extracellular and intracellular pathogens. Moreover, intracellular pathogens modify the structure/stability of their membrane-bound compartments and escape degradation from phagocytic or autophagic pathways. Here, we review the manipulation of host membranes by Listeria monocytogenes, Francisella tularensis, Shigella flexneri and Yersinia spp. These four bacterial model pathogens exemplify generalized strategies as well as specific features observed during bacterial infection processes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. CpG oligodeoxyribonucleotides protect mice from Burholderia pseudomallei but not Francisella tularensis Schu 54 aersols

    DTIC Science & Technology

    2010-01-01

    Agency, Department of Defense, project number ZZ0001_06_RD_B. This project was supported in part by appointments to the Internship /Research Participation...formerly Francisella novicida) associated with human disease. Journal of clinical microbiology 1989, 27(7):1601-1608. doi:10.1186/1476-8518-8-2 Cite

  2. The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion

    PubMed Central

    de Bruin, Olle M.; Duplantis, Barry N.; Ludu, Jagjit S.; Hare, Rebekah F.; Nix, Eli B.; Schmerk, Crystal L.; Robb, Craig S.; Boraston, Alisdair B.; Hueffer, Karsten

    2011-01-01

    The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nano-tubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure. PMID:21980115

  3. Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds: Inhibition of response regulator QseB by antibiofilm compounds

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Milton, Morgan E.; Allen, C. Leigh; Feldmann, Erik A.

    With antibiotic resistance increasing at alarming rates, targets for new antimicrobial therapies must be identified. A particularly promising target is the bacterial two-component system. Two-component systems allow bacteria to detect, evaluate and protect themselves against changes in the environment, such as exposure to antibiotics and also to trigger production of virulence factors. Drugs that target the response regulator portion of two-component systems represent a potent new approach so far unexploited. Here, we focus efforts on the highly virulent bacterium Francisella tularensis tularensis. Francisella contains only three response regulators, making it an ideal system to study. In this study, we initiallymore » present the structure of the N-terminal domain of QseB, the response regulator responsible for biofilm formation. Subsequently, using binding assays, computational docking and cellular studies, we show that QseB interacts with2-aminoimidazole based compounds that impede its function. This information will assist in tailoring compounds to act as adjuvants that will enhance the effect of antibiotics.« less

  4. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fischer, N. O.

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the third quarter of the third year, F344 rats vaccinated with adjuvanted NLP formulations were challenged with F. tularensis SCHU S4 at Battelle. Preliminary data indicate that up to 65% of females vaccinated intranasally with an NLP-based formulation survived this challenge, compared to only 20% survival of naïve animals. In addition, NLPs were successfully formulated with Burkholderia protein antigens. IACUC approvalmore » for immunological assessments in BALB/c mice was received and we anticipate that these assessments will begin by March 2015, pending ACURO approval.« less

  5. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.

    Francisella tularensis is a highly infectious bacterium that has the potential of causing high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

  6. CpG Oligodeoxyribonucleotides Protect Mice from Burkholderia Pseudomallei but not Francisella Tularensis Schu S4 Aerosols

    DTIC Science & Technology

    2010-01-01

    of Defense, project number ZZ0001_06_RD_B. This project was supported in part by appointments to the Internship /Research Participation Program for...Francisella novicida) associated with human disease. Journal of clinical microbiology 1989, 27(7):1601-1608. doi:10.1186/1476-8518-8-2 Cite this article

  7. Lipophilic Prodrugs of FR900098 Are Antimicrobial against Francisella novicida In Vivo and In Vitro and Show GlpT Independent Efficacy

    PubMed Central

    McKenney, Elizabeth S.; Sargent, Michelle; Khan, Hameed; Uh, Eugene; Jackson, Emily R.; Jose, Géraldine San; Couch, Robin D.; Dowd, Cynthia S.; van Hoek, Monique L.

    2012-01-01

    Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP) pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase). MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC50 = 230 nM). Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed “compound 1,” was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad

  8. Glycogen synthase kinase-3β (GSK3β) inhibition suppresses the inflammatory response to Francisella infection and protects against tularemia in mice

    PubMed Central

    Zhang, Ping; Katz, Jenny; Michalek, Suzanne M.

    2011-01-01

    Francisella tularensis, the causative agent of tularemia, is currently considered a category A bioterrorism agent due to its high virulence. Infection with F. tularensis results in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms regulating this response are poorly understood. Glycogen synthase kinase-3β (GSK3β) is a serine/threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. In this study, we investigated the effect of GSK3β inhibition in regulating F. tularensis LVS-induced inflammatory responses. F. tularensis LVS infection of murine peritoneal macrophages induced a TLR2 dependent phosphorylation of GSK3β. Inhibition of GSK3β resulted in a significant decrease in the production of pro-inflammatory cytokine IL-6, IL-12p40 and TNF-α, as well as a significant increase in the production of the anti-inflammatory cytokine IL-10. GSK3β regulated the F. tularensis LVS-induced cytokine response by differentially affecting the activation of transcription factors NF-κB and CREB. Inhibition of GSK3β by lithium in vivo suppressed the inflammatory response in mice infected with F. tularensis LVS and conferred a survival advantage. In addition, we show that the production of IFN-γ contributed to the development of tularemia and to the fatal outcome of the infected animals, depending on the timing and the relative level of the IFN-γ produced. IFN-γ potentiated F. tularensis LVS-induced cytokine production by increasing GSK3β activity and the nuclear translocation of NF-κB. Taken together, these results demonstrate a regulatory function of GSK3β in modulating inflammatory responses that can be detrimental to the host during an F. tularensis LVS infection, and suggest that inhibition of GSK3β may represent a novel therapeutic approach in the treatment of tularemia. PMID:18929413

  9. Characterization of Francisella species isolated from the cooling water of an air conditioning system.

    PubMed

    Gu, Quan; Li, Xunde; Qu, Pinghua; Hou, Shuiping; Li, Juntao; Atwill, Edward R; Chen, Shouyi

    2015-01-01

    Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

  10. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fischer, N. O.

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the second quarter of the third year, LLNL finalized all immunological assessments of NLP vaccine formulations in the F344 model. Battelle has immunized rats with three unique NLP formulations by either intramuscular or intranasal administration. All inoculations have been completed, and protective efficacy against an aerosolized challenge will begin at the end of October, 2014.

  11. Francisella guangzhouensis sp. nov., isolated from air-conditioning systems.

    PubMed

    Qu, Ping-Hua; Chen, Shou-Yi; Scholz, Holger C; Busse, Hans-Jürgen; Gu, Quan; Kämpfer, Peter; Foster, Jeffrey T; Glaeser, Stefanie P; Chen, Cha; Yang, Zhi-Chong

    2013-10-01

    Four strains (08HL01032(T), 09HG994, 10HP82-6 and 10HL1960) were isolated from water of air-conditioning systems of various cooling towers in Guangzhou city, China. Cells were Gram-stain-negative coccobacilli without flagella, catalase-positive and oxidase-negative, showing no reduction of nitrate, no hydrolysis of urea and no production of H2S. Growth was characteristically enhanced in the presence of l-cysteine, which was consistent with the properties of members of the genus Francisella. The quinone system was composed of ubiquinone Q-8 with minor amounts of Q-9. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids (PL2, PL3), an unidentified aminophospholipid and an unidentified glycolipid (GL2). The polyamine pattern consisted of the major compounds spermidine, cadaverine and spermine. The major cellular fatty acids were C10 : 0, C14 : 0, C16 : 0, C18 : 1ω9c and C18 : 1 3-OH. A draft whole-genome sequence of the proposed type strain 08HL01032(T) was generated. Comparative sequence analysis of the complete 16S and 23S rRNA genes confirmed affiliation to the genus Francisella, with 95 % sequence identity to the closest relatives in the database, the type strains of Francisella philomiragia and Francisella noatunensis subsp. orientalis. Full-length deduced amino acid sequences of various housekeeping genes, recA, gyrB, groEL, dnaK, rpoA, rpoB, rpoD, rpoH, fopA and sdhA, exhibited similarities of 67-92 % to strains of other species of the genus Francisella. Strains 08HL01032(T), 09HG994, 10HP82-6 and 10HL1960 exhibited highly similar pan-genome PCR profiles. Both the phenotypic and molecular data support the conclusion that the four strains belong to the genus Francisella but exhibit considerable divergence from all recognized Francisella species. Therefore, we propose the name Francisella guangzhouensis sp

  12. Novel genomic tools for specific and real-time detection of biothreat and frequently encountered foodborne pathogens.

    PubMed

    Woubit, Abdela; Yehualaeshet, Teshome; Habtemariam, Tsegaye; Samuel, Temesgen

    2012-04-01

    The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia, and Francisella include important food safety and biothreat agents. By extensive mining of the whole genome and protein databases of diverse, closely and distantly related bacterial species and strains, we have identified novel genome regions, which we utilized to develop a rapid detection platform for these pathogens. The specific genomic targets we have identified to design the primers in Francisella tularensis subsp. tularensis, F. tularensis subsp. novicida, Shigella dysenteriae, Salmonella enterica serovar Typhimurium, Vibrio cholerae, Yersinia pestis, and Yersinia pseudotuberculosis contained either known genes or putative proteins. Primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in silico PCR against whole-genome sequences of different species, subspecies, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (Escherichia coli O157:H7 strain EDL 933, Shigella dysenteriae, S. enterica serovar Typhi, F. tularensis subsp. tularensis, V. cholerae, and Y. pestis) and six foodborne pathogens (Salmonella Typhimurium, Salmonella Saintpaul, Shigella sonnei, F. tularensis subsp. novicida, Vibrio parahaemolyticus, and Y. pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed with purified DNA showed the lowest detection limit of 128 fg of DNA/μl for F. tularensis subsp. tularensis. A preliminary test to detect Shigella organisms in a milk matrix also enabled the detection of 6 to 60 CFU/ml. These new tools could ultimately be used to develop platforms to simultaneously detect these pathogens.

  13. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Annual Technical Report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fischer, N. O.

    The goal of this proposal is to demonstrate that co-localization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of recombinant subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. NLPs are are biocompatible, high-density lipoprotein mimetics that are amenable to the incorporation of multiple, chemically-disparate adjuvant and antigen molecules. We hypothesize that the ability to co-localize optimized adjuvant formulations with subunit antigens within a single particle will enhance the stimulation and activation of key immune effector cells, increasing the protective efficacy of subunit antigen-based vaccines. While Burkholderia spp. and F.more » tularensis subunit antigens are the focus of this proposal, we anticipate that this approach is applicable to a wide range of DOD-relevant biothreat agents. The F344 rat aerosol challenge model for F. tularensis has been successfully established at Battelle under this contract, and Year 3 efficacy studies performed at Battelle demonstrated that an NLP vaccine formulation was able to enhance survival of female F344 rats relative to naïve animals. In addition, Year 3 focused on the incorporation of multiple Burkholderia antigens (both polysaccharides and proteins) onto adjuvanted NLPs, with immunological analysis poised to begin in the next quarter.« less

  14. Immunotherapy for tularemia.

    PubMed

    Skyberg, Jerod A

    2013-11-15

    Francisella tularensis is a gram-negative bacterium that causes the zoonotic disease tularemia. Francisella is highly infectious via the respiratory route (~10 CFUs) and pulmonary infections due to type A strains of F. tularensis are highly lethal in untreated patients (> 30%). In addition, no vaccines are licensed to prevent tularemia in humans. Due to the high infectivity and mortality of pulmonary tularemia, F. tularensis has been weaponized, including via the introduction of antibiotic resistance, by several countries. Because of the lack of efficacious vaccines, and concerns about F. tularensis acquiring resistance to antibiotics via natural or illicit means, augmentation of host immunity, and humoral immunotherapy have been investigated as countermeasures against tularemia. This manuscript will review advances made and challenges in the field of immunotherapy against tularemia.

  15. Immunotherapy for tularemia

    PubMed Central

    Skyberg, Jerod A.

    2013-01-01

    Francisella tularensis is a gram-negative bacterium that causes the zoonotic disease tularemia. Francisella is highly infectious via the respiratory route (~10 CFUs) and pulmonary infections due to type A strains of F. tularensis are highly lethal in untreated patients (>30%). In addition, no vaccines are licensed to prevent tularemia in humans. Due to the high infectivity and mortality of pulmonary tularemia, F. tularensis has been weaponized, including via the introduction of antibiotic resistance, by several countries. Because of the lack of efficacious vaccines, and concerns about F. tularensis acquiring resistance to antibiotics via natural or illicit means, augmentation of host immunity, and humoral immunotherapy have been investigated as countermeasures against tularemia. This manuscript will review advances made and challenges in the field of immunotherapy against tularemia. PMID:23959031

  16. Understanding Virulence in the Brucellae and Francisellae: Towards Efficacious Treatments for Two Potential Biothreat Agents

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rasley, A; Parsons, D A; El-Etr, S

    2009-12-30

    Francisella tularensis, Yersinia pestis and Brucellae species are highly infectious pathogens classified as select agents by the Centers for Disease Control and Prevention (CDC) with the potential for use in bioterrorism attacks. These organisms are known to be facultative intracellular pathogens that preferentially infect human monocytes. As such, understanding how the host responds to infection with these organisms is paramount in detecting and combating human disease. We have compared the ability of fully virulent strains of each pathogen and their non-pathogenic near neighbors to enter and survive inside the human monocytic cell line THP-1 and have quantified the cellular responsemore » to infection with the goal of identifying both unique and common host response patterns. We expanded the scope of these studies to include experiments with pathogenic and non-pathogenic strains of Y. pestis, the causative agent of plague. Nonpathogenic strains of each organism were impaired in their ability to survive intracellularly compared with their pathogenic counterparts. Furthermore, infection of THP-1 cells with pathogenic strains of Y. pestis and F. tularensis resulted in marked increases in the secretion of the inflammatory chemokines IL-8, RANTES, and MIP-1{beta}. In contrast, B. melitensis infection failed to elicit any significant increases in a panel of cytokines tested. These differences may underscore distinct strategies in pathogenic mechanisms employed by these pathogens.« less

  17. Determination of the Persistence of Non-Spore-Forming ...

    EPA Pesticide Factsheets

    Report This report presents the results of an investigation to evaluate the persistence (or natural attenuation) of Yersinia pestis (Y. pestis), Francisella tularensis (F. tularensis), and Burkholderia mallei (B. mallei) on glass and soil under multiple environmental conditions and time points.

  18. Feasibility of Screening for Antibiotic Resistance-Part II

    DTIC Science & Technology

    2005-08-01

    fluoroquinolones were introduced on the market. The first two generations are not widely used anymore, because of widespread resistance or ineffectiveness...Mutations that confer resistance against ciprofloxacin have been found in all four genes encoding subunits of Topoisomerase H and IV, and also in an entirely...aerogenes, Erwinia carotovora, Escherichia coli K12, Francisella tularensis, Klebsiella pneumoniae , Neisseria gonorrhoeae, Providencia stuartii

  19. Francisella marina sp. nov., etiologic agent of systemic disease in cultured spotted rose snapper (Lutjanus guttatus) in Central America.

    PubMed

    Soto, Esteban; Griffin, Matt J; Morales, Juan Alberto; Barquero Calvo, Elías; de Alexandre Sebastião, Fernanda; Lopez Porras, Adrián; Víquez-Rodríguez, Xindy; Reichley, Stephen R; Rosser, Thomas G; Ware, Cynthia; Byrne, Barbara A; García, Julio C; LaFrentz, Benjamin R; Camus, Alvin C

    2018-06-18

    Historically, piscine francisellosis in various warm, temperate and coldwater fish hosts has been attributed to Francisella noatunensis From 2015-2016, an undescribed Francisella sp. was recovered during mortality events in cultured spotted rose snapper ( Lutjanus guttatus ) off the Pacific coast of Central America. Despite high mortality and emaciation, limited gross findings were observed in affected fish. Histological examination revealed multifocal granulomatous lesions, with the presence of numerous small, pleomorphic coccobacilli, predominantly in the peritoneum, spleen, kidneys, liver, pancreas, heart, and intestine. Sequencing of an ∼1400 bp fragment of the 16S rRNA gene demonstrated these isolates to be most similar (99.9% identity) to Francisella sp. isolate TX077308 cultured from sea water in the Gulf of Mexico, while sharing <99% similarity to other Fransicella spp. Biochemical analysis, multi-locus sequence comparisons of select housekeeping-genes, repetitive extragenic palindromic PCR fingerprinting, matrix-assisted laser-desorption ionization-time of flight mass spectrometry, and fatty acid methyl esters analysis revealed marked differences between these isolates and other described members of the genus. Koch's postulates were fulfilled by experimental intracoelomic injection and immersion trials using Nile ( Oreochromis niloticus ) and blue tilapia ( O. aureus ). Based on observed phenotypic and genotypic differences from recognized Francisella spp., the name Francisella marina sp. nov. (NRRL # B-65518) is proposed to accommodate these novel strains. Importance Finfish aquaculture is the fastest growing global food production sector. Infectious disease, particularly emergent pathogens, pose a significant threat to established and nascent aquaculture industries worldwide. Herein, we characterize a novel pathogen isolated from mortality events in cultured spotted rose snapper in Central America. The bacteria recovered from these outbreaks were

  20. Diagnostic procedures in tularaemia with special focus on molecular and immunological techniques.

    PubMed

    Splettstoesser, W D; Tomaso, H; Al Dahouk, S; Neubauer, H; Schuff-Werner, P

    2005-08-01

    Tularaemia is a severe bacterial zoonosis caused by the highly infectious agent Francisella tularensis. It is endemic in countries of the northern hemisphere ranging from North America to Europe, Asia and Japan. Very recently, Francisella-like strains causing disease in humans were described from tropical northern Australia. In the last decade, efforts have been made to develop sensitive and specific immunological and molecular techniques for the laboratory diagnosis of tularaemia and also for the definite identification of members of the species F. tularensis and its four subspecies. Screening for the keyword 'Francisella' a Medline search over the last decade was performed and articles describing diagnostic methods for tularaemia and its causative agent were selected. Besides classical microbiological techniques (cultivation, biochemical profiling, susceptibility testing) several new immunological and molecular approaches to identify F. tularensis have been introduced employing highly specific antibodies and various polymerase chain reaction (PCR)-based methods. Whereas direct antigen detection by enzyme-linked immunosorbent assay (ELISA) or immunofluorescence might allow early presumptive diagnosis of tularaemia, these methods--like all PCR techniques--still await further evaluation. Therefore, diagnosis of tularaemia still relies mainly on the demonstration of specific antibodies in the host. ELISA and immunoblot methods started to replace the standard tube or micro-agglutination assays. However, the diagnostic value of antibody detection in the very early clinical phase of tularaemia is limited. Francisella tularensis is regarded as a 'highest priority' biological agent (category 'A' according to the CDC, Atlanta, GA, USA), thus rapid and reliable diagnosis of tularaemia is required not only for a timely onset of therapy, the handling of outbreak investigations but also for the surveillance of endemic foci. Only very recently, evaluated test kits for

  1. [Tularaemia - an overview of the current knowledge].

    PubMed

    Lukásová, Eva; Cermák, Pavel; Smelá, Gabriela; Jedlicková, Anna

    2010-02-01

    Francisella tularensis belongs to the family Francisellaceae. It is the aetiological agent of a zoonosis called tularaemia, spread throughout the northern hemisphere. Currently, several subspecies of F. tularensis may be distinguished with various pathogenicity and geographical distribution. In human medicine, only sporadic infections or local epidemics are reported. Given the fact that F. tularensis is highly pathogenic for humans and is easily spread by aerosol, water or food, it may be exploited as a biological weapon. It belongs to fastidious strains requiring specially prepared culture media.

  2. Tularemia vaccine: Safety, reactogenicity, "Take" skin reactions, and antibody responses following vaccination with a new lot of the Francisella tularensis live vaccine strain - A phase 2 randomized clinical Trial.

    PubMed

    Mulligan, Mark J; Stapleton, Jack T; Keitel, Wendy A; Frey, Sharon E; Chen, Wilbur H; Rouphael, Nadine; Edupuganti, Srilatha; Beck, Allison; Winokur, Patricia L; El Sahly, Hana M; Patel, Shital M; Atmar, Robert L; Graham, Irene; Anderson, Edwin; El-Kamary, Samer S; Pasetti, Marcela F; Sztein, Marcelo B; Hill, Heather; Goll, Johannes B

    2017-08-24

    Tularemia is caused by Francisella tularensis, a gram-negative bacterium that has been weaponized as an aerosol. For protection of personnel conducting biodefense research, the United States Army required clinical evaluation of a new lot of tularemia live vaccine strain manufactured in accordance with Current Good Manufacturing Practices. A phase 2 randomized clinical trial compared the new lot (DVC-LVS) to the existing vaccine that has been in use for decades (USAMRIID-LVS). The vaccines were delivered by scarification to 228 participants. Safety, reactogenicity, take and/or antibody levels were assessed on days 0, 1, 2, 8, 14, 28, 56, and 180. Both vaccines were safe and had acceptable reactogenicity profiles during six months of follow-up. There were no serious or grade 3 and 4 laboratory adverse events. Moderate systemic reactogenicity (mostly headache or feeling tired) was reported by ∼23% of participants receiving either vaccine. Injection site reactogenicity was mostly mild itchiness and pain. The frequencies of vaccine take skin reactions were 73% (95% CI, 64, 81) for DVC-LVS and 80% (95% CI, 71, 87) for USAMRIID-LVS. The 90% CI for the difference in proportions was -6.9% (-16.4, 2.6). The rates of seroconversion measured by microagglutination assay on days 28 or 56 were 94% (95% CI, 88, 98; n=98/104) for DVC-LVS and 94% (95% CI, 87, 97; n=103/110) for USAMRIID-LVS (p=1.00). Day 14 sera revealed more rapid seroconversion for DVC-LVS relative to USAMRIID-LVS: 82% (95% CI, 73, 89) versus 55% (95% CI, 45, 65), respectively (p<0.0001). The DVC-LVS vaccine had similar safety, reactogenicity, take and antibody responses compared to the older USAMRIID vaccine, and was superior for early (day 14) antibody production. Vaccination take was not a sensitive surrogate for seroconversion in a multi-center study where personnel at five research clinics performed assessments. ClinicalTrials.gov identifier NCT01150695. Copyright © 2017 The Authors. Published by Elsevier

  3. Shared Features of Cryptic Plasmids from Environmental and Pathogenic Francisella Species

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Challacombe, Jean Faust; Pillai, Segaran; Kuske, Cheryl R.

    The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We havemore » identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. In conclusion, genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.« less

  4. Shared Features of Cryptic Plasmids from Environmental and Pathogenic Francisella Species

    DOE PAGES

    Challacombe, Jean Faust; Pillai, Segaran; Kuske, Cheryl R.

    2017-08-24

    The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We havemore » identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. In conclusion, genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.« less

  5. Tularemia blood test

    MedlinePlus

    Tularemia test; Serology for Francisella tularensis ... This blood test is done when tularemia is suspected. ... Elsevier; 2017:chap 44. Chernecky CC, Berger BJ. Tularemia agglutinins - serum. In: Chernecky CC, Berger BJ, eds. ...

  6. Performance of seven serological assays for diagnosing tularemia

    PubMed Central

    2014-01-01

    Background Tularemia is a rare zoonotic disease caused by the Gram-negative bacterium Francisella tularensis. Serology is frequently the preferred diagnostic approach, because the pathogen is highly infectious and difficult to cultivate. The aim of this retrospective study was to determine the diagnostic accuracy of tularemia specific tests. Methods The Serazym®Anti-Francisella tularensis ELISA, Serion ELISA classic Francisella tularensis IgG/IgM, an in-house ELISA, the VIRapid® Tularemia immunochromatographic test, an in-house antigen microarray, and a Western Blot (WB) assay were evaluated. The diagnosis tularemia was established using a standard micro-agglutination assay. In total, 135 sera from a series of 110 consecutive tularemia patients were tested. Results The diagnostic sensitivity and diagnostic specificity of the tests were VIRapid (97.0% and 84.0%), Serion IgG (96.3% and 96.8%), Serion IgM (94.8% and 96.8%), Serazym (97.0% and 91.5%), in-house ELISA (95.6% and 76.6%), WB (93.3% and 83.0%), microarray (91.1% and 97.9%). Conclusions The diagnostic value of the commercial assays was proven, because the diagnostic accuracy was >90%. The diagnostic sensitivity of the in-house ELISA and the WB were acceptable, but the diagnostic accuracy was <90%. Interestingly, the antigen microarray test was very specific and had a very good positive predictive value. PMID:24885274

  7. CHLORINE INACTIVATION OF CATEGORY "A" BIO-TERRORISM AGENTS

    EPA Science Inventory

    This poster presents information on the inactivation of select bioterrorist agents. Information will be presented on chlorine disinfection of vegetative cells of Brucella suis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis and endos...

  8. Customizable PCR-microplate array for differential identification of multiple pathogens

    PubMed Central

    Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

    2014-01-01

    Customizable PCR-microplate arrays were developed for the rapid identification of Francisella tularensis subsp. tularensis, Salmonella Typhi, Shigella dysenteriae, Yersinia pestis, Vibrio cholerae Escherichia coli O157:H7, Salmonella Typhimurium, Salmonella Saintpaul, Francisella tularensis subsp. novicida, Vibrio parahaemolyticus, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of the pathogens above. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers. A mixed aliquot of genomic DNA from 38 different strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Results show specific amplifications on all the three custom plates. In a preliminary test to evaluate the sensitivity of these assays in laboratory-inoculated samples, detection limits as low as 9 cfu/g/ml S. Typhimurium were obtained from beef hot dog, and 78 cfu/ml from milk. Such microplate arrays could serve as valuable tools for initial identification or secondary confirmation of these pathogens. PMID:24215700

  9. Customizable PCR-microplate array for differential identification of multiple pathogens.

    PubMed

    Woubit, Abdela; Yehualaeshet, Teshome; Roberts, Sherrelle; Graham, Martha; Kim, Moonil; Samuel, Temesgen

    2013-11-01

    Customizable PCR-microplate arrays were developed for the rapid identification of Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Typhi, Shigella dysenteriae, Escherichia coli O157:H7, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. novicida, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, and Yersinia pseudotuberculosis. Previously, we identified highly specific primers targeting each of these pathogens. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers identified. A mixed aliquot of genomic DNA from 38 strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Specific amplifications were obtained on all three custom plates. In preliminary tests performed to evaluate the sensitivity of these assays in samples inoculated in the laboratory with Salmonella Typhimurium, amplifications were obtained from 1 g of beef hot dog inoculated at as low as 9 CFU/ml or from milk inoculated at as low as 78 CFU/ml. Such microplate arrays could be valuable tools for initial identification or secondary confirmation of contamination by these pathogens.

  10. Evaluation of In-House and Commercial Serological Tests for Diagnosis of Human Tularemia

    PubMed Central

    Yanes, Hadjila; Hennebique, Aurélie; Pelloux, Isabelle; Boisset, Sandrine; Bicout, Dominique J.; Caspar, Yvan

    2017-01-01

    ABSTRACT Tularemia is a zoonosis caused by the bacterium Francisella tularensis. Its specific diagnosis remains based on serological methods, while F. tularensis is rarely detected in clinical samples by culture or PCR. The aim of the present study was to evaluate the performance of the Serion enzyme-linked immunosorbent assay (ELISA) classic Francisella tularensis IgG and IgM tests (Virion/Serion GmbH Institute, Würzburg, Germany) and the VIRapid tularemia immunochromatographic test (ICT) (Vircell, Granada, Spain) compared to that of the in-house microagglutination test (MAT) and indirect immunofluorescence assay (IFA) currently used at the French National Reference Center for Francisella. We evaluated 256 consecutive sera from 208 patients, including 51 confirmed and 23 probable tularemia cases, and 134 control patients not infected with F. tularensis. The IFA tests displayed 72.5% sensitivity for IgM (cutoff titer ≥80) and 74.5% for IgG (cutoff titer ≥160), and 99.3% specificity for both IgM and IgG. Using cutoffs advocated by the manufacturer, the Serion ELISAs displayed 88.2% sensitivity for IgM and 86.3% for IgG antibodies; specificity was 94.8% for IgM and 95.5% for IgG. Compared to MAT and IFA tests, the Serion ELISAs allowed earlier detection of specific antibodies (1 to 2 weeks versus 2 to 3 weeks after the onset of symptoms). The ICT sensitivity and specificity were 90% and 83.6%, respectively, when considering the cutoff advocated by the manufacturer. In conclusion, the Serion ELISAs are useful as screening tests for tularemia diagnosis, but additional confirmatory tests (such as MAT and IFA) are needed, especially in areas of low endemicity. PMID:29118164

  11. Detection of Multiple Waterborne Pathogens Using Microsequencing Arrays

    EPA Science Inventory

    Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically dete...

  12. Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens.

    PubMed

    Ruppitsch, W; Stöger, A; Indra, A; Grif, K; Schabereiter-Gurtner, C; Hirschl, A; Allerberger, F

    2007-03-01

    In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.

  13. Synthetic Promoters Functional in Francisella novicida and Escherichia coli

    PubMed Central

    McWhinnie, Ralph L.

    2014-01-01

    In this work, we describe the identification of synthetic, controllable promoters that function in the bacterial pathogen Francisella novicida, a model facultative intracellular pathogen. Synthetic DNA fragments consisting of the tetracycline operator (tetO) flanked by a random nucleotide sequence were inserted into a Francisella novicida shuttle plasmid upstream of a promoterless artificial operon containing the reporter genes cat and lacZ. Fragments able to promote transcription were selected for based on their ability to drive expression of the cat gene, conferring chloramphenicol resistance. Promoters of various strengths were found, many of which were repressed in the presence of the tetracycline repressor (TetR) and promoted transcription only in the presence of the TetR inducer anhydrotetracycline. A subset of both constitutive and inducible synthetic promoters were characterized to find their induction ratios and to identify their transcription start sites. In cases where tetO was located between or downstream of the −10 and −35 regions of the promoter, control by TetR was observed. If the tetO region was upstream of the −35 region by more than 9 bp, it did not confer TetR control. We found that three of three promoters isolated in F. novicida functioned at a comparable level in E. coli; however, none of the 10 promoters isolated in E. coli functioned at a significant level in F. novicida. Our results allowed us to isolate minimal F. novicida promoters of 47 and 48 bp in length. PMID:24141126

  14. Simultaneous, specific and real-time detection of biothreat and frequently encountered food-borne pathogens

    PubMed Central

    Woubit, Abdela Salah; Yehualaeshet, Teshome; Habtemariam, Tsegaye; Samuel, Temesgen

    2012-01-01

    The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia and Francisella include important food safety and biothreat agents causing food-related and other human illnesses worldwide. We aimed to develop rapid methods with the capability to simultaneously and differentially detect all six pathogens in one run. Our initial experiments to use previously reported sets of primers revealed non-specificity of some of the sequences when tested against a broader array of pathogens, or proved not optimal for simultaneous detection parameters. By extensive mining of the whole genome and protein databases of diverse closely and distantly related bacterial species and strains, we have identified unique genome regions, which we utilized to develop a detection platform. Twelve of the specific genomic targets we have identified to design the primers in F. tularensis ssp. tularensis, F. tularensis ssp. novicida, S. dysentriae, S. typhimurium, V. cholera, Y. pestis, and Y. pseudotuberculosis contained either hypothetical or putative proteins, the functions of which have not been clearly defined. Corresponding primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in-silico PCR against whole genome sequences of different species, sub-species, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (E.coli O157:H7 strain EDL 933, Shigella dysentriae, Salmonella typhi, Francisella tularensis ssp. tularensis, Vibrio cholera, and Yersinia pestis) and six foodborne pathogens (Salmonella typhimurium, Salmonella saintpaul, Shigella sonnei, Francisella novicida, Vibrio parahemolytica and Yersinia pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed using purified DNA showed the lowest detection limit of 640 fg

  15. A Spontaneous Mutation in kdsD, a Biosynthesis Gene for 3-Deoxy-D-manno-Octulosonic Acid, Occurred in a Ciprofloxacin Resistant Strain of Francisella tularensis and Causes a High Level of Attenuation in Murine Models of Tularemia

    DTIC Science & Technology

    2016-08-30

    tularemia. As a facultative intracellular pathogen, its ability to replicate 72 within various host cells , such as macrophages, dendritic cells ...neutrophils, and epithelial cells is 73 well documented and essential for virulence (1-11). F. tularensis is able to infect a wide range 74 of animal...contrast, 94 lipid A moieties from other gram-negative bacteria are able to interact with the Toll-like-receptor 95 4, activating the innate immune

  16. Tularaemia in European Brown Hares (Lepus europaeus) and Mountain Hares (Lepus timidus) Characterized by Histopathology and Immunohistochemistry: Organ Lesions and Suggestions of Routes of Infection and Shedding.

    PubMed

    Hestvik, G; Uhlhorn, H; Södersten, F; Åkerström, S; Karlsson, E; Westergren, E; Gavier-Widén, D

    Tularaemia is an emerging zoonotic infectious disease caused by the bacterium Francisella tularensis. In Sweden, hares are considered to be key species in the epidemiology of tularaemia. The aim of this study was to characterize the pathology of natural tularaemia infection in European brown hares (EBHs) (Lepus europaeus) and mountain hares (MHs) (Lepus timidus) in Sweden, in order to better understand the presentation of disease and the routes of infection, body dissemination and shedding of F. tularensis. During 2000-2013, 49 EBHs and 37 MHs were diagnosed with tularaemia. Enlargement of the spleen was seen in 80% of EBHs and 62% of MHs. Necrosis was often obvious in the bone marrow, liver, lung and spleen, but 30% of the hares had no lesions or minimal gross lesions. On microscopical examination of tissues from 27 EBHs and three MHs, necrosis was seen in the majority of samples of liver, spleen, bone marrow, lymph node and adrenal glands and was common in the lungs and brain meninges. Immunohistochemistry for Francisella spp. detected bacteria in association with necrosis and inflammation. In several cases, Francisella spp. were also found inside blood vessels, in the renal pelvis, in lactating mammary glands, in bronchioles and in the skin, associated with tick bites. Using quantitative polymerase chain reaction, two genotypes of F. tularensis subsp. holarctica were found; canSNP group B.6, all belonging to subgroup B.7, and canSNP group B.12. There were no differences in pathology between the genotypes. Our results indicate that the urinary tract and mammary glands are important routes for the shedding of F. tularensis. Hunters may not be aware of the risks of contracting tularaemia while handling hares, since infected hares do not always show noticeable gross lesions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Host immune response and acute disease in a zebrafish model of francisella pathogenesis

    USGS Publications Warehouse

    Vojtech, L.N.; Sanders, G.E.; Conway, C.; Ostland, V.; Hansen, J.D.

    2009-01-01

    Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebraflsh were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebraflsh following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebraflsh mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-l?? (IL-1??), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebraflsh to heat-killed bacteria demonstrated that the significant induction of IL-?? was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebraflsh share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

  18. Molecular detection of Rickettsia, Anaplasma, Coxiella and Francisella bacteria in ticks collected from Artiodactyla in Thailand.

    PubMed

    Sumrandee, Chalao; Baimai, Visut; Trinachartvanit, Wachareeporn; Ahantarig, Arunee

    2016-07-01

    A total of 79 ticks collected from Sambar deer (Cervus unicolor), Barking deer (Muntiacus muntjak) and Wild boar (Sus scrofa) were examined by PCR for the presence of Rickettsia, Anaplasma, Coxiella, and Francisella bacteria. Of the 79 ticks, 13% tested positive for Rickettsia, 15% tested positive for Anaplasma, 4% tested positive for Coxiella, and 3% tested positive for Francisella. Interestingly, triple infection with Anaplasma, Rickettsia and Francisella was determined in a Dermacentor auratus tick. Moreover, another triple infection with Rickettsia, Anaplasma, and Coxiella was found in a Haemaphysalis lagrangei tick. Double infection of Rickettsia with Coxiella was also detected in another H. lagrangei tick. From the phylogenetic analyses, we found a Rickettsia sp. with a close evolutionary relationship to Rickettsia bellii in the H. lagrangei tick. We also found the first evidence of a Rickettsia sp. that is closely related to Rickettsia tamurae in Rhipicephalus (Boophilus) microplus ticks from Thailand. H. lagrangei and Haemaphysalis obesa ticks collected from Sambar deer tested positive for Anaplasma species form the same clade with Anaplasma bovis. In contrast, other H. lagrangei ticks collected from Sambar deer and D. auratus ticks collected from Wild boar were also reported for the first time to be infected with an Anaplasma species that is closely related to Anaplasma platys. The phylogenetic analysis of the 16S rRNA gene of Coxiella bacteria revealed that Coxiella symbionts from H. lagrangei formed a distinctly different lineage from Coxiella burnetii (a human pathogen). Additionally, Francisella bacteria identified in D. auratus ticks were found to be distantly related to a group of pathogenic Francisella species. The identification of these bacteria in several feeding ticks suggests the risk of various emerging tick-borne diseases and endosymbionts in humans, wildlife, and domestic animals in Thailand. Copyright © 2016 Elsevier GmbH. All rights

  19. Expansion and retention of pulmonary CD4+ T cells after prime boost vaccination correlates with improved longevity and strength of immunity against tularemia.

    PubMed

    Roberts, Lydia M; Wehrly, Tara D; Crane, Deborah D; Bosio, Catharine M

    2017-05-02

    Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent intracellular bacterium. Inhalation of 10 or fewer organisms results in an acute and potentially lethal disease called pneumonic tularemia. Ftt infections occur naturally in the U.S. and Ftt was developed as a bioweapon. Thus, there is a need for vaccines that protect against this deadly pathogen. Although a live vaccine strain of Francisella tularensis (LVS) exists, LVS fails to generate long-lived protective immunity against modest challenge doses of Ftt. We recently identified an important role for high avidity CD4 + T cells in short-term protection and hypothesized that expanding this pool of cells would improve overall vaccine efficacy with regard to longevity and challenge dose. In support of our hypothesis, application of a prime/boost vaccination strategy increased the pool of high avidity CD4 + T cells which correlated with improved survival following challenge with either increased doses of virulent Ftt or at late time points after vaccination. In summary, we demonstrate that both epitope selection and vaccination strategies that expand antigen-specific T cells correlate with superior immunity to Ftt as measured by survival. Copyright © 2017. Published by Elsevier Ltd.

  20. Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015

    PubMed Central

    Janse, Ingmar; Maas, Miriam; Rijks, Jolianne M; Koene, Miriam; van der Plaats, Rozemarijn QJ; Engelsma, Marc; van der Tas, Peter; Braks, Marieta; Stroo, Arjan; Notermans, Daan W; de Vries, Maaike C; Reubsaet, Frans; Fanoy, Ewout; Swaan, Corien; Kik, Marja JL; IJzer, Jooske; Jaarsma, Ryanne I; van Wieren, Sip; de Roda-Husman, Ana Maria; van Passel, Mark; Roest, Hendrik-Jan; van der Giessen, Joke

    2017-01-01

    Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife. PMID:28877846

  1. [THE DEVELOPMENT OF IMMUNE ENZYME AND IMMUNE CHROMATOGRAPHIC MONOCLONAL TEST-SYSTEM FOR DETECTING TULAREMIA AGENT].

    PubMed

    Eremkin, A V; Elagin, G D; Petchenkin, D V; Fomenkov, O O; Bogatcheva, N V; Kitmanov, A A; Kuklina, G V; Tikhvinskaya, O V

    2016-03-01

    The immune enzyme and immunochromatographic test-systems for detecting tularemia agent were developed on the basis of selected set of monoclonal antibodies having immunochemical activity to antigens Francisella tularensis. The evaluation of sensitivity and specificity of developed test-systems demonstrated that samples provided detection of strains of F. tularensis in concentration from 5.0 x 105 mkxcm-3 to 1.0 x 106 mkxcm-3 and gave no false positive results in analysis of heterologous microorganisms in concentration of 1.0 x 108 mkxcm-3.

  2. [Microbiological and clinical aspects of tularaemia].

    PubMed

    Pavliš, Oto; Pohanka, Miroslav

    2011-10-01

    Francisella tularensis belongs to the most important biological agents potentially applicable in biological warfare and bioterrorism. High virulence, easy and rapid spread among individual vectors, stability of the cells in aerosol and good penetration into the lungs make F. tularensis one of the most important biological warfare agents in both human and veterinary medicine. The text provides comprehensive data about tularaemia and outlines the fate of the pathogen in the host. Special attention is paid to immunological aspects of the disease, therapy, and diagnostic methods.

  3. Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

    PubMed

    Janse, Ingmar; Maas, Miriam; Rijks, Jolianne M; Koene, Miriam; van der Plaats, Rozemarijn Qj; Engelsma, Marc; van der Tas, Peter; Braks, Marieta; Stroo, Arjan; Notermans, Daan W; de Vries, Maaike C; Reubsaet, Frans; Fanoy, Ewout; Swaan, Corien; Kik, Marja Jl; IJzer, Jooske; Jaarsma, Ryanne I; van Wieren, Sip; de Roda-Husman, Ana Maria; van Passel, Mark; Roest, Hendrik-Jan; van der Giessen, Joke

    2017-08-31

    Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife. This article is copyright of The Authors, 2017.

  4. Tularaemia: clinical aspects in Europe.

    PubMed

    Maurin, Max; Gyuranecz, Miklós

    2016-01-01

    Tularaemia is a zoonotic disease caused by Francisella tularensis, a Gram-negative, facultative intracellular bacterium. Typically, human and animal infections are caused by F tularensis subspecies tularensis (type A) strains mainly in Canada and USA, and F tularensis subspecies holarctica (type B) strains throughout the northern hemisphere, including Europe. In the past, the epidemiological, clinical, therapeutic, and prognostic aspects of tularaemia reported in the English medical literature were mainly those that had been reported in the USA, where the disease was first described. Tularaemia has markedly changed in the past decade, and a large number of studies have provided novel data for the disease characteristics in Europe. In this Review we aim to emphasise the specific and variable aspects of tularaemia in different European countries. In particular, two natural lifecycles of F tularensis have been described in this continent, although not fully characterised, which are associated with different modes of transmission, clinical features, and public health burdens of tularaemia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Tularaemia in southwest Germany: Three cases of tick-borne transmission.

    PubMed

    Boone, I; Hassler, D; Nguyen, T; Splettstoesser, W D; Wagner-Wiening, C; Pfaff, G

    2015-07-01

    Tularaemia, caused by Francisella tularensis, is an endemic zoonosis frequently occurring in southwest Germany. Since 2005 there is an increase in the number of reported cases of tularaemia in Germany. We report on two cases of ulceroglandular tularaemia and one case of glandular tularaemia that occurred in the summer of 2012 and 2013 in two counties in the Federal State of Baden-Wuerttemberg. Bacteria were transmitted through tick bites, which to date has only rarely been reported in Germany. Inadequate treatment of the patients and an aggravation of clinical symptoms were caused by a delay between disease onset and the detection of the pathogen. Although contact to or consumption of infected hares are the most often reported transmission routes of tularaemia in Germany, tick-bites should also be taken into account. Health professionals should include Francisella tularensis in the differential diagnosis of patients with fever and/or ulcerative lymphadenopathy following a tick bite. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Treatment of Tularemia in Patient with Chronic Graft-versus-Host Disease

    PubMed Central

    Seibold, Erik; Knabbe, Cornelius; Kaufmann, Martin; Splettstoesser, Wolf

    2013-01-01

    We describe a case of human tularemia caused by Francisella tularensis subsp. holarctica in a stem cell transplant recipient with chronic graft-versus-host disease who was receiving levofloxacin prophylaxis. The infection was characterized by pneumonia with septic complications. The patient was successfully treated with doxycycline. PMID:23647853

  7. Serologic survey for viral and bacterial infections in western populations of Canada Lynx (Lynx canadensis)

    Treesearch

    Roman Biek; Randall L. Zarnke; Colin Gillin; Margaret Wild; John R. Squires; Mary Poss

    2002-01-01

    A serologic survey for exposure to pathogens in Canada lynx (Lynx canadensis) in western North America was conducted. Samples from 215 lynx from six study areas were tested for antibodies to feline parvovirus (FPV), feline coronavirus, canine distemper virus, feline calicivirus, feline herpesvirus, Yersinia pestis, and Francisella tularensis. A subset of...

  8. The metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic Francisella.

    PubMed

    Ziveri, Jason; Tros, Fabiola; Guerrera, Ida Chiara; Chhuon, Cerina; Audry, Mathilde; Dupuis, Marion; Barel, Monique; Korniotis, Sarantis; Fillatreau, Simon; Gales, Lara; Cahoreau, Edern; Charbit, Alain

    2017-10-11

    The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response.The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.

  9. Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations.

    PubMed

    Sjöstedt, Anders

    2007-06-01

    Francisella tularensis has been recognized as a human pathogen for almost 100 years and is the etiological agent of the zoonotic disease tularemia. Soon after its discovery, it became recognized as an important pathogen in several parts of the world, for example, in the United States and Soviet Union. The number of tularemia cases in the two countries peaked in the 1940s and has thereafter steadily declined. Despite this decline, there was still much interest in the pathogen in the 1950s and 1960s since it is highly infectious and transmissible by aerosol, rendering it a potent biothreat agent. In fact, it was one of the agents that was given the highest priority in the offensive programs of the United States and Soviet Union. After termination of the offensive programs in the 1960s, the interest in F. tularensis diminished significantly and little research was carried out for several decades. Outbreaks of tularemia during the last decade in Europe, for example, in Kosovo, Spain, and Scandinavia, led to a renewed public interest in the disease. This, together with a massive increase in the research funding, in particular in the United States since 2001, has resulted in a significant increase in the number of active Francisella researchers. This article summarizes, predominantly with a historical perspective, the epidemiology and clinical manifestations of tularemia and the physiology of F. tularensis.

  10. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Linxiao; Valentine, Jenny L.; Huang, Chung-Jr

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. In this paper, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Usingmore » this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Finally, given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.« less

  11. Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

    DOE PAGES

    Chen, Linxiao; Valentine, Jenny L.; Huang, Chung-Jr; ...

    2016-06-06

    The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. In this paper, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Usingmore » this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Finally, given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.« less

  12. Incidence and seroprevalence of tularaemia in Finland, 1995 to 2013: regional epidemics with cyclic pattern.

    PubMed

    Rossow, H; Ollgren, J; Hytonen, J; Rissanen, H; Huitu, O; Henttonen, H; Kuusi, M; Vapalahti, O

    2015-08-20

    We studied the incidence of reported tularaemia by year and region and the prevalence of antibodies against Francisella tularensis in the adult general population in Finland. Moreover, we assessed the correlation between vole population cycles and human tularaemia outbreaks. The seroprevalence study made use of serum samples from a nationwide population-based health survey (Health 2000). The samples of 1,045 randomly selected persons, representative for the Finnish population in each region, were screened with an enzyme-linked immunosorbent assay (ELISA) for the presence of IgG antibodies against F. tularensis, and positive results were further confirmed by immunoblotting. A serological response to F. tularensis was found in 2% (95% confidence interval: 1.1–3.5) of the population. Incidence and seroprevalence were highest in the same areas, and vole population peaks clearly preceded tularaemia outbreaks one year later.

  13. Waterborne outbreak of tularemia associated with crayfish fishing.

    PubMed Central

    Anda, P.; Segura del Pozo, J.; Díaz García, J. M.; Escudero, R.; García Peña, F. J.; López Velasco, M. C.; Sellek, R. E.; Jiménez Chillarón, M. R.; Sánchez Serrano, L. P.; Martínez Navarro, J. F.

    2001-01-01

    In 1997, an outbreak of human tularemia associated with hare-hunting in central Spain affected 585 patients. We describe the identification of Francisella tularensis biovar palaearctica in a second outbreak of ulceroglandular tularemia associated with crayfish (Procambarus clarkii) fishing in a contaminated freshwater stream distant from the hare-associated outbreak. The second outbreak occurred 1 year after the first. PMID:11485678

  14. Prevalence of tick-borne pathogens in Ixodes ricinus and Dermacentor reticulatus ticks from different geographical locations in Belarus.

    PubMed

    Reye, Anna L; Stegniy, Valentina; Mishaeva, Nina P; Velhin, Sviataslau; Hübschen, Judith M; Ignatyev, George; Muller, Claude P

    2013-01-01

    Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5-17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.

  15. Pneumonic Tularemia in Rabbits Resembles the Human Disease as Illustrated by Radiographic and Hematological Changes after Infection

    PubMed Central

    Reed, Douglas S.; Smith, Le'Kneitah; Dunsmore, Tammy; Trichel, Anita; Ortiz, Luis A.; Cole, Kelly Stefano; Barry, Eileen

    2011-01-01

    Background Pneumonic tularemia is caused by inhalation of the gram negative bacterium, Francisella tularensis. Because of concerns that tularemia could be used as a bioterrorism agent, vaccines and therapeutics are urgently needed. Animal models of pneumonic tularemia with a pathophysiology similar to the human disease are needed to evaluate the efficacy of these potential medical countermeasures. Principal Findings Rabbits exposed to aerosols containing Francisella tularensis strain SCHU S4 developed a rapidly progressive fatal pneumonic disease. Clinical signs became evident on the third day after exposure with development of a fever (>40.5°C) and a sharp decline in both food and water intake. Blood samples collected on day 4 found lymphopenia and a decrease in platelet counts coupled with elevations in erythrocyte sedimentation rate, alanine aminotransferase, cholesterol, granulocytes and monocytes. Radiographs demonstrated the development of pneumonia and abnormalities of intestinal gas consistent with ileus. On average, rabbits were moribund 5.1 days after exposure; no rabbits survived exposure at any dose (190–54,000 cfu). Gross evaluation of tissues taken at necropsy showed evidence of pathology in the lungs, spleen, liver, kidney and intestines. Bacterial counts confirmed bacterial dissemination from the lungs to the liver and spleen. Conclusions/Significance The pathophysiology of pneumonic tularemia in rabbits resembles what has been reported for humans. Rabbits therefore are a relevant model of the human disease caused by type A strains of F. tularensis. PMID:21931798

  16. Landscape Epidemiology of Tularemia Outbreaks in Sweden

    PubMed Central

    Svensson, Kerstin; Bäck, Erik; Eliasson, Henrik; Berglund, Lennart; Granberg, Malin; Karlsson, Linda; Larsson, Pär; Forsman, Mats

    2009-01-01

    Summer outbreaks of tularemia that occurred from 1995 through 2005 in 2 locations in Sweden affected 441 persons. We performed an epidemiologic investigation of these outbreaks using a novel strategy, involving high-resolution genotyping of Francisella tularensis isolates obtained from 136 patients (using 18 genetic markers developed from 6 F. tularensis genome sequences) and interviews with the patients. Strong spatial associations were found between F. tularensis subpopulations and the places of disease transmission; infection by some subpopulations occurred within areas as small as 2 km2, indicating unidentified environmental point sources of tularemia. In both locations, disease clusters were associated with recreational areas beside water, and genetic subpopulations were present throughout the tularemia season and persisted over years. High-resolution genotyping in combination with patients’ statements about geographic places of disease transmission provided valuable indications of likely sources of infection and the causal genotypes during these tularemia outbreaks. PMID:19961673

  17. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification.

    PubMed

    Janse, Ingmar; Hamidjaja, Raditijo A; Bok, Jasper M; van Rotterdam, Bart J

    2010-12-08

    Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum.

  18. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

    PubMed Central

    2010-01-01

    Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

  19. Public Health Surveillance: A Local Health Department Perspective

    DTIC Science & Technology

    2002-04-03

    vomiting – Diarrhea (+/-bloody) • Rash and fever – Vesicular – Petechial • Neurologic – cranial nerve palsies, HA, fever , confusion • Septic Shock...Francisella tularensis (tularemia) • Viral hemorrhagic fever Agents of Concern: CDC Category B • Coxiella burnetti (Q fever ) • Brucella species...Concern: CDC Category C • Nipah virus • hantaviruses • tickborne hemorrhagic fever viruses • yellow fever • multidrug-resistant tuberculosis

  20. Technologies and Techniques for Early Warning Systems to Monitor and Evaluate Drinking Water Quality: A State-of-the-Art Review

    DTIC Science & Technology

    2005-08-25

    tests currently available are the same as BTA with the inclusion of Vibrio cholerae O1. The detection limits of bacteria are 10 cfu/mL and for...spp., Burkholderia spp., Campylobacter spp., Clostridium perfringens, E. coli O157:H7, Francisella tularensis, Salmonella typhi, Shigella spp., Vibrio ... cholerae O1, Yersinia pestis, Y. enterocolitica Viruses Caliciviruses, Enteroviruses, Hepatitis A/E, Variola, Venezuelan equine encephalitis virus

  1. Tularaemia in Berlin - two independent cases in travellers returning from central Anatolia, Turkey, February 2011.

    PubMed

    Schubert, A; Splettstoesser, W; Bätzing-Feigenbaum, J

    2011-05-05

    Tularaemia, though rare, has recently been increasingly reported in Germany. Most cases are indigenous infections. This report describes two epidemiologically independent infections with Francisella tularensis subspecies holarctica detected in Berlin in February 2011 that were acquired in central Anatolia, Turkey. In Turkey, there have been repeated tularaemia outbreaks since 2000 and the disease should therefore be considered as a differential diagnosis in travellers returning from that country.

  2. Immunization to Protect the U.S. Armed Forces: Heritage, Current Practice, Prospects

    DTIC Science & Technology

    2005-01-01

    population . (4, 12, 16, 163, 164) This vaccine was derived from virus- infected mouse brain. Immunization to Protect the U.S. Armed Forces 14...individual health and to keep units strong so they can accomplish their military missions. The FDA-licensed vaccines selected protect against infections ...CA. Passive protection of mice against lethal Francisella tularensis (live tularemia vaccine strain) infection by the sera of human recipients of

  3. Use of essential gene, encoding prophobilinogen deaminase from extreme psychrophilic Colwellia sp. C1, to generate temperature-sensitive strain of Francisella novicida.

    PubMed

    Pankowski, J A

    2016-08-01

    Previously, several essential genes from psychrophilic bacteria have been substituted for their homologues in mesophilic bacterial pathogens to make the latter temperature sensitive. It has been noted that an essential ligA gene from an extreme psychrophile, Colwellia sp. C1, yielded a gene product that is inactivated at 27°C, the lowest that has been observed for any psychrophilic enzyme, and hypothesized that other essential proteins of that strain would also have low inactivation temperatures. This work describes the partial sequencing of the genome of Colwellia sp. C1 strain and the identification of 24 open reading frames encoding homologues of highly conserved bacterial essential genes. The gene encoding porphobilinogen deaminase (hemC), which is involved in the pathway of haem synthesis, has been tested for its ability to convert Francisella novicida into a temperature-sensitive strain. The hybrid strain carrying the C1-derived hemC gene exhibited a temperature-sensitive phenotype with a restrictive temperature of 36°C. These results support the conclusion that Colwellia sp. C1 is a rich source of heat-labile enzymes. The issue of biosafety is often raised when it comes to work with pathogenic organisms. The main concern is caused by the risk of researchers being exposed to infectious doses of dangerous microbes. This paper analyses essential genes identified in partial genomic sequence of the psychrophilic bacterium Collwelia sp. C1. These sequences can be used as a mean of generating temperature-sensitive strains of pathogenic bacteria. Such strains are incapable of surviving at the temperature of human body. This means they could be applied as vaccines or for safer work with dangerous organisms. © 2016 The Society for Applied Microbiology.

  4. Tularaemia seroprevalence of captured and wild animals in Germany: the fox (Vulpes vulpes) as a biological indicator.

    PubMed

    Kuehn, A; Schulze, C; Kutzer, P; Probst, C; Hlinak, A; Ochs, A; Grunow, R

    2013-04-01

    A total of 2475 animals from Germany, both captive and wild, were tested for antibodies against Francisella tularensis to obtain more knowledge about the presence of this pathogen in Germany. An indirect and a competitive ELISA served as screening methods, positive and inconclusive samples were confirmed by Western blot. Of the zoo animals sampled between 1992 and 2007 (n = 1122), three (0·3%) were seropositive. The seroconversion of a hippopotamus in Berlin Zoo was documented. From 1353 serum samples of wild foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and wild boars (Sus scrofa), collected between 2005 and 2009 in the federal state of Brandenburg (surrounding Berlin), a total of 101 (7·5%) tested positive for antibodies to F. tularensis lipopolysaccharide. Our results indicate a higher seroprevalence of F. tularensis in wildlife in eastern Germany than commonly assumed. Furthermore, we found foxes and raccoon dogs to be biological indicators for tularaemia.

  5. Francisella marina sp. nov., etiologic agent of systemic disease in cultured spotted rose snapper (Lutjanus guttatus) in Central America

    USDA-ARS?s Scientific Manuscript database

    Historically, piscine francisellosis in various warm, temperate and coldwater fish hosts has been attributed to Francisella noatunensis. From 2015-2016, an undescribed Francisella sp. was recovered during mortality events in cultured spotted rose snapper (Lutjanus guttatus) off the Pacific coast of ...

  6. New England Bioterrorism Preparedness Workshop

    DTIC Science & Technology

    2002-04-04

    Hypoxia • GI – Fever – Nausea/vomiting – Diarrhea (+/-bloody) • Rash and fever – Vesicular – Petechial • Neurologic – cranial nerve palsies, HA...plague) • variola major (smallpox) • Francisella tularensis (tularemia) • Viral hemorrhagic fever Agents of Concern: CDC Category B • Coxiella...burnetti (Q fever ) • Brucella species (brucellosis) • Burkholderia mallei (glanders) • ricin toxin from Ricinus communis (castor beans) • epsilon toxin of

  7. Assessing the Effectiveness of Cumulative Sum Normal- and Poisson-Based Tests for Detecting Rare Diseases

    DTIC Science & Technology

    2010-12-01

    The Francisella tularensis is one of these and is the causal agent of the tularemia disease. Tularemia is used as the motivating problem to evaluate...PAGES 79 14. SUBJECT TERMS Biosurveillance, Rare Disease, Tularemia , Cumulative Sum, CUSUM 16. PRICE CODE 17. SECURITY CLASSIFICATION OF REPORT...is one of these, and is the causal agent of the tularemia disease. Tularemia is used as the motivating problem to evaluate and compare the

  8. Entry of Yersinia Pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

    DTIC Science & Technology

    2011-03-16

    to enter the VBNC state [17,18,19,20]. In addition, many other bacterial pathogens, including Francisella tularensis, Vibrio cholerae, and Escherichia...sterilized distilled water microcosms at 4uC after 21 days [31]. Likewise, Linder and Oliver reported that Vibrio vulnificus entered the VBNC state... Vibrio cholerae, and Escherichia coli enter the VBNC state [12,21]. In addition, the two closest pathogenic relatives of Y. pestis, namely Y

  9. Survival and persistence of nonspore-forming biothreat agents in water.

    PubMed

    Gilbert, S E; Rose, L J

    2012-09-01

    To determine whether nonspore-forming biothreat agents can survive and persist in potable water that does not contain a disinfectant.  Autoclaved, de-chlorinated Atlanta municipal water was inoculated with eight isolates of bacterial biothreat agents (10⁶ CFU ml⁻¹). The inoculated water samples were incubated at 5, 8 (Francisella tularensis only) or 25°C and assayed for viability by culture and by the presence of metabolic activity as measured by esterase activity (ScanRDI, AES Chemunex). Viability as determined by culture varied from 1 to 30 days, depending upon the organism and the temperature of the water. All organisms were determined viable as measured by esterase activity for the entire 30 days, regardless of the incubation temperature.  Francisella tularensis was culturable for at least 21 days if held at 8°C. The remaining nonspore-forming bacterial biothreat agents were found to be metabolically active for at least 30 days in water held at 5 or 25°C.  The data can assist public health officials to determine the safety of drinking water after contamination with a biothreat agent. No claim to US Government works. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  10. [Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

    PubMed

    Wen, Hai-yan; Wang, Jing; Liu, Heng-chuan; Sun, Xiao-hong; Yang, Yu; Hu, Kong-xin; Shan, Lin-jun

    2009-10-01

    To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

  11. Disease Surveillance of California Ground Squirrels ( Spermophilus beecheyi ) in a Drive-through Zoo in Oregon, USA.

    PubMed

    Beest, Julia Ter; Cushing, Andrew; McClean, Modesto; Hsu, Wendy; Bildfell, Robert

    2017-07-01

    Rodents and other small wild mammals are often considered to be pests and vectors for disease in zoos that house small populations of valuable threatened and endangered animals. In 2005, three nonhuman primates at a drive-through zoo in Oregon, US, acquired tularemia from an unknown source. Due to an abundance of California ground squirrels ( Spermophilus beecheyi ) on zoo grounds, we instituted serosurveillance of this species from July through September 2008 to determine the prevalence of antibodies against pathogens considered to be potentially transmissible to collection animals. Serologic testing was performed for Francisella tularensis ; Leptospira interrogans serovars Canicola, Grippotyphosa, Hardjo, Icterohemorrhagiae, and Pomona; Toxoplasma gondii ; and Yersinia pestis . All squirrels were seronegative for Yersinia pestis (0%; 0/45) and Toxoplasma gondii (0%; 0/20); there was a prevalence of 2% (1/45) for Francisella tularensis antibodies and 57% (24/42) were positive for various Leptospira serovars. Although it remains unclear whether ground squirrels present a significant risk for transmission of disease to zoo animals, vaccination of high-risk zoo animals against leptospirosis warrants consideration. Beyond this, continued vigilance and persistence with various forms of pest control may reduce the likelihood of disease transmission from wildlife hosts to animals in human care.

  12. Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp. orientalis

    PubMed Central

    Gustavo Ramirez-Paredes, Jose; Harold, Graham; Lopez-Jimena, Benjamin; Adams, Alexandra; Weidmann, Manfred

    2018-01-01

    Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7–3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through

  13. Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp. orientalis.

    PubMed

    Shahin, Khalid; Gustavo Ramirez-Paredes, Jose; Harold, Graham; Lopez-Jimena, Benjamin; Adams, Alexandra; Weidmann, Manfred

    2018-01-01

    Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7-3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through

  14. Tularaemia in a brown hare (Lepus europaeus) in 2013: first case in the Netherlands in 60 years.

    PubMed

    Rijks, J M; Kik, M; Koene, M G; Engelsma, M Y; van Tulden, P; Montizaan, M G; Oomen, T; Spierenburg, M A; Ijzer, J; van der Giessen, J W; Gröne, A; Roest, H J

    2013-12-05

    Tularaemia has not been reported in Dutch wildlife since 1953. To enhance detection, as of July 2011, brown hares (Lepus europaeus) submitted for postmortem examination in the context of non-targeted wildlife disease surveillance, were routinely tested for tularaemia by polymerase chain reaction (PCR). Francisella tularensis subspecies holarctica infection was confirmed in a hare submitted in May 2013. The case occurred in Limburg, near the site of the 1953 case. Further surveillance should clarify the significance of this finding.

  15. Prevalence of Rickettsia species in Dermacentor variabilis ticks from Ontario, Canada.

    PubMed

    Wood, Heidi; Dillon, Liz; Patel, Samir N; Ralevski, Filip

    2016-07-01

    Relatively little is known about the prevalence of rickettsial species in Dermacentor ticks in eastern Canada. In this study, Dermacentor ticks from the province of Ontario, Canada, were tested for the presence of spotted fever group rickettsial (SFGR) species, Coxiella burnetii and Francisella tularensis. Rickettsia rickettsii was not detected in any ticks tested, but R. montanensis was detected at a prevalence of 2.2% in D. variabilis (17/778). Two other SFGR species, R. parkeri and Candidatus R. andeanae, were detected individually in 2 Amblyomma maculatum ticks. Rickettsia peacockii, a non-pathogenic endosymbiont, was detected in two D. andersonii ticks. Given the highly abundant nature of D. variabilis, surveillance for human pathogens in this species of tick has important public health implications, but the lack of detection of known human pathogens indicates a low risk of infection via this tick species in Ontario. However, the detection of R. parkeri in an adventive A. maculatum tick indicates that health care providers should be aware of the possibility of spotted fever rickettsioses in individuals with a history of travel outside of Ontario and symptoms compatible with a spotted fever rickettsiosis. Coxiella burnetii and Francisella tularensis, human pathogens also potentially transmitted by D. variabilis, were not detected in a subset of the ticks. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration

    USGS Publications Warehouse

    Francy, D.S.; Bushon, R.N.; Brady, A.M.G.; Bertke, E.E.; Kephart, C.M.; Likirdopulos, C.A.; Mailot, B.E.; Schaefer, F. W.; Lindquist, H.D. Alan

    2009-01-01

    Aims: To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large-volume drinking-water samples concentrated by ultrafiltration (UF). Methods and Results: Drinking-water samples (100 l) were seeded with Bacillus anthracis, Cryptospordium parvum, Francisella tularensis, Salmonella Typhi, and Vibrio cholerae and concentrated by UF. Recoveries by traditional methods were variable between samples and between some replicates; recoveries were not determined by qPCR. Francisella tularensis and V. cholerae were detected in all 14 samples after UF, B. anthracis was detected in 13, and C. parvum was detected in 9 out of 14 samples. Numbers found by qPCR after UF were significantly or nearly related to those found by traditional methods for all organisms except for C. parvum. A qPCR assay for S. Typhi was not available. Conclusions: qPCR can be used to rapidly detect biological agents after UF as well as traditional methods, but additional work is needed to improve qPCR assays for several biological agents, determine recoveries by qPCR, and expand the study to other areas. Significance and Impact of the Study: To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples. ?? 2009 The Society for Applied Microbiology.

  17. Tularemia type A in captive Bornean orangutans (Pongo pygmaeus pygmaeus).

    PubMed

    Ketz-Riley, Cornelia J; Kennedy, George A; Carpenter, James W; Zeidner, Nordin S; Petersen, Jeannine M

    2009-06-01

    In 2003, tularemia was suspected to be the cause of severe illness in two orangutans (Pongo pygmaeus pygmaeus) and the cause of death in a third orangutan at an urban zoo. The two sick orangutans were treated two times under chemical immobilization with i.v. doxycycline, fluids, and antipyretic drugs, followed by a sustained course of oral doxycycline. The rest of the orangutan group was treated prophylactically with oral doxycycline. Postmortem diagnosis was obtained via immunohistochemistry and bacterial culture that revealed Francisella tularensis type A. Tularemia was also confirmed in the two surviving orangutans via paired serology testing. In addition, F. tularensis was identified in two wild rabbit carcasses submitted during a die-off, several weeks prior to the tularemia outbreak in the apes, indicating that rabbits were possibly a reservoir for tularemia within the zoo premises.

  18. Tularaemia: a challenging zoonosis.

    PubMed

    Carvalho, C L; Lopes de Carvalho, I; Zé-Zé, L; Núncio, M S; Duarte, E L

    2014-03-01

    In recent years, several emerging zoonotic vector-borne infections with potential impact on human health have been identified in Europe, including tularaemia, caused by Francisella tularensis. This remarkable pathogen, one of the most virulent microorganisms currently known, has been detected in increasingly new settings and in a wide range of wild species, including lagomorphs, rodents, carnivores, fish and invertebrate arthropods. Also, a renewed concern has arisen with regard to F. tularensis: its potential use by bioterrorists. Based on the information published concerning the latest outbreaks, the aim of this paper is to review the main features of the agent, its biology, immunology and epidemiology. Moreover, special focus will be given to zoonotic aspects of the disease, as tularaemia outbreaks in human populations have been frequently associated with disease in animals. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. [Evaluation of modern epizootic activity of natural tularemia foci in Voronezh region using immune-serological and molecular-genetic study of main carriers of the disease].

    PubMed

    Meshcheriakova, I S; Trankvilevskiĭ, D V; Kvasov, D A; Mikhaĭlova, T V; Kormilitsina, M I; Demidova, T N; Stepkin, Iu I; Zhukov, V I

    2015-01-01

    Improvement of monitoring and prognosis of epidemic manifestations of natural foci of tularemia on the territory of Voronezh region using immune-serological and molecular-genetic study of main carriers of the disease. 539 small mammals captured during summer period of 2011 in 4 districts of North-Eastern part of Voronezh region were studied. Animal organs were studied by serologic (search for Francisella tularensis antigens) and molecular-biologic (detection of F. tularensis DNA) methods. Tularemia antigen was detected using passive hemagglutination reaction (PHAR) with erythrocytic tularemia immunoglobulin diagnosticum. Real-time polymerase chain reaction (RT-PCR) was applied for detection of tularemia causative agent DNA. Complex study revealed epizootic activity of natural foci of tularemia in the examined territory. F. tularensis antigen and/or DNA were detected in 82 objects (15.2%). Use of RT-PCR allowed to additionally detect samples with relatively low content of F. tularensis DNA substrate, when antigen was not detected in samples. High sensitivity and specificity of the RT-PCR was ensured by inclusion of specific probes (tu14-PR2 and ISFTu2P). The results obtained give evidence on functioning and epizootic activity of natural foci of tularemia in Voronezh region that requires constant monitoring of the territory and prophylaxis measures, first of all vaccination of risk groups by live tularemia vaccine.

  20. Bis-indolic compounds as potential new therapeutic alternatives for tularaemia

    PubMed Central

    Caspar, Yvan; Sutera, Vivien; Boisset, Sandrine; Denis, Jean-Noël; Maurin, Max

    2014-01-01

    Francisella tularensis is the etiological agent of tularaemia and a CDC class A biological threat agent. Few antibiotic classes are currently useful in treating tularaemia, including the aminoglycosides gentamicin and streptomycin, fluoroquinolones, and tetracyclines. However, treatment failures and relapses remain frequent and F. tularensis strains resistant to antibiotics have been easily selected in vitro. In this study, we evaluated the activity of new synthetic bis-indole derivatives against this pathogen. Minimum inhibitory concentrations (MICs) of four compounds (dcm01 to dcm04) were determined for the reference strains F. tularensis subsp. holarctica LVS NCTC10857, F. tularensis subsp. novicida CIP56.12 and F. philomiragia ATCC25015, and for 41 clinical strains of F. tularensis subsp. holarctica isolated in France. Minimal bactericidal concentrations (MBCs) were determined for the dcm02 and dcm04 compounds for the LVS and two clinical strains. Killing curves were also determined for the same three strains exposed to dcm04. All tested bis-indole compounds were bacteriostatic against F. tularensis subsp. holarctica strains, with a MIC90 of 8 μg/mL for dcm01, dcm02, and dcm03, and 2 μg/mL for dcm04. Only one strain was resistant to both dcm01 and dcm03, with MICs > 32 μg/mL. In contrast, F. tularensis subsp. novicida was resistant to all derivatives and F. philomiragia was only susceptible to dcm02 and dcm04, with MICs of 16 and 4 μg/mL, respectively. MBC and killing curve experiments revealed significant bactericidal activity (i.e., 3-log reduction of the bacterial inoculum) of the dcm02 and dcm04 compounds only for the LVS strain. In conclusion, we have identified novel synthetic bis-indole compounds that are active against F. tularensis subsp. holarctica. They may be drug candidates for the development of new therapeutic alternatives for tularaemia treatment. Their further characterization is needed, especially identification of their bacterial targets

  1. Bis-indolic compounds as potential new therapeutic alternatives for tularaemia.

    PubMed

    Caspar, Yvan; Sutera, Vivien; Boisset, Sandrine; Denis, Jean-Noël; Maurin, Max

    2014-01-01

    Francisella tularensis is the etiological agent of tularaemia and a CDC class A biological threat agent. Few antibiotic classes are currently useful in treating tularaemia, including the aminoglycosides gentamicin and streptomycin, fluoroquinolones, and tetracyclines. However, treatment failures and relapses remain frequent and F. tularensis strains resistant to antibiotics have been easily selected in vitro. In this study, we evaluated the activity of new synthetic bis-indole derivatives against this pathogen. Minimum inhibitory concentrations (MICs) of four compounds (dcm01 to dcm04) were determined for the reference strains F. tularensis subsp. holarctica LVS NCTC10857, F. tularensis subsp. novicida CIP56.12 and F. philomiragia ATCC25015, and for 41 clinical strains of F. tularensis subsp. holarctica isolated in France. Minimal bactericidal concentrations (MBCs) were determined for the dcm02 and dcm04 compounds for the LVS and two clinical strains. Killing curves were also determined for the same three strains exposed to dcm04. All tested bis-indole compounds were bacteriostatic against F. tularensis subsp. holarctica strains, with a MIC90 of 8 μg/mL for dcm01, dcm02, and dcm03, and 2 μg/mL for dcm04. Only one strain was resistant to both dcm01 and dcm03, with MICs > 32 μg/mL. In contrast, F. tularensis subsp. novicida was resistant to all derivatives and F. philomiragia was only susceptible to dcm02 and dcm04, with MICs of 16 and 4 μg/mL, respectively. MBC and killing curve experiments revealed significant bactericidal activity (i.e., 3-log reduction of the bacterial inoculum) of the dcm02 and dcm04 compounds only for the LVS strain. In conclusion, we have identified novel synthetic bis-indole compounds that are active against F. tularensis subsp. holarctica. They may be drug candidates for the development of new therapeutic alternatives for tularaemia treatment. Their further characterization is needed, especially identification of their bacterial targets.

  2. Finished genome assembly of warm spring isolate Francisella novicida DPG 3A-IS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Johnson, Shannon L.; Minogue, Timothy D.; Daligault, Hajnalka E.

    2015-09-17

    We sequenced the complete genome of Francisella novicida DPG 3A-IS to closed and finished status. This is a warm spring isolate recovered from Hobo Warm Spring (Utah, USA). The last assembly is available in NCBI under accession number CP012037.

  3. [Tularemia in a jogger woman after the attack by a common buzzard (Buteo buteo): A "One Health" case report].

    PubMed

    Ehrensperger, F; Riederer, L; Friedl, A

    2018-03-01

    A female jogger was attacked by a common buzzard (Buteo buteo) and was scratched lightly at the back of the head. One week later she was taken ill with high fever and headache which was later diagnosed as ulcero-glandular tularemia in regional lymph nodes, caused by Francisella tularensis. Recovery was only achieved after several weeks of systemic antibiotic treatment (Gentamicin/ Ciprofloxacine). Tularemia is a well known zoonotic disease, called "rabbit fever", mainly affecting rabbits and hares, but also small rodents. Human infection occurs often following tick bites or bloodsucking insects, or in hunters or slaughterers handling infected animals. Bites by mice have also been reported as a cause of tularemia. For the first time we report this case of tularemia as a result of an attack by a bird of prey. We assume that the bird acted as a vector just carrying the F. tularensis on its claws or beak, but we cannot exclude an infection of the bird itself. Several other joggers had also been attacked by a common buzzard in the same area shortly after the above described event and one of these also became infected with F. tularensis.

  4. Comparison of experimental respiratory tularemia in three nonhuman primate species.

    PubMed

    Glynn, Audrey R; Alves, Derron A; Frick, Ondraya; Erwin-Cohen, Rebecca; Porter, Aimee; Norris, Sarah; Waag, David; Nalca, Aysegul

    2015-04-01

    Tularemia is a zoonotic disease caused by Francisella tularensis, which is transmitted to humans most commonly by contact with infected animals, tick bites, or inhalation of aerosolized bacteria. F. tularensis is highly infectious via the aerosol route; inhalation of as few as 10-50 organisms can cause pneumonic tularemia. Left untreated, the pneumonic form has more than >30% case-fatality rate but with early antibiotic intervention can be reduced to 3%. This study compared tularemia disease progression across three species of nonhuman primates [African green monkey (AGM), cynomolgus macaque (CM), and rhesus macaque (RM)] following aerosolized F. tularensis Schu S4 exposure. Groups of the animals exposed to various challenge doses were observed for clinical signs of infection and blood samples were analyzed to characterize the disease pathogenesis. Whereas the AGMs and CMs succumbed to disease following challenge doses of 40 and 32 colony forming units (CFU), respectively, the RM lethal dose was 276,667 CFU. Following all challenge doses that caused disease, the NHPs experienced weight loss, bacteremia, fever as early as 4 days post exposure, and tissue burden. Necrotizing-to-pyogranulomatous lesions were observed most commonly in the lung, lymph nodes, spleen, and bone marrow. Overall, the CM model consistently manifested pathological responses similar to those resulting from inhalation of F. tularensis in humans and thereby most closely emulates human tularemia disease. The RM model displayed a higher tolerance to infection and survived exposures of up to 15,593 CFU of aerosolized F. tularensis. Published by Elsevier Ltd.

  5. Rapid Generation of Specific Protective Immunity to Francisella tularensis

    DTIC Science & Technology

    1992-11-01

    4571-4577 Vol. 60, No. 11 0019-9567/92/114571-07$02.00/0 Copyright © 1992, American Society for Microbiology Rapid Generation of Specific Protective... tesied the effect on the actual W118 i.p. LDSf on day 4 after sublethal LVS priming i.d., a time when LVS 100 priming resulted in the maximal increase

  6. Bacterial biofilms of importance to medicine and bioterrorism: proteomic techniques to identify novel vaccine components and drug targets.

    PubMed

    Hassett, Daniel J; Limbach, Patrick A; Hennigan, Robert F; Klose, Karl E; Hancock, Robert E W; Platt, Mark D; Hunt, Donald F

    2003-12-01

    Biofilms are highly ordered microbial communities enmeshed in a carefully sculpted matrix designed for survival of organisms either in multi- or mono-genus/species in a specific microniche. In human disease, biofilm infections are some of the most recalcitrant to treat. Even with rigorous antibiotic regimens, some biofilms, such as those within the thick airway mucus of cystic fibrosis (CF) patients, persist throughout the course of the disease process. In this editorial, discussion will cover the utility of using advanced proteomic techniques to help identify potential weaknesses in the already impressive defensive armamentarium of biofilm bacteria. Two biofilm systems will be discussed herein, one of which is that of Pseudomonas aeruginosa biofilms within CF airway biofilms. The other is referred to as persistent 'bioterrorist agent biofilms' in which Francisella tularensis can grow on surfaces where environmental amoeba can phagocytose them, allowing for growth of F. tularensis within the amoebae.

  7. Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching

    PubMed Central

    Hevener, Kirk E.; Mehboob, Shahila; Su, Pin-Chih; Truong, Kent; Boci, Teuta; Deng, Jiangping; Ghassemi, Mahmood; Cook, James L.; Johnson, Michael E.

    2011-01-01

    Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with sub-micromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented. PMID:22098466

  8. First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

    PubMed

    Ortega, Cesar; Mancera, Gerardo; Enríquez, Ricardo; Vargas, Augusto; Martínez, Simón; Fajardo, Raúl; Avendaño-Herrera, Ruben; Navarrete, María José; Romero, Alex

    2016-08-09

    Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry.

  9. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mehboob, Shahila; Hevener, Kirk E.; Truong, Kent

    Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motifmore » of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.« less

  10. [Tularaemia in a boy following participation in a mud race].

    PubMed

    Zijlstra, M; Hulsker, C C C; Fanoy, E B; Pijnacker, R; Kraaijeveld, A; Koene, M G J; Wolfs, T F W

    2017-01-01

    Tularaemia is a rare disease. In Europe it mostly occurs in Scandinavia. Since 2011 more cases are being reported in the Netherlands. Tularaemia may manifest itself in various ways. It is important to take strict precautions during biopsy, drainage and biopsy processing in order to prevent transmission. A 10-year-old boy presented to the paediatrician with a left inguinal lymphadenitis. A week before the onset of symptoms he had participated in a children's mud race. Serology and PCR of pus from the lymph node tested positive for Francisella tularensis. Treatment with ciprofloxacin was insufficiently effective, so surgical drainage of the gland was performed under strict isolation conditions. Water from the mud race location contained genetic material from F. tularensis. Given the rising incidence of tularaemia in the Netherlands, it is important to consider 'tularaemia' in the differential diagnosis in patients with lymphadenitis and epidemiological clues in their case history. Since 1 November 2016 it has been mandatory to report tularaemia in the Netherlands.

  11. DNA capture elements for rapid detection and identification of biological agents

    NASA Astrophysics Data System (ADS)

    Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

    2004-08-01

    DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

  12. Computer simulation of the processes of inactivation of bacterial cells by dynamic low-coherent speckles

    NASA Astrophysics Data System (ADS)

    Ulianova, Onega V.; Ulyanov, Sergey S.; Sazanova, Elena V.; Zhihong, Zhang; Sibo, Zhou; Luo, Qingming; Zudina, Irina; Bednov, Andrey

    2006-05-01

    Biochemical, biophysical and optical aspects of interaction of low-coherent light with bacterial cells have been discussed. Influence of low-coherent speckles on the colonies grows is investigated. It has been demonstrated that effects of light on the inhibition of cells (Francisella Tularensis) are connected with speckle dynamics. The regimes of illumination of cell suspension with purpose of devitalization of hazard bacteria, caused very dangerous infections, such as tularemia, are found. Mathematical model of interaction of low-coherent laser radiation with bacteria suspension has been proposed. Computer simulations of the processes of laser-cells interaction have been carried out.

  13. Genetic analysis and antimicrobial susceptibility of Francisella noatunensis subsp. orientalis (sun. F. asiatica) isolates from fish

    USDA-ARS?s Scientific Manuscript database

    Francisella noatunensis subsp. orientalis (syn. F. asiatica) (Fno) is an emergent fish pathogen that causes acute to chronic disease in a wide variety of freshwater, brackish and marine fish. Due to the emergent nature of this bacterium, established protocols to measure antimicrobial susceptibility ...

  14. Clinical Characterization of Aerosolized Francisella tularensis Infection in Cynomolgus macaques

    DTIC Science & Technology

    2016-11-21

    2Molecular Toxicology Division, 3Pathology Division U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland...Corresponding author. Address: Center for Aerobiological Sciences, U.S. Army Medical Research Institute of Infectious Diseases , 1425 Porter Street...16-186 DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. UNCLASSIFIED 2 Abstract The disease progression and

  15. Tularaemia in Norwegian dogs.

    PubMed

    Nordstoga, Anne; Handeland, Kjell; Johansen, Tone Bjordal; Iversen, Lena; Gavier-Widén, Dolores; Mattsson, Roland; Wik-Larssen, Kjersti; Afset, Jan Egil; Næverdal, Rune; Lund, Arve

    2014-10-10

    We describe tularaemia in a Norwegian dog caused by Francisella tularensis subspecies holarctica. A Hamilton Hound and his owner developed tulaeremia after hunting an infected mountain hare (Lepus timidus). The dog showed signs of lethargy, anorexia and fever during a period two to four days after hunting and thereafter fully recovered. Its antibody titers increased 32-fold from one to three weeks post exposure. Thereafter, the titer declined and leveled off at moderate positive values up to one year after exposure (end of study). This is believed to be the first case report of clinical F. tularensis subspecies holarctica infection in a European dog. In 2011, enormous numbers of Norway lemmings (Lemmus lemmus) occurred in Finnmark, the northernmost county of Norway and many dogs caught and swallowed lemmings. Some of these dogs developed non-specific signs of disease and the owners consulted a veterinary surgeon, who suspected tularaemia. In order to investigate this hypothesis, serum samples from 33 dogs were examined for antibodies to F. tularensis. The dogs were allocated into three groups: Dogs from Finnmark that became sick (Group 1) or remained healthy following contact with lemmings (Group 2), and healthy control dogs from Oslo without known contact with lemmings (Group 3). All the serum samples were analyzed with a tube agglutination assay. Among dogs exposed to lemmings, 10/11 and 3/12 were antibody positive in Group 1 and Group 2, respectively, whereas none of the control dogs (n=10) were positive for antibodies against F. tularensis. These results strongly indicate that the non-specific disease seen in the dogs in Finnmark was linked to F. tularensis infection acquired through contact with lemmings. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. [Mechanisms for the formation of a combination of the natural foci of trematodiases and tularemia in the floodplain-river ecosystem of the Konda River in different periods of epizootic activity of an infection focus].

    PubMed

    Ushakov, A V

    2011-01-01

    The basis of the mechanism responsible for the formation of a combination of the natural foci of trematodiases and tularemia in the infection foci in the interepizootic periods is their association with the morphological structure of the floodplain-river landscape of the Konda River. The landscape's facias and holes, which are common to the foci, are integrated, by predetermining the territorial combination. The formation of a closed trinomial Francisella tularensis parasite system integrating these foci serves as a basis for the mechanism of the formation of a combination of the natural foci of trematodiases and tularemia on transition of the latter from the interepizootic state to epizooty. The absence of host populations linking the foci of methorchiasis (M. xanthosomus), bilharziasis, and tularemia determines their combination both in the interepizootic periods and at the level of the landscape morphological structure. The basis for the mechanism of the formation of a combination of the natural foci of trematodiases and tularemia in the periods of diffuse epizooties in the infection foci is the formation of an open binomial F. tularensis parasite system and the infestation of the co-members of F. tularensis trematode parasite systems. By penetrating into them, the pathogen forces the F. tularensis parasite system to enter the trematode parasite systems, causing their complete (opisthorchiases, methorchiases) or partial (bilharziasis) coincidence. Thus, these foci are combined at the level of parasitocenoses of the parasite systems of infections and infestations, by giving rise to pseudosystem combined foci. The natural foci of B. polonica and F. tularensis are combined at the level of parasitocenosis of the coacting hemipopulation of trematode parthenitas, F. tularensis subpopulations and intermediate host population, by forming an autoecious population-combination bilharziasis-tularemia focus. The formation of territorial-combination, autoecious population

  17. Dynamics of a Tularemia Outbreak in a Closely Monitored Free-Roaming Population of Wild House Mice.

    PubMed

    Dobay, Akos; Pilo, Paola; Lindholm, Anna K; Origgi, Francesco; Bagheri, Homayoun C; König, Barbara

    2015-01-01

    Infectious disease outbreaks can be devastating because of their sudden occurrence, as well as the complexity of monitoring and controlling them. Outbreaks in wildlife are even more challenging to observe and describe, especially when small animals or secretive species are involved. Modeling such infectious disease events is relevant to investigating their dynamics and is critical for decision makers to accomplish outbreak management. Tularemia, caused by the bacterium Francisella tularensis, is a potentially lethal zoonosis. Of the few animal outbreaks that have been reported in the literature, only those affecting zoo animals have been closely monitored. Here, we report the first estimation of the basic reproduction number R0 of an outbreak in wildlife caused by F. tularensis using quantitative modeling based on a susceptible-infected-recovered framework. We applied that model to data collected during an extensive investigation of an outbreak of tularemia caused by F. tularensis subsp. holarctica (also designated as type B) in a closely monitored, free-roaming house mouse (Mus musculus domesticus) population in Switzerland. Based on our model and assumptions, the best estimated basic reproduction number R0 of the current outbreak is 1.33. Our results suggest that tularemia can cause severe outbreaks in small rodents. We also concluded that the outbreak self-exhausted in approximately three months without administrating antibiotics.

  18. Dynamics of a Tularemia Outbreak in a Closely Monitored Free-Roaming Population of Wild House Mice

    PubMed Central

    Dobay, Akos; Pilo, Paola; Lindholm, Anna K.; Origgi, Francesco; Bagheri, Homayoun C.; König, Barbara

    2015-01-01

    Infectious disease outbreaks can be devastating because of their sudden occurrence, as well as the complexity of monitoring and controlling them. Outbreaks in wildlife are even more challenging to observe and describe, especially when small animals or secretive species are involved. Modeling such infectious disease events is relevant to investigating their dynamics and is critical for decision makers to accomplish outbreak management. Tularemia, caused by the bacterium Francisella tularensis, is a potentially lethal zoonosis. Of the few animal outbreaks that have been reported in the literature, only those affecting zoo animals have been closely monitored. Here, we report the first estimation of the basic reproduction number R 0 of an outbreak in wildlife caused by F. tularensis using quantitative modeling based on a susceptible-infected-recovered framework. We applied that model to data collected during an extensive investigation of an outbreak of tularemia caused by F. tularensis subsp. holarctica (also designated as type B) in a closely monitored, free-roaming house mouse (Mus musculus domesticus) population in Switzerland. Based on our model and assumptions, the best estimated basic reproduction number R 0 of the current outbreak is 1.33. Our results suggest that tularemia can cause severe outbreaks in small rodents. We also concluded that the outbreak self-exhausted in approximately three months without administrating antibiotics. PMID:26536232

  19. Tularaemia outbreaks in Sakarya, Turkey: case-control and environmental studies.

    PubMed

    Meric, M; Sayan, M; Dundar, D; Willke, A

    2010-08-01

    Tularaemia is an important zoonotic disease that leads to outbreaks. This study aimed to compare the epidemiological characteristics of two tularaemia outbreaks that occurred in the Sakarya region of Turkey, analyse the risk factors for the development of outbreaks and identify Francisella (F.) tularensis in the water samples. Two tularaemia outbreaks occurred in the Kocadongel village in 2005 and 2006. A field investigation and a case-control study with 47 cases and 47 healthy households were performed during the second outbreak. Clinical samples from the patients and filtrated water samples were analysed for F. tularensis via real-time polymerase chain reaction. From the two outbreaks, a total of 58 patients were diagnosed with oropharyngeal tularaemia based on their clinical and serological results. Both outbreaks occurred between the months of January and April, and the number of patients peaked in February. Logistic regression analysis revealed that the consumption of natural spring water was the only significant risk factor for tularaemia infection (odds ratio 3.5, confidence interval 1.23-10.07). F. tularensis was detected in eight clinical samples and in the filtrated natural spring water. This study is the first report of tularaemia from this region. The results show that both tularaemia outbreaks were related to the consumption of untreated natural spring water. To prevent waterborne tularaemia, community water supplies should be treated and checked periodically.

  20. Single vector platform vaccine protects against lethal respiratory challenge with Tier 1 select agents of anthrax, plague, and tularemia.

    PubMed

    Jia, Qingmei; Bowen, Richard; Dillon, Barbara Jane; Masleša-Galić, Saša; Chang, Brennan T; Kaidi, Austin C; Horwitz, Marcus A

    2018-05-03

    Bacillus anthracis, Yersinia pestis, and Francisella tularensis are the causative agents of Tier 1 Select Agents anthrax, plague, and tularemia, respectively. Currently, there are no licensed vaccines against plague and tularemia and the licensed anthrax vaccine is suboptimal. Here we report F. tularensis LVS ΔcapB (Live Vaccine Strain with a deletion in capB)- and attenuated multi-deletional Listeria monocytogenes (Lm)-vectored vaccines against all three aforementioned pathogens. We show that LVS ΔcapB- and Lm-vectored vaccines express recombinant B. anthracis, Y. pestis, and F. tularensis immunoprotective antigens in broth and in macrophage-like cells and are non-toxic in mice. Homologous priming-boosting with the LVS ΔcapB-vectored vaccines induces potent antigen-specific humoral and T-cell-mediated immune responses and potent protective immunity against lethal respiratory challenge with all three pathogens. Protection against anthrax was far superior to that obtained with the licensed AVA vaccine and protection against tularemia was comparable to or greater than that obtained with the toxic and unlicensed LVS vaccine. Heterologous priming-boosting with LVS ΔcapB- and Lm-vectored B. anthracis and Y. pestis vaccines also induced potent protective immunity against lethal respiratory challenge with B. anthracis and Y. pestis. The single vaccine platform, especially the LVS ΔcapB-vectored vaccine platform, can be extended readily to other pathogens.

  1. Environmental and behavioral changes may influence the exposure of an Arctic apex predator to pathogens and contaminants

    USGS Publications Warehouse

    Atwood, Todd C.; Duncan, Colleen G.; Patyk, Kelly A.; Nol, Pauline; Rhyan, Jack; McCollum, Matthew; McKinney, Melissa A.; Ramey, Andy M.; Cerqueira-Cezar, Camila; Kwok, Oliver C H; Dubey, Jitender P; Hennager, S.G.

    2017-01-01

    Recent decline of sea ice habitat has coincided with increased use of land by polar bears (Ursus maritimus) from the southern Beaufort Sea (SB), which may alter the risks of exposure to pathogens and contaminants. We assayed blood samples from SB polar bears to assess prior exposure to the pathogens Brucella spp., Toxoplasma gondii, Coxiella burnetii, Francisella tularensis, and Neospora caninum, estimate concentrations of persistent organic pollutants (POPs), and evaluate risk factors associated with exposure to pathogens and POPs. We found that seroprevalence of Brucella spp. and T. gondii antibodies likely increased through time, and provide the first evidence of exposure of polar bears to C. burnetii, N. caninum, and F. tularensis. Additionally, the odds of exposure to T. gondii were greater for bears that used land than for bears that remained on the sea ice during summer and fall, while mean concentrations of the POP chlordane (ΣCHL) were lower for land-based bears. Changes in polar bear behavior brought about by climate-induced modifications to the Arctic marine ecosystem may increase exposure risk to certain pathogens and alter contaminant exposure pathways.

  2. Cervical lymphadenitis: tuberculosis or tularaemia?

    PubMed

    Karabay, O; Kilic, S; Gurcan, S; Pelitli, T; Karadenizli, A; Bozkurt, H; Bostanci, S

    2013-02-01

    Both tuberculosis cervical lymphadenitis (TCL) and oropharyngeal tularaemia (OT) have similar signs, symptoms and pathological findings. We aimed to investigate the frequency of tularaemia antibodies in patients diagnosed with TCL. Using data from the Tuberculosis Control Dispensaries between the years of 2008 and 2011 in Turkey, all patients diagnosed with TCL were informed about and included in the study. Control group subjects were selected from healthy blood donors who lived in the same region. After informed consent was obtained, the sera obtained from volunteer TCL patients and the control group were tested with a microagglutination technique for Francisella tularensis. Antibodies to Brucella were also investigated with a tube agglutination test for cross-reactivity in sera that were seropositive for tularaemia. Sera were obtained from a total of 1170 individuals in the TCL group and 596 in the control group from 67 of 81 provinces in Turkey. Francisella tularensis-positive antibodies were found in 79 (6.75%) cases in the TCL group and two (0.33%) cases in the control group with a titre of ≥1:80 (p < 0.01). When the presence of antibody of any titre was considered, the ratio became 8.2% (96/1170) in the TCL group and 0.67% (4/596) in the control group (p < 0.001). For the first time, with this study, tularaemia serology was found to be positive in a significant portion (6.75%) of diagnosed cases of TCL. In tularaemia endemic regions, it was concluded that tularaemia serology should be investigated in patients suspected of having TCL. © 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

  3. Francisella-Like Endosymbionts and Rickettsia Species in Local and Imported Hyalomma Ticks.

    PubMed

    Azagi, Tal; Klement, Eyal; Perlman, Gidon; Lustig, Yaniv; Mumcuoglu, Kosta Y; Apanaskevich, Dmitry A; Gottlieb, Yuval

    2017-09-15

    Hyalomma ticks (Acari: Ixodidae) are hosts for Francisella -like endosymbionts (FLE) and may serve as vectors of zoonotic disease agents. This study aimed to provide an initial characterization of the interaction between Hyalomma and FLE and to determine the prevalence of pathogenic Rickettsia in these ticks. Hyalomma marginatum , Hyalomma rufipes , Hyalomma dromedarii , Hyalomma aegyptium , and Hyalomma excavatum ticks, identified morphologically and molecularly, were collected from different hosts and locations representing the distribution of the genus Hyalomma in Israel, as well as from migratory birds. A high prevalence of FLE was found in all Hyalomma species (90.6%), as well as efficient maternal transmission of FLE (91.8%), and the localization of FLE in Malpighian tubules, ovaries, and salivary glands in H. marginatum Furthermore, we demonstrated strong cophylogeny between FLE and their host species. Contrary to FLE, the prevalence of Rickettsia ranged from 2.4% to 81.3% and was significantly different between Hyalomma species, with a higher prevalence in ticks collected from migratory birds. Using ompA gene sequences, most of the Rickettsia spp. were similar to Rickettsia aeschlimannii , while a few were similar to Rickettsia africae of the spotted fever group (SFG). Given their zoonotic importance, 249 ticks were tested for Crimean Congo hemorrhagic fever virus infection, and all were negative. The results imply that Hyalomma and FLE have obligatory symbiotic interactions, indicating a potential SFG Rickettsia zoonosis risk. A further understanding of the possible influence of FLE on Hyalomma development, as well as on its infection with Rickettsia pathogens, may lead to novel ways to control tick-borne zoonoses. IMPORTANCE This study shows that Francisella -like endosymbionts were ubiquitous in Hyalomma , were maternally transmitted, and cospeciated with their hosts. These findings imply that the interaction between FLE and Hyalomma is of an obligatory

  4. Transmission of tularemia from a water source by transstadial maintenance in a mosquito vector

    NASA Astrophysics Data System (ADS)

    Bäckman, Stina; Näslund, Jonas; Forsman, Mats; Thelaus, Johanna

    2015-01-01

    Mosquitoes are thought to function as mechanical vectors of Francisella tularensis subspecies holarctica (F. t. holarctica) causing tularemia in humans. We investigated the clinical relevance of transstadially maintained F. t. holarctica in mosquitoes. Aedes egypti larvae exposed to a fully virulent F. t. holarctica strain for 24 hours, were allowed to develop into adults when they were individually homogenized. Approximately 24% of the homogenates tested positive for F. t. DNA in PCR. Mice injected with the mosquito homogenates acquired tularemia within 5 days. This novel finding demonstrates the possibility of transmission of bacteria by adult mosquitoes having acquired the pathogen from their aquatic larval habitats.

  5. Increasing role of arthropod bites in tularaemia transmission in Poland - case reports and diagnostic methods.

    PubMed

    Formińska, Kamila; Zasada, Aleksandra A; Rastawicki, Waldemar; Śmietańska, Karolina; Bander, Dorota; Wawrzynowicz-Syczewska, Marta; Yanushevych, Mariya; Niścigórska-Olsen, Jolanta; Wawszczak, Marek

    2015-01-01

    The study describes four cases of tularaemia - one developed after contact with rabbits and three developed after an arthropod bite. Due to non-specific clinical symptoms, accurate diagnosis of tularaemia may be difficult. The increasing contribution of the arthropod vectors in the transmission of the disease indicates that special effort should be made to apply sensitive and specific diagnostic methods for tularaemia, and to remind health-care workers about this route of Francisella tularensis infections. The advantages and disadvantages of various diagnostic methods - molecular, serological and microbiological culture - are discussed. The PCR as a rapid and proper diagnostic method for ulceroglandular tularaemia is presented.

  6. Efficacy of florfenicol for control of mortality with Francisella noatunensis subsp. orientalis in Nile tilapia, oreochromis niloticus (L.)

    USDA-ARS?s Scientific Manuscript database

    Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram-negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments of vaccines. The objective of this project was ...

  7. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA

    PubMed Central

    Montoya, Guillermo; Stella, Stefano

    2017-01-01

    Bacteria and archaea use the CRISPR–Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR–Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5′-TTN-3′ protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1–crRNA–DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C2221, with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments. PMID:28695850

  8. Multiple-locus, variable number of tandem repeat analysis (MLVA) of the fish-pathogen Francisella noatunensis

    PubMed Central

    2011-01-01

    Background Since Francisella noatunensis was first isolated from cultured Atlantic cod in 2004, it has emerged as a global fish pathogen causing disease in both warm and cold water species. Outbreaks of francisellosis occur in several important cultured fish species making a correct management of this disease a matter of major importance. Currently there are no vaccines or treatments available. A strain typing system for use in studies of F. noatunensis epizootics would be an important tool for disease management. However, the high genetic similarity within the Francisella spp. makes strain typing difficult, but such typing of the related human pathogen Francisella tullarensis has been performed successfully by targeting loci with higher genetic variation than the traditional signature sequences. These loci are known as Variable Numbers of Tandem Repeat (VNTR). The aim of this study is to identify possible useful VNTRs in the genome of F. noatunensis. Results Seven polymorphic VNTR loci were identified in the preliminary genome sequence of F. noatunensis ssp. noatunensis GM2212 isolate. These VNTR-loci were sequenced in F. noatunensis isolates collected from Atlantic cod (Gadus morhua) from Norway (n = 21), Three-line grunt (Parapristipoma trilineatum) from Japan (n = 1), Tilapia (Oreochromis spp.) from Indonesia (n = 3) and Atlantic salmon (Salmo salar) from Chile (n = 1). The Norwegian isolates presented in this study show both nine allelic profiles and clades, and that the majority of the farmed isolates belong in two clades only, while the allelic profiles from wild cod are unique. Conclusions VNTRs can be used to separate isolates belonging to both subspecies of F. noatunensis. Low allelic diversity in F. noatunensis isolates from outbreaks in cod culture compared to isolates wild cod, indicate that transmission of these isolates may be a result of human activity. The sequence based MLVA system presented in this study should provide a good starting point for

  9. The biblical plague of the Philistines now has a name, tularemia.

    PubMed

    Trevisanato, Siro Igino

    2007-01-01

    An epidemic thought to have been the first instance of bubonic plague in the Mediterranean reveals to have been an episode of tularemia. The deadly epidemic took place in the aftermath of the removal of a wooden box from an isolated Hebrew sanctuary. Death, tumors, and rodents thereafter plagued Philistine country. Unlike earlier explanations proposed, tularemia caused by Francisella tularensis exhaustively explains the outbreak. Tularemia fits all the requirements indicated in the biblical text: it is carried by animals, is transmitted to humans, results in the development of ulceroglandular formations, often misdiagnosed for bubonic plague, and is fatal. Moreover, there is the evidence from the box and rodents: mice, which are known carrier for F. tularensis and can communicate it to humans, were credited by the very Philistines to be linked to the outbreak, and are small enough to nest in the box. Mice also explain the otherwise odd statement in the biblical text of a small Philistine idol repeatedly falling on the floor at night in the building where the Philistines had stored the box as mice exiting the box would easily have tipped over the statuette. Tularemia scores yet another point: an episode of the disease is known to have originated in Canaan and spread to Egypt around 1715 BC, indicating recurrence for the disease, and suggesting Canaan was a reservoir for F. tularensis in the 2nd millennium BC.

  10. Systematic detection of positive selection in the human-pathogen interactome and lasting effects on infectious disease susceptibility.

    PubMed

    Corona, Erik; Wang, Liuyang; Ko, Dennis; Patel, Chirag J

    2018-01-01

    Infectious disease has shaped the natural genetic diversity of humans throughout the world. A new approach to capture positive selection driven by pathogens would provide information regarding pathogen exposure in distinct human populations and the constantly evolving arms race between host and disease-causing agents. We created a human pathogen interaction database and used the integrated haplotype score (iHS) to detect recent positive selection in genes that interact with proteins from 26 different pathogens. We used the Human Genome Diversity Panel to identify specific populations harboring pathogen-interacting genes that have undergone positive selection. We found that human genes that interact with 9 pathogen species show evidence of recent positive selection. These pathogens are Yersenia pestis, human immunodeficiency virus (HIV) 1, Zaire ebolavirus, Francisella tularensis, dengue virus, human respiratory syncytial virus, measles virus, Rubella virus, and Bacillus anthracis. For HIV-1, GWAS demonstrate that some naturally selected variants in the host-pathogen protein interaction networks continue to have functional consequences for susceptibility to these pathogens. We show that selected human genes were enriched for HIV susceptibility variants (identified through GWAS), providing further support for the hypothesis that ancient humans were exposed to lentivirus pandemics. Human genes in the Italian, Miao, and Biaka Pygmy populations that interact with Y. pestis show significant signs of selection. These results reveal some of the genetic footprints created by pathogens in the human genome that may have left lasting marks on susceptibility to infectious disease.

  11. The impact of "omic" and imaging technologies on assessing the host immune response to biodefence agents.

    PubMed

    Tree, Julia A; Flick-Smith, Helen; Elmore, Michael J; Rowland, Caroline A

    2014-01-01

    Understanding the interactions between host and pathogen is important for the development and assessment of medical countermeasures to infectious agents, including potential biodefence pathogens such as Bacillus anthracis, Ebola virus, and Francisella tularensis. This review focuses on technological advances which allow this interaction to be studied in much greater detail. Namely, the use of "omic" technologies (next generation sequencing, DNA, and protein microarrays) for dissecting the underlying host response to infection at the molecular level; optical imaging techniques (flow cytometry and fluorescence microscopy) for assessing cellular responses to infection; and biophotonic imaging for visualising the infectious disease process. All of these technologies hold great promise for important breakthroughs in the rational development of vaccines and therapeutics for biodefence agents.

  12. Natural coinfection by Streptococcus agalactiae and Francisella noatunensis subsp. orientalis in farmed Nile tilapia (Oreochromis niloticus L.).

    PubMed

    Assis, G B N; Tavares, G C; Pereira, F L; Figueiredo, H C P; Leal, C A G

    2017-01-01

    Streptococcus agalactiae and Francisella noatunensis subsp. orientalis (Fno) are important pathogens for farm-raised tilapia worldwide. There are no reports of coinfection caused by S. agalactiae and Fno in fish. This study aimed to determine the aetiology of atypical mortalities in a cage farm of Nile tilapia and to characterize the genetic diversity of the isolates. Fifty-two fish were sampled and subjected to parasitological and bacteriological examination. The S. agalactiae and Fno isolates were genotyped using MLST and REP-PCR, respectively. Whole-genome sequencing was performed to confirm the MLST results. Seven fish were shown coinfected by S. agalactiae and Fno. Chronic hypoxia and a reduction in the water temperature were determined as risk factors for coinfection. Fno isolates were shown clonally related in REP-PCR. The MLST analysis revealed that the S. agalactiae isolates from seven coinfected fish were negative for the glcK gene; however, these were determined to be members of clonal complex CC-552. This is the first description of coinfection by S. agalactiae and Fno in farm-raised Nile tilapia. The coinfection was predisposed by chronic hypoxia and was caused by the main genotypes of S. agalactiae and Fno reported in Brazil. Finally, a new S. agalactiae genotype with glcK gene partially deleted was described. © 2016 John Wiley & Sons Ltd.

  13. Surveillance of tularaemia in Kosovo, 2001 to 2010.

    PubMed

    Grunow, R; Kalaveshi, A; Kühn, A; Mulliqi-Osmani, G; Ramadani, N

    2012-07-12

    Tularaemia, caused by Francisella tularensis, had not been registered in Kosovo before an outbreak in 1999 and 2000. A national surveillance system has been implemented in Kosovo since 2000 to monitor a number of diseases, including tularaemia. Antibody detection in human sera was used for laboratory diagnosis of tularaemia and F. tularensis lipopolysaccharide antigen was used as a marker of infection. The purpose of this study is to describe the incidence of tularaemia in Kosovo after the 1999-00 outbreak. In 2001 and 2002, a second outbreak occurred, with 327 serologically confirmed cases. From 2001 to 2010, 25-327 cases were registered per year, giving a mean annual incidence of 5.2 per 100,000 population. The most likely sources of infection were contaminated drinking water and food. The dominant clinical manifestations were the glandular (79%) and ulcero-glandular (21%) forms. By 2010, the disease had spread throughout Kosovo. Presumably as a result of war and subsequent environmental disruption, mass population displacement and breakdown of sanitation and hygiene, the two major outbreaks of tularaemia resulted in the establishment of an active endemic area of tularaemia in Kosovo.

  14. Rapid Countermeasure Discovery against Francisella tularensis Based on a Metabolic Network Reconstruction

    DTIC Science & Technology

    2013-05-21

    minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished... molecular weight, was non-toxic, and abolished bacterial growth at 13 mM, with putative activity against pantetheine-phosphate adenylyltransferase, an...time period. Metabolic genome-scale models of bacteria have provided a computational framework for in silico simulations to evaluate how metabolic

  15. Detection of Staphylococcus Aureus Enterotoxin A and B Genes with PCR-EIA and a Hand-Held Electrochemical Sensor

    DTIC Science & Technology

    2004-06-11

    Streptococcus pneumoniae 33400 Enterobacter cloaceae 49141 S. pyogenes 19615 E. aerogenes m10822 Vibrio cholerae N16961 Enterococcus durans 6056 Yersinia...identified. Thus the sensitivity for both assays was 100%. Of the 56 samples that lacked sea or seb genes, two false positives ( Enterobacter aerogenes ...Comanonas, Enterobacter , Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella, Listeria, Moraxella, Neisseria, Proteus, Pseudomonas, Salmonella

  16. Novel utilization of the outer membrane proteins for the identification and differentiation of pathogenic versus nonpathogenic microbial strains using mass spectrometry-based proteomics approach

    NASA Astrophysics Data System (ADS)

    Jabbour, Rabih E.; Wade, Mary; Deshpande, Samir V.; McCubbin, Patrick; Snyder, A. Peter; Bevilacqua, Vicky

    2012-06-01

    Mass spectrometry based proteomic approaches are showing promising capabilities in addressing various biological and biochemical issues. Outer membrane proteins (OMPs) are often associated with virulence in gram-negative pathogens and could prove to be excellent model biomarkers for strain level differentiation among bacteria. Whole cells and OMP extracts were isolated from pathogenic and non-pathogenic strains of Francisella tularensis, Burkholderia thailandensis, and Burkholderia mallei. OMP extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest-neighbor database strains. This study addresses the comparative experimental proteome analyses of OMPs vs. whole cell lysates on the strain-level discrimination among gram negative pathogenic and non-pathogenic strains.

  17. [Tularemia in Germany].

    PubMed

    Kohlmann, R; Geis, G; Gatermann, S G

    2014-07-01

    The bacterium Francisella tularensis is known for more than 100 years by now as the etiological agent of the disease tularemia, a zoonotic infection with a worldwide distribution in the Northern Hemisphere. The prevalence of tularemia shows a wide geographic variation, being comparably infrequent in Germany. Tularemia can present itself with multiple clinical manifestations including ulceroglandular, glandular, oropharyngeal, oculoglandular, respiratory and typhoidal forms. Due to the low prevalence and the unspecific symptomatology, a rapid diagnosis and early start of an effective therapy are rarely obtained. Thus, in this article we summarize important aspects concerning etiology, ecology and routes of transmission, recent epidemiologic situation, clinical picture, diagnostics and treatment of tularemia, focusing on the situation in Germany. © Georg Thieme Verlag KG Stuttgart · New York.

  18. Atypical Presentations of Tularemia.

    PubMed

    Odegaard, Karah; Boersma, Beth; Keegan, James

    2017-05-01

    Francisella tularensis is a gram-negative coccobacillus that causes a condition commonly referred to as tularemia. There has been a dramatic increase in tularemia cases reported in South Dakota, many of which were challenging to diagnose due to atypical clinical manifestations. We describe an interesting case of pneumonic tularemia and summarize six similar cases, several of which presented with lung nodules suggestive of malignancy. According to the literature, this is only the third outbreak of pneumonic tularemia reported in the U.S. We believe it is important for clinicians to be aware of the increased incidence of tularemia in the area and to be vigilant in the diagnosis and management of these atypically presenting cases. Copyright© South Dakota State Medical Association.

  19. Biofilm formation of Francisella noatunensis subsp. orientalis

    USGS Publications Warehouse

    Soto, Esteban; Halliday-Wimmonds, Iona; Francis , Stewart; Kearney, Michael T.; Hansen, John D.

    2015-01-01

    Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC)and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon®, bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in theiglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

  20. The Mycobacterium tuberculosis Complex has a Pathway for the Biosynthesis of 4-Formamido-4,6-Dideoxy-d-Glucose.

    PubMed

    Brown, Haley A; Vinogradov, Evgeny; Gilbert, Michel; Holden, Hazel M

    2018-05-15

    Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

  1. The status of tularemia in Europe in a one-health context: a review.

    PubMed

    Hestvik, G; Warns-Petit, E; Smith, L A; Fox, N J; Uhlhorn, H; Artois, M; Hannant, D; Hutchings, M R; Mattsson, R; Yon, L; Gavier-Widen, D

    2015-07-01

    The bacterium Francisella tularensis causes the vector-borne zoonotic disease tularemia, and may infect a wide range of hosts including invertebrates, mammals and birds. Transmission to humans occurs through contact with infected animals or contaminated environments, or through arthropod vectors. Tularemia has a broad geographical distribution, and there is evidence which suggests local emergence or re-emergence of this disease in Europe. This review was developed to provide an update on the geographical distribution of F. tularensis in humans, wildlife, domestic animals and vector species, to identify potential public health hazards, and to characterize the epidemiology of tularemia in Europe. Information was collated on cases in humans, domestic animals and wildlife, and on reports of detection of the bacterium in arthropod vectors, from 38 European countries for the period 1992-2012. Multiple international databases on human and animal health were consulted, as well as published reports in the literature. Tularemia is a disease of complex epidemiology that is challenging to understand and therefore to control. Many aspects of this disease remain poorly understood. Better understanding is needed of the epidemiological role of animal hosts, potential vectors, mechanisms of maintenance in the different ecosystems, and routes of transmission of the disease.

  2. Necroptotic debris including damaged mitochondria elicits sepsis-like syndrome during late-phase tularemia.

    PubMed

    Singh, Anju; Periasamy, Sivakumar; Malik, Meenakshi; Bakshi, Chandra Shekhar; Stephen, Laurie; Ault, Jeffrey G; Mannella, Carmen A; Sellati, Timothy J

    2017-01-01

    Infection with Francisella tularensis ssp. tularensis ( Ft ) strain SchuS4 causes an often lethal disease known as tularemia in rodents, non-human primates, and humans. Ft subverts host cell death programs to facilitate their exponential replication within macrophages and other cell types during early respiratory infection (⩽72 h). The mechanism(s) by which cell death is triggered remains incompletely defined, as does the impact of Ft on mitochondria, the host cell's organellar 'canary in a coal mine'. Herein, we reveal that Ft infection of host cells, particularly macrophages and polymorphonuclear leukocytes, drives necroptosis via a receptor-interacting protein kinase 1/3-mediated mechanism. During necroptosis mitochondria and other organelles become damaged. Ft -induced mitochondrial damage is characterized by: (i) a decrease in membrane potential and consequent mitochondrial oncosis or swelling, (ii) increased generation of superoxide radicals, and (iii) release of intact or damaged mitochondria into the lung parenchyma. Host cell recognition of and response to released mitochondria and other damage-associated molecular patterns engenders a sepsis-like syndrome typified by production of TNF, IL-1 β , IL-6, IL-12p70, and IFN- γ during late-phase tularemia (⩾72 h), but are absent early during infection.

  3. Structure and Engineering of Francisella novicida Cas9

    PubMed Central

    Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro; Abudayyeh, Omar O.; Kimura, Mika; Hsu, Patrick D.; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-01-01

    Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA, and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  4. Structure and Engineering of Francisella novicida Cas9.

    PubMed

    Hirano, Hisato; Gootenberg, Jonathan S; Horii, Takuro; Abudayyeh, Omar O; Kimura, Mika; Hsu, Patrick D; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-02-25

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Depletion of alveolar macrophages in CD11c diphtheria toxin receptor mice produces an inflammatory response

    PubMed Central

    Roberts, Lydia M; Ledvina, Hannah E; Tuladhar, Shraddha; Rana, Deepa; Steele, Shaun P; Sempowski, Gregory D; Frelinger, Jeffrey A

    2015-01-01

    Alveolar macrophages play a critical role in initiating the immune response to inhaled pathogens and have been shown to be the first cell type infected following intranasal inoculation with several pathogens, including Francisella tularensis. In an attempt to further dissect the role of alveolar macrophages in the immune response to Francisella, we selectively depleted alveolar macrophages using CD11c.DOG mice. CD11c.DOG mice express the diphtheria toxin receptor (DTR) under control of the full CD11c promoter. Because mice do not express DTR, tissue restricted expression of the primate DTR followed by treatment with diphtheria toxin (DT) has been widely used as a tool in immunology to examine the effect of acute depletion of a specific immune subset following normal development. We successfully depleted alveolar macrophages via intranasal administration of DT. However, alveolar macrophage depletion was accompanied by many other changes to the cellular composition and cytokine/chemokine milieu in the lung that potentially impact innate and adaptive immune responses. Importantly, we observed a transient influx of neutrophils in the lung and spleen. Our experience serves as a cautionary note to other researchers using DTR mice given the complex changes that occur following DT treatment that must be taken into account when analyzing data. PMID:26029367

  6. An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia

    PubMed Central

    Periasamy, Sivakumar; Avram, Dorina; McCabe, Amanda; MacNamara, Katherine C.; Sellati, Timothy J.; Harton, Jonathan A.

    2016-01-01

    Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection. PMID:27015566

  7. Ocular Manifestations of Mosquito-Transmitted Diseases.

    PubMed

    Karesh, James W; Mazzoli, Robert A; Heintz, Shannon K

    2018-03-01

    Of the 3,548 known mosquito species, about 100 transmit human diseases. Mosquitoes are distributed globally throughout tropical and temperate regions where standing water sources are available for egg laying and the maturation of larva. Female mosquitoes require blood meals for egg production. This is the main pathway for disease transmission. Mosquitoes carry several pathogenic organisms responsible for significant ocular pathology and vision loss including West Nile, Rift Valley, chikungunya, dengue viruses, various encephalitis viruses, malarial parasites, Francisella tularensis, microfilarial parasites, including Dirofilaria, Wuchereria, and Brugia spp., and human botfly larvae. Health care providers may not be familiar with many of these mosquito-transmitted diseases or their associated ocular findings delaying diagnosis, treatment, and recovery of visual function. This article aims to provide an overview of the ocular manifestations associated with mosquito-transmitted diseases.

  8. The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism.

    PubMed

    Feng, Youjun; Chin, Chui-Yoke; Chakravartty, Vandana; Gao, Rongsui; Crispell, Emily K; Weiss, David S; Cronan, John E

    2015-06-09

    The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. Our findings show that Francisella novicida has evolved two functional biotin protein ligases, BplA and BirA. BplA is a much more efficient enzyme than BirA, and its expression is significantly induced upon infection of macrophages. Only BplA is required for F. novicida pathogenicity, whereas BirA prevents wasteful biotin synthesis. These data

  9. Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia

    PubMed Central

    Tadin, Ante; Tokarz, Rafal; Markotić, Alemka; Margaletić, Josip; Turk, Nenad; Habuš, Josipa; Svoboda, Petra; Vucelja, Marko; Desai, Aaloki; Jain, Komal; Ian Lipkin, W.

    2016-01-01

    Croatia is a focus for many rodent-borne zoonosis. Here, we report a survey of 242 rodents and small mammals, including 43 Myodes glareolus, 131 Apodemus flavicollis, 53 Apodemus agrarius, three Apodemus sylvaticus, six Sorex araneus, four Microtus arvalis, one Microtus agrestis, and one Muscardinus avellanarius, collected at eight sites in Croatia over an 8-year period. Multiplex MassTag polymerase chain reaction (PCR) was used for detection of Borrelia, Rickettsia, Bartonella, Babesia, Ehrlichia, Anaplasma, Francisella tularensis, and Coxiella burnetii. Individual PCR assays were used for detection of Leptospira, lymphocytic choriomeningitis virus, orthopoxviruses, flaviviruses, hantaviruses, and Toxoplasma gondii. Of the rodents, 52 (21.5%) were infected with Leptospira, 9 (3.7%) with Borrelia miyamotoi, 5 (2%) with Borrelia afzelii, 29 (12.0%) with Bartonella, 8 (3.3%) with Babesia microti, 2 (0.8%) with Ehrlichia, 4 (1.7%) with Anaplasma, 2 (0.8%) with F. tularensis, 43 (17.8%) with hantaviruses, and 1 (0.4%) with an orthopoxvirus. Other agents were not detected. Multiple infections were found in 32 rodents (13.2%): dual infections in 26 rodents (10.7%), triple infections in four rodents (2.9%), and quadruple infections in two rodents (0.8%). Our findings indicate that rodents in Croatia harbor a wide range of bacteria and viruses that are pathogenic to humans. PMID:26711522

  10. Longitudinal study on the seroprevalence of avian influenza, leptospirosis, and tularemia in an urban population of raccoons (Procyon lotor) in Ontario, Canada.

    PubMed

    Jardine, Claire; Lindsay, L Robbin; Nicholson, Vivian M; Ojkic, Davor; Prescott, John F

    2011-01-01

    Raccoons (Procyon lotor) live at high densities, often in close association with people, in urban areas in Ontario and have been implicated as potential reservoirs of numerous zoonotic disease agents. We collected 137 blood samples from 61 apparently healthy raccoons in a small area of Toronto, Ontario, from June to October 2007 as part of a longitudinal study to determine the seasonal patterns of seroprevalence of Francisella tularensis, avian influenza, and Leptospira. In addition, we collected 35 urine samples by cystocentesis from 23 animals to look for evidence of urinary shedding of Leptospira. All samples were serologically negative for F. tularensis and avian influenza. Nineteen of 61 animals (31%) were positive for Leptospira antibodies in one or more trapping periods. The seroprevalence of Leptospira increased from 5% in June to 38% in October. Of the 19 positive animals, 14 were seropositive for serogroup Grippotyphosa, 4 for serogroup Pomona, and 1 for both serogroups Australis and Grippotyphosa. Raccoons were seronegative to serovars representative of serogroups Autumnalis, Canicola, Icterohaemorrhagiae, and Sejroe. Only one urine sample was culture positive for Leptospira (2.9%). Although we found evidence that raccoons in this study were exposed to leptospires belonging to serogroup Grippotyphosa, likely serovar Grippotyphosa, during the summer and able to shed leptospires in urine, further work is required to determine the importance of raccoons as reservoirs of Leptospira in Ontario.

  11. A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents.

    PubMed

    Mechaly, Adva; Cohen, Hila; Cohen, Ofer; Mazor, Ohad

    2016-08-01

    Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein-protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10(4) pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Respiratory and oral vaccination improves protection conferred by the live vaccine strain against pneumonic tularemia in the rabbit model

    PubMed Central

    Stinson, Elizabeth; Smith, Le'Kneitah P.; Cole, Kelly Stefano; Barry, Eileen M.; Reed, Douglas S.

    2016-01-01

    Tularemia is a severe, zoonotic disease caused by a gram-negative bacterium, Francisella tularensis. We have previously shown that rabbits are a good model of human pneumonic tularemia when exposed to aerosols containing a virulent, type A strain, SCHU S4. We further demonstrated that the live vaccine strain (LVS), an attenuated type B strain, extended time to death when given by scarification. Oral or aerosol vaccination has been previously shown in humans to offer superior protection to parenteral vaccination against respiratory tularemia challenge. Both oral and aerosol vaccination with LVS were well tolerated in the rabbit with only minimal fever and no weight loss after inoculation. Plasma antibody titers against F. tularensis were higher in rabbits that were vaccinated by either oral or aerosol routes compared to scarification. Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4. LVS given by scarification extended time to death compared to mock-vaccinated controls. One orally vaccinated rabbit did survive aerosol challenge, however, only aerosol vaccination extended time to death significantly compared to scarification. These results further demonstrate the utility of the rabbit model of pneumonic tularemia in replicating what has been reported in humans and macaques as well as demonstrating the utility of vaccination by oral and respiratory routes against an aerosol tularemia challenge. PMID:27511964

  13. Organs of BALB/c mice can be injured in course of tularemia.

    PubMed

    Pavlis, Oto; Kusakova, Eva; Novotny, Ladislav; Pohanka, Miroslav

    2014-12-01

    Francisella tularensis is a biological agent exploitable for bioterrorism and biological warfare purposes due to serious pathogenic progression and easy dissemination. Despite intensive research in the past, some adverse consequences remain unclear. One consequence of this pathogen is oxidative stress. The aim of this study was to undertake ex vivo assays for monitoring the disease in mice and increase our knowledge of the oxidative stress induced by tularemia. The mouse BALB/c model was chosen and the animals were infected by a dose 10(4) CFU of F. tularensis. After five days, the animals were euthanized. Blood immediately processed in plasma, spleen and liver were sampled from the cadavers. Oxidative stress markers, cytokines and histopathological were undertaken. There was a significant link between oxidative stress and tularemia. Particularly elevated levels of malondialdehyde and decreased levels of low molecular weight antioxidants were found in the liver and spleen of tularemia-infected animals. The histopathological findings correlated well with the oxidative stress markers. The liver and spleen were proven to be significantly at risk from the disease and an association between stress and neutrophils in the affected organs was found. The histopathology excluded risk to other organs such as the kidney and or heart. Oxidative stress plays a significant role in tularemia infection in mice and this was confirmed by the histology.

  14. Rabbit hunter uveitis: case report of tularemia uveitis.

    PubMed

    Terrada, Céline; Azza, Said; Bodaghi, Bahram; Le Hoang, Phuc; Drancourt, Michel

    2016-09-01

    Literature reports on ophthalmological manifestations related to tularemia, a zoonose caused by the bacterium Francisella tularensis, largely refer to Parinaud's oculoglandular syndrome, which consists of the association of conjunctivitis with preauricular lymphadenitis. In this paper, we report a case of intraocular inflammation during tularemia infection. A 52-year-old Caucasian man was diagnosed with unilateral uveitis. The uveitis was posterior, with a 2+ vitritis and a large yellowish lesion involving the macula with an overlying sub-retinal detachment, extending inferiorly, and subretinal hemorrhages. Fluorescein angiography showed a late hyperfluorescence with focal vascular leakage. Ultrasound biomicroscopy confirmed the presence of a 3.8 mm parietal granuloma with a few calcifications in the left eye. While extensive work-up eliminated any other infectious and non-infectious etiology, tularemia was diagnosed by advanced serology consisting of two-dimensional Western-immunoblotting. The patient, a hunter, recalled having killed rabbits in the days before the symptoms appeared. Uveitis was rapidly controlled following treatment with doxycycline, yet three years after initiation of the treatment, the patient still complained of loss of vision in the left eye with a central scotoma. Posterior uveitis may be an infrequent manifestation of tularemia infection, and therefore this infection should be considered in the differential diagnosis of intraocular inflammation in areas where F. tularensis is endemic.

  15. Respiratory and oral vaccination improves protection conferred by the live vaccine strain against pneumonic tularemia in the rabbit model.

    PubMed

    Stinson, Elizabeth; Smith, Le'Kneitah P; Cole, Kelly Stefano; Barry, Eileen M; Reed, Douglas S

    2016-10-01

    Tularemia is a severe, zoonotic disease caused by a gram-negative bacterium, Francisella tularensis We have previously shown that rabbits are a good model of human pneumonic tularemia when exposed to aerosols containing a virulent, type A strain, SCHU S4. We further demonstrated that the live vaccine strain (LVS), an attenuated type B strain, extended time to death when given by scarification. Oral or aerosol vaccination has been previously shown in humans to offer superior protection to parenteral vaccination against respiratory tularemia challenge. Both oral and aerosol vaccination with LVS were well tolerated in the rabbit with only minimal fever and no weight loss after inoculation. Plasma antibody titers against F. tularensis were higher in rabbits that were vaccinated by either oral or aerosol routes compared to scarification. Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4. LVS given by scarification extended time to death compared to mock-vaccinated controls. One orally vaccinated rabbit did survive aerosol challenge, however, only aerosol vaccination extended time to death significantly compared to scarification. These results further demonstrate the utility of the rabbit model of pneumonic tularemia in replicating what has been reported in humans and macaques as well as demonstrating the utility of vaccination by oral and respiratory routes against an aerosol tularemia challenge. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Tularemia in Germany—A Re-emerging Zoonosis

    PubMed Central

    Faber, Mirko; Heuner, Klaus; Jacob, Daniela; Grunow, Roland

    2018-01-01

    Tularemia, also known as “rabbit fever,” is a zoonosis caused by the facultative intracellular, gram-negative bacterium Francisella tularensis. Infection occurs through contact with infected animals (often hares), arthropod vectors (such as ticks or deer flies), inhalation of contaminated dust or through contaminated food and water. In this review, we would like to provide an overview of the current epidemiological situation in Germany using published studies and case reports, an analysis of recent surveillance data and our own experience from the laboratory diagnostics, and investigation of cases. While in Germany tularemia is a rarely reported disease, there is evidence of recent re-emergence. We also describe some peculiarities that were observed in Germany, such as a broad genetic diversity, and a recently discovered new genus of Francisella and protracted or severe clinical courses of infections with the subspecies holarctica. Because tularemia is a zoonosis, we also touch upon the situation in the animal reservoir and one-health aspects of this disease. Apparently, many pieces of the puzzle need to be found and put into place before the complex interaction between wildlife, the environment and humans are fully understood. Funding for investigations into rare diseases is scarce. Therefore, combining efforts in several countries in the framework of international projects may be necessary to advance further our understanding of this serious but also scientifically interesting disease. PMID:29503812

  17. Imaging Findings of Ulceroglandular Tularemia.

    PubMed

    Anand, Neil; Deochand, Osmani; Murphy, Robyn

    2017-01-01

    Francisella tularensis, the causative organism in Tularemia, is a relatively rare disease. There are a few radiological clues to elucidate its presence when suspicion arises. There should be strong consideration for Tularemia in the differential of any patient with its classic symptoms, diffuse cervical lymphadenopathy with evidence of necrosis, and enlarged adenoids. Ultrasound may demonstrate suppurative lymphadenopathy suggestive of infection, as in the case presented. CT often will demonstrate the extent of lymphadenopathy. On chest radiography, tularemia pneumonia is often the presenting finding, which may demonstrate bilateral or lobar infiltrates. Additionally, hilar lymphadenopathy and pleural effusions are often associated findings. Cavitary lesions may be present, which are better delineated on CT scan. We present a case of a 7-year-old male who presented with a painful right-sided palpable neck mass for 9 days, who was diagnosed with Tularemia after numerous admissions.

  18. Hunting for tularaemia - a review of cases in North Carolina.

    PubMed

    Rimawi, R H; Shah, K B; Chowdhary, R A; Cook, P P

    2015-05-01

    Human infections with Francisella tularensis can be acquired via numerous routes, including ingestion, inhalation, arthropod bite or direct contact with infected animals. Since 1991, there have been 25 reported cases of tularaemia in North Carolina, most of which were associated with rabbit hunting or cat bites. We present two adults cases of pulmonary and oropharyngeal tularaemia and review the reported cases since 1991-2013. We also present the fifth case of pulmonary empyema. While cavitary pneumonias are primarily treated with drainage, we illustrate a case of cavitary pneumonia associated with tularaemia successfully treated with oral ciprofloxacin after drainage. Tularaemia should be considered in patients with a perplexing radiographic image, animal exposure and lack of response to conventional empiric broad-spectrum antibiotics. Even in serious cases of pneumonic tularaemia, fluoroquinolones may provide a suitable alternative to aminoglycosides. © 2014 Blackwell Verlag GmbH.

  19. The Cynomolgus Macaque Natural History Model of Pneumonic Tularemia for Predicting Clinical Efficacy Under the Animal Rule

    PubMed Central

    Guina, Tina; Lanning, Lynda L.; Omland, Kristian S.; Williams, Mark S.; Wolfraim, Larry A.; Heyse, Stephen P.; Houchens, Christopher R.; Sanz, Patrick; Hewitt, Judith A.

    2018-01-01

    Francisella tularensis is a highly infectious Gram-negative bacterium that is the etiologic agent of tularemia in animals and humans and a Tier 1 select agent. The natural incidence of pneumonic tularemia worldwide is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCM) in human populations. Development and licensure of tularemia therapeutics and vaccines need to occur under the Food and Drug Administration's (FDA's) Animal Rule under which efficacy studies are conducted in well-characterized animal models that reflect the pathophysiology of human disease. The Tularemia Animal Model Qualification (AMQ) Working Group is seeking qualification of the cynomolgus macaque (Macaca fascicularis) model of pneumonic tularemia under Drug Development Tools Qualification Programs with the FDA based upon the results of studies described in this manuscript. Analysis of data on survival, average time to death, average time to fever onset, average interval between fever and death, and bacteremia; together with summaries of clinical signs, necropsy findings, and histopathology from the animals exposed to aerosolized F. tularensis Schu S4 in five natural history studies and one antibiotic efficacy study form the basis for the proposed cynomolgus macaque model. Results support the conclusion that signs of pneumonic tularemia in cynomolgus macaques exposed to 300–3,000 colony forming units (cfu) aerosolized F. tularensis Schu S4, under the conditions described herein, and human pneumonic tularemia cases are highly similar. Animal age, weight, and sex of animals challenged with 300–3,000 cfu Schu S4 did not impact fever onset in studies described herein. This study summarizes critical parameters and endpoints of a well-characterized cynomolgus macaque model of pneumonic tularemia and demonstrates this model is appropriate for qualification, and for testing efficacy of tularemia therapeutics under Animal Rule. PMID

  20. Seroprevalence of seven zoonotic infections in Nunavik, Quebec (Canada).

    PubMed

    Messier, V; Lévesque, B; Proulx, J-F; Rochette, L; Serhir, B; Couillard, M; Ward, B J; Libman, M D; Dewailly, E; Déry, S

    2012-03-01

    In Nunavik, common practices and food habits such as consumption of raw meat and untreated water place the Inuit at risk for contracting zoonotic diseases. The aim of this study was to determine the seroprevalence of seven zoonotic infections among the permanent residents of Nunavik. The study was conducted in the fall 2004 as part of the Nunavik Health Survey. Blood samples from adults aged 18-74 years (n = 917) were collected and analysed for the presence of antibodies against Trichinella spp., Toxocara canis, Echinococcus granulosus, Brucella spp., Coxiella burnetii, Leptospira spp. and Francisella tularensis. Information on sociodemographic characteristics, traditional activities, drinking water supply and nutrition was gathered using english/inuktitut bilingual questionnaires. The chi-squared test was used to evaluate associations between seropositivity and other measured variables. Statistically significant variables were included in a multivariate logistic regression model to control for confounding factors. Estimated seroprevalences were 8.3% for E. granulosus, 3.9% for T. canis, 5.9% for Leptospira spp. and 18.9% for F. tularensis. Seroprevalence was ≤ 1% for Trichinella spiralis, Brucella spp. and C. burnetii. For most infections, seropositivity tended to increase with age. In multivariate analyses, seroprevalence was positively (i.e. directly) associated with age and residence in the Ungava coast area for F. tularensis; age and residence in the Hudson coast area for T. canis; female gender, lower level of schooling and frequent cleaning of water reservoirs for E. granulosus. No risk factor for Leptospira spp. infection was identified. No associations were detected with regards to food habits or environmental exposures. A small but significant portion of the Nunavik population has serologic evidence of exposure to at least one of the pathogenic microorganisms investigated. Further studies are needed to better understand the mechanisms for transmission

  1. Structure and biosynthesis of free lipid A molecules that replace lipopolysaccharide in Francisella novicida

    PubMed Central

    Wang, Xiaoyuan; Ribeiro, Anthony A.; Guan, Ziqiang; McGrath, Sara C.; Cotter, Robert J.; Raetz, Christian R. H.

    2008-01-01

    Francisella novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15 % of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. novicida U112, less than 5 % of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2′, and a secondary palmitoyl residue at position 2′. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4′- and 3′-positions of A1 and A2 are not derivatized, and Kdo is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an α-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An α-linked glucose moiety is attached to the 6′-position of A2. The lipid A released by mild acid hydrolysis of F. novicida lipopolysaccharide consists solely of component A1. F. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism. PMID:17128982

  2. Large Direct Repeats Flank Genomic Rearrangements between a New Clinical Isolate of Francisella tularensis subsp. tularensis A1 and Schu S4

    DTIC Science & Technology

    2010-02-03

    occur at locations 1,374,336–1,408,246 ( DRI ) and 1,767,671– 1,801,581 (DRII) in Schu S4. In addition, a 5358 bp segment of Table 2. VNTR markers and...iglABCD operon were found in NE061598. The location of DRI and DRII in both Schu S4 and NE061598 are shown in figures 2A and 2B. In addition, DRIII (III...loss of the first 207 bp in DRI of NE061598 (Figure 2). Similar chromosomal changes mediated by these duplicated regions were also observed between Schu

  3. Transcriptome Analysis of Human Immune Responses Following Live Vaccine Strain (LVS) Francisella Tularensis Vaccination

    DTIC Science & Technology

    2007-03-08

    with CD3D 50848 PAR1/UBE3A Prader–Willi syndrome chromosome region 1, GMCSFRalpha precursor, IL3Ralpha precursor (CD123) Brain development...intervention programs justifiable? Emerg. Infect. Dis. 3, 83–94. iebel, U., Kindler , B., Pepperkok, R., 2004. ‘Harvester’: a fast meta search engine of human...protein resources. Bioinformatics 20, 1962–1963. iebel, U., Kindler , B., Pepperkok, R., 2005. Bioinformatic “Harvester”: a search engine for genome

  4. Discrimination of Pathogenic vs. Nonpathogenic Francisella tularensis and Burkholderia pseudomallei Using Proteomics Mass Spectrometry

    DTIC Science & Technology

    2011-03-01

    GroEL AhpC/TSA family protein hypothetical protein FTL0617 heat shock protein DnaK succinyl-CoA synthetase subunit beta hypothetical protein...lipoprotein chaperonin GroEL co-chaperonin GroES DNA-directed RNA polymerase subunit beta intracellular growth locus, subunit C 3.2 Differentiation...thailandensis E264 Unique Proteins Whole Cell Lysates OMPs putative lipoprotein glucan 1,4-a-glucosidase glycosy hydrolase family protein putative

  5. Decontamination of materials contaminated with Francisella philomiragia or MS2 bacteriophage using PES-Solid, a solid source of peracetic acid.

    PubMed

    Buhr, T L; Young, A A; Johnson, C A; Minter, Z A; Wells, C M

    2014-08-01

    The aim of the study was to develop test methods and evaluate survival of Francisella philomiragia cells and MS2 bacteriophage after exposure to PES-Solid (a solid source of peracetic acid) formulations with or without surfactants. Francisella philomiragia cells (≥7·6 log10 CFU) or MS2 bacteriophage (≥6·8 log10 PFU) were deposited on seven different test materials and treated with three different PES-Solid formulations, three different preneutralized samples and filter controls at room temperature for 15 min. There were 0-1·3 log10 CFU (<20 cells) of cell survival, or 0-1·7 log10 (<51 PFU) of bacteriophage survival in all 21 test combinations (organism, formulation and substrate) containing reactive PES-Solid. In addition, the microemulsion (Dahlgren Surfactant System) showed ≤2 log10 (100 cells) of viable F. philomiragia cells, indicating the microemulsion achieved <2 log10 CFU on its own. Three PES-Solid formulations and one microemulsion system (DSS) inactivated F. philomiragia cells and/or MS2 bacteriophage that were deposited on seven different materials. A test method was developed to show that reactive PES-Solid formulations and a microemulsion system (DSS) inactivated >6 log10 CFU/PFU F. philomiragia cells and/or MS2 bacteriophage on different materials. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  6. Molecular Investigation of Tularemia Outbreaks, Spain, 1997–2008

    PubMed Central

    Ariza-Miguel, Jaime; Johansson, Anders; Fernández-Natal, María Isabel; Martínez-Nistal, Carmen; Orduña, Antonio; Rodríguez-Ferri, Elías F.; Hernández, Marta

    2014-01-01

    Tularemia outbreaks occurred in northwestern Spain in 1997–1998 and 2007–2008 and affected >1,000 persons. We assessed isolates involved in these outbreaks by using pulsed-field gel electrophoresis with 2 restriction enzymes and multilocus variable number tandem repeat analysis of 16 genomic loci of Francisella tularensis, the cause of this disease. Isolates were divided into 3 pulsotypes by pulsed-field gel electrophoresis and 8 allelic profiles by multilocus variable number tandem repeat analysis. Isolates obtained from the second tularemia outbreak had the same genotypes as isolates obtained from the first outbreak. Both outbreaks were caused by genotypes of genetic subclade B.Br:FTNF002–00, which is widely distributed in countries in central and western Europe. Thus, reemergence of tularemia in Spain was not caused by the reintroduction of exotic strains, but probably by persistence of local reservoirs of infection. PMID:24750848

  7. Rare Cause of Pleuropnemonia: Tularemia Disease.

    PubMed

    Agca, Meltem; Duman, Dildar; Sulu, Ebru; Ozbaki, Fatma; Barkay, Orcun; Ozturk, Derya; Yarkin, Tulay

    2017-09-01

    Tularemia is a zoonotic infection which is caused by gram negative coccobacilli, Francisella tularensis. The disease occurs after contact with blood and body fluids of infected animals, bites and ingestion of infected food and water. Although it commonly presents with skin lesions, there may also be serious organ involvements. A55-year woman was consulted for presumptive diagnosis of tuberculosis. Multiple lymphadenopathy in right cervical area was present on physical examination. Pleural effusion on left side was detected with computed tomography. In detailed history, knowledge of a family member with the diagnosis of tularemia was obtained. Both of them had the history of contact with infected animals. Diagnosis of tularemia was confirmed with microagglutination test. With this patient who was initially presumptively diagnosed as tuberculosis, we aim to draw attention to diagnosis of tularemia in the presence of pleuropnemonia and peripheral lymphadenopathy and emphasize importance of detailed patient history.

  8. Tularemia in Children, Turkey, September 2009–November 2012

    PubMed Central

    Tezer, Hasan; Aykan, Hakan; Erkocoglu, Mustafa; Gülhan, Belgin; Demir, Ahmet; Kanik-Yuksek, Saliha; Tapisiz, Anil; Polat, Meltem; Kara, Soner; Devrim, Ilker; Kilic, Selcuk

    2015-01-01

    Tularemia, a zoonotic disease caused by Francisella tularensis, is found throughout most of the Northern Hemisphere. It is not well known and is often misdiagnosed in children. Our aim with this study was to evaluate the diagnosis, treatment, and prognosis for 100 children with tularemia in Turkey. The mean patient age was 10.1 ± 3.5 years (range 3–18 years), and most (63%) patients were male. The most common physical signs and laboratory findings were cervical lymphadenopathy (92%) and elevated erythrocyte sedimentation rate (89%). Treatment response was higher and rate of relapse lower for children 5–10 years of age than for those in other age groups. Associated with treatment failure were female sex, treatment delay of ≥16 days, and use of doxycycline. Tularemia is endemic to Turkey, and the number of cases has been increasing among children as well as adults. PMID:25529639

  9. Imaging Findings of Ulceroglandular Tularemia

    PubMed Central

    Anand, Neil; Deochand, Osmani; Murphy, Robyn

    2017-01-01

    Francisella tularensis, the causative organism in Tularemia, is a relatively rare disease. There are a few radiological clues to elucidate its presence when suspicion arises. There should be strong consideration for Tularemia in the differential of any patient with its classic symptoms, diffuse cervical lymphadenopathy with evidence of necrosis, and enlarged adenoids. Ultrasound may demonstrate suppurative lymphadenopathy suggestive of infection, as in the case presented. CT often will demonstrate the extent of lymphadenopathy. On chest radiography, tularemia pneumonia is often the presenting finding, which may demonstrate bilateral or lobar infiltrates. Additionally, hilar lymphadenopathy and pleural effusions are often associated findings. Cavitary lesions may be present, which are better delineated on CT scan. We present a case of a 7-year-old male who presented with a painful right-sided palpable neck mass for 9 days, who was diagnosed with Tularemia after numerous admissions. PMID:28580063

  10. [Severe tularaemia mimicking glandular tuberculosis during adalimumab therapy].

    PubMed

    Konstantinou, M-P; Abecassis-Cotta, S; Valeyrie-Allanore, L; Ortonne, N; Maurin, M; Roujeau, J-C; Revuz, J; Bagot, M

    2009-10-01

    Tularaemia is an anthropozoonosis, transmitted by small mammals (hares) and arthropods (ticks, horseflies). The causative agent is Francisella tularensis, a facultatively intracellular Gram-negative bacillus. We report a case of tularaemia in its ulceroglandular form occurring during methotrexate and adalimumab treatment (Humira) for rheumatoid arthritis. A 58-year-old man with a history of primary tuberculosis receiving adalimumab in combination with methotrexate for rheumatoid arthritis for almost 1 year consulted for a febrile inflammatory plaque on the left leg with a small central necrotic area. An enlarged left inguinal lymph node was present. Doxycycline has previously been prescribed for a tick bite. The lymphadenopathy gradually became enlarged resulting in skin fistulisation. After surgical excision, histopathology revealed epithelioid granulomas accompanied by giant cells and central necrosis. Mycobacterial cultures were negative. Positive tularaemia serology at significant titres suggested a diagnosis of tularaemia, with probable transmission via a tick bite. The diagnosis was confirmed by F. tularensis DNA amplification using PCR on a lymph node biopsy. Doxycycline was continued for a further 6 weeks. One year later, no relapse had occurred. A febrile adenopathy presenting the histological features of necrotic granulomas in a patient receiving anti-TNF alpha treatment initially suggested reactivation of tuberculosis. However, the history of tick bite and failure to isolate mycobacteria from different tissue specimens prompted screening for a tick-borne disease, finally leading to a diagnosis of tularaemia. We discuss the possible relationship between immunosuppression and the clinical course of this rare infection.

  11. Treatment of Francisella infections via PLGA- and lipid-based nanoparticle delivery of antibiotics in a zebrafish model.

    PubMed

    Ulanova, Lilia S; Pinheiro, Marina; Vibe, Carina; Nunes, Claudia; Misaghian, Dorna; Wilson, Steven; Zhu, Kaizheng; Fenaroli, Federico; Winther-Larsen, Hanne C; Reis, Salette; Griffiths, Gareth

    2017-06-19

    We tested the efficiency of 2 different antibiotics, rifampicin and oxolinic acid, against an established infection caused by fish pathogen Francisella noatunensis ssp. orientalis (F.n.o.) in zebrafish. The drugs were tested in the free form as well as encapsulated into biodegradable nanoparticles, either polylactic-co-glycolic acid (PLGA) nanoparticles or nanostructured lipid carriers. The most promising therapies were PLGA-rifampicin nanoparticles and free oxolinic acid; the PLGA nanoparticles significantly delayed embryo mortality while free oxolinic acid prevented it. Encapsulation of rifampicin in both PLGA and nanostructured lipid carriers enhanced its efficiency against F.n.o. infection relative to the free drug. We propose that the zebrafish model is a robust, rapid system for initial testing of different treatments of bacterial diseases important for aquaculture.

  12. Pathogen and rodenticide exposure in American badgers (Taxidea taxus) in California.

    PubMed

    Quinn, Jessica H; Girard, Yvette A; Gilardi, Kirsten; Hernandez, Yvette; Poppenga, Robert; Chomel, Bruno B; Foley, Janet E; Johnson, Christine K

    2012-04-01

    Urban and agricultural land use may increase the risk of disease transmission among wildlife, domestic animals, and humans as we share ever-shrinking and fragmented habitat. American badgers (Taxidae taxus), a species of special concern in California, USA, live in proximity to urban development and often share habitat with livestock and small peridomestic mammals. As such, they may be susceptible to pathogens commonly transmitted at this interface and to anticoagulant rodenticides used to control nuisance wildlife on agricultural lands. We evaluated free-ranging badgers in California for exposure to pathogens and anticoagulant rodenticides that pose a risk to wildlife, domestic animals, or public health. We found serologic evidence of badger exposure to Francisella tularensis, Toxoplasma gondii, Anaplasma phagocytophilum, canine distemper virus, and three Bartonella species: B. henselae, B. clarridgeiae, and B. vinsonii subsp. berkhoffii. Badger tissues contained anticoagulant rodenticides brodifacoum and bromadiolone, commonly used to control periurban rodent pests. These data provide a preliminary investigation of pathogen and toxicant exposure in the wild badger population.

  13. Membrane-Targeting DCAP Analogues with Broad-Spectrum Antibiotic Activity against Pathogenic Bacteria

    PubMed Central

    2015-01-01

    We performed a structure–activity relationship study of 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), which is an antibacterial agent that disrupts the membrane potential and permeability of bacteria. The stereochemistry of DCAP had no effect on the biological activity of DCAP. The aromaticity and electronegativity of the chlorine-substituted carbazole was required for activity, suggesting that its planar and dipolar characteristics orient DCAP in membranes. Increasing the hydrophobicity of the tail region of DCAP enhanced its antibiotic activity. Two DCAP analogues displayed promising antibacterial activity against the BSL-3 pathogens Bacillus anthracis and Francisella tularensis. Codosing DCAP analogues with ampicillin or kanamycin increased their potency. These studies demonstrate that DCAP and its analogues may be a promising scaffold for developing chemotherapeutic agents that bind to bacterial membranes and kill strains of slow-growing or dormant bacteria that cause persistent infections. PMID:25941556

  14. Seroprevalence of selected disease agents from free-ranging black bears in Florida.

    PubMed

    Dunbar, M R; Cunningham, M W; Roof, J C

    1998-07-01

    Sera obtained from 66 free-ranging Florida black bears (Ursus americanus floridanus) from three geographic areas of Florida (USA) between November 1993 and August 1995 were tested for antibodies to 13 disease agents. Antibody prevalences were 3 positive of 37 tested (8%) Coxiella burnetti, 37 of 66 (56%) Toxoplasma gondii, 3 of 61 (5%) bluetongue virus/epizootic hemorrhagic disease virus (BTV/EHDV), 4 of 66 (6%) canine adenovirus-type 1, 5 of 66 (8%) canine distemper virus (CDV), 10 of 62 (16%) canine parvovirus (CPV), 7 of 66 (11%) eastern equine encephalitis virus, 4 of 66 (6%) western equine encephalitis virus, 2 of 66 (3%) Venezuelan equine encephalitis virus, and 11 of 66 (17%) St. Louis encephalitis virus. No samples had serologic evidence of exposure to Brucella spp. (n = 37), Francisella tularensis (n = 40), or pseudorabies virus (n = 37). This is the first known published report of antibodies to BTV/EHDV, CDV, and CPV in black bears.

  15. Tularemia Outbreaks and Common Vole (Microtus arvalis) Irruptive Population Dynamics in Northwestern Spain, 1997-2014.

    PubMed

    Luque-Larena, Juan José; Mougeot, François; Roig, Dolors Vidal; Lambin, Xavier; Rodríguez-Pastor, Ruth; Rodríguez-Valín, Elena; Anda, Pedro; Escudero, Raquel

    2015-09-01

    During the last decades, large tularemia outbreaks in humans have coincided in time and space with population outbreaks of common voles in northwestern Spain, leading us to hypothesize that this rodent species acts as a key spillover agent of Francisella tularensis in the region. Here, we evaluate for the first time a potential link between irruptive vole numbers and human tularemia outbreaks in Spain. We compiled vole abundance estimates obtained through live-trapping monitoring studies and official reports of human tularemia cases during the period 1997-2014. We confirm a significant positive association between yearly cases of tularemia infection in humans and vole abundance. High vole densities during outbreaks (up to 1000 voles/hectare) may therefore enhance disease transmission and spillover contamination in the environment. If this ecological link is further confirmed, the apparent multiannual cyclicity of common vole outbreaks might provide a basis for forecasting the risk of tularemia outbreaks in northwestern Spain.

  16. Tularaemia presenting as parapharyngeal abscess: case presentation.

    PubMed

    Koc, S; Gürbüzler, L; Yaman, H; Eyibilen, A; Salman, N; Ekici, A

    2012-05-01

    We report an extremely rare case of the oropharyngeal form of tularaemia, causing a parapharyngeal abscess. A 48-year-old woman presented with fever, sore throat, breathing difficulty and a right-sided neck swelling. This mass had previously been treated with penicillin without response, and had already been surgically drained once in another hospital. On physical examination, the tonsils were exudative and hypertrophic and the pharynx was hyperaemic. A fluctuant, 4 × 4 cm mass was seen on endoscopic examination, originating from the left parapharyngeal area and protruding towards the pyriform sinus, and partly obstructing the airway. Microagglutination test antibody titres for Francisella tularensis were positive (1/1280). The patient healed completely after definitive drainage of the abscess and antimicrobial therapy for 14 days (streptomycin, 2 × 1 g intramuscularly). Tularaemia should be considered in the differential diagnosis of patients presenting with tonsillopharyngitis, cervical lymphadenitis and parapharyngeal abscess who do not respond to treatment with penicillin, even if they do not live in an endemic region.

  17. A rare cause of abdominal lymphadenopathy--tularemia: report of two pediatric cases.

    PubMed

    Gülhan, Belgin; Tezer, Hasan; Kanık-Yüksek, Saliha; Kılıç, Selçuk; Senel, Emrah

    2014-01-01

    Tularemia caused by Francisella tularensis occurs worldwide in the northern hemisphere, with great variation in geographic and temporal occurrence. It generally presents as an acute febrile disease with the major clinical presentations including the six classic forms of tularemia: ulceroglandular, glandular, oculoglandular, oropharyngeal, typhoidal, and pneumonic. In contrast to European countries, where the ulceroglandular form is more prominent, the oropharyngeal form is the most common presentation in Turkey. We present rare cases of oropharyngeal tularemia in a 16-year-old boy and nine-year-old girl. To the best of our knowledge, these are the firstly described abdominal lymphadenopathy cases from Turkey. The second case was admitted with erythema nodosum, and abdominal lymphadenopathy was detected during the investigation. Excisional lymph node biopsy revealed abdominal tularemia. It is necessary to consider tularemia in the differential diagnosis of abdominal lymphadenopathy in tularemia regions. We also conclude that oropharyngeal tularemia can cause lymphadenopathy in any part of the gastrointestinal tract.

  18. The microfluidic bioagent autonomous networked detector (M-BAND): an update. Fully integrated, automated, and networked field identification of airborne pathogens

    NASA Astrophysics Data System (ADS)

    Sanchez, M.; Probst, L.; Blazevic, E.; Nakao, B.; Northrup, M. A.

    2011-11-01

    We describe a fully automated and autonomous air-borne biothreat detection system for biosurveillance applications. The system, including the nucleic-acid-based detection assay, was designed, built and shipped by Microfluidic Systems Inc (MFSI), a new subsidiary of PositiveID Corporation (PSID). Our findings demonstrate that the system and assay unequivocally identify pathogenic strains of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei. In order to assess the assay's ability to detect unknown samples, our team also challenged it against a series of blind samples provided by the Department of Homeland Security (DHS). These samples included natural occurring isolated strains, near-neighbor isolates, and environmental samples. Our results indicate that the multiplex assay was specific and produced no false positives when challenged with in house gDNA collections and DHS provided panels. Here we present another analytical tool for the rapid identification of nine Centers for Disease Control and Prevention category A and B biothreat organisms.

  19. 3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

    DTIC Science & Technology

    2013-07-01

    FabI, but share low sequence identity and are poorly inhibited by triclosan.25,26 S. pneumoniae and P. aeruginosa contain FabK,24 and Vibrio cholerae,27...with 0.2 mM IPTG. The cells were harvested after an overnight induction period at 17 °C. The cells were lysed and sonicated and loaded onto a nickel...of enoyl- (acyl-carrier protein) reductase, FabV, from Vibrio fischeri. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2012, 68, 78−80. (27

  20. Transcriptional Profiling of Francisella tularensis Infected Peripheral Blood Monomuclear Cells: A Predictive Tool for Tularemia

    DTIC Science & Technology

    2008-01-01

    1.42 0.08 AA897516 PTGER4 0.99 0.02 1.04 0.02 1.09 0.02 1.150.03 1.19 0.03 Apoptosis (BP) BC006121 AIFM2 1.02 0.05 1.05 0.03 1.10 0.04 1.27... PTGER4 ) increased signifi- cantly (P 0.05) starting at 8 h. Although not statistically significant (P = 0.065), the expression of prostaglandin

  1. Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated Neorickettsia sp. in Europe.

    PubMed

    Hornok, Sándor; Szőke, Krisztina; Estók, Péter; Krawczyk, Aleksandra; Haarsma, Anne-Jifke; Kováts, Dávid; Boldogh, Sándor A; Morandini, Pál; Szekeres, Sándor; Takács, Nóra; Kontschán, Jenő; Meli, Marina L; Fernández de Mera, Isabel G; de la Fuente, José; Gyuranecz, Miklós; Sulyok, Kinga M; Weibel, Beatrice; Gönczi, Enikő; de Bruin, Arnout; Sprong, Hein; Hofmann-Lehmann, Regina

    2018-02-28

    In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.

  2. Evaluation of tularemia cases focusing on the oculoglandular form.

    PubMed

    Eren Gok, Sebnem; Kocagul Celikbas, Aysel; Baykam, Nurcan; Atay Buyukdemirci, Ayse; Eroglu, Mustafa Necati; Evren Kemer, Ozlem; Dokuzoguz, Basak

    2014-10-15

    Tularemia is a zoonotic disease caused by Francisella tularensis. The oculoglandular form is one of the rarest forms. In this study, evaluated tularemia patients, focusing on the ocular form and the efficacy of early antibiotic therapy. During a tularemia outbreak, the epidemiological and clinical findings, laboratory assays, and drugs used for the treatment of 48 patients were recorded prospectively. The diagnosis of tularemia was confirmed with microagglutination test (MAT) as well as clinical findings. The mean age of the subject was 48.6 years; 23 (47.9%) of them were female. Thirty-six (81.25%) patients had clinical presentation compatible with oropharyngeal tularemia, seven (14.58%) with oculoglandular tularemia, and two (4.1%) with ulceroglandular tularemia. The most common symptoms were fever (91.6%) and sore throat (81.2%), and the most common findings were lymphadenopathy (91.6%) and tonsillopharyngitis (81.2%). In the oculoglandular form, fever, lymphadenopathy, periorbital edema, conjunctival injection, and chemosis were found. The most distinctive ophthalmic feature was follicular conjunctivitis and conjunctival epithelial defects. Forty-five cases had positive serological results with MAT. All the patients were treated with antibiotics considered effective against F. tularensis, and topical antimicrobial treatment was given to the patients with oculoglandular tularemia. Twenty-six (54.16%) patients, who were admitted within three weeks of the onset of symptoms, recovered without sequel. During tularemia outbreaks, ocular involvement should be considered carefully. The early administration of appropriate treatment will be more effective in resolving the infection and preventing complications. Along with systemic antibiotic therapy, topical treatment will help recovery.

  3. Did an epidemic of tularemia in Ancient Egypt affect the course of world history?

    PubMed

    Trevisanato, Siro I

    2004-01-01

    The dynamics of the spreading, and the identity of a virulent epidemic, similar to bubonic plague or typhus, which hit Ancient Egypt in the middle of the Bronze Age, are hereby presented. Documented in medical papyri as well as archaeological findings, and re-echoed in biblical texts, a plague entered Egypt's main harbor, Avaris, around 1715 BC. As a result, the country was severely weakened at a time when it was already facing serious sociopolitical issues: crumbling central government, immigration, foreign influence, and the rise in power of the army and of warlords. The Hebrews, one of the ethnic groups within the Avaris area, appeared immune amidst the devastation of the plague. This immunity was key to identify the etiological agent of the epidemic: Francisella tularensis, the Gram-negative bacterium passed by ticks, and that causes tularemia. The disease manifests itself as either an ulceroglandular or a typhoid syndrome, accounting for its similarity with bubonic plague and typhus. Cellular immunity provides the main defense against F. tularensis, and is achieved through continuous contact with avirulent strains of the bacterium. Urban dwellers of Avaris, who had a limited contact to animals, would have been defenseless against the virulent strains, unlike the Hebrews, who lived off sheep and other animals. Attempting to consolidate Egypt's central government in the aftermath of the tularemia epidemic, led among others to the internment of the Hebrews in labor camps. The repressive measures against the Hebrews left traces in the subsequent archaeological layer (stratum F) at Avaris. As a corollary, the enslavement would set the stage for the exodus of the Hebrew community from Egypt at a later time.

  4. Bioterrorism and the Role of the Clinical Microbiology Laboratory

    PubMed Central

    2015-01-01

    SUMMARY Regular review of the management of bioterrorism is essential for maintaining readiness for these sporadically occurring events. This review provides an overview of the history of biological disasters and bioterrorism. I also discuss the recent recategorization of tier 1 agents by the U.S. Department of Health and Human Services, the Laboratory Response Network (LRN), and specific training and readiness processes and programs, such as the College of American Pathologists (CAP) Laboratory Preparedness Exercise (LPX). LPX examined the management of cultivable bacterial vaccine and attenuated strains of tier 1 agents or close mimics. In the LPX program, participating laboratories showed improvement in the level of diagnosis required and referral of isolates to an appropriate reference laboratory. Agents which proved difficult to manage in sentinel laboratories included the more fastidious Gram-negative organisms, especially Francisella tularensis and Burkholderia spp. The recent Ebola hemorrhagic fever epidemic provided a check on LRN safety processes. Specific guidelines and recommendations for laboratory safety and risk assessment in the clinical microbiology are explored so that sentinel laboratories can better prepare for the next biological disaster. PMID:26656673

  5. Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

    NASA Astrophysics Data System (ADS)

    Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

    2004-12-01

    Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

  6. [Mission oriented diagnostic real-time PCR].

    PubMed

    Tomaso, Herbert; Scholz, Holger C; Al Dahouk, Sascha; Splettstoesser, Wolf D; Neubauer, Heinrich; Pfeffer, Martin; Straube, Eberhard

    2007-01-01

    In out of area military missions soldiers are potentially exposed to bacteria that are endemic in tropical areas and can be used as biological agents. It can be difficult to culture these bacteria due to sample contamination, low number of bacteria or pretreatment with antibiotics. Commercial biochemical identification systems are not optimized for these agents which can result in misidentification. Immunological assays are often not commercially available or not specific. Real-time PCR assays are very specific and sensitive and can shorten the time required to establish a diagnosis markedly. Therefore, real-time PCRs for the identification of Bacillus anthracis, Brucella spp., Burkholderia mallei und Burkholderia pseudomallei, Francisella tularensis und Yersinia pestis have been developed. PCR results can be false negative due to inadequate clinical samples, low number of bacteria in samples, DNA degradation, inhibitory substances and inappropriate DNA preparation. Hence, it is crucial to cultivate the organisms as a prerequisite for adequate antibiotic therapy and typing of the agent. In a bioterrorist scenario samples have to be treated according to rules applied in forensic medicine and documentation has to be flawless.

  7. Nine-analyte detection using an array-based biosensor

    NASA Technical Reports Server (NTRS)

    Taitt, Chris Rowe; Anderson, George P.; Lingerfelt, Brian M.; Feldstein, s. Mark. J.; Ligler, Frances S.

    2002-01-01

    A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.

  8. Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

    PubMed

    Yang, Yu; Wang, Jing; Wen, Haiyan; Liu, Hengchuan

    2012-01-01

    We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

  9. Immunoproteomic analysis of the human antibody response to natural tularemia infection with Type A or Type B strains or LVS vaccination

    PubMed Central

    Fulton, Kelly M.; Zhao, Xigeng; Petit, Mireille D.; Kilmury, Sara L.N; Wolfraim, Lawrence A.; House, Robert V.; Sjostedt, Anders; Twine, Susan M.

    2011-01-01

    Francisella tularensis is pathogenic for many mammalian species including humans, causing a spectrum of diseases called tularemia. The highly virulent Type A strains have associated mortality rates of up to 60% if inhaled. An attenuated live vaccine strain (LVS) is the only vaccine to show efficacy in humans, but suffers several barriers to licensure, including the absence of a correlate of protection. An immunoproteomics approach was used to survey the repertoire of antibodies in sera from individuals who had contracted tularemia during two outbreaks and individuals from two geographical areas who had been vaccinated with NDBR Lot 11 or Lot 17 LVS. These data showed a large overlap in the antibodies generated in response to tularemia infection or LVS vaccination. A total of seven proteins were observed to be reactive with 60 % or more sera from vaccinees and convalescents. A further four proteins were recognised by 30–60 % of the sera screened. These proteins have the potential to serve as markers of vaccination or candidates for subunit vaccines. PMID:21873113

  10. Safety and Accuracy of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Highly Pathogenic Organisms.

    PubMed

    Rudrik, James T; Soehnlen, Marty K; Perry, Michael J; Sullivan, Maureen M; Reiter-Kintz, Wanda; Lee, Philip A; Pettit, Denise; Tran, Anthony; Swaney, Erin

    2017-12-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) sample preparation methods, including the direct, on-plate formic acid, and ethanol/formic acid tube extraction methods, were evaluated for their ability to render highly pathogenic organisms nonviable and safe for handling in a biosafety level 2 laboratory. Of these, the tube extraction procedure was the most successful, with none of the tested strains surviving this sample preparation method. Tube extracts from several agents of bioterrorism and their near neighbors were analyzed in an eight-laboratory study to examine the utility of the Bruker Biotyper and Vitek MS MALDI-TOF MS systems and their in vitro diagnostic (IVD), research-use-only, and Security-Relevant databases, as applicable, to accurately identify these agents. Forty-six distinct strains of Bacillus anthracis , Yersinia pestis , Francisella tularensis , Burkholderia mallei , Burkholderia pseudomallei , Clostridium botulinum , Brucella melitensis , Brucella abortus , Brucella suis , and Brucella canis were extracted and distributed to participating laboratories for analysis. A total of 35 near-neighbor isolates were also analyzed. Copyright © 2017 Rudrik et al.

  11. Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens

    NASA Astrophysics Data System (ADS)

    Seo, Youngmin; Kim, Ji-Eun; Jeong, Yoon; Lee, Kwan Hong; Hwang, Jangsun; Hong, Jongwook; Park, Hansoo; Choi, Jonghoon

    2016-01-01

    Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens.

  12. Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11.

    PubMed

    Lagrange, Brice; Benaoudia, Sacha; Wallet, Pierre; Magnotti, Flora; Provost, Angelina; Michal, Fanny; Martin, Amandine; Di Lorenzo, Flaviana; Py, Bénédicte F; Molinaro, Antonio; Henry, Thomas

    2018-01-16

    Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

  13. Genetic and Biochemical Characterization of Peptidoglycan Synthesis in Chlamydia

    DTIC Science & Technology

    2005-09-01

    and Delcour, J. (1997) The alanine racemase gene is essential for growth of Lactobacillus plantarum . J Bacteriol 179: 3804-3807. 225 Holtje, J...including Lactobacillus spp. and Francisella, only one alanine racemase gene (alr) is present in the genome and is essential for growth (Bron et al

  14. Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

    PubMed

    Yang, Samuel; Rothman, Richard E; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A

    2008-04-01

    To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

  15. The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

    PubMed Central

    Feng, Youjun; Chin, Chui-Yoke; Chakravartty, Vandana; Gao, Rongsui; Crispell, Emily K.

    2015-01-01

    ABSTRACT The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. PMID:26060274

  16. Tularemia, a re-emerging infectious disease in Iran and neighboring countrie

    PubMed Central

    Zargar, Afsaneh; Maurin, Max; Mostafavi, Ehsan

    2015-01-01

    OBJECTIVES: Tularemia is a zoonotic disease transmitted by direct contact with infected animals and through arthropod bites, inhalation of contaminated aerosols, ingestion of contaminated meat or water, and skin contact with any infected material. It is widespread throughout the northern hemisphere, including Iran and its neighbors to the north, northeast, and northwest. METHODS: In this paper, the epidemiology of tularemia as a re-emerging infectious disease in the world with a focus on Iran and the neighboring countries is reviewed. RESULTS: In Iran, positive serological tests were first reported in 1973, in wildlife and domestic livestock in the northwestern and southeastern parts of the country. The first human case was reported in 1980 in the southwest of Iran, and recent studies conducted among at-risk populations in the western, southeastern, and southwestern parts of Iran revealed seroprevalences of 14.4, 6.52, and 6%, respectively. CONCLUSIONS: Several factors may explain the absence of reported tularemia cases in Iran since 1980. Tularemia may be underdiagnosed in Iran because Francisella tularensis subspecies holarctica is likely to be the major etiological agent and usually causes mild to moderately severe disease. Furthermore, tularemia is not a disease extensively studied in the medical educational system in Iran, and empirical therapy may be effective in many cases. Finally, it should be noted that laboratories capable of diagnosing tularemia have only been established in the last few years. Since both recent and older studies have consistently found tularemia antibodies in humans and animals, the surveillance of this disease should receive more attention. In particular, it would be worthwhile for clinical researchers to confirm tularemia cases more often by isolating F. tularensis from infected humans and animals. PMID:25773439

  17. A high-throughput fluorescence polarization assay for inhibitors of gyrase B.

    PubMed

    Glaser, Bryan T; Malerich, Jeremiah P; Duellman, Sarah J; Fong, Julie; Hutson, Christopher; Fine, Richard M; Keblansky, Boris; Tang, Mary J; Madrid, Peter B

    2011-02-01

    DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-µL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

  18. Climate change and infectious diseases in the Arctic: establishment of a circumpolar working group

    PubMed Central

    Parkinson, Alan J.; Evengard, Birgitta; Semenza, Jan C.; Ogden, Nicholas; Børresen, Malene L.; Berner, Jim; Brubaker, Michael; Sjöstedt, Anders; Evander, Magnus; Hondula, David M.; Menne, Bettina; Pshenichnaya, Natalia; Gounder, Prabhu; Larose, Tricia; Revich, Boris; Hueffer, Karsten; Albihn, Ann

    2014-01-01

    The Arctic, even more so than other parts of the world, has warmed substantially over the past few decades. Temperature and humidity influence the rate of development, survival and reproduction of pathogens and thus the incidence and prevalence of many infectious diseases. Higher temperatures may also allow infected host species to survive winters in larger numbers, increase the population size and expand their habitat range. The impact of these changes on human disease in the Arctic has not been fully evaluated. There is concern that climate change may shift the geographic and temporal distribution of a range of infectious diseases. Many infectious diseases are climate sensitive, where their emergence in a region is dependent on climate-related ecological changes. Most are zoonotic diseases, and can be spread between humans and animals by arthropod vectors, water, soil, wild or domestic animals. Potentially climate-sensitive zoonotic pathogens of circumpolar concern include Brucella spp., Toxoplasma gondii, Trichinella spp., Clostridium botulinum, Francisella tularensis, Borrelia burgdorferi, Bacillus anthracis, Echinococcus spp., Leptospira spp., Giardia spp., Cryptosporida spp., Coxiella burnetti, rabies virus, West Nile virus, Hantaviruses, and tick-borne encephalitis viruses. PMID:25317383

  19. Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

    PubMed Central

    Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T.

    2013-01-01

    Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. PMID:23345286

  20. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    USGS Publications Warehouse

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W.; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  1. Classification of select category A and B bacteria by Fourier transform infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Samuels, Alan C.; Snyder, A. Peter; St. Amant, Diane; Emge, Darren K.; Minter, Jennifer; Campbell, Mark; Tripathi, Ashish

    2008-04-01

    Relatively few reports have investigated the determination and classification of pathogens such as the National Institute of Allergy and Infectious Diseases (NIAID) Category A Bacillus anthracis spores and cells (BA), Yersinia species, Francisella tularensis (FT), and Category B Brucella species from FTIR spectra. We investigated the classification ability of the Fourier transform infrared (FTIR) spectra of viable pathogenic and non-pathogenic NIAID Category A and B bacteria. The impact of different growth media, growth time and temperature, rolling circle filter of the data, and wavelength range were investigated for their microorganism differentiation. Various 2-D PC plots provided differential degrees of separation with respect to the four viable, bacterial genera including the BA sub-categories of pathogenic spores, vegetative cells, and nonpathogenic vegetative cells. FT spectra were separated from that of the three other genera. The BA pathogenic spore strains 1029, LA1, and Ames were clearly differentiated from the rest of the dataset. Yersinia species were distinctly separated from the remaining dataset and could also be classified by growth media. This work provided evidence that FTIR spectroscopy can separate the four major pathogenic bacterial genera of NIAID Category A and B biological threat agents.

  2. An overview: tularemia and travel medicine.

    PubMed

    Ulu-Kilic, Aysegul; Doganay, Mehmet

    2014-01-01

    Tularemia is a bacterial zoonotic infection. The disease is endemic in most parts of the world, has been reported through the northern hemisphere between 30 and 71° N latitude. Francisella tularensis causes infection in a wide range of vertebrates (rodents, lagomorphs) and invertebrates (ticks, mosquitoes and other arthropods). Humans can acquire this infection through several routes including; a bite from an infected tick, deerfly or mosquito, contact with an infected animal or its dead body. It can also be spread to human by drinking contaminated water or breathing contaminated dirt or aerosol. Clinical manifestation of this disease varies depending on the biotype, inoculum and port of entry. Infection is potentially life threatening, but can effectively be treated with antibiotics. Travelers visiting rural and agricultural areas in endemic countries may be at greater risk. Appropriate clothing and use of insect repellants is essential to prevent tick borne illness. Travelers also should be aware of food and waterborne disease; avoid consuming potentially contaminated water and uncooked meat. Physicians should be aware of any clinical presentation of tularemia in the patients returning from endemic areas. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Molecular strategy for the diagnosis of infectious lymphadenitis.

    PubMed

    Prudent, Elsa; La Scola, Bernard; Drancourt, Michel; Angelakis, Emmanouil; Raoult, Didier

    2018-06-01

    Molecular methods have been considered to be the gold standard for the diagnosis of infectious lymphadenitis. However, culture remains critical in the case of low bacterial concentrations. We used molecular assays and culture to examine fresh lymph node biopsies from patients with suspected infectious lymphadenopathy. We analyzed 1762 lymph node biopsies of which 522 (30%) samples were found positive by real-time PCR; the most commonly amplified bacteria were Bartonella henselae (n = 438, 84%), Francisella tularensis (n = 46, 9%), and Mycobacterium spp. (n = 29, 6%). PCR amplification and sequencing of the 16S rDNA were positive for 359 (20%) lymph node specimens including mainly B. henselae (n = 167, 47%), Staphylococcus spp. (n = 77, 21%), and Streptococcus spp. (n = 41, 11%). In total, 351 lymph nodes were cultured on agar plates and 77 (22%) were positive. Significantly more lymph nodes infected by Gram-positive easy-growing agents were diagnosed by culture (n = 45) than by 16S rDNA PCR (p = 0.02). Culture remains critical for the diagnosis of easy-growing bacteria and mycobacteria; clinicians should be aware that a negative molecular result does not imply absence of infection.

  4. Gene repression via multiplex gRNA strategy in Y. lipolytica.

    PubMed

    Zhang, Jin-Lai; Peng, Yang-Zi; Liu, Duo; Liu, Hong; Cao, Ying-Xiu; Li, Bing-Zhi; Li, Chun; Yuan, Ying-Jin

    2018-04-20

    The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest. In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized. Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for

  5. [Tularemia is spreading from north to south side of Turkey: a small outbreak in Kahramanmaras, Turkey].

    PubMed

    Bozkurt, İlkay; Kiliç, Selçuk

    2014-07-01

    Tularemia is a zoonotic disease caused by Francisella tularensis. Sporadic tularemia cases have been increasingly reported particularly from provinces located at northwest and central regions of Turkey especially during last two decades, as well as waterborne outbreaks reported from almost all regions. Transmission most often occurs through consumption of contaminated water and food, thus, oropharyngeal form is the most common clinical presentation in our country. The aim of this study was to present a small outbreak experience in Afsin, country of Kahramanmaras province located at southern part of Turkey. A total of 10 patients (5 male, 5 female; age range 2-68 years; mean age 25 years) who were admitted to Afsin State Hospital with the complaints of swollen neck between 21 October 2013-22 January 2014, were evaluated considering their clinical findings and treatment outcomes. Following the diagnosis of the first tularemia case coming from Nadir village, a field investigation was performed. All villagers were informed about the disease and water samples from the possible sources of outbreak were collected by provincial health authorities. Lymph node aspirate and serum samples were sent for culture and serologic investigation and the environmental water samples were sent for molecular analysis to the National Tularemia Reference Laboratory at Public Health Institution of Turkey. Six out of 10 patients' sera were found positive in terms of F.tularensis antibodies between the titers of 1/320-1/1280 by microagglutination test (MAT) and diagnosis of oropharyngeal tularemia was based on the clinical and serological findings. One of the patients also presented with oculoglandular form accompanying oropharyngeal form. Cultures from aspirate samples that could be obtained from only two patients yielded negative results. Three out of six patients' lymph nodes were drained surgically and one was drained by ultrasound-guided needle. In one case lymph node suppuration occured

  6. The Genome of the Amoeba Symbiont “Candidatus Amoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria ▿ †‡

    PubMed Central

    Schmitz-Esser, Stephan; Tischler, Patrick; Arnold, Roland; Montanaro, Jacqueline; Wagner, Michael; Rattei, Thomas; Horn, Matthias

    2010-01-01

    Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “Candidatus Amoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans. PMID:20023027

  7. The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" reveals common mechanisms for host cell interaction among amoeba-associated bacteria.

    PubMed

    Schmitz-Esser, Stephan; Tischler, Patrick; Arnold, Roland; Montanaro, Jacqueline; Wagner, Michael; Rattei, Thomas; Horn, Matthias

    2010-02-01

    Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of "Candidatus Amoebophilus asiaticus," an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. "Ca. Amoebophilus asiaticus" encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of "Ca. Amoebophilus asiaticus". More generally, we show that the eukaryotic domains identified in "Ca. Amoebophilus asiaticus" are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.

  8. Dermacentor andersoni transmission of Francisella tularensis subsp. novicida reflects bacterial colonization, dissemination and replication coordinated with tick feeding.

    USDA-ARS?s Scientific Manuscript database

    Ticks serve as biological vectors for a wide variety of bacterial pathogens which must be able to efficiently colonize specific tick tissues prior to transmission. The bacterial determinants of tick colonization are largely unknown, a knowledge gap attributed in large part to the paucity of tools t...

  9. Performance of Traditional and Molecular Methods for Detecting Biological Agents in Drinking Water

    USGS Publications Warehouse

    Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Bertke, Erin E.; Kephart, Christopher M.; Likirdopulos, Christina A.; Mailot, Brian E.; Schaefer, Frank W.; Lindquist, H.D. Alan

    2009-01-01

    To reduce the impact from a possible bioterrorist attack on drinking-water supplies, analytical methods are needed to rapidly detect the presence of biological agents in water. To this end, 13 drinking-water samples were collected at 9 water-treatment plants in Ohio to assess the performance of a molecular method in comparison to traditional analytical methods that take longer to perform. Two 100-liter samples were collected at each site during each sampling event; one was seeded in the laboratory with six biological agents - Bacillus anthracis (B. anthracis), Burkholderia cepacia (as a surrogate for Bu. pseudomallei), Francisella tularensis (F. tularensis), Salmonella Typhi (S. Typhi), Vibrio cholerae (V. cholerae), and Cryptospordium parvum (C. parvum). The seeded and unseeded samples were processed by ultrafiltration and analyzed by use of quantiative polymerase chain reaction (qPCR), a molecular method, and culture methods for bacterial agents or the immunomagnetic separation/fluorescent antibody (IMS/FA) method for C. parvum as traditional methods. Six replicate seeded samples were also processed and analyzed. For traditional methods, recoveries were highly variable between samples and even between some replicate samples, ranging from below detection to greater than 100 percent. Recoveries were significantly related to water pH, specific conductance, and dissolved organic carbon (DOC) for all bacteria combined by culture methods, but none of the water-quality characteristics tested were related to recoveries of C. parvum by IMS/FA. Recoveries were not determined by qPCR because of problems in quantifying organisms by qPCR in the composite seed. Instead, qPCR results were reported as detected, not detected (no qPCR signal), or +/- detected (Cycle Threshold or 'Ct' values were greater than 40). Several sample results by qPCR were omitted from the dataset because of possible problems with qPCR reagents, primers, and probes. For the remaining 14 qPCR results

  10. Interaction of Francisella noatunensis subsp. orientalis with Oreochromis mossambicus bulbus arteriosus cell line

    USGS Publications Warehouse

    Soto, Esteban; Yun, Susan; Lewis, J.; Kearney, Michael T.; Hansen, John D.

    2017-01-01

    Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent warmwater fish pathogen and the causative agent of piscine francisellosis. Although Fno causes septicemia and can live extracellularly in infected tilapia (Oreochromis spp.), the early interaction of Fno with vasculature endothelium is unknown. In the present study, we examined the interaction of wild-type Fno (WT) and two Fno knockout [intracellular growth loci C (ΔiglC) and pathogenicity determinant protein A (ΔpdpA)] strains with the endothelial O. mossambicus bulbus arteriosus cell line (TmB) at 25 °C and 30 °C. Similar amounts of WT, ΔiglC, and ΔpdpA attached and were detected intracellularly after 5 h of incubation at both temperatures; however temperature affected attachment and uptake. While significantly greater amounts of Fno (WT, ΔiglC, and ΔpdpA) were detected intracellularly when TmB cells were incubated at 30 °C, bacteria attached to TmBs at greater levels at 25 °C. Only WT Fno was able to replicate intracellularly at 25 °C, which resulted in Fno mediated cytotoxicity and apoptosis at 24 and 72 h post-infection. WT Fno incubated at 30 °C as well as ΔiglC,and ΔpdpA incubated at 25 °C and 30 °C were all defective for survival, replication, and the ability to cause cytotoxicity in TmB. Taken together, these results demonstrate that temperature plays a vital role for Fno intracellular survival, persistence and cytotoxicity.

  11. Final Report for LDRD Project 02-ERD-069: Discovering the Unknown Mechanism(s) of Virulence in a BW, Class A Select Agent

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chain, P; Garcia, E

    2003-02-06

    The goal of this proposed effort was to assess the difficulty in identifying and characterizing virulence candidate genes in an organism for which very limited data exists. This was accomplished by first addressing the finishing phase of draft-sequenced F. tularensis genomes and conducting comparative analyses to determine the coding potential of each genome; to discover the differences in genome structure and content, and to identify potential genes whose products may be involved in the F. tularensis virulence process. The project was divided into three parts: (1) Genome finishing: This part involves determining the order and orientation of the consensus sequencesmore » of contigs obtained from Phrap assemblies of random draft genomic sequences. This tedious process consists of linking contig ends using information embedded in each sequence file that relates the sequence to the original cloned insert. Since inserts are sequenced from both ends, we can establish a link between these paired-ends in different contigs and thus order and orient contigs. Since these genomes carry numerous copies of insertion sequences, these repeated elements ''confuse'' the Phrap assembly program. It is thus necessary to break these contigs apart at the repeated sequences and individually join the proper flanking regions using paired-end information, or using results of comparisons against a similar genome. Larger repeated elements such as the small subunit ribosomal RNA operon require verification with PCR. Tandem repeats require manual intervention and typically rely on single nucleotide polymorphisms to be resolved. Remaining gaps require PCR reactions and sequencing. Once the genomes have been ''closed'', low quality regions are addressed by resequencing reactions. (2) Genome analysis: The final consensus sequences are processed by combining the results of three gene modelers: Glimmer, Critica and Generation. The final gene models are submitted to a battery of homology searches

  12. Joint USGS/USEPA Pathogens in Soils Geographic ...

    EPA Pesticide Factsheets

    Online interactive maps In order to protect the environment from current and potential threats posed by uncontrolled intentional releases of hazardous substances, pollutants, and contaminants, the biothreat research community recognizes the needs to be able to detect threats in the appropriate matrices and also consider whether a detected constituent is naturally occurring or a contaminant associated with an accidental or purposeful release. Therefore, sensitive and specific methods for processing and analyzing environmental samples as well as methods to determine the existing risk to the public from endemic microorganisms are needed. Background data is also an important variable for assessing and managing the risks posed by a contaminated site. The EPA has collaborated with the USGS to analyze over 4800 soil samples collected during the USGS North American Soil Geochemical Landscapes Project for the presence of Bacillus anthracis and a subset of those samples for the presence of Yersinia pestis, and Francisella tularensis. EPA and USGS scientists correlated occurrences with geochemical constituents (> 40 major and trace elements), historical outbreak data, and climate data by creating online interactive maps using a Geographic Information Systems (GIS) platform. This on-going nationwide survey can be used as an investigative tool by animal and public health scientists and emergency responders determine the potential for disease outbreaks and persistenc

  13. Cationic host defense peptides; novel antimicrobial therapeutics against Category A pathogens and emerging infections.

    PubMed

    Findlay, Fern; Proudfoot, Lorna; Stevens, Craig; Barlow, Peter G

    2016-01-01

    Cationic Host Defense Peptides (HDP, also known as antimicrobial peptides) are crucial components of the innate immune system and possess broad-spectrum antibacterial, antiviral, and immunomodulatory activities. They can contribute to the rapid clearance of biological agents through direct killing of the organisms, inhibition of pro-inflammatory mediators such as lipopolysaccharide, and by modulating the inflammatory response to infection. Category A biological agents and materials, as classified by the United States National Institutes for Health, the US Centers for Disease Control and Prevention, and the US Department of Homeland Security, carry the most severe threat in terms of human health, transmissibility, and preparedness. As such, there is a pressing need for novel frontline approaches for prevention and treatment of diseases caused by these organisms, and exploiting the broad antimicrobial activity exhibited by cationic host defense peptides represents an exciting priority area for clinical research. This review will summarize what is known about the antimicrobial and antiviral effects of the two main families of cationic host defense peptides, cathelicidins, and defensins in the context of Category A biological agents which include, but are not limited to; anthrax (Bacillus anthracis), plague (Yersinia pestis), smallpox (Variola major), tularemia (Francisella tularensis). In addition, we highlight priority areas, particularly emerging viral infections, where more extensive research is urgently required.

  14. YopP-expressing variant of Y. pestis activates a potent innate immune response affording cross-protection against yersiniosis and tularemia [corrected].

    PubMed

    Zauberman, Ayelet; Flashner, Yehuda; Levy, Yinon; Vagima, Yaron; Tidhar, Avital; Cohen, Ofer; Bar-Haim, Erez; Gur, David; Aftalion, Moshe; Halperin, Gideon; Shafferman, Avigdor; Mamroud, Emanuelle

    2013-01-01

    Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53ΔJ+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53ΔJ+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-γ dependent. Moreover, inoculation of mice with Y. pestis Kim53ΔJ+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment.

  15. A microfluidic platform with integrated arrays for immunologic assays for biological pathogen detection

    NASA Astrophysics Data System (ADS)

    Klemm, Richard; Becker, Holger; Hlawatsch, Nadine; Julich, Sandra; Miethe, Peter; Moche, Christian; Schattschneider, Sebastian; Tomaso, Herbert; Gärtner, Claudia

    2014-05-01

    The ability to integrate complete assays on a microfluidic chip helps to greatly simplify instrument requirements and allows the use of lab-on-a-chip technology in the field. A core application for such field-portable systems is the detection of pathogens in a CBRN scenario such as permanent monitoring of airborne pathogens, e.g. in subway stations or hospitals etc. An immunological assay was chosen as method for the pathogen identification. The conceptual approach was its realization as a lab-on-a-chip system, enabling an easy handling of the sample in an automated manner. The immunological detection takes place on an antibody array directly implemented in the microfluidic network. Different immobilization strategies will be presented showing the performance of the system. Central elements of the disposable microfluidic device like fluidic interface, turning valves, liquid introduction and waste storage, as well as the architecture of measurement and control fluidic network, will be introduced. Overall process times of about 30 minutes were achieved and assays for the detection of Francisella tularensis and Yersinia pestis are presented. An important feature of the integrated lab-on-a-chip approach is that all waste liquids remain on-chip and contamination risks can be avoided.

  16. DOE Office of Scientific and Technical Information (OSTI.GOV)

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discoveredmore » two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

  17. DOE Office of Scientific and Technical Information (OSTI.GOV)

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we havemore » discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

  18. Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa.

    PubMed

    Salih, Osman; He, Shaoda; Planamente, Sara; Stach, Lasse; MacDonald, James T; Manoli, Eleni; Scheres, Sjors H W; Filloux, Alain; Freemont, Paul S

    2018-02-06

    Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Water quality monitoring using an automated portable fiber optic biosensor: RAPTOR

    NASA Astrophysics Data System (ADS)

    Anderson, George P.; Rowe-Taitt, Chris A.

    2001-03-01

    The RAPTOR is a portable, automated biosensor capable of performing rapid, ten-minute assays on a sample for four target analytes simultaneously. Samples are analyzed using a fluorescent sandwich immunoassay on the surface of short polystyrene optical probes with capture antibody adsorbed to the probe surface. Target analytes bound to the fiber by capture antibodies are detected with fluorescently labeled tracer antibodies, which are held in a separate reservoir. Since target recognition is a two-step process, selectivity is enhanced, and the optical probes can be reused up to forty times, or until a positive result is obtained. This greatly reduces the logistical burden for field operations. Numerous assays for toxins, such as SEB and ricin, and bacteria, such as Bacillus anthracis and Francisella tularensis, have been developed for the RAPTOR. An assay of particular interest for water quality monitoring and the screening of fruits and vegetables is detection of Giardia cysts. Giardia lamblia is a parasitic protozoan common in the developing world that causes severe intestinal infections. Thus, a simple field assay for screening water supplies would be highly useful. Such an assay has been developed using the RAPTOR. The detection limit for Giardia cysts was 5x104/ml for a 10-minute assay.

  20. Development and Comparison of Two Assay Formats for Parallel Detection of Four Biothreat Pathogens by Using Suspension Microarrays

    PubMed Central

    Janse, Ingmar; Bok, Jasper M.; Hamidjaja, Raditijo A.; Hodemaekers, Hennie M.; van Rotterdam, Bart J.

    2012-01-01

    Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics. PMID:22355407

  1. A case of typhoidal tularemia in a male Japanese farmer.

    PubMed

    Nakamura, Kiwamu; Fujita, Hiromi; Miura, Tomoya; Igata, Yu; Narita, Masashi; Monma, Naota; Hara, Yasuka; Saito, Kyoichi; Matsumoto, Akinori; Kanemitsu, Keiji

    2018-06-01

    In Japan, most tularemia cases occur after contact with hares (hunting, cooking) and involve the glandular or ulceroglandular form. Here, we present a case of typhoidal tularemia in a 72-year-old Japanese male farmer who presented with fever, fatigue, and right lower abdominal pain. Computed tomography revealed intestinal wall thickening at the ascending colon, pleural effusion, and ascites. Following an initial diagnosis of bacterial enteric infection, his symptoms deteriorated after a week-long cephalosporin treatment course. The patient lived in an area endemic for scrub typhus; the antibiotic was changed to a tetracycline on suspicion of scrub typhus infection. His symptoms rapidly improved after initiation of minocycline treatment. Later, blood tests revealed marked increases in serological tests against Francisella tularensis exclusively, and the patient was diagnosed with typhoidal tularemia. Typhoidal tularemia may be characterized by any combination of general symptoms, but does not exhibit the local manifestations associated with other forms of tularemia. The patient, in this case, had no direct contact with hares or other wild animals and did not present with local manifestations of tularemia. Physicians should consider this disease, especially when tick-borne disease is suspected in the absence of local wounds, eschar, ulcers, or lymphadenopathy. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  2. Defective anti-polysaccharide IgG vaccine responses in IgA deficient mice.

    PubMed

    Furuya, Yoichi; Kirimanjeswara, Girish S; Roberts, Sean; Racine, Rachael; Wilson-Welder, Jennifer; Sanfilippo, Alan M; Salmon, Sharon L; Metzger, Dennis W

    2017-09-05

    We report that IgA -/- mice exhibit specific defects in IgG antibody responses to various polysaccharide vaccines (Francisella tularensis LPS and Pneumovax), but not protein vaccines such as Fluzone. This defect further included responses to polysaccharide-protein conjugate vaccines (Prevnar and Haemophilus influenzae type b-tetanus toxoid vaccine). In agreement with these findings, IgA -/- mice were protected from pathogen challenge with protein- but not polysaccharide-based vaccines. Interestingly, after immunization with live bacteria, IgA +/+ and IgA -/- mice were both resistant to lethal challenge and their IgG anti-polysaccharide antibody responses were comparable. Immunization with live bacteria, but not purified polysaccharide, induced production of serum B cell-activating factor (BAFF), a cytokine important for IgG class switching; supplementing IgA -/- cell cultures with BAFF enhanced in vitro polyclonal IgG production. Taken together, these findings show that IgA deficiency impairs IgG class switching following vaccination with polysaccharide antigens and that live bacterial immunization can overcome this defect. Since IgA deficient patients also often show defects in antibody responses following immunization with polysaccharide vaccines, our findings could have relevance to the clinical management of this population. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Risk Prioritization Tool to Identify the Public Health Risks of Wildlife Trade: The Case of Rodents from Latin America.

    PubMed

    Bueno, I; Smith, K M; Sampedro, F; Machalaba, C C; Karesh, W B; Travis, D A

    2016-06-01

    Wildlife trade (both formal and informal) is a potential driver of disease introduction and emergence. Legislative proposals aim to prevent these risks by banning wildlife imports, and creating 'white lists' of species that are cleared for importation. These approaches pose economic harm to the pet industry, and place substantial burden on importers and/or federal agencies to provide proof of low risk for importation of individual species. As a feasibility study, a risk prioritization tool was developed to rank the pathogens found in rodent species imported from Latin America into the United States with the highest risk of zoonotic consequence in the United States. Four formally traded species and 16 zoonotic pathogens were identified. Risk scores were based on the likelihood of pathogen release and human exposure, and the severity of the disease (consequences). Based on the methodology applied, three pathogens (Mycobacterium microti, Giardia spp. and Francisella tularensis) in one species (Cavia porcellus) were ranked as highest concern. The goal of this study was to present a methodological approach by which preliminary management resources can be allocated to the identified high-concern pathogen-species combinations when warranted. This tool can be expanded to other taxa and geographic locations to inform policy surrounding the wildlife trade. © 2015 Blackwell Verlag GmbH.

  4. Potential targets for next generation antimicrobial glycoconjugate vaccines

    PubMed Central

    Micoli, Francesca; Costantino, Paolo; Adamo, Roberto

    2018-01-01

    Abstract Cell surface carbohydrates have been proven optimal targets for vaccine development. Conjugation of polysaccharides to a carrier protein triggers a T-cell-dependent immune response to the glycan moiety. Licensed glycoconjugate vaccines are produced by chemical conjugation of capsular polysaccharides to prevent meningitis caused by meningococcus, pneumococcus and Haemophilus influenzae type b. However, other classes of carbohydrates (O-antigens, exopolysaccharides, wall/teichoic acids) represent attractive targets for developing vaccines. Recent analysis from WHO/CHO underpins alarming concern toward antibiotic-resistant bacteria, such as the so called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) and additional pathogens such as Clostridium difficile and Group A Streptococcus. Fungal infections are also becoming increasingly invasive for immunocompromised patients or hospitalized individuals. Other emergencies could derive from bacteria which spread during environmental calamities (Vibrio cholerae) or with potential as bioterrorism weapons (Burkholderia pseudomallei and mallei, Francisella tularensis). Vaccination could aid reducing the use of broad-spectrum antibiotics and provide protection by herd immunity also to individuals who are not vaccinated. This review analyzes structural and functional differences of the polysaccharides exposed on the surface of emerging pathogenic bacteria, combined with medical need and technological feasibility of corresponding glycoconjugate vaccines. PMID:29547971

  5. Familial tularaemia.

    PubMed

    Peker, E; Ayaydin, A; Duran, N

    2009-01-01

    Tularaemia is a zoonotic disease caused by Francisella tularensis . In this report, we have presented an early stage case of tularemia with fever and pharyngitis and two cases from the same non-endemic region with typical lymphadenitis. All three patients were treated with non-specific medications in healthcare centres, the treatment being directed towards symptoms resembling those of upper respiratory tract infections. However, there was no regression in their complaints. Because the first case had been treated earlier, his lymphadenopaties regressed and there was no suppuration. The other two cases, which had been suspected to be exposed to the same pathogen based on their histories, were at a mild acute phase and presented to our clinic with typical lymphadenitis. The diagnoses of each of the three patients were made serologically. An early clinical recovery was achieved in the first patient with streptomycin (1 x 1 g/day im) and doxycyline (2 x 100 mg/day peroral) therapy. The therapy was prolonged to 4 weeks in the other two cases according to lymph node response and no complications occurring in their follow-ups. It can be concluded that tularaemia should be considered in the differential diagnosis of patients with fever, pharyngitis, conjunctivitis and cervical lymphadenopathies that do not respond to beta-lactam antibiotics.

  6. Tularemia: potential role of cytopathology in differential diagnosis of cervical lymphadenitis: multicenter experience in 53 cases and literature review.

    PubMed

    Tuncer, Ersin; Onal, Binnur; Simsek, Gulcin; Elagoz, Sahande; Sahpaz, Ahmet; Kilic, Selcuk; Altuntas, Emine Elif; Ulu Kilic, Aysegul

    2014-03-01

    Tularemia is a zoonosis caused by Francisella tularensis. Tularemia outbreaks occurred in Central Anatolia during 2009 and 2011. We evaluated the clinical characteristics and cytomorphologies of fine needle aspirations (FNAs) from cervical lymph nodes in serologically confirmed tularemia cases. To our knowledge, this is the first large series concerning FNA morphology of Tularemia. FNA smears of 53 patients of the 290, diagnosed by microagglutination tests and PCR, were evaluated at three Pathology centers. FNAs were performed by cytopathologists or ear-nose-throat surgeons. Of all patients, 17 had also lymph node resections. FNAs showed the presence of suppuration and abscess. Rare epithelioid histiocytes and granulomas, seldom phagocytosed bacilli-like microorganisms were observed. On histopathology; granulomas, necrosis, and suppurative inflammation extending extracapsular areas were seen. Tularemia is endemic in certain areas of the Northern Hemisphere. The benefit from cytopathology is limited and cytological suspicion should be confirmed by serology. However FNA cytology is helpful in differential diagnosis of tularemia and other diseases presented with suppurative, granulomatous cervical lymphadenitis. It is also useful in providing the material for PCR and culture in early phase when the serology is negative and the treatment is more effective. © 2013 APMIS. Published by John Wiley & Sons Ltd.

  7. Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays.

    PubMed

    Janse, Ingmar; Bok, Jasper M; Hamidjaja, Raditijo A; Hodemaekers, Hennie M; van Rotterdam, Bart J

    2012-01-01

    Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.

  8. Physiologic Reference Ranges for Captive Black-Tailed Prairie Dogs (Cynomys ludovicianus)

    PubMed Central

    Keckler, M Shannon; Gallardo-Romero, Nadia F; Langham, Gregory L; Damon, Inger K; Karem, Kevin L; Carroll, Darin S

    2010-01-01

    The black-tailed prairie dog (Cynomys ludovicianus) is a member of the order Rodentia and the family Sciuridae. Ecologically, prairie dogs are a keystone species in prairie ecology. This species is used as an animal model for human gallbladder disease and diseases caused by infection with Clostridium difficile, Yersinia pestis, Francisella tularensis, and most recently, Orthopoxvirus. Despite increasing numbers of prairie dogs used in research and kept as pets, few data are available on their baseline physiology in animal facility housing conditions. To establish baseline physiologic reference ranges, we designed a study using 18 wild-caught black-tailed prairie dogs. Telemetry data were analyzed to establish circadian rhythms for activity and temperature. In addition, hematologic and serum chemistry analyses were performed. Baseline measurements were used to establish the mean for each animal, which then were compiled and analyzed to determine the reference ranges. Here we present physiologic data on serum chemistry and hematology profiles, as well as weight, core body temperature, and daily activity patterns for black-tailed prairie dogs. These results reflect the use of multiple measurements from species- and age-matched prairie dogs and likely will be useful to ecologists, scientists interested in using this animal model in research, and veterinarians caring for pet prairie dogs. PMID:20587156

  9. Roles of inflammatory caspases during processing of zebrafish interleukin-1β in Francisella noatunensis infection

    USGS Publications Warehouse

    Vojtech, Lucia N.; Scharping, Nichole; Woodson, James C.; Hansen, John D.

    2012-01-01

    The interleukin-1 family of cytokines are essential for the control of pathogenic microbes but are also responsible for devastating autoimmune pathologies. Consequently, tight regulation of inflammatory processes is essential for maintaining homeostasis. In mammals, interleukin-1 beta (IL-1β) is primarily regulated at two levels, transcription and processing. The main pathway for processing IL-1β is the inflammasome, a multiprotein complex that forms in the cytosol and which results in the activation of inflammatory caspase (caspase 1) and the subsequent cleavage and secretion of active IL-1β. Although zebrafish encode orthologs of IL-1β and inflammatory caspases, the processing of IL-1β by activated caspase(s) has never been examined. Here, we demonstrate that in response to infection with the fish-specific bacterial pathogen Francisella noatunensis, primary leukocytes from adult zebrafish display caspase-1-like activity that results in IL-1β processing. Addition of caspase 1 or pancaspase inhibitors considerably abrogates IL-1β processing. As in mammals, this processing event is concurrent with the secretion of cleaved IL-1β into the culture medium. Furthermore, two putative zebrafish inflammatory caspase orthologs, caspase A and caspase B, are both able to cleave IL-1β, but with different specificities. These results represent the first demonstration of processing and secretion of zebrafish IL-1β in response to a pathogen, contributing to our understanding of the evolutionary processes governing the regulation of inflammation.                   

  10. Host Genes and Resistance/Sensitivity to Military Priority Pathogens

    DTIC Science & Technology

    2011-06-01

    tularensis (FT Schu S4) that yields a significantly different outcome to infection in B6 and D2 mice. Both strains succumb to infection at essentially the...Figure 2). Some of the group sizes are too small to yield statistically relevant findings, and additional studies will be performed with these strains as...generated approximately 100-fold coverage of the DBA/2J genome (Table 2) and sequenced 99.96% of the DBA/2J genome (excluding gaps in the reference

  11. DESTRUCTION OF FRANCISELLA TULARENSIS AND YERSINIA PESTIS PERSISTENCE OF BACILLUS ANTHRACIS SPORES AND CLOSTRIDIUM BOTULINUM IN MUNICIPAL SOLID LANDFILL LEACHATES

    EPA Science Inventory

    The United States Environmental Protection Agency Office of Research and Development National Homeland Security Research Center (NHSRC) in collaboration with the Department of Defense Edgewood Chemical Biological Center (ECBC) are evaluating the permanence of biological and chemi...

  12. Cationic host defense peptides; novel antimicrobial therapeutics against Category A pathogens and emerging infections

    PubMed Central

    Findlay, Fern; Proudfoot, Lorna; Stevens, Craig

    2016-01-01

    Cationic Host Defense Peptides (HDP, also known as antimicrobial peptides) are crucial components of the innate immune system and possess broad-spectrum antibacterial, antiviral, and immunomodulatory activities. They can contribute to the rapid clearance of biological agents through direct killing of the organisms, inhibition of pro-inflammatory mediators such as lipopolysaccharide, and by modulating the inflammatory response to infection. Category A biological agents and materials, as classified by the United States National Institutes for Health, the US Centers for Disease Control and Prevention, and the US Department of Homeland Security, carry the most severe threat in terms of human health, transmissibility, and preparedness. As such, there is a pressing need for novel frontline approaches for prevention and treatment of diseases caused by these organisms, and exploiting the broad antimicrobial activity exhibited by cationic host defense peptides represents an exciting priority area for clinical research. This review will summarize what is known about the antimicrobial and antiviral effects of the two main families of cationic host defense peptides, cathelicidins, and defensins in the context of Category A biological agents which include, but are not limited to; anthrax (Bacillus anthracis), plague (Yersinia pestis), smallpox (Variola major), tularemia (Francisella tularensis). In addition, we highlight priority areas, particularly emerging viral infections, where more extensive research is urgently required. PMID:27315342

  13. Ticks and tick-borne pathogens and putative symbionts of black bears (Ursus americanus floridanus) from Georgia and Florida.

    PubMed

    Yabsley, Michael J; Nims, Todd N; Savage, Mason Y; Durden, Lance A

    2009-10-01

    Ticks were collected from 38 black bears (Ursus americanus floridanus) from northwestern Florida (n = 18) from 2003 to 2005 and southern Georgia (n = 20) in 2006. Five species (Amblyomma americanum, A. maculatum, Dermacentor variabilis, Ixodes scapularis, and I. affinis) were collected from Florida bears, and 4 species (A. americanum, A. maculatum, D. variabilis, I. scapularis) were collected from bears in Georgia. Ixodes scapularis was the most frequently collected tick, followed by D. variabilis, A. americanum, A. maculatum, and I. affinis. The collection of I. affinis from a Florida bear represents a new host record. A subset of ticks was screened for pathogens and putative symbionts by polymerase chain reaction (PCR). The zoonotic tick-borne pathogens Ehrlichia chaffeensis and Rickettsia parkeri were detected in 1 of 23 (4.3%) A. americanum and 1 of 12 (8.3%) A. maculatum, respectively. The putative zoonotic pathogen "Rickettsia amblyommii" was detected in 4 (17.4%) A. americanum and 1 (8.3%) A. maculatum. Other putative symbiotic rickettsiae detected included R. bellii and R. montanensis in D. variabilis, a Rickettsia cooleyi-like sp. and Rickettsia sp. Is-1 in I. scapularis, and Rickettsia TR39-like sp. in I. scapularis and A. americanum. All ticks were PCR-negative for Anaplasma phagocytophilum, Panola Mountain Ehrlichia sp., E. ewingii, Francisella tularensis, and Borrelia spp.

  14. Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection.

    PubMed

    Song, Linan; Ahn, Soohyoun; Walt, David R

    2006-02-15

    We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.

  15. Crystal structure and tartrate inhibition of Legionella pneumophila histidine acid phosphatase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Reilly, Thomas J; Tanner, John J

    2015-11-01

    Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. A stable live bacterial vaccine.

    PubMed

    Kunda, Nitesh K; Wafula, Denis; Tram, Meilinn; Wu, Terry H; Muttil, Pavan

    2016-06-01

    Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Dermatological aspects of tularaemia: a study of 168 cases.

    PubMed

    Polat, M; Karapınar, T; Sırmatel, F

    2018-05-14

    Tularaemia is a zoonotic infectious disease caused by Francisella tularensis, an aerobic, uncapsulated, gram-negative coccobacillus. Several case reports have appeared on the dermatological manifestations of tularaemia, but relatively few longer-term studies are available. To identify skin features of tularaemia that aid in its diagnosis. In total, 168 patients (68 male, 100 female) diagnosed with tularaemia were retrospectively examined. All dermatological data for these patients were evaluated. Of the 168 patients, 149 (88.69%) had tularaemia of the oropharyngeal type, 12 (7.73%) had the ulceroglandular type, 5 (2.9%) had the oculoglandular type and 2 (0.59%) had the pulmonary type. Secondary skin manifestations were found in 26 patients (15.47%). Sweet syndrome (SS) was found in 11 patients (6.54%), most of whom presented with the oropharyngeal form, while erythema nodosum (EN) was found in 7 patients (4.16%), dermatitis in 2 (1.19%), urticaria in 2 (1.19%), acneiform eruptions in 1 (0.59%), vasculitis-like eruptions in 1 (0.59%) and SS + EN in 1 (0.59%). Patients with the oropharyngeal form had a statistically significant (P < 0.001) higher number of skin findings than patients with the other forms. In clinical practice, tularaemia may present with various cutaneous manifestations, and dermatologists who work in endemic regions must be aware of the possibility of this disease. © 2018 British Association of Dermatologists.

  18. Tularemia, plague, yersiniosis, and Tyzzer’s disease in wild rodents and lagomorphs in Canada: A review

    PubMed Central

    Wobeser, Gary; Campbell, G. Douglas; Dallaire, André; McBurney, Scott

    2009-01-01

    Information related to infection of wild rodents or lagomorphs in Canada by Francisella tularensis, Yersinia pestis, other Yersinia spp., and Clostridium piliforme was searched for this study. Reports on tularemia in humans linked to these species came from diagnostic databases, literature, wildlife health specialists, and public health agencies. Tularemia has been diagnosed in 8 species of wild rodent and 2 species in the genus Lepus in Canada. Tularemia occurred in wild animals, or in humans associated with these species, in all jurisdictions except the Yukon and Nunavut. Tularemia was diagnosed most frequently in beaver, muskrats, and snowshoe hares, and although tularemia is closely linked to cottontail rabbits in the USA, it has not been reported in cottontails in Canada. Tularemia in humans was associated with muskrats and hares more commonly than with beaver. Plague was diagnosed in bushy-tailed woodrats in British Columbia in 1988. Based on surveys, Y. pestis may occur enzootically in southern Alberta, Saskatchewan, and British Columbia. Infection with Yersinia pseudotuberculosis and Y. enterocolitica has been diagnosed in beaver, muskrats, and snowshoe hares in many provinces. Tyzzer’s disease has been diagnosed in muskrats in British Columbia, Saskatchewan, Ontario, and Quebec and in snowshoe hares in Ontario. Infection with these bacteria is likely much more frequent than indicated by diagnostic records. PMID:20190973

  19. Pilot Cross-Sectional Study of Three Zoonoses (Lyme Disease, Tularaemia, Leptospirosis) among Healthy Blood Donors in Eastern Slovakia.

    PubMed

    Zákutná, Ľubica; Dorko, Erik; Rimárová, Kvetoslava; Kizeková, Marianna

    2015-06-01

    The aim of the study was to determine the seroprevalence of three zoonotic infections among healthy blood donors/volunteers in Eastern Slovakia. Sera from 124 blood donors were investigated for the presence of antibodies against Borrelia burgdorferi, Francisella tularensis and Leptospira pomona. The participants also completed the questionnaire about demographic, exposure and epidemiological characteristics. Two serological methods were used for the diagnosis: the enzyme linked protein A/G assay (ELPAGA) and the Western blot (WB). First, sera were screened by ELPAGA (except for leptospirosis). The observed seroprevalence was 15% for Lyme borreliosis (LB) and 4% for tularaemia (TUL). The results were confirmed by WB. Positive IgG antibodies (WB method) were detected only in 1.6% of examined for LB and 0.8% for TUL. Our results did not identify any antibodies against Leptospira pomona agent in the examined healthy blood donors group. ELPAGA seroprevalence for TUL was significantly higher in blood donors working in the agricultural area in the direct contact with hay, straw, manure, and agricultural land. Our outputs determine tick bite as a significant risk factor for LB. The study confirms the explosion of tick-borne diseases in the healthy population of people. The exposure risk for leptospirosis seems to be minimal. Copyright© by the National Institute of Public Health, Prague 2015.

  20. Spatio-temporal dynamics of tularemia in French wildlife: 2002-2013.

    PubMed

    Moinet, Marie; Decors, Anouk; Mendy, Christiane; Faure, Eva; Durand, Benoit; Madani, Nora

    2016-08-01

    Tularemia, caused by Francisella tularensis, is endemic in France. The surveillance of this disease in wildlife is operated by the SAGIR Network and by the National Reference Laboratory for Tularemia. Wild animals found dead or dying collected by the SAGIR network are necropsied and when tularemia is suspected culture and/or PCR are performed to confirm the diagnosis. The aim of this study was to present the results of tularemia surveillance in wildlife and to investigate the spatial and temporal pattern of tularemia observed between the 2002-2003 and 2012-2013 hunting seasons in French wildlife. Fourty-one to 121 cases were collected each hunting season for a total of 693 confirmed cases and 46 additional suspected cases. The main species affected was the European Brown Hare (Lepus europaeus) but 4 rabbits (Oryctolagus cuniculus), 2 roe deer (Capreolus capreolus) and one wild boar (Sus scrofa) were also found positive. The Standard Mortality Ratio was mapped and Kulldorff's retrospective space-time scan statistic was implemented and allowed the detection of several clusters: the nationwide outbreak of 2007-2008; 2 clusters in northern and central-western France in high hare-abundance areas and another in North-eastern France where the abundance of hares is low. Our results confirm the usefulness of brown hare as a sentinel of environmental risk. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

    PubMed

    Del Río, Jonathan Sabaté; Lobato, Ivan Magriñà; Mayboroda, Olena; Katakis, Ioanis; O'Sullivan, Ciara K

    2017-05-01

    Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10 -15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

  2. [Investigation of the presence of Mycobacterium tuberculosis in the lymph node aspirates of the suspected tularemia lymphadenitis cases].

    PubMed

    Albayrak, Nurhan; Celebi, Bekir; Kavas, Semra; Simşek, Hülya; Kılıç, Selçuk; Sezen, Figen; Arslantürk, Ahmet

    2014-01-01

    Recently reports of cervical tuberculous lymphadenitis and oropharyngeal tularemia which are the most common infectious causes of granulomatous lymphadenitis, have been significantly increased in Turkey. The differentiation of cervical tuberculous lymphadenitis and oropharyngeal tularemia is usually confusing on the basis of clinical and histopathological findings. Thus, in tularemia endemic areas, the patients are more commonly evaluated in terms of tularemia lymphadenitis leaving tuberculosis out. The aim of this study was to investigate the presence of Mycobacterium tuberculosis in cervical lymph node aspirates, obtained from tularemia suspected cases. A total of 105 oropharyngeal tularemia-suspected cases which were found negative for Francisella tularensis by bacteriological (culture), molecular (PCR) and serological (microagglutination) methods, were included in the study. The samples had been previously studied at National Tularemia Reference Laboratory, Turkish Public Health Institution, between 2009-2011. The study samples were evaluated in terms of M.tuberculosis by culture and real-time PCR (rtPCR) methods in the National Tuberculosis Reference Laboratory. Both Lowenstein-Jensen (LJ) medium and liquid-based MGIT (BD, USA) automated culture system were used for mycobacterial culture. Samples that yielded mycobacterial growth were identified as M.tuberculosis by immunochromotographic test (BD, USA). The lymph node aspirates of 65 patients who were F.tularensis PCR negative but antibody positive, were used as the control group. As a result, M.tuberculosis was found to be positive in 9 (8.6%) of 105 tularemia-negative lymph node aspirates, sent to our laboratory from different geographic regions for the investigation of tularemia. Six of the M.tuberculosis positive cases were male and the age range of the patients was 26-85 years. The presence of M.tuberculosis was detected only by culture in two samples, only by rtPCR in five samples and both by culture and

  3. Automated Lipid A Structure Assignment from Hierarchical Tandem Mass Spectrometry Data

    NASA Astrophysics Data System (ADS)

    Ting, Ying S.; Shaffer, Scott A.; Jones, Jace W.; Ng, Wailap V.; Ernst, Robert K.; Goodlett, David R.

    2011-05-01

    Infusion-based electrospray ionization (ESI) coupled to multiple-stage tandem mass spectrometry (MS n ) is a standard methodology for investigating lipid A structural diversity (Shaffer et al. J. Am. Soc. Mass. Spectrom. 18(6), 1080-1092, 2007). Annotation of these MS n spectra, however, has remained a manual, expert-driven process. In order to keep up with the data acquisition rates of modern instruments, we devised a computational method to annotate lipid A MS n spectra rapidly and automatically, which we refer to as hierarchical tandem mass spectrometry (HiTMS) algorithm. As a first-pass tool, HiTMS aids expert interpretation of lipid A MS n data by providing the analyst with a set of candidate structures that may then be confirmed or rejected. HiTMS deciphers the signature ions (e.g., A-, Y-, and Z-type ions) and neutral losses of MS n spectra using a species-specific library based on general prior structural knowledge of the given lipid A species under investigation. Candidates are selected by calculating the correlation between theoretical and acquired MS n spectra. At a false discovery rate of less than 0.01, HiTMS correctly assigned 85% of the structures in a library of 133 manually annotated Francisella tularensis subspecies novicida lipid A structures. Additionally, HiTMS correctly assigned 85% of the structures in a smaller library of lipid A species from Yersinia pestis demonstrating that it may be used across species.

  4. Pathobiology and management of laboratory rodents administered CDC category A agents.

    PubMed

    He, Yongqun; Rush, Howard G; Liepman, Rachel S; Xiang, Zuoshuang; Colby, Lesley A

    2007-02-01

    The Centers for Disease Control and Prevention Category A infectious agents include Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), variola major virus (smallpox), Francisella tularensis (tularemia), and the filoviruses and arenaviruses that induce viral hemorrhagic fevers. These agents are regarded as having the greatest potential for adverse impact on public health and therefore are a focus of renewed attention in infectious disease research. Frequently rodent models are used to study the pathobiology of these agents. Although much is known regarding naturally occurring infections in humans, less is documented on the sources of exposures and potential risks of infection to researchers and animal care personnel after the administration of these hazardous substances to laboratory animals. Failure to appropriately manage the animals can result both in the creation of workplace hazards if human exposures occur and in disruption of the research if unintended animal exposures occur. Here we review representative Category A agents, with a focus on comparing the biologic effects in naturally infected humans and rodent models and on considerations specific to the management of infected rodent subjects. The information reviewed for each agent has been curated manually and stored in a unique Internet-based database system called HazARD (Hazards in Animal Research Database, http://helab.bioinformatics.med.umich.edu/hazard/) that is designed to assist researchers, administrators, safety officials, Institutional Biosafety Committees, and veterinary personnel seeking information on the management of risks associated with animal studies involving hazardous substances.

  5. Molecular architecture of an N-formyltransferase from Salmonella enterica O60.

    PubMed

    Woodford, Colin R; Thoden, James B; Holden, Hazel M

    2017-12-01

    N-formylated sugars are found on the lipopolysaccharides of various pathogenic Gram negative bacteria including Campylobacter jejuni 81116, Francisella tularensis, Providencia alcalifaciens O30, and Providencia alcalifaciens O40. The last step in the biosynthetic pathways for these unusual sugars is catalyzed by N-formyltransferases that utilize N 10 -formyltetrahydrofolate as the carbon source. The substrates are dTDP-linked amino sugars with the functional groups installed at either the C-3' or C-4' positions of the pyranosyl rings. Here we describe a structural and enzymological investigation of the putative N-formyltransferase, FdtF, from Salmonella enterica O60. In keeping with its proposed role in the organism, the kinetic data reveal that the enzyme is more active with dTDP-3-amino-3,6-dideoxy-d-galactose than with dTDP-3-amino-3,6-dideoxy-d-glucose. The structural data demonstrate that the enzyme contains, in addition to the canonical N-formyltransferase fold, an ankyrin repeat moiety that houses a second dTDP-sugar binding pocket. This is only the second time an ankyrin repeat has been shown to be involved in small molecule binding. The research described herein represents the first structural analysis of a sugar N-formyltransferase that specifically functions on dTDP-3-amino-3,6-dideoxy-d-galactose in vivo and thus adds to our understanding of these intriguing enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Treatment failure of gentamicin in pediatric patients with oropharyngeal tularemia

    PubMed Central

    Kaya, Ali; Uysal, İsmail Önder; Güven, Ahmet Sami; Engin, Aynur; Gültürk, Abdulaziz; İçağasıoğlu, Füsun Dilara; Cevit, Ömer

    2011-01-01

    Summary Background Tularemia is a zoonotic infection, and the causative agent is Francisella tularensis. A first-line therapy for treating tularemia is aminoglycosides (streptomycin or, more commonly, gentamicin), and treatment duration is typically 7 to 10 days, with longer courses for more severe cases. Material/Methods We evaluated 11 patients retrospectively. Failure of the therapy was defined by persistent or recurrent fever, increased size or appearance of new lymphadenopathies and persistence of the constitutional syndrome with elevation of the levels of the proteins associated with the acute phase of infection. Results We observed fluctuating size of lymph nodes of 4 patients who were on the 7th day of empirical therapy. The therapy was switched to streptomycin alone and continued for 14 days. The other 7 patients, who had no complications, were on cefazolin and gentamycin therapy until the serologic diagnosis. Then we evaluated them again and observed that none of their lymph nodes regressed. We also switched their therapy to 14 days of streptomycin. After the 14 days on streptomycin therapy, we observed all the lymph nodes had recovered or regressed. During a follow-up 3 weeks later, we observed that all their lymph nodes had regressed to the clinically non-significant dimensions (<1 cm). Conclusions All patients were first treated with gentamicin, but were than given streptomycin after failure of gentamicin. This treatment was successful in all patients. The results of our study suggest that streptomycin is an effective choice of first-line treatment for pediatric oropharyngeal tularemia patients. PMID:21709631

  7. Structure-Based Design and Synthesis of a Small Molecule that Exhibits Anti-inflammatory Activity by Inhibition of MyD88-mediated Signaling to Bacterial Toxin Exposure.

    PubMed

    Alam, Shahabuddin; Javor, Sacha; Degardin, Melissa; Ajami, Dariush; Rebek, Mitra; Kissner, Teri L; Waag, David M; Rebek, Julius; Saikh, Kamal U

    2015-08-01

    Both Gram-positive and Gram-negative pathogens or pathogen-derived components, such as staphylococcal enterotoxins (SEs) and endotoxin (LPS) exposure, activate MyD88-mediated pro-inflammatory cellular immunity for host defense. However, dysregulated MyD88-mediated signaling triggers exaggerated immune response that often leads to toxic shock and death. Previously, we reported a small molecule compound 1 mimicking BB-loop structure of MyD88 was capable of inhibiting pro-inflammatory response to SEB exposure in mice. In this study, we designed a dimeric structure compound 4210 covalently linked with compound 1 by a non-polar cyclohexane linker which strongly inhibited the production of pro-inflammatory cytokines in human primary cells to SEB (IC50 1-50 μm) or LPS extracted from Francisella tularensis, Escherichia coli, or Burkholderia mallei (IC50 10-200 μm). Consistent with cytokine inhibition, in a ligand-induced cell-based reporter assay, compound 4210 inhibited Burkholderia mallei or LPS-induced MyD88-mediated NF-kB-dependent expression of reporter activity (IC50 10-30 μm). Furthermore, results from a newly expressed MyD88 revealed that 4210 inhibited MyD88 dimer formation which is critical for pro-inflammatory signaling. Importantly, a single administration of compound 4210 in mice showed complete protection from lethal toxin challenge. Collectively, these results demonstrated that compound 4210 inhibits toxin-induced inflated pro-inflammatory immune signaling, thus displays a potential bacterial toxin therapeutic. © 2014 John Wiley & Sons A/S.

  8. A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents

    PubMed Central

    Madrid, Peter B.; Chopra, Sidharth; Manger, Ian D.; Gilfillan, Lynne; Keepers, Tiffany R.; Shurtleff, Amy C.; Green, Carol E.; Iyer, Lalitha V.; Dilks, Holli Hutcheson; Davey, Robert A.; Kolokoltsov, Andrey A.; Carrion, Ricardo; Patterson, Jean L.; Bavari, Sina; Panchal, Rekha G.; Warren, Travis K.; Wells, Jay B.; Moos, Walter H.; Burke, RaeLyn L.; Tanga, Mary J.

    2013-01-01

    Background The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency. Methodology/Principal Findings A large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo. Conclusions/Significance The feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses. PMID:23577127

  9. Evaluating Thermodynamic Integration Performance of the New Amber Molecular Dynamics Package and Assess Potential Halogen Bonds of Enoyl-ACP Reductase (FabI) Benzimidazole Inhibitors

    PubMed Central

    Su, Pin-Chih; Johnson, Michael E.

    2015-01-01

    Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the para-halogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme. PMID:26666582

  10. All-time high tularaemia incidence in Norway in 2011: report from the national surveillance.

    PubMed

    Larssen, K W; Bergh, K; Heier, B T; Vold, L; Afset, J E

    2014-11-01

    Tularaemia has mainly been a sporadic disease in Norway. In 2011, 180 persons (3.7 per 100,000 population) were diagnosed with tularaemia. This article describes the epidemiological and clinical features of tularaemia cases during a year with exceptionally high tularaemia incidence. Data from the national reference laboratory for tularaemia combined with epidemiological data from the Norwegian Surveillance System for Communicable Diseases (MSIS) were used. The incidence of tularaemia varied greatly between counties, but almost every county was involved. The majority (77.8 %) of the cases were diagnosed during the autumn and winter months. The geographic distribution also showed seasonal patterns. Overall, oropharyngeal tularaemia (41.1 %) was the most common clinical presentation, followed by glandular (14.4 %), typhoidal (14.4 %), respiratory (13.3 %) and ulceroglandular (12.8 %) tularaemia. From January to April, oropharyngeal tularaemia dominated, from May to September, ulceroglandular tularaemia was most common, whereas from October to December, there was an almost even distribution between several clinical forms of tularaemia. Eighty-five (47.2 %) of all tularaemia cases were admitted to, or seen as outpatients in, hospitals. An unexpectedly high number (3.9 %) of the patients had positive blood culture with Francisella tularensis. The clinical manifestations of tularaemia in Norway in 2011 were diverse, and changing throughout the year. Classification was sometimes difficult due to uncharacteristic symptoms and unknown mode of transmission. In rodent years, tularaemia is an important differential diagnosis to keep in mind at all times of the year for a variety of clinical symptoms.

  11. Seroepidemiological survey of tularemia among different groups in western Iran.

    PubMed

    Esmaeili, Saber; Gooya, Mohammad Mehdi; Shirzadi, Mohammad Reza; Esfandiari, Behzad; Amiri, Fahimeh Bagheri; Behzadi, Manijeh Yousefi; Banafshi, Omid; Mostafavi, Ehsan

    2014-01-01

    The first human case of tularemia in Iran was reported in 1980 and there have been no subsequent reports of tularemia in the country. The aim of this study was to carry out a survey of tularemia among different groups in the province of Kurdistan in western Iran. The following information was collected by means of an in-house questionnaire: participant demographic characteristics, exposure to risks, and use of appropriate personal protective equipment and disinfectant in their occupation. A blood sample was collected from each participant. Sera were tested using an ELISA kit (Virion\\Serion) to detect specific IgG antibodies against Francisella tularensis. Of a total of 250 serum samples, 14.40% had anti-tularemia IgG antibodies. The highest seroprevalence was found in hunters (18%) and the lowest in health care workers (12%). Age had a significant positive association with tularemia seroprevalence (p<0.001). The seroprevalence of tularemia in people exposed to foxes (hunting or eating the meat) (25%) was significantly higher than in others (8.65%) (p = 0.01). According to the findings of this study, it is highly recommended that physicians and health care workers are informed about bacteria circulating in this area. By sensitizing the health system, it is expected that some cases of the clinical disease will be reported in the near future. Similar studies in other parts of the country and on domestic and wild animals will clarify the epidemiology of tularemia in Iran. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. New therapeutic approaches for treatment of tularaemia: a review.

    PubMed

    Boisset, Sandrine; Caspar, Yvan; Sutera, Vivien; Maurin, Max

    2014-01-01

    Antibiotic treatment of tularaemia is based on a few drugs, including the fluoroquinolones (e.g., ciprofloxacin), the tetracyclines (e.g., doxycycline), and the aminoglycosides (streptomycin and gentamicin). Because no effective and safe vaccine is currently available, tularaemia prophylaxis following proven exposure to F. tularensis also relies on administration of antibiotics. A number of reasons make it necessary to search for new therapeutic alternatives: the potential toxicity of first-line drugs, especially in children and pregnant women; a high rate of treatment relapses and failures, especially for severe and/or suppurated forms of the disease; and the possible use of antibiotic-resistant strains in the context of a biological threat. This review presents novel therapeutic approaches that have been explored in recent years to improve tularaemia patients' management and prognosis. These new strategies have been evaluated in vitro, in axenic media and cell culture systems and/or in animal models. First, the activities of newly available antibiotic compounds were evaluated against F. tularensis, including tigecycline (a glycylcycline), ketolides (telithromycin and cethromycin), and fluoroquinolones (moxifloxacin, gatifloxacin, trovafloxacin and grepafloxacin). The liposome delivery of some antibiotics was evaluated. The effect of antimicrobial peptides against F. tularensis was also considered. Other drugs were evaluated for their ability to suppress the intracellular multiplication of F. tularensis. The effects of the modulation of the innate immune response (especially via TLR receptors) on the course of F. tularensis infection was characterized. Another approach was the administration of specific antibodies to induce passive resistance to F. tularensis infection. All of these studies highlight the need to develop new therapeutic strategies to improve the management of patients with tularaemia. Many possibilities exist, some unexplored. Moreover, it is

  13. New therapeutic approaches for treatment of tularaemia: a review

    PubMed Central

    Boisset, Sandrine; Caspar, Yvan; Sutera, Vivien; Maurin, Max

    2014-01-01

    Antibiotic treatment of tularaemia is based on a few drugs, including the fluoroquinolones (e.g., ciprofloxacin), the tetracyclines (e.g., doxycycline), and the aminoglycosides (streptomycin and gentamicin). Because no effective and safe vaccine is currently available, tularaemia prophylaxis following proven exposure to F. tularensis also relies on administration of antibiotics. A number of reasons make it necessary to search for new therapeutic alternatives: the potential toxicity of first-line drugs, especially in children and pregnant women; a high rate of treatment relapses and failures, especially for severe and/or suppurated forms of the disease; and the possible use of antibiotic-resistant strains in the context of a biological threat. This review presents novel therapeutic approaches that have been explored in recent years to improve tularaemia patients' management and prognosis. These new strategies have been evaluated in vitro, in axenic media and cell culture systems and/or in animal models. First, the activities of newly available antibiotic compounds were evaluated against F. tularensis, including tigecycline (a glycylcycline), ketolides (telithromycin and cethromycin), and fluoroquinolones (moxifloxacin, gatifloxacin, trovafloxacin and grepafloxacin). The liposome delivery of some antibiotics was evaluated. The effect of antimicrobial peptides against F. tularensis was also considered. Other drugs were evaluated for their ability to suppress the intracellular multiplication of F. tularensis. The effects of the modulation of the innate immune response (especially via TLR receptors) on the course of F. tularensis infection was characterized. Another approach was the administration of specific antibodies to induce passive resistance to F. tularensis infection. All of these studies highlight the need to develop new therapeutic strategies to improve the management of patients with tularaemia. Many possibilities exist, some unexplored. Moreover, it is

  14. Literature Review on Processing and Analytical Methods for ...

    EPA Pesticide Factsheets

    Report The purpose of this report was to survey the open literature to determine the current state of the science regarding the processing and analytical methods currently available for recovery of F. tularensis from water and soil matrices, and to determine what gaps remain in the collective knowledge concerning F. tularensis identification from environmental samples.

  15. Advances with using CRISPR/Cas-mediated gene editing to treat infections with hepatitis B virus and hepatitis C virus.

    PubMed

    Moyo, Buhle; Bloom, Kristie; Scott, Tristan; Ely, Abdullah; Arbuthnot, Patrick

    2018-01-15

    Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal. Disabling cccDNA is thus key to curing HBV infections and application of gene editing technology, such as harnessing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, has curative potential. Several studies have reported good efficacy when employing CRISPR/Cas technologies to disable HBV replication in cultured cells and in hydrodynamically injected mice. Recent advances with HCV drug development have revolutionized treatment of the infection. Nevertheless, individuals may be refractory to treatment. Targeting RNA from HCV with CRISPR/Cas isolated from Francisella novicida may have therapeutic utility. Although preclinical work shows that CRISPR/Cas technology has potential to overcome infection with HBV and HCV, significant challenges need to be met. Ensuring specificity for viral targets and efficient delivery of the gene editing sequences to virus-infected cells are particularly important. The field is at an interesting stage and the future of curative antiviral drug regimens, particularly for treatment of chronic HBV infection, may well entail use of combinations that include derivatives of CRISPR/Cas. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Biocidal and Sporicidal Efficacy of Pathoster® 0.35% and Pathoster® 0.50% Against Bacterial Agents in Potential Bioterrorism Use

    PubMed Central

    Candeliere, Antonio; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster® 0.35% and Pathoster® 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster® 0.35% was stable until 16±1 days, while Pathoster® 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

  17. Biocidal and Sporicidal Efficacy of Pathoster(®) 0.35% and Pathoster(®) 0.50% Against Bacterial Agents in Potential Bioterrorism Use.

    PubMed

    Candeliere, Antonio; Campese, Emanuele; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster(®) 0.35% and Pathoster(®) 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster(®) 0.35% was stable until 16±1 days, while Pathoster(®) 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid.

  18. Considerations in detecting CDC select agents under field conditions

    NASA Astrophysics Data System (ADS)

    Spinelli, Charles; Soelberg, Scott; Swanson, Nathaneal; Furlong, Clement; Baker, Paul

    2008-04-01

    Surface Plasmon Resonance (SPR) has become a widely accepted technique for real-time detection of interactions between receptor molecules and ligands. Antibody may serve as receptor and can be attached to the gold surface of the SPR device, while candidate analyte fluids contact the detecting antibody. Minute, but detectable, changes in refractive indices (RI) indicate that analyte has bound to the antibody. A decade ago, an inexpensive, robust, miniature and fully integrated SPR chip, called SPREETA, was developed. University of Washington (UW) researchers subsequently developed a portable, temperature-regulated instrument, called SPIRIT, to simultaneously use eight of these three-channel SPREETA chips. A SPIRIT prototype instrument was tested in the field, coupled to a remote reporting system on a surrogate unmanned aerial vehicle (UAV). Two target protein analytes were released sequentially as aerosols with low analyte concentration during each of three flights and were successfully detected and verified. Laboratory experimentation with a more advanced SPIRIT instrument demonstrated detection of very low levels of several select biological agents that might be employed by bioterrorists. Agent detection under field-like conditions is more challenging, especially as analyte concentrations are reduced and complex matricies are introduced. Two different sample preconditioning protocols have been developed for select agents in complex matrices. Use of these preconditioning techniques has allowed laboratory detection in spiked heavy mud of Francisella tularensis at 10 3 CFU/ml, Bacillus anthracis spores at 10 3 CFU/ml, Staphylococcal enterotoxin B (SEB) at 1 ng/ml, and Vaccinia virus (a smallpox simulant) at 10 5 PFU/ml. Ongoing experiments are aimed at simultaneous detection of multiple agents in spiked heavy mud, using a multiplex preconditioning protocol.

  19. Evaluating thermodynamic integration performance of the new amber molecular dynamics package and assess potential halogen bonds of enoyl-ACP reductase (FabI) benzimidazole inhibitors.

    PubMed

    Su, Pin-Chih; Johnson, Michael E

    2016-04-05

    Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the parahalogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme. © 2015 Wiley Periodicals, Inc.

  20. Genus Francisella.

    DTIC Science & Technology

    1981-05-15

    temperatures for 30 min-. Cool to 45 - 48°C. Aseptically add 25 ml of packed, human blood cells or 50 ml of defibrinated rabbit or sheep blood. Mix...now included in var. palaearctica, is reported to ferment glycerol but not maltose (Kunitsa et al., 1972). Growth in litmus milk is scant; slight...muskrats, water rats, beavers, squirrels;, woodclhocks, sheep , mice, voles and game birds as well as biting insects (usuallv ticks or deer flies

  1. Effect of size and temperature at vaccination on immunization and protection conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid tilapia.

    PubMed

    Soto, Esteban; Brown, Nicholas; Gardenfors, Zackarias O; Yount, Shaun; Revan, Floyd; Francis, Stewart; Kearney, Michael T; Camus, Alvin

    2014-12-01

    Francisella noatunensis subsp. orientalis (Fno) is a pleomorphic, facultative intracellular, Gram-negative, emerging bacterial pathogen of marine and fresh water fish with worldwide distribution. In this study, the efficacy of an attenuated Fno intracellular growth locus C (iglC) mutant was evaluated for use as a live immersion vaccine, when administered to hybrid tilapia at two different stages of growth (5 g fry and 10 g fingerlings) and at two temperatures (25 °C and 30 °C). To determine vaccine efficacy, mortality, days to first death, and Fno genome equivalents (GE) in the spleens of survivors, as well as serum and mucus antibody levels, were evaluated after 30 d in fish challenged with a wild type virulent strain. Both size and temperature at vaccination played an important role in immunization and protection. Fry vaccinated at 25 °C were not protected when compared to non-vaccinated fry at 25 °C (p = 0.870). In contrast, 5 g fry vaccinated at 30 °C were significantly protected compared to non-vaccinated fry at 30 °C (p = 0.038). Although lower mortalities occurred, 10 g fingerlings vaccinated at 25 °C were not protected, compared to non-vaccinated fingerlings at 25 °C (p = 0.328), while, 10 g fingerlings vaccinated at 30 °C were significantly protected, compared to non-vaccinated fingerlings at 30 °C (p = 0.038). Additionally, overall mortality of 5 g fish was significantly higher than in 10 g fish. Mortality was also significantly higher in fish subjected to a 30 to 25 °C temperature change one week prior to challenge, than in fish maintained at the same temperature during vaccination and challenge. This information demonstrates that both temperature and size at vaccination are important factors when implementing immunization prophylaxis in cultured tilapia.

  2. Glycogen Synthase Kinase-3 Is an Early Determinant in the Differentiation of Pathogenic Th17 Cells

    PubMed Central

    Beurel, Eléonore; Yeh, Wen-I; Michalek, Suzanne M.; Harrington, Laurie E.; Jope, Richard S.

    2011-01-01

    CD4+ T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4+ T cells was recently revised by the identification of the Th17 lineage of CD4+ T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4+ T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4+ T cells to Th17 cells increased by 10-fold the expression of GSK3β protein levels in Th17 cells, whereas GSK3β was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases. PMID:21191064

  3. Prioritizing Risks and Uncertainties from Intentional Release of Selected Category A Pathogens

    PubMed Central

    Hong, Tao; Gurian, Patrick L.; Huang, Yin; Haas, Charles N.

    2012-01-01

    This paper synthesizes available information on five Category A pathogens (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Variola major and Lassa) to develop quantitative guidelines for how environmental pathogen concentrations may be related to human health risk in an indoor environment. An integrated model of environmental transport and human health exposure to biological pathogens is constructed which 1) includes the effects of environmental attenuation, 2) considers fomite contact exposure as well as inhalational exposure, and 3) includes an uncertainty analysis to identify key input uncertainties, which may inform future research directions. The findings provide a framework for developing the many different environmental standards that are needed for making risk-informed response decisions, such as when prophylactic antibiotics should be distributed, and whether or not a contaminated area should be cleaned up. The approach is based on the assumption of uniform mixing in environmental compartments and is thus applicable to areas sufficiently removed in time and space from the initial release that mixing has produced relatively uniform concentrations. Results indicate that when pathogens are released into the air, risk from inhalation is the main component of the overall risk, while risk from ingestion (dermal contact for B. anthracis) is the main component of the overall risk when pathogens are present on surfaces. Concentrations sampled from untracked floor, walls and the filter of heating ventilation and air conditioning (HVAC) system are proposed as indicators of previous exposure risk, while samples taken from touched surfaces are proposed as indicators of future risk if the building is reoccupied. A Monte Carlo uncertainty analysis is conducted and input-output correlations used to identify important parameter uncertainties. An approach is proposed for integrating these quantitative assessments of parameter uncertainty with broader

  4. Structural and Functional Features of a Developmentally Regulated Lipopolysaccharide-Binding Protein

    PubMed Central

    Krasity, Benjamin C.; Troll, Joshua V.; Lehnert, Erik M.; Hackett, Kathleen T.; Dillard, Joseph P.; Apicella, Michael A.; Goldman, William E.

    2015-01-01

    ABSTRACT Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. PMID:26463160

  5. [Tularaemia - a forgotten disease? Own experience].

    PubMed

    Yanushevych, Mariya; Komorowska-Piotrowska, Anna; Feleszko, Wojciech

    2013-01-01

    Tularaemia is an acute, animal-born disease, caused by Gram-negative aerobic bacilli - Francisella tularensis. Due to its high infectiousness and virulence it remains an epidemiological issue in some countries. The prevalence rate of tularaemia in the Scandinavian region, where most of the cases among European countries have been reported, it has remained stable since 2006, whereas in Poland a small, but stable increase has been observed. The first case of tularaemia in Poland was diagnosed in 1949 and since then more than 600 cases have been reported, mainly in the north-eastern and north-western regions of the country. In the majority of cases the infection has been transmitted by ixode bites or through direct contact with infected animals. Depending on the route of transmission, tularaemia can develop in different forms with a characteristic sudden onset of high fever and local lymphadenopathy. In Europe 95% of cases account for the ulceroglandular type, which characteristically presents with skin ulceration and inflammation of regional tissues and lymph nodes. This is the reason why it should always be taken into consideration in differential diagnosis of lymphadenitis, especially if its course is atypical or it does not respond to antibiotic therapy. The above mentioned atypical course of the disease and resistance to empirical treatment contribute to the challenge encountered by physicians in the process of diagnosis and treatment. The aim of the study is to present the most important data on tularaemia as an animal-born, infectious disease and to convince physicians to take it into consideration in the differential diagnosis of ymphadenopathy. We illustrate this article with the case of ulceroglandular type of tularemia diagnosed in a 5 year-old boy and describe the encountered diagnostic and therapeutic problems.

  6. Dermatologic manifestations of tularemia: a study of 151 cases in the mid-Anatolian region of Turkey.

    PubMed

    Şenel, Engin; Satılmış, Özgür; Acar, Bilal

    2015-01-01

    Tularemia is a serious and potentially life-threatening zoonosis caused by Francisella tularensis, a highly infective, gram-negative coccobacillus. Although there are plenty of case reports and studies of tularemia outbreaks, the literature is lacking in reports on dermatologic manifestations of the disease. This study aimed to identify skin manifestations in clinical forms of tularemia. A total of 151 patients diagnosed with tularemia at Çankırı State Hospital, Çankırı, Turkey, were retrospectively examined. Dermatologic data for these patients were assessed. The most frequent clinical manifestation of tularemia was the glandular form (49.7%), followed by the oropharyngeal, ulceroglandular, and oculoglandular forms (39.1, 6.0, and 5.3%, respectively). Physical manifestations were observed in 64.5% of females and 56.9% of males. Lymphadenopathy and tonsillitis were the most frequent physical findings and were noted in 57.6 and 25.2% of patients, respectively. Erythema multiforme was found in 17 patients (11.3%), most of whom presented with the oropharyngeal and glandular forms, and was followed by ulcer (6.0%), urticaria (3.3%), erythema nodosum (2.6%), and cellulitis (0.7%). However, it should be noted that this study was retrospective and that its patient sample demonstrated four of the six clinical forms of tularemia. Patients with the oropharyngeal form of tularemia had statistically significantly more physical findings than those with other clinical forms of the disease (P < 0.001). There were statistically more skin findings in the ulceroglandular form (P < 0.001). There was no statistical correlation between serum antibody titers and cutaneous findings (P = 0.585). Although the literature reports that skin lesions are observed more frequently in women than in men, we did not find any statistically significant difference between the sexes in any type of skin lesion. © 2014 The International Society of Dermatology.

  7. Lipoxin A4, a 5-lipoxygenase pathway metabolite, modulates immune response during acute respiratory tularemia.

    PubMed

    Singh, Anju; Rahman, Tabassum; Bartiss, Rose; Arabshahi, Alireza; Prasain, Jeevan; Barnes, Stephen; Musteata, Florin Marcel; Sellati, Timothy J

    2017-02-01

    Respiratory infection with Francisella tularensis (Ft) is characterized by a muted, acute host response, followed by sepsis-like syndrome that results in death. Infection with Ft establishes a principally anti-inflammatory environment that subverts host-cell death programs to facilitate pathogen replication. Although the role of cytokines has been explored extensively, the role of eicosanoids in tularemia pathogenesis is not fully understood. Given that lipoxin A 4 (LXA 4 ) has anti-inflammatory properties, we investigated whether this lipid mediator affects host responses manifested early during infection. The addition of exogenous LXA 4 inhibits PGE 2 release by Ft-infected murine monocytes in vitro and diminishes apoptotic cell death. Tularemia pathogenesis was characterized in 5‑lipoxygenase-deficient (Alox5 -/- ) mice that are incapable of generating LXA 4 Increased release of proinflammatory cytokines and chemokines, as well as increased apoptosis, was observed in Alox5 -/- mice as compared with their wild-type counterparts. Alox5 -/- mice also exhibited elevated recruitment of neutrophils during the early phase of infection and increased resistance to lethal challenge. Conversely, administration of exogenous LXA 4 to Alox5 -/- mice made them more susceptible to infection thus mimicking wild-type animals. Taken together, our results suggest that 5-LO activity is a critical regulator of immunopathology observed during the acute phase of respiratory tularemia, regulating bacterial burden and neutrophil recruitment and production of proinflammatory modulators and increasing morbidity and mortality. These studies identify a detrimental role for the 5-LO-derived lipid mediator LXA 4 in Ft-induced immunopathology. Targeting this pathway may have therapeutic benefit as an adjunct to treatment with antibiotics and conventional antimicrobial peptides, which often have limited efficacy against intracellular bacteria. © Society for Leukocyte Biology.

  8. Vaccination evokes gender-dependent protection against tularemia infection in C57BL/6Tac mice.

    PubMed

    Sunagar, Raju; Kumar, Sudeep; Franz, Brian J; Gosselin, Edmund J

    2016-06-17

    Francisella tularensis (Ft) is a Category A biothreat agent for which there currently is no FDA-approved vaccine. Thus, there is a substantial effort underway to develop an effective tularemia vaccine. While it is well established that gender can significantly impact susceptibility to primary infection, the impact of gender on vaccine efficacy is not well established. Thus, development of a successful vaccine against tularemia will require an understanding of the impact gender has on vaccine-induced protection against this organism. In this study, a role for gender in vaccine-induced protection following Ft challenge is identified for the first time. In the present study, mucosal vaccination with inactivated Ft (iFt) LVS elicited gender-based protection in C57BL/6Tac mice against respiratory challenge with Ft LVS. Specifically, vaccinated male mice were more susceptible to subsequent Ft LVS challenge. This increased susceptibility in male mice correlated with increased bacterial burden, increased tissue inflammation, and increased proinflammatory cytokine production late in post-challenge infection. In contrast, improved survival of iFt-vaccinated female mice correlated with reduced bacterial burden and enhanced levels of Ft-specific Abs in serum and broncho-alveolar lavage (BAL) fluid post-challenge. Furthermore, vaccination with a live attenuated vaccine consisting of an Ft LVS superoxide dismutase (SodB) mutant, which has proven efficacious against the highly virulent Ft SchuS4 strain, demonstrated similar gender bias in protection post-Ft SchuS4 challenge. Of particular significance is the fact that these are the first studies to demonstrate that gender differences impact disease outcome in the case of lethal respiratory tularemia following mucosal vaccination. In addition, these studies further emphasize the fact that gender differences must be a serious consideration in any future tularemia vaccine development studies. Copyright © 2016 Elsevier Ltd. All

  9. Identification of Ciprofloxacin Resistance by SimpleProbe (trademark), High Resolution Melt and Pyrosequencing (trademark) Nucleic Acid Analysis in Biothreat Agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis

    DTIC Science & Technology

    2010-01-01

    A266/ C gyrB: F1163-BIO GTG TTG CAG CGA AAA AAG C R1469 ATA TCA AAA TCT CCG CCA ATG T S1309 50-ATC CAC CGG CAG AGT-30 G1309/ A S1431 AAT AAT TGT ACG...CAC TTC AT A1423/G parC: F177 AGC GTT CCG TAA GTC GGC TAA A R342-BIO CGG ATC CCC GTC AAC ACT S227 ACC CGC ACG GTG ATT C242/ Te Y. pestis KIM5 gryA...ACA TGG CAT TTT GAA AC R694-BIO GGA GTG TTT CAG CTT CTA GTT TAT GGT S625 AAG CTT ACA TGG CAT TTT GAA AC del: bp 653e657 (TTAAA) a F e Forward (Upstream

  10. Immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model.

    PubMed

    Lagos, Leidy; Tandberg, Julia I; Becker, María Inés; Winther-Larsen, Hanne C

    2017-08-01

    The development of vaccines for aquaculture has been an important milestone in providing a continuous and sustainable production. Most of the vaccines currently on the market for aquaculture include oil as adjuvant. Nevertheless, several studies reported an occurrence of side effects after their use in farmed fish. As a result, there is a need for new and improved adjuvants that can stimulate the immune system while causing as few side-effects as possible. Hemocyanins are versatile macromolecules with strong immunogenic and immunomodulatory properties. Due to these characteristics, hemocyanin from Concholepas concholepas (CCH) has been biochemically characterized and evaluated as vaccine adjuvant in mice and humans. Francisellosis is a chronic granulomatous disease, which can result in high mortality depending on the host. The disease is caused by the facultative intracellular Gram-negative bacteria Francisella noatunensis, which remains an unsolved problem for the aquaculture, as no efficient vaccines are available. The aim of the present work was to investigate the immunoregulatory properties of CCH against francisellosis in an experimental zebrafish model. When immunized with CCH, zebrafish were protected from subsequent challenge with a lethal dose of Francisella noatunensis subsp. orientalis. Subsequently the mRNA expression levels of several immune-related genes were studied, including mhcii, il12a, tnfα and ifng1-1. Taken together, the data report the immunoregulatory properties of CCH and its potential use as a vaccine adjuvant for aquaculture. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. A snapshot of the microbiome of Amblyomma tuberculatum ticks infesting the gopher tortoise, an endangered species.

    PubMed

    Budachetri, Khemraj; Gaillard, Daniel; Williams, Jaclyn; Mukherjee, Nabanita; Karim, Shahid

    2016-10-01

    The gopher tortoise tick, Amblyomma tuberculatum, has a unique relationship with the gopher tortoise, Gopherus polyphemus, found in sandy habitats across the southeastern United States. We aimed to understand the overall bacterial community associated with A. tuberculatum while also focusing on spotted fever group Rickettsia. These tortoises in the Southern Mississippi region are a federally threatened species; therefore, we have carefully trapped the tortoises and removed the species-specific ticks attached to them. Genomic DNA was extracted from individual ticks and used to explore overall bacterial load using pyrosequencing of bacterial 16S rRNA on 454-sequencing platform. The spotted fever group of Rickettsia was explored by amplifying rickettsial outer membrane protein A (rompA) gene by nested PCR. Sequencing results revealed 330 bacterial operational taxonomic units (OTUs) after all the necessary curation of sequences. Four whole A. tuberculatum ticks showed Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes as the most dominant phyla with a total of 74 different bacterial genera detected. Together Rickettsiae and Francisella showed >85% abundance, thus dominating the bacterial community structure. Partial sequences obtained from ompA amplicons revealed the presence of an uncharacterized Rickettsia similar to the Rickettsial endosymbiont of A. tuberculatum. This is the first preliminary profile of a complete bacterial community from gopher tortoise ticks and warrants further investigation regarding the functional role of Rickettsial and Francisella-like endosymbionts in tick physiology. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. Genes and Gene Therapy

    MedlinePlus

    ... a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  13. Gene network biological validity based on gene-gene interaction relevance.

    PubMed

    Gómez-Vela, Francisco; Díaz-Díaz, Norberto

    2014-01-01

    In recent years, gene networks have become one of the most useful tools for modeling biological processes. Many inference gene network algorithms have been developed as techniques for extracting knowledge from gene expression data. Ensuring the reliability of the inferred gene relationships is a crucial task in any study in order to prove that the algorithms used are precise. Usually, this validation process can be carried out using prior biological knowledge. The metabolic pathways stored in KEGG are one of the most widely used knowledgeable sources for analyzing relationships between genes. This paper introduces a new methodology, GeneNetVal, to assess the biological validity of gene networks based on the relevance of the gene-gene interactions stored in KEGG metabolic pathways. Hence, a complete KEGG pathway conversion into a gene association network and a new matching distance based on gene-gene interaction relevance are proposed. The performance of GeneNetVal was established with three different experiments. Firstly, our proposal is tested in a comparative ROC analysis. Secondly, a randomness study is presented to show the behavior of GeneNetVal when the noise is increased in the input network. Finally, the ability of GeneNetVal to detect biological functionality of the network is shown.

  14. A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival.

    PubMed

    Chen, Grischa Y; McDougal, Courtney E; D'Antonio, Marc A; Portman, Jonathan L; Sauer, John-Demian

    2017-03-21

    Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity. IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis , are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role

  15. Fc receptor-targeting of immunogen as a strategy for enhanced antigen loading, vaccination, and protection using intranasally administered antigen-pulsed dendritic cells.

    PubMed

    Pham, Giang H; Iglesias, Bibiana V; Gosselin, Edmund J

    2014-09-08

    Dendritic cells (DCs) play a critical role in the generation of adaptive immunity via the efficient capture, processing, and presentation of antigen (Ag) to naïve T cells. Administration of Ag-pulsed DCs is also an effective strategy for enhancing immunity to tumors and infectious disease organisms. Studies have also demonstrated that targeting Ags to Fcγ receptors (FcγR) on Ag presenting cells can enhance humoral and cellular immunity in vitro and in vivo. Specifically, our studies using a Francisella tularensis (Ft) infectious disease vaccine model have demonstrated that targeting immunogens to FcγR via intranasal (i.n.) administration of monoclonal antibody (mAb)-inactivated Ft (iFt) immune complexes (ICs) enhances protection against Ft challenge. Ft is the causative agent of tularemia, a debilitating disease of humans and other mammals and a category A biothreat agent for which there is no approved vaccine. Therefore, using iFt Ag as a model immunogen, we sought to determine if ex vivo targeting of iFt to FcγR on DCs would enhance the potency of i.n. administered iFt-pulsed DCs. In this study, bone marrow-derived DCs (BMDCs) were pulsed ex vivo with iFt or mAb-iFt ICs. Intranasal administration of mAb-iFt-pulsed BMDCs enhanced humoral and cellular immune responses, as well as protection against Ft live vaccine strain (LVS) challenge. Increased protection correlated with increased iFt loading on the BMDC surface as a consequence of FcγR-targeting. However, the inhibitory FcγRIIB had no impact on this enhancement. In conclusion, targeting Ag ex vivo to FcγR on DCs provides a method for enhanced Ag loading of DCs ex vivo, thereby reducing the amount of Ag required, while also avoiding the inhibitory impact of FcγRIIB. Thus, this represents a simple and less invasive strategy for increasing the potency of ex vivo-pulsed DC vaccines against chronic infectious diseases and cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Secretion of TcpF by the Vibrio cholerae Toxin-Coregulated Pilus Biogenesis Apparatus Requires an N-Terminal Determinant

    PubMed Central

    Megli, Christina J.

    2013-01-01

    Type IV pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. We and others have shown that type IV pili are important for protein secretion across the outer membrane, similar to type II secretion systems. This study explored the relationship between protein secretion and pilus formation in Vibrio cholerae. The toxin-coregulated pilus (TCP), a type IV pilus required for V. cholerae pathogenesis, is necessary for the secretion of the colonization factor TcpF (T. J. Kirn, N. Bose, and R. K. Taylor, Mol. Microbiol. 49:81–92, 2003). This phenomenon is not unique to V. cholerae; secreted virulence factors that are dependent on the presence of components of the type IV pilus biogenesis apparatus for secretion have been reported with Dichelobacter nodosus (R. M. Kennan, O. P. Dhungyel, R. J. Whittington, J. R. Egerton, and J. I. Rood, J. Bacteriol. 183:4451–4458, 2001) and Francisella tularensis (A. J. Hager et al., Mol. Microbiol. 62:227–237, 2006). Using site-directed mutagenesis, we demonstrated that the secretion of TcpF is dependent on the presence of selected amino acid R groups at position five. We were unable to find other secretion determinants, suggesting that Y5 is the major secretion determinant within TcpF. We also report that proteins secreted in a type IV pilus biogenesis apparatus-dependent manner have a YXS motif within the first 15 amino acids following the Sec cleavage site. The YXS motif is not present in proteins secreted by type II secretion systems, indicating that this is unique to type IV pilus-mediated secretion. Moreover, we show that TcpF interacts with the pilin TcpA, suggesting that these proteins are secreted by the type IV pilus biogenesis system. These data provide a starting point for understanding how type IV pili can mediate secretion of virulence factors important for bacterial pathogenesis. PMID:23564177

  17. Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

    PubMed

    Silbereisen, Angelika; Tamborrini, Marco; Wittwer, Matthias; Schürch, Nadia; Pluschke, Gerd

    2015-10-05

    Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural

  18. Q fever community-acquired pneumonia in a patient with Crohn's disease on immunosuppressive therapy.

    PubMed

    Nausheen, Sara; Cunha, Burke A

    2007-01-01

    Community-acquired pneumonia (CAP) may be caused by typical or atypical pathogens. The three most common zoonotic atypical pathogens are Chlamydophila psittaci (psittacosis), Francisella tularensis (tularemia), and Coxiella burnetii (Q fever). Atypical CAPs are suggested by a distinctive pattern of extrapulmonary organ involvement. Zoonotic CAP may be differentiated from nonzoonotic CAP (Chlamydia pneumoniae, Mycoplasma pneumoniae, Legionnaire's disease) by a recent zoonotic vector contact history. Zoonotic atypical CAP occurs sporadically, but not randomly, and require close association with the appropriate zoonotic vector to transmit the infection. CAP accompanied by the extrapulmonary finding of splenomegaly in a normal host limits differential diagnostic possibilities to Q fever and psittacosis. Splenomegaly does not occur with other typical or atypical CAP. Another common extrapulmonary finding occurs with some atypical pneumonias, that is, Q fever, psittacosis, and Legionnaire's disease is early mild/transient elevations of serum transaminases indicative of (hepatic) extrapulmonary organ involvement. The case presented is a middle-aged man with longstanding Crohn's disease who was further immunosuppressed by chronic prednisone therapy. The patient presented with CAP and extrapulmonary findings, that is, splenomegaly and increased serum transaminases. He denied recent contact with birds or animals. Because Crohn's disease and Q fever CAP may be accompanied by splenomegaly, the cause of his splenomegaly was a diagnostic dilemma. The patient was treated with levofloxacin. Serologic tests for atypical pathogens (Q fever, psittacosis, Legionnaire's disease, C. pneumoniae, and M. pneumoniae) were ordered. Enzyme-linked immunosorbent assay serology for Q fever was positive with elevated acute immunoglobulin-M (phase II) titers. Re-questioning of the patient revealed a recent exposure to a neighbor's parturient cat, providing the necessary zoonotic vector contact

  19. Tuberculosis or tularemia? A molecular study in cervical lymphadenitis.

    PubMed

    Yıldırım, Sükrü; Turhan, Vedat; Karadenizli, Aynur; Önem, Yalçın; Karagöz, Ergenekon; Eroğlu, Cafer; Çiftçi, Faruk

    2014-01-01

    Over the last two to three decades there has been a marked decrease in certain bacterial infections in Turkey. One of them is tuberculosis. Of note, statistics published by the Turkish Ministry of Health (MoH) show decreasing pulmonary tuberculosis (PTB), but on the other hand, increasing extrapulmonary tuberculosis (EPTB). The most common form of EPTB is tuberculous cervical lymphadenitis (TCL). The increase in the number of TCL cases despite the decline in cases of PTB is seen as a paradoxical issue. In contrast there has been an increase in the number of oropharyngeal tularemia cases in the last decade in Turkey. The aim of this study was to draw attention to the importance of differentiating between TCL and tularemia lymphadenitis, because these diseases may have a similar histopathological appearance. Thirty-two cases diagnosed as TCL were identified from the archives of a pathology laboratory (Patomer Pathology Laboratory, Bursa, Turkey). PCR tests for Francisella tularensis and Mycobacterium tuberculosis were carried out on the paraffin tissue blocks of these cases. At the same time, statistical data on PTB and EPTB for the period 1996-2010 were retrieved from the MoH and reviewed. Statistics related to tularemia, which has been diagnosed since 1988 and has been increasing in the last 10 years, were obtained from the Department of Zoonoses of the MoH. Six out of 32 (19%) cases who had previously been diagnosed with 'casseifying granulomatous lymphadenitis consistent with tuberculosis' were found to be positive for tularemia by PCR test of the cervical lymph nodes. Oropharyngeal tularemia should be kept in mind in the differential diagnosis of cervical lymphadenitis in widespread geographic regions of the Northern Hemisphere, including the Asian continent. In particular, if granulomatous inflammation with necrosis is seen histopathologically, tularemia should be excluded before making the diagnosis of TCL. Copyright © 2013 The Authors. Published by Elsevier

  20. Bacterial and protozoal pathogens found in ticks collected from humans in Corum province of Turkey

    PubMed Central

    Karasartova, Djursun; Gureser, Ayse Semra; Gokce, Tuncay; Celebi, Bekir; Yapar, Derya; Keskin, Adem; Celik, Selim; Ece, Yasemin; Erenler, Ali Kemal; Usluca, Selma; Mumcuoglu, Kosta Y.

    2018-01-01

    Background Tick-borne diseases are increasing all over the word, including Turkey. The aim of this study was to determine the bacterial and protozoan vector-borne pathogens in ticks infesting humans in the Corum province of Turkey. Methodology/Principal findings From March to November 2014 a total of 322 ticks were collected from patients who attended the local hospitals with tick bites. Ticks were screened by real time-PCR and PCR, and obtained amplicons were sequenced. The dedected tick was belonging to the genus Hyalomma, Haemaphysalis, Rhipicephalus, Dermacentor and Ixodes. A total of 17 microorganism species were identified in ticks. The most prevalent Rickettsia spp. were: R. aeschlimannii (19.5%), R. slovaca (4.5%), R. raoultii (2.2%), R. hoogstraalii (1.9%), R. sibirica subsp. mongolitimonae (1.2%), R. monacensis (0.31%), and Rickettsia spp. (1.2%). In addition, the following pathogens were identified: Borrelia afzelii (0.31%), Anaplasma spp. (0.31%), Ehrlichia spp. (0.93%), Babesia microti (0.93%), Babesia ovis (0.31%), Babesia occultans (3.4%), Theileria spp. (1.6%), Hepatozoon felis (0.31%), Hepatozoon canis (0.31%), and Hemolivia mauritanica (2.1%). All samples were negative for Francisella tularensis, Coxiella burnetii, Bartonella spp., Toxoplasma gondii and Leishmania spp. Conclusions/Significance Ticks in Corum carry a large variety of human and zoonotic pathogens that were detected not only in known vectors, but showed a wider vector diversity. There is an increase in the prevalence of ticks infected with the spotted fever group and lymphangitis-associated rickettsiosis, while Ehrlichia spp. and Anaplasma spp. were reported for the first time from this region. B. microti was detected for the first time in Hyalomma marginatum infesting humans. The detection of B. occultans, B. ovis, Hepatozoon spp., Theileria spp. and Hemolivia mauritanica indicate the importance of these ticks as vectors of pathogens of veterinary importance, therefore patients with

  1. Bacterial and protozoal pathogens found in ticks collected from humans in Corum province of Turkey.

    PubMed

    Karasartova, Djursun; Gureser, Ayse Semra; Gokce, Tuncay; Celebi, Bekir; Yapar, Derya; Keskin, Adem; Celik, Selim; Ece, Yasemin; Erenler, Ali Kemal; Usluca, Selma; Mumcuoglu, Kosta Y; Taylan-Ozkan, Aysegul

    2018-04-01

    Tick-borne diseases are increasing all over the word, including Turkey. The aim of this study was to determine the bacterial and protozoan vector-borne pathogens in ticks infesting humans in the Corum province of Turkey. From March to November 2014 a total of 322 ticks were collected from patients who attended the local hospitals with tick bites. Ticks were screened by real time-PCR and PCR, and obtained amplicons were sequenced. The dedected tick was belonging to the genus Hyalomma, Haemaphysalis, Rhipicephalus, Dermacentor and Ixodes. A total of 17 microorganism species were identified in ticks. The most prevalent Rickettsia spp. were: R. aeschlimannii (19.5%), R. slovaca (4.5%), R. raoultii (2.2%), R. hoogstraalii (1.9%), R. sibirica subsp. mongolitimonae (1.2%), R. monacensis (0.31%), and Rickettsia spp. (1.2%). In addition, the following pathogens were identified: Borrelia afzelii (0.31%), Anaplasma spp. (0.31%), Ehrlichia spp. (0.93%), Babesia microti (0.93%), Babesia ovis (0.31%), Babesia occultans (3.4%), Theileria spp. (1.6%), Hepatozoon felis (0.31%), Hepatozoon canis (0.31%), and Hemolivia mauritanica (2.1%). All samples were negative for Francisella tularensis, Coxiella burnetii, Bartonella spp., Toxoplasma gondii and Leishmania spp. Ticks in Corum carry a large variety of human and zoonotic pathogens that were detected not only in known vectors, but showed a wider vector diversity. There is an increase in the prevalence of ticks infected with the spotted fever group and lymphangitis-associated rickettsiosis, while Ehrlichia spp. and Anaplasma spp. were reported for the first time from this region. B. microti was detected for the first time in Hyalomma marginatum infesting humans. The detection of B. occultans, B. ovis, Hepatozoon spp., Theileria spp. and Hemolivia mauritanica indicate the importance of these ticks as vectors of pathogens of veterinary importance, therefore patients with a tick infestation should be followed for a variety of pathogens

  2. Occurrence and Control of Tularemia in Drinking Water ...

    EPA Pesticide Factsheets

    Journal Article This review article will focus on tularemia acquired from contaminated drinking water and the adequacy of control measures for preventing waterborne transmission. Contaminated drinking water can serve as a vehicle for the spread of tularemia. Disease occurrence has been most commonly associated with untreated community and domestic water supplies. F. tularensis is of interest both from the standpoint of natural occurring waterborne outbreaks and in regards to its potential use as a bio-threat organism. The organism can persist in the aquatic environment and methods exist for detection in water. While capable of being inactivated by commonly used drinking water disinfectants, F. tularensis does exhibit an increased resistance to chlorination in comparison to other waterborne vegetative bacterial pathogens.

  3. Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes.

    PubMed

    De Feyter, R; Yang, Y; Gabriel, D W

    1993-01-01

    Six plasmid-borne avirulence (avr) genes were previously cloned from strain XcmH of the cotton pathogen, Xanthomonas campestris pv. malvacearum. We have now localized all six avr genes on the cloned fragments by subcloning and Tn5-gusA insertional mutagenesis. None of these avr genes appeared to exhibit exclusively gene-for-gene patterns of interactions with cotton R genes, and avrB4 was demonstrated to confer avr gene-for-R genes (plural) avirulence to X. c. pv. malvacearum on congenic cotton lines carrying either of two different resistance loci, B1 or B4. Furthermore, the B1 locus appeared to confer R gene-for-avr genes resistance to cotton against isogenic X. c. pv. malvacearum strains carrying any one of three avr genes: avrB4, avrb6, or avrB102. Restriction enzyme, Southern blot hybridization, and DNA sequence analyses showed that the XcmH avr genes are all highly similar to each other, to avrBs3 and avrBsP from the pepper pathogen X. c. pv. vesicatoria, and to the host-specific virulence gene pthA from the citrus pathogen X. citri. The XcmH avr genes differed primarily in the multiplicity of a tandemly repeated 102-base pair motif within the central portions of the genes, repeated from 14 to 23 times in members of this gene family. The complete nucleotide sequence of avrb6 revealed that it is 97% identical in DNA sequence to avrB4, avrBs3, avrBsP, and pthA and that 62-bp inverted terminal repeats mark the boundaries of homology between avrb6 and all members of this Xanthomonas virulence/avirulence gene family sequenced to date. The terminal 38 bp of both inverted repeats are highly similar to the 38-bp consensus terminal sequence of the Tn3 family of transposons. Up to 11 members of the avr gene family appear to be present in North American strains of X. c. pv. malvacearum, including XcmH. The high level of homology observed among these avr genes and their presence in multiple copies may explain the gene-for-genes interactions and also the observed high

  4. [REAL TIME POLYMERASE CHAIN REACTION IN TULAREMIA LABORATORY DIAGNOSTICS].

    PubMed

    Kormilitsyna, M I; Mescheryakova, I S; Mikhailova, T V; Dobrovolsky, A A

    2015-01-01

    Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR). 39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tu14GF/R+tul4-PR2. Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4G F/R+tul4-PR2 combination--92% of sera. The data were obtained when DNA was isolated from sera using "Proba Rapid" express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3-4 weeks after the onset of the disease. RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3-4 weeks after the onset of the disease.

  5. Multiple-Locus VNTR Analysis (MLVA) for Bacterial Strain Identification - Quarterly Progress Report for the period 7/1/00 to 10/30/00

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dr. Paul Keim

    2000-11-07

    Multiple locus VNTR analysis (MLVA) systems are being developed for B. anthracis, Y. pestis and F. tularensis. These are high resolution DNA fingerprinting systems that will allow for molecular epidemiology and forensic analysis of these pathogens.

  6. Multiple-Locus VNTR Analysis (MLVA) for Bacterial Strain Identification - Quarterly Progress Report for the Period 4/1/00 to 6/30/00

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dr. Paul Keim

    2000-11-07

    Multiple locus VNTR analysis (MLVA) systems are being developed for B. anthracis, Y. pestis and F. tularensis. These are high resolution DNA fingerprinting systems that will allow for molecular epidemiology and forensic analysis of these pathogens.

  7. Avirulence Genes in Cereal Powdery Mildews: The Gene-for-Gene Hypothesis 2.0.

    PubMed

    Bourras, Salim; McNally, Kaitlin E; Müller, Marion C; Wicker, Thomas; Keller, Beat

    2016-01-01

    The gene-for-gene hypothesis states that for each gene controlling resistance in the host, there is a corresponding, specific gene controlling avirulence in the pathogen. Allelic series of the cereal mildew resistance genes Pm3 and Mla provide an excellent system for genetic and molecular analysis of resistance specificity. Despite this opportunity for molecular research, avirulence genes in mildews remain underexplored. Earlier work in barley powdery mildew (B.g. hordei) has shown that the reaction to some Mla resistance alleles is controlled by multiple genes. Similarly, several genes are involved in the specific interaction of wheat mildew (B.g. tritici) with the Pm3 allelic series. We found that two mildew genes control avirulence on Pm3f: one gene is involved in recognition by the resistance protein as demonstrated by functional studies in wheat and the heterologous host Nicotiana benthamiana. A second gene is a suppressor, and resistance is only observed in mildew genotypes combining the inactive suppressor and the recognized Avr. We propose that such suppressor/avirulence gene combinations provide the basis of specificity in mildews. Depending on the particular gene combinations in a mildew race, different genes will be genetically identified as the "avirulence" gene. Additionally, the observation of two LINE retrotransposon-encoded avirulence genes in B.g. hordei further suggests that the control of avirulence in mildew is more complex than a canonical gene-for-gene interaction. To fully understand the mildew-cereal interactions, more knowledge on avirulence determinants is needed and we propose ways how this can be achieved based on recent advances in the field.

  8. Avirulence Genes in Cereal Powdery Mildews: The Gene-for-Gene Hypothesis 2.0

    PubMed Central

    Bourras, Salim; McNally, Kaitlin E.; Müller, Marion C.; Wicker, Thomas; Keller, Beat

    2016-01-01

    The gene-for-gene hypothesis states that for each gene controlling resistance in the host, there is a corresponding, specific gene controlling avirulence in the pathogen. Allelic series of the cereal mildew resistance genes Pm3 and Mla provide an excellent system for genetic and molecular analysis of resistance specificity. Despite this opportunity for molecular research, avirulence genes in mildews remain underexplored. Earlier work in barley powdery mildew (B.g. hordei) has shown that the reaction to some Mla resistance alleles is controlled by multiple genes. Similarly, several genes are involved in the specific interaction of wheat mildew (B.g. tritici) with the Pm3 allelic series. We found that two mildew genes control avirulence on Pm3f: one gene is involved in recognition by the resistance protein as demonstrated by functional studies in wheat and the heterologous host Nicotiana benthamiana. A second gene is a suppressor, and resistance is only observed in mildew genotypes combining the inactive suppressor and the recognized Avr. We propose that such suppressor/avirulence gene combinations provide the basis of specificity in mildews. Depending on the particular gene combinations in a mildew race, different genes will be genetically identified as the “avirulence” gene. Additionally, the observation of two LINE retrotransposon-encoded avirulence genes in B.g. hordei further suggests that the control of avirulence in mildew is more complex than a canonical gene-for-gene interaction. To fully understand the mildew–cereal interactions, more knowledge on avirulence determinants is needed and we propose ways how this can be achieved based on recent advances in the field. PMID:26973683

  9. Method development for optimum recovery of Yersinia pestis ...

    EPA Pesticide Factsheets

    Report The primary goal of this project was to determine the best combination of sampling swab, pre-moistening agent, transport media, and extraction method for a high efficiency recovery of Y. pestis and F. tularensis vegetative cells.

  10. Converting cancer genes into killer genes.

    PubMed Central

    Da Costa, L T; Jen, J; He, T C; Chan, T A; Kinzler, K W; Vogelstein, B

    1996-01-01

    Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8633039

  11. GoGene: gene annotation in the fast lane.

    PubMed

    Plake, Conrad; Royer, Loic; Winnenburg, Rainer; Hakenberg, Jörg; Schroeder, Michael

    2009-07-01

    High-throughput screens such as microarrays and RNAi screens produce huge amounts of data. They typically result in hundreds of genes, which are often further explored and clustered via enriched GeneOntology terms. The strength of such analyses is that they build on high-quality manual annotations provided with the GeneOntology. However, the weakness is that annotations are restricted to process, function and location and that they do not cover all known genes in model organisms. GoGene addresses this weakness by complementing high-quality manual annotation with high-throughput text mining extracting co-occurrences of genes and ontology terms from literature. GoGene contains over 4,000,000 associations between genes and gene-related terms for 10 model organisms extracted from more than 18,000,000 PubMed entries. It does not cover only process, function and location of genes, but also biomedical categories such as diseases, compounds, techniques and mutations. By bringing it all together, GoGene provides the most recent and most complete facts about genes and can rank them according to novelty and importance. GoGene accepts keywords, gene lists, gene sequences and protein sequences as input and supports search for genes in PubMed, EntrezGene and via BLAST. Since all associations of genes to terms are supported by evidence in the literature, the results are transparent and can be verified by the user. GoGene is available at http://gopubmed.org/gogene.

  12. Down-weighting overlapping genes improves gene set analysis

    PubMed Central

    2012-01-01

    Background The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set. Results In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights are designed to emphasize the genes appearing in few gene sets, versus genes that appear in many gene sets. We demonstrate the usefulness of the method when analyzing gene sets that correspond to the KEGG pathways, and hence we called our method Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). Unlike most gene set analysis methods which are validated through the analysis of 2-3 data sets followed by a human interpretation of the results, the validation employed here uses 24 different data sets and a completely objective assessment scheme that makes minimal assumptions and eliminates the need for possibly biased human assessments of the analysis results. Conclusions PADOG significantly improves gene set ranking and boosts sensitivity of analysis using information already available in the gene expression profiles and the collection of gene sets to be analyzed. The advantages of PADOG over other existing approaches are shown to be stable to changes in the database of gene sets to be analyzed. PADOG was implemented as an R package available at: http://bioinformaticsprb.med.wayne.edu/PADOG/or http://www.bioconductor.org. PMID:22713124

  13. Lest We Forget: A Critical Analysis of Bioterrorist Incidents, National Exercises, and U.S. Prevention, Response and Recovery Strategies

    DTIC Science & Technology

    2011-04-01

    American Type Culture Collection (ATCC), including Salmonella typhi (causes typhoid fever), Fancisella tularensis (causes tularemia ), Salmonella...incident, the Rajneesh cult obtained the agents on which it experimented, and Iraq obtained some of its lethal strains of anthrax, tularemia and

  14. Gene-gene interactions and gene polymorphisms of VEGFA and EG-VEGF gene systems in recurrent pregnancy loss.

    PubMed

    Su, Mei-Tsz; Lin, Sheng-Hsiang; Chen, Yi-Chi; Kuo, Pao-Lin

    2014-06-01

    Both vascular endothelial growth factor A (VEGFA) and endocrine gland-derived vascular endothelial growth factor (EG-VEGF) systems play major roles in angiogenesis. A body of evidence suggests VEGFs regulate critical processes during pregnancy and have been associated with recurrent pregnancy loss (RPL). However, little information is available regarding the interaction of these two major major angiogenesis-related systems in early human pregnancy. This study was conducted to investigate the association of gene polymorphisms and gene-gene interaction among genes in VEGFA and EG-VEGF systems and idiopathic RPL. A total of 98 women with history of idiopathic RPL and 142 controls were included, and 5 functional SNPs selected from VEGFA, KDR, EG-VEGF (PROK1), PROKR1 and PROKR2 were genotyped. We used multifactor dimensionality reduction (MDR) analysis to choose a best model and evaluate gene-gene interactions. Ingenuity pathways analysis (IPA) was introduced to explore possible complex interactions. Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL (P<0.01). The MDR test revealed that the KDR (Q472H) polymorphism was the best loci to be associated with RPL (P=0.02). IPA revealed EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3 signaling pathways. Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL. EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3.

  15. FunGene: the functional gene pipeline and repository.

    PubMed

    Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R

    2013-01-01

    Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

  16. Mining disease genes using integrated protein-protein interaction and gene-gene co-regulation information.

    PubMed

    Li, Jin; Wang, Limei; Guo, Maozu; Zhang, Ruijie; Dai, Qiguo; Liu, Xiaoyan; Wang, Chunyu; Teng, Zhixia; Xuan, Ping; Zhang, Mingming

    2015-01-01

    In humans, despite the rapid increase in disease-associated gene discovery, a large proportion of disease-associated genes are still unknown. Many network-based approaches have been used to prioritize disease genes. Many networks, such as the protein-protein interaction (PPI), KEGG, and gene co-expression networks, have been used. Expression quantitative trait loci (eQTLs) have been successfully applied for the determination of genes associated with several diseases. In this study, we constructed an eQTL-based gene-gene co-regulation network (GGCRN) and used it to mine for disease genes. We adopted the random walk with restart (RWR) algorithm to mine for genes associated with Alzheimer disease. Compared to the Human Protein Reference Database (HPRD) PPI network alone, the integrated HPRD PPI and GGCRN networks provided faster convergence and revealed new disease-related genes. Therefore, using the RWR algorithm for integrated PPI and GGCRN is an effective method for disease-associated gene mining.

  17. Raptor: A Portable Biosensor Upgraded for Reliability and Sensitivity

    DTIC Science & Technology

    2003-07-01

    Research Center for Molecular Diagnostics and Therapy. The F. tularensis antigen (killed vaccine strain), affinity-purified rabbit anti-B...Russian Research Center of Molecular Diagnostics and Therapies, for their generous donations materials. The views expressed here are those of the

  18. Gene-gene, gene-environment, gene-nutrient interactions and single nucleotide polymorphisms of inflammatory cytokines.

    PubMed

    Nadeem, Amina; Mumtaz, Sadaf; Naveed, Abdul Khaliq; Aslam, Muhammad; Siddiqui, Arif; Lodhi, Ghulam Mustafa; Ahmad, Tausif

    2015-05-15

    Inflammation plays a significant role in the etiology of type 2 diabetes mellitus (T2DM). The rise in the pro-inflammatory cytokines is the essential step in glucotoxicity and lipotoxicity induced mitochondrial injury, oxidative stress and beta cell apoptosis in T2DM. Among the recognized markers are interleukin (IL)-6, IL-1, IL-10, IL-18, tissue necrosis factor-alpha (TNF-α), C-reactive protein, resistin, adiponectin, tissue plasminogen activator, fibrinogen and heptoglobins. Diabetes mellitus has firm genetic and very strong environmental influence; exhibiting a polygenic mode of inheritance. Many single nucleotide polymorphisms (SNPs) in various genes including those of pro and anti-inflammatory cytokines have been reported as a risk for T2DM. Not all the SNPs have been confirmed by unifying results in different studies and wide variations have been reported in various ethnic groups. The inter-ethnic variations can be explained by the fact that gene expression may be regulated by gene-gene, gene-environment and gene-nutrient interactions. This review highlights the impact of these interactions on determining the role of single nucleotide polymorphism of IL-6, TNF-α, resistin and adiponectin in pathogenesis of T2DM.

  19. Neighboring Genes Show Correlated Evolution in Gene Expression.

    PubMed

    Ghanbarian, Avazeh T; Hurst, Laurence D

    2015-07-01

    When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  20. Neighboring Genes Show Correlated Evolution in Gene Expression

    PubMed Central

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  1. Gene doping: gene delivery for olympic victory

    PubMed Central

    Gould, David

    2013-01-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

  2. Structural and functional features of a developmentally regulated lipopolysaccharide-binding protein.

    PubMed

    Krasity, Benjamin C; Troll, Joshua V; Lehnert, Erik M; Hackett, Kathleen T; Dillard, Joseph P; Apicella, Michael A; Goldman, William E; Weiss, Jerrold P; McFall-Ngai, Margaret J

    2015-10-13

    Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. Mammalian lipopolysaccharide (LPS)-binding protein (LBP) is implicated in conveying LPS to host cells and potentiating its signaling activity. In certain disease states, such as obesity, the overproduction of this protein has been a

  3. GeneSigDB—a curated database of gene expression signatures

    PubMed Central

    Culhane, Aedín C.; Schwarzl, Thomas; Sultana, Razvan; Picard, Kermshlise C.; Picard, Shaita C.; Lu, Tim H.; Franklin, Katherine R.; French, Simon J.; Papenhausen, Gerald; Correll, Mick; Quackenbush, John

    2010-01-01

    The primary objective of most gene expression studies is the identification of one or more gene signatures; lists of genes whose transcriptional levels are uniquely associated with a specific biological phenotype. Whilst thousands of experimentally derived gene signatures are published, their potential value to the community is limited by their computational inaccessibility. Gene signatures are embedded in published article figures, tables or in supplementary materials, and are frequently presented using non-standard gene or probeset nomenclature. We present GeneSigDB (http://compbio.dfci.harvard.edu/genesigdb) a manually curated database of gene expression signatures. GeneSigDB release 1.0 focuses on cancer and stem cells gene signatures and was constructed from more than 850 publications from which we manually transcribed 575 gene signatures. Most gene signatures (n = 560) were successfully mapped to the genome to extract standardized lists of EnsEMBL gene identifiers. GeneSigDB provides the original gene signature, the standardized gene list and a fully traceable gene mapping history for each gene from the original transcribed data table through to the standardized list of genes. The GeneSigDB web portal is easy to search, allows users to compare their own gene list to those in the database, and download gene signatures in most common gene identifier formats. PMID:19934259

  4. Gene doping: gene delivery for olympic victory.

    PubMed

    Gould, David

    2013-08-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. © 2012 The Author. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

  5. Gene-Gene and Gene-Environment Interactions in Ulcerative Colitis

    PubMed Central

    Wang, Ming-Hsi; Fiocchi, Claudio; Zhu, Xiaofeng; Ripke, Stephan; Kamboh, M. Ilyas; Rebert, Nancy; Duerr, Richard H.; Achkar, Jean-Paul

    2014-01-01

    Genome-wide association studies (GWAS) have identified at least 133 ulcerative colitis (UC) associated loci. The role of genetic factors in clinical practice is not clearly defined. The relevance of genetic variants to disease pathogenesis is still uncertain because of not characterized gene-gene and gene-environment interactions. We examined the predictive value of combining the 133 UC risk loci with genetic interactions in an ongoing inflammatory bowel disease (IBD) GWAS. The Wellcome Trust Case-Control Consortium (WTCCC) IBD GWAS was used as a replication cohort. We applied logic regression (LR), a novel adaptive regression methodology, to search for high order interactions. Exploratory genotype correlations with UC sub-phenotypes (extent of disease, need of surgery, age of onset, extra-intestinal manifestations and primary sclerosing cholangitis (PSC)) were conducted. The combination of 133 UC loci yielded good UC risk predictability (area under the curve [AUC] of 0.86). A higher cumulative allele score predicted higher UC risk. Through LR, several lines of evidence for genetic interactions were identified and successfully replicated in the WTCCC cohort. The genetic interactions combined with the gene-smoking interaction significantly improved predictability in the model (AUC, from 0.86 to 0.89, P=3.26E-05). Explained UC variance increased from 37% to 42% after adding the interaction terms. A within case analysis found suggested genetic association with PSC. Our study demonstrates that the LR methodology allows the identification and replication of high order genetic interactions in UC GWAS datasets. UC risk can be predicted by a 133 loci and improved by adding gene-gene and gene-environment interactions. PMID:24241240

  6. Gene doping.

    PubMed

    Azzazy, Hassan M E

    2010-01-01

    Gene doping abuses the legitimate approach of gene therapy. While gene therapy aims to correct genetic disorders by introducing a foreign gene to replace an existing faulty one or by manipulating existing gene(s) to achieve a therapeutic benefit, gene doping employs the same concepts to bestow performance advantages on athletes over their competitors. Recent developments in genetic engineering have contributed significantly to the progress of gene therapy research and currently numerous clinical trials are underway. Some athletes and their staff are probably watching this progress closely. Any gene that plays a role in muscle development, oxygen delivery to tissues, neuromuscular coordination, or even pain control is considered a candidate for gene dopers. Unfortunately, detecting gene doping is technically very difficult because the transgenic proteins expressed by the introduced genes are similar to their endogenous counterparts. Researchers today are racing the clock because assuring the continued integrity of sports competition depends on their ability to develop effective detection strategies in preparation for the 2012 Olympics, which may mark the appearance of genetically modified athletes.

  7. Tularemia without lesions in grey tree squirrels: A diagnostic challenge

    USDA-ARS?s Scientific Manuscript database

    Fifteen cases of Francisella tularenesis infection (tularemia) were identified in western grey (Sciurus griseus) and eastern grey (Sciurus carolinesis) squirrels submitted to the Washington Animal Disease Diagnostic Laboratory between 2008 and 2011. All of the squirrels originated in Washington stat...

  8. Gene trap and gene inversion methods for conditional gene inactivation in the mouse

    PubMed Central

    Xin, Hong-Bo; Deng, Ke-Yu; Shui, Bo; Qu, Shimian; Sun, Qi; Lee, Jane; Greene, Kai Su; Wilson, Jason; Yu, Ying; Feldman, Morris; Kotlikoff, Michael I.

    2005-01-01

    Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene. PMID:15659575

  9. Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network.

    PubMed

    Jiang, Xue; Zhang, Han; Quan, Xiongwen

    2016-01-01

    Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets.

  10. Hox genes and study of Hox genes in crustacean

    NASA Astrophysics Data System (ADS)

    Hou, Lin; Chen, Zhijuan; Xu, Mingyu; Lin, Shengguo; Wang, Lu

    2004-12-01

    Homeobox genes have been discovered in many species. These genes are known to play a major role in specifying regional identity along the anterior-posterior axis of animals from a wide range of phyla. The products of the homeotic genes are a set of evolutionarily conserved transcription factors that control elaborate developmental processes and specify cell fates in metazoans. Crustacean, presenting a variety of body plans not encountered in any other class or phylum of the Metazoa, has been shown to possess a single set of homologous Hox genes like insect. The ancestral crustacean Hox gene complex comprised ten genes: eight homologous to the hometic Hox genes and two related to nonhomeotic genes presented within the insect Hox complexes. The crustacean in particular exhibits an abundant diversity segment specialization and tagmosis. This morphological diversity relates to the Hox genes. In crustacean body plan, different Hox genes control different segments and tagmosis.

  11. Serological survey for diseases in free-ranging coyotes (Canis latrans) in Yellowstone National Park, Wyoming.

    PubMed

    Gese, E M; Schultz, R D; Johnson, M R; Williams, E S; Crabtree, R L; Ruff, R L

    1997-01-01

    From October 1989 to June 1993, we captured and sampled 110 coyotes (Canis latrans) for various diseases in Yellowstone National Park, Wyoming (USA). Prevalence of antibodies against canine parvovirus (CPV) was 100% for adults (> 24 months old), 100% for yearlings (12 to 24 months old), and 100% for old pups (4 to 12 months old); 0% of the young pups (< 3 months old) had antibodies against CPV. Presence of antibodies against canine distemper virus (CDV) was associated with the age of the coyote, with 88%, 54%, 23%, and 0% prevalence among adults, yearlings, old pups, and young pups, respectively. Prevalence of CDV antibodies declined over time from 100% in 1989 to 33% in 1992. The prevalence of canine infectious hepatitis (ICH) virus antibodies was 97%, 82%, 54%, and 33%, for adults, yearlings, old pups, and young pups, respectively. The percentage of coyotes with ICH virus antibodies also declined over time from a high of 100% in 1989 to 31% in 1992, and 42% in 1993. Prevalence of antibodies against Yersinia pestis was 86%, 33%, 80%, and 7%, for adults, yearlings, old pups, and young pups, respectively, and changed over time from 57% in 1991 to 0% in 1993. The prevalence of antibodies against Francisella tularensis was 21%, 17%, 10%, and 20%, for adults, yearlings, old pups, and young pups, respectively. No coyotes had serologic evidence of exposure to brucellosis, either Brucella abortus or Brucella canis. No coyotes were seropositive to Leptospira interrogans (serovars canicola, hardjo, and icterohemorrhagiae). Prevalence of antibodies against L. interrogans serovar pomona was 7%, 0%, 0%, and 9%, for adults, yearlings, old pups, and young pups, respectively. Antibodies against L. interrogans serovar grippotyphosa were present in 17% of adults and 0% of yearlings, old pups, and young pups. Many infectious canine pathogens (CPV, CDV, ICH virus) are prevalent in coyotes in Yellowstone National Park, with CPV influencing coyote pup survival during the first 3 months

  12. Factors associated with tick bites and pathogen prevalence in ticks parasitizing humans in Georgia, USA.

    PubMed

    Gleim, Elizabeth R; Garrison, Laurel E; Vello, Marianne S; Savage, Mason Y; Lopez, Gaylord; Berghaus, Roy D; Yabsley, Michael J

    2016-03-02

    The incidence and emergence of tick-borne diseases has increased dramatically in the United States during the past 30 years, yet few large-scale epidemiological studies have been performed on individuals bitten by ticks. Epidemiological information, including disease development, may provide valuable information regarding effectiveness of tick bite prevention education, pathogen transmission, human-disease dynamics, and potential implications for under reporting of tick-borne diseases. Ticks found attached to Georgia residents were submitted for identification and polymerase chain reaction (PCR) testing for Francisella tularensis, Ehrlichia, Anaplasma, Borrelia, and Rickettsia spp. Tick bite victims were interviewed three weeks after the tick bite to identify various epidemiologic factors associated with infestation and if signs suggestive of a tick-borne disease had developed. Fisher's exact test of independence was used to evaluate associations between various factors evaluated in the study. A multivariable logistic regression model was used for the prediction of non-specific illness post-tick bite. From April 2005-December 2006, 444 participants submitted 597 ticks (426 Amblyomma americanum, 142 Dermacentor variabilis, 19 A. maculatum, 7 Ixodes scapularis, 3 Amblyomma sp.) which originated from 95 counties. Only 25 (34 %) of 74 interviewed individuals purposely took tick bite prevention measures. Ticks that were PCR positive for bacterial organisms were attached to 136 participants. Of the 77 participants who developed non-specific illness, 50 did not have PCR positive ticks, whereas 27 did have PCR positive tick (s). Of those 27 individuals, 12 fit the criteria for a possible tick-borne illness (i.e., tick attached >6 h [if known], ≥4 day incubation period, and the individual exhibited clinical symptoms typical of a tick-borne illness without exhibiting cough, sore throat, or sinus congestion). Ticks from these individuals were positive for R. amblyommii (n

  13. The atypical pneumonias: clinical diagnosis and importance.

    PubMed

    Cunha, B A

    2006-05-01

    The most common atypical pneumonias are caused by three zoonotic pathogens, Chlamydia psittaci (psittacosis), Francisella tularensis (tularemia), and Coxiella burnetii (Q fever), and three nonzoonotic pathogens, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella. These atypical agents, unlike the typical pathogens, often cause extrapulmonary manifestations. Atypical CAPs are systemic infectious diseases with a pulmonary component and may be differentiated clinically from typical CAPs by the pattern of extrapulmonary organ involvement which is characteristic for each atypical CAP. Zoonotic pneumonias may be eliminated from diagnostic consideration with a negative contact history. The commonest clinical problem is to differentiate legionnaire's disease from typical CAP as well as from C. pneumoniae or M. pneumonia infection. Legionella is the most important atypical pathogen in terms of severity. It may be clinically differentiated from typical CAP and other atypical pathogens by the use of a weighted point system of syndromic diagnosis based on the characteristic pattern of extrapulmonary features. Because legionnaire's disease often presents as severe CAP, a presumptive diagnosis of Legionella should prompt specific testing and empirical anti-Legionella therapy such as the Winthrop-University Hospital Infectious Disease Division's weighted point score system. Most atypical pathogens are difficult or dangerous to isolate and a definitive laboratory diagnosis is usually based on indirect, i.e., direct flourescent antibody (DFA), indirect flourescent antibody (IFA). Atypical CAP is virtually always monomicrobial; increased IFA IgG tests indicate past exposure and not concurrent infection. Anti-Legionella antibiotics include macrolides, doxycycline, rifampin, quinolones, and telithromycin. The drugs with the highest level of anti-Legionella activity are quinolones and telithromycin. Therapy is usually continued for 2 weeks if potent anti-Legionella drugs are

  14. GeneBuilder: interactive in silico prediction of gene structure.

    PubMed

    Milanesi, L; D'Angelo, D; Rogozin, I B

    1999-01-01

    Prediction of gene structure in newly sequenced DNA becomes very important in large genome sequencing projects. This problem is complicated due to the exon-intron structure of eukaryotic genes and because gene expression is regulated by many different short nucleotide domains. In order to be able to analyse the full gene structure in different organisms, it is necessary to combine information about potential functional signals (promoter region, splice sites, start and stop codons, 3' untranslated region) together with the statistical properties of coding sequences (coding potential), information about homologous proteins, ESTs and repeated elements. We have developed the GeneBuilder system which is based on prediction of functional signals and coding regions by different approaches in combination with similarity searches in proteins and EST databases. The potential gene structure models are obtained by using a dynamic programming method. The program permits the use of several parameters for gene structure prediction and refinement. During gene model construction, selecting different exon homology levels with a protein sequence selected from a list of homologous proteins can improve the accuracy of the gene structure prediction. In the case of low homology, GeneBuilder is still able to predict the gene structure. The GeneBuilder system has been tested by using the standard set (Burset and Guigo, Genomics, 34, 353-367, 1996) and the performances are: 0.89 sensitivity and 0.91 specificity at the nucleotide level. The total correlation coefficient is 0.88. The GeneBuilder system is implemented as a part of the WebGene a the URL: http://www.itba.mi. cnr.it/webgene and TRADAT (TRAncription Database and Analysis Tools) launcher URL: http://www.itba.mi.cnr.it/tradat.

  15. Gene: a gene-centered information resource at NCBI.

    PubMed

    Brown, Garth R; Hem, Vichet; Katz, Kenneth S; Ovetsky, Michael; Wallin, Craig; Ermolaeva, Olga; Tolstoy, Igor; Tatusova, Tatiana; Pruitt, Kim D; Maglott, Donna R; Murphy, Terence D

    2015-01-01

    The National Center for Biotechnology Information's (NCBI) Gene database (www.ncbi.nlm.nih.gov/gene) integrates gene-specific information from multiple data sources. NCBI Reference Sequence (RefSeq) genomes for viruses, prokaryotes and eukaryotes are the primary foundation for Gene records in that they form the critical association between sequence and a tracked gene upon which additional functional and descriptive content is anchored. Additional content is integrated based on the genomic location and RefSeq transcript and protein sequence data. The content of a Gene record represents the integration of curation and automated processing from RefSeq, collaborating model organism databases, consortia such as Gene Ontology, and other databases within NCBI. Records in Gene are assigned unique, tracked integers as identifiers. The content (citations, nomenclature, genomic location, gene products and their attributes, phenotypes, sequences, interactions, variation details, maps, expression, homologs, protein domains and external databases) is available via interactive browsing through NCBI's Entrez system, via NCBI's Entrez programming utilities (E-Utilities and Entrez Direct) and for bulk transfer by FTP. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  16. Gene-gene and gene-environment interactions: new insights into the prevention, detection and management of coronary artery disease.

    PubMed

    Lanktree, Matthew B; Hegele, Robert A

    2009-02-26

    Despite the recent success of genome-wide association studies (GWASs) in identifying loci consistently associated with coronary artery disease (CAD), a large proportion of the genetic components of CAD and its metabolic risk factors, including plasma lipids, type 2 diabetes and body mass index, remain unattributed. Gene-gene and gene-environment interactions might produce a meaningful improvement in quantification of the genetic determinants of CAD. Testing for gene-gene and gene-environment interactions is thus a new frontier for large-scale GWASs of CAD. There are several anecdotal examples of monogenic susceptibility to CAD in which the phenotype was worsened by an adverse environment. In addition, small-scale candidate gene association studies with functional hypotheses have identified gene-environment interactions. For future evaluation of gene-gene and gene-environment interactions to achieve the same success as the single gene associations reported in recent GWASs, it will be important to pre-specify agreed standards of study design and statistical power, environmental exposure measurement, phenomic characterization and analytical strategies. Here we discuss these issues, particularly in relation to the investigation and potential clinical utility of gene-gene and gene-environment interactions in CAD.

  17. Sexy gene conversions: locating gene conversions on the X-chromosome.

    PubMed

    Lawson, Mark J; Zhang, Liqing

    2009-08-01

    Gene conversion can have a profound impact on both the short- and long-term evolution of genes and genomes. Here, we examined the gene families that are located on the X-chromosomes of human (Homo sapiens), chimpanzee (Pan troglodytes), mouse (Mus musculus) and rat (Rattus norvegicus) for evidence of gene conversion. We identified seven gene families (WD repeat protein family, Ferritin Heavy Chain family, RAS-related Protein RAB-40 family, Diphosphoinositol polyphosphate phosphohydrolase family, Transcription Elongation Factor A family, LDOC1-related family, Zinc Finger Protein ZIC, and GLI family) that show evidence of gene conversion. Through phylogenetic analyses and synteny evidence, we show that gene conversion has played an important role in the evolution of these gene families and that gene conversion has occurred independently in both primates and rodents. Comparing the results with those of two gene conversion prediction programs (GENECONV and Partimatrix), we found that both GENECONV and Partimatrix have very high false negative rates (i.e. failed to predict gene conversions), which leads to many undetected gene conversions. The combination of phylogenetic analyses with physical synteny evidence exhibits high resolution in the detection of gene conversions.

  18. Gene doping.

    PubMed

    Harridge, Stephen D R; Velloso, Cristiana P

    2008-01-01

    Gene doping is the misuse of gene therapy to enhance athletic performance. It has recently been recognised as a potential threat and subsequently been prohibited by the World Anti-Doping Agency. Despite concerns with safety and efficacy of gene therapy, the technology is progressing steadily. Many of the genes/proteins which are involved in determining key components of athletic performance have been identified. Naturally occurring mutations in humans as well as gene-transfer experiments in adult animals have shown that altered expression of these genes does indeed affect physical performance. For athletes, however, the gains in performance must be weighed against the health risks associated with the gene-transfer process, whereas the detection of such practices will provide new challenges for the anti-doping authorities.

  19. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    PubMed

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

  20. Reconstructing directed gene regulatory network by only gene expression data.

    PubMed

    Zhang, Lu; Feng, Xi Kang; Ng, Yen Kaow; Li, Shuai Cheng

    2016-08-18

    Accurately identifying gene regulatory network is an important task in understanding in vivo biological activities. The inference of such networks is often accomplished through the use of gene expression data. Many methods have been developed to evaluate gene expression dependencies between transcription factor and its target genes, and some methods also eliminate transitive interactions. The regulatory (or edge) direction is undetermined if the target gene is also a transcription factor. Some methods predict the regulatory directions in the gene regulatory networks by locating the eQTL single nucleotide polymorphism, or by observing the gene expression changes when knocking out/down the candidate transcript factors; regrettably, these additional data are usually unavailable, especially for the samples deriving from human tissues. In this study, we propose the Context Based Dependency Network (CBDN), a method that is able to infer gene regulatory networks with the regulatory directions from gene expression data only. To determine the regulatory direction, CBDN computes the influence of source to target by evaluating the magnitude changes of expression dependencies between the target gene and the others with conditioning on the source gene. CBDN extends the data processing inequality by involving the dependency direction to distinguish between direct and transitive relationship between genes. We also define two types of important regulators which can influence a majority of the genes in the network directly or indirectly. CBDN can detect both of these two types of important regulators by averaging the influence functions of candidate regulator to the other genes. In our experiments with simulated and real data, even with the regulatory direction taken into account, CBDN outperforms the state-of-the-art approaches for inferring gene regulatory network. CBDN identifies the important regulators in the predicted network: 1. TYROBP influences a batch of genes that are

  1. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

    PubMed Central

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

  2. Gene Architectures that Minimize Cost of Gene Expression.

    PubMed

    Frumkin, Idan; Schirman, Dvir; Rotman, Aviv; Li, Fangfei; Zahavi, Liron; Mordret, Ernest; Asraf, Omer; Wu, Song; Levy, Sasha F; Pilpel, Yitzhak

    2017-01-05

    Gene expression burdens cells by consuming resources and energy. While numerous studies have investigated regulation of expression level, little is known about gene design elements that govern expression costs. Here, we ask how cells minimize production costs while maintaining a given protein expression level and whether there are gene architectures that optimize this process. We measured fitness of ∼14,000 E. coli strains, each expressing a reporter gene with a unique 5' architecture. By comparing cost-effective and ineffective architectures, we found that cost per protein molecule could be minimized by lowering transcription levels, regulating translation speeds, and utilizing amino acids that are cheap to synthesize and that are less hydrophobic. We then examined natural E. coli genes and found that highly expressed genes have evolved more forcefully to minimize costs associated with their expression. Our study thus elucidates gene design elements that improve the economy of protein expression in natural and heterologous systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Gene transfer and gene mapping in mammalian cells in culture.

    PubMed

    Shows, T B; Sakaguchi, A Y

    1980-01-01

    The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being empolyed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probles. Recently, single genes have been cloned into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed.

  4. Patenting human genes: Chinese academic articles' portrayal of gene patents.

    PubMed

    Du, Li

    2018-04-24

    The patenting of human genes has been the subject of debate for decades. While China has gradually come to play an important role in the global genomics-based testing and treatment market, little is known about Chinese scholars' perspectives on patent protection for human genes. A content analysis of academic literature was conducted to identify Chinese scholars' concerns regarding gene patents, including benefits and risks of patenting human genes, attitudes that researchers hold towards gene patenting, and any legal and policy recommendations offered for the gene patent regime in China. 57.2% of articles were written by law professors, but scholars from health sciences, liberal arts, and ethics also participated in discussions on gene patent issues. While discussions of benefits and risks were relatively balanced in the articles, 63.5% of the articles favored gene patenting in general and, of the articles (n = 41) that explored gene patents in the Chinese context, 90.2% supported patent protections for human genes in China. The patentability of human genes was discussed in 33 articles, and 75.8% of these articles reached the conclusion that human genes are patentable. Chinese scholars view the patent regime as an important legal tool to protect the interests of inventors and inventions as well as the genetic resources of China. As such, many scholars support a gene patent system in China. These attitudes towards gene patents remain unchanged following the court ruling in the Myriad case in 2013, but arguments have been raised about the scope of gene patents, in particular that the increasing numbers of gene patents may negatively impact public health in China.

  5. Gene Circuit Analysis of the Terminal Gap Gene huckebein

    PubMed Central

    Ashyraliyev, Maksat; Siggens, Ken; Janssens, Hilde; Blom, Joke; Akam, Michael; Jaeger, Johannes

    2009-01-01

    The early embryo of Drosophila melanogaster provides a powerful model system to study the role of genes in pattern formation. The gap gene network constitutes the first zygotic regulatory tier in the hierarchy of the segmentation genes involved in specifying the position of body segments. Here, we use an integrative, systems-level approach to investigate the regulatory effect of the terminal gap gene huckebein (hkb) on gap gene expression. We present quantitative expression data for the Hkb protein, which enable us to include hkb in gap gene circuit models. Gap gene circuits are mathematical models of gene networks used as computational tools to extract regulatory information from spatial expression data. This is achieved by fitting the model to gap gene expression patterns, in order to obtain estimates for regulatory parameters which predict a specific network topology. We show how considering variability in the data combined with analysis of parameter determinability significantly improves the biological relevance and consistency of the approach. Our models are in agreement with earlier results, which they extend in two important respects: First, we show that Hkb is involved in the regulation of the posterior hunchback (hb) domain, but does not have any other essential function. Specifically, Hkb is required for the anterior shift in the posterior border of this domain, which is now reproduced correctly in our models. Second, gap gene circuits presented here are able to reproduce mutants of terminal gap genes, while previously published models were unable to reproduce any null mutants correctly. As a consequence, our models now capture the expression dynamics of all posterior gap genes and some variational properties of the system correctly. This is an important step towards a better, quantitative understanding of the developmental and evolutionary dynamics of the gap gene network. PMID:19876378

  6. Gene circuit analysis of the terminal gap gene huckebein.

    PubMed

    Ashyraliyev, Maksat; Siggens, Ken; Janssens, Hilde; Blom, Joke; Akam, Michael; Jaeger, Johannes

    2009-10-01

    The early embryo of Drosophila melanogaster provides a powerful model system to study the role of genes in pattern formation. The gap gene network constitutes the first zygotic regulatory tier in the hierarchy of the segmentation genes involved in specifying the position of body segments. Here, we use an integrative, systems-level approach to investigate the regulatory effect of the terminal gap gene huckebein (hkb) on gap gene expression. We present quantitative expression data for the Hkb protein, which enable us to include hkb in gap gene circuit models. Gap gene circuits are mathematical models of gene networks used as computational tools to extract regulatory information from spatial expression data. This is achieved by fitting the model to gap gene expression patterns, in order to obtain estimates for regulatory parameters which predict a specific network topology. We show how considering variability in the data combined with analysis of parameter determinability significantly improves the biological relevance and consistency of the approach. Our models are in agreement with earlier results, which they extend in two important respects: First, we show that Hkb is involved in the regulation of the posterior hunchback (hb) domain, but does not have any other essential function. Specifically, Hkb is required for the anterior shift in the posterior border of this domain, which is now reproduced correctly in our models. Second, gap gene circuits presented here are able to reproduce mutants of terminal gap genes, while previously published models were unable to reproduce any null mutants correctly. As a consequence, our models now capture the expression dynamics of all posterior gap genes and some variational properties of the system correctly. This is an important step towards a better, quantitative understanding of the developmental and evolutionary dynamics of the gap gene network.

  7. Genes from scratch--the evolutionary fate of de novo genes.

    PubMed

    Schlötterer, Christian

    2015-04-01

    Although considered an extremely unlikely event, many genes emerge from previously noncoding genomic regions. This review covers the entire life cycle of such de novo genes. Two competing hypotheses about the process of de novo gene birth are discussed as well as the high death rate of de novo genes. Despite the high death rate, some de novo genes are retained and remain functional, even in distantly related species, through their integration into gene networks. Further studies combining gene expression with ribosome profiling in multiple populations across different species will be instrumental for an improved understanding of the evolutionary processes operating on de novo genes. Copyright © 2015 The Author. Published by Elsevier Ltd.. All rights reserved.

  8. Initial description of primate-specific cystine-knot Prometheus genes and differential gene expansions of D-dopachrome tautomerase genes

    PubMed Central

    Premzl, Marko

    2015-01-01

    Using eutherian comparative genomic analysis protocol and public genomic sequence data sets, the present work attempted to update and revise two gene data sets. The most comprehensive third party annotation gene data sets of eutherian adenohypophysis cystine-knot genes (128 complete coding sequences), and d-dopachrome tautomerases and macrophage migration inhibitory factor genes (30 complete coding sequences) were annotated. For example, the present study first described primate-specific cystine-knot Prometheus genes, as well as differential gene expansions of D-dopachrome tautomerase genes. Furthermore, new frameworks of future experiments of two eutherian gene data sets were proposed. PMID:25941635

  9. GenePRIMP: A Gene Prediction Improvement Pipeline For Prokaryotic Genomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kyrpides, Nikos C.; Ivanova, Natalia N.; Pati, Amrita

    2010-07-08

    GenePRIMP (Gene Prediction Improvement Pipeline, Http://geneprimp.jgi-psf.org), a computational process that performs evidence-based evaluation of gene models in prokaryotic genomes and reports anomalies including inconsistent start sites, missing genes, and split genes. We show that manual curation of gene models using the anomaly reports generated by GenePRIMP improves their quality and demonstrate the applicability of GenePRIMP in improving finishing quality and comparing different genome sequencing and annotation technologies. Keywords in context: Gene model, Quality Control, Translation start sites, Automatic correction. Hardware requirements; PC, MAC; Operating System: UNIX/LINUX; Compiler/Version: Perl 5.8.5 or higher; Special requirements: NCBI Blast and nr installation; File Types:more » Source Code, Executable module(s), Sample problem input data; installation instructions other; programmer documentation. Location/transmission: http://geneprimp.jgi-psf.org/gp.tar.gz« less

  10. Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms.

    PubMed

    Li, Zhen; Defoort, Jonas; Tasdighian, Setareh; Maere, Steven; Van de Peer, Yves; De Smet, Riet

    2016-02-01

    Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of "gene duplicability" is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes. © 2016 American Society of Plant Biologists. All rights reserved.

  11. Cytokine-related genes and oxidation-related genes detected in preeclamptic placentas.

    PubMed

    Lee, Gui Se Ra; Joe, Yoon Seong; Kim, Sa Jin; Shin, Jong Chul

    2010-10-01

    To investigate cytokine- and oxidation-related genes for preeclampsia using DNA microarray analysis. Placentas were collected from 13 normal pregnancies and 13 patients with preeclampsia. Gene expression was studied using DNA microarray. Among significantly expressed genes, we focused on genes associated with cytokines and oxidation, and the results were confirmed using quantitative real time-polymerase chain reaction (QRT-PCR). 415 genes out of 30,940 genes were altered by > or =2-fold in the microarray analysis. 121 up-regulated genes and 294 down-regulated genes were found to be in preeclamptic placenta. Six cytokine-related genes and 5 oxidation-related genes were found from among the 121 up-regulated genes. The cytokine-related genes studied included oncostatin M (OSM), fms-related tyrosine kinase (FLT1) and vascular endothelial growth factor A (VEGFA), and the oxidation-related genes studied included spermine oxidase (SMOX), l cytochrome P450, family 26, subfamily A, polypeptide 1 (CYP26A1), acetate dehydrogenase A (LDHA). These six genes were also significantly higher in placentas from patients with preeclampsia than in those from women with normal pregnancies. The placental tissue of patients with preeclampsia showed significantly higher mRNA expression of these six genes than the normal group, using QRT-PCR. DNA microarray analysis is one of the great methods for simultaneously detecting the functionally associated genes of preeclampsia. The cytokine-related genes such as OSM, FLT1 and VEGFA, and the oxidation-related genes such as LDHA, CYP26A1 and SMOX might prove to be the starting point in the elucidation of the pathogenesis of preeclampsia.

  12. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.

    PubMed

    Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D

    2017-04-07

    Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

  13. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

    PubMed Central

    Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D.

    2017-01-01

    Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification. PMID:28387220

  14. Diversity of bacteria in ships ballast water as revealed by next generation DNA sequencing.

    PubMed

    Brinkmeyer, Robin

    2016-06-15

    The bacterial diversity in ballast water from five general cargo ships calling at the Port of Houston was determined with ion semiconductor DNA sequencing (Ion Torrent PGM) of PCR amplified 16S rRNA genes. Phylogenetic analysis revealed that the composition of bacteria in ballast water did not resemble that of typical marine habitats or even open ocean waters where BWEs occur. The predominant group of bacteria in ships conducting BWEs was the Roseobacter clade within the Alphaproteobacteria. In contrast, Gammaproteobacteria were predominant in the ship that did not conduct a BWE. All the ships contained human, fish, and terrestrial plant pathogens as well as bacteria indicative of fecal or activated sludge contamination. Most of the 60 pathogens had not been detected in ballast water previously. Among these were the human pathogens Corynebacterium diptheriae and several Legionella species and the fish pathogens Francisella piscicida and Piscirickettsia salmonis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    NASA Astrophysics Data System (ADS)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  16. Discovering Implicit Entity Relation with the Gene-Citation-Gene Network

    PubMed Central

    Song, Min; Han, Nam-Gi; Kim, Yong-Hwan; Ding, Ying; Chambers, Tamy

    2013-01-01

    In this paper, we apply the entitymetrics model to our constructed Gene-Citation-Gene (GCG) network. Based on the premise there is a hidden, but plausible, relationship between an entity in one article and an entity in its citing article, we constructed a GCG network of gene pairs implicitly connected through citation. We compare the performance of this GCG network to a gene-gene (GG) network constructed over the same corpus but which uses gene pairs explicitly connected through traditional co-occurrence. Using 331,411 MEDLINE abstracts collected from 18,323 seed articles and their references, we identify 25 gene pairs. A comparison of these pairs with interactions found in BioGRID reveal that 96% of the gene pairs in the GCG network have known interactions. We measure network performance using degree, weighted degree, closeness, betweenness centrality and PageRank. Combining all measures, we find the GCG network has more gene pairs, but a lower matching rate than the GG network. However, combining top ranked genes in both networks produces a matching rate of 35.53%. By visualizing both the GG and GCG networks, we find that cancer is the most dominant disease associated with the genes in both networks. Overall, the study indicates that the GCG network can be useful for detecting gene interaction in an implicit manner. PMID:24358368

  17. Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents

    PubMed Central

    Fishbein, Ilia; Chorny, Michael; Adamo, Richard F; Forbes, Scott P; Corrales, Ricardo A; Alferiev, Ivan S; Levy, Robert J

    2015-01-01

    A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation. PMID:26225356

  18. Androgen-responsive gene database: integrated knowledge on androgen-responsive genes.

    PubMed

    Jiang, Mei; Ma, Yunsheng; Chen, Congcong; Fu, Xuping; Yang, Shu; Li, Xia; Yu, Guohua; Mao, Yumin; Xie, Yi; Li, Yao

    2009-11-01

    Androgen signaling plays an important role in many biological processes. Androgen Responsive Gene Database (ARGDB) is devoted to providing integrated knowledge on androgen-controlled genes. Gene records were collected on the basis of PubMed literature collections. More than 6000 abstracts and 950 original publications were manually screened, leading to 1785 human genes, 993 mouse genes, and 583 rat genes finally included in the database. All the collected genes were experimentally proved to be regulated by androgen at the expression level or to contain androgen-responsive regions. For each gene important details of the androgen regulation experiments were collected from references, such as expression change, androgen-responsive sequence, response time, tissue/cell type, experimental method, ligand identity, and androgen amount, which will facilitate further evaluation by researchers. Furthermore, the database was integrated with multiple annotation resources, including National Center for Biotechnology Information, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway, to reveal the biological characteristics and significance of androgen-regulated genes. The ARGDB web site is mainly composed of the Browse, Search, Element Scan, and Submission modules. It is user friendly and freely accessible at http://argdb.fudan.edu.cn. Preliminary analysis of the collected data was performed. Many disease pathways, such as prostate carcinogenesis, were found to be enriched in androgen-regulated genes. The discovered androgen-response motifs were similar to those in previous reports. The analysis results are displayed in the web site. In conclusion, ARGDB provides a unified gateway to storage, retrieval, and update of information on androgen-regulated genes.

  19. Allelic-based gene-gene interaction associated with quantitative traits.

    PubMed

    Jung, Jeesun; Sun, Bin; Kwon, Deukwoo; Koller, Daniel L; Foroud, Tatiana M

    2009-05-01

    Recent studies have shown that quantitative phenotypes may be influenced not only by multiple single nucleotide polymorphisms (SNPs) within a gene but also by the interaction between SNPs at unlinked genes. We propose a new statistical approach that can detect gene-gene interactions at the allelic level which contribute to the phenotypic variation in a quantitative trait. By testing for the association of allelic combinations at multiple unlinked loci with a quantitative trait, we can detect the SNP allelic interaction whether or not it can be detected as a main effect. Our proposed method assigns a score to unrelated subjects according to their allelic combination inferred from observed genotypes at two or more unlinked SNPs, and then tests for the association of the allelic score with a quantitative trait. To investigate the statistical properties of the proposed method, we performed a simulation study to estimate type I error rates and power and demonstrated that this allelic approach achieves greater power than the more commonly used genotypic approach to test for gene-gene interaction. As an example, the proposed method was applied to data obtained as part of a candidate gene study of sodium retention by the kidney. We found that this method detects an interaction between the calcium-sensing receptor gene (CaSR), the chloride channel gene (CLCNKB) and the Na, K, 2Cl cotransporter gene (CLC12A1) that contributes to variation in diastolic blood pressure.

  20. A hybrid approach of gene sets and single genes for the prediction of survival risks with gene expression data.

    PubMed

    Seok, Junhee; Davis, Ronald W; Xiao, Wenzhong

    2015-01-01

    Accumulated biological knowledge is often encoded as gene sets, collections of genes associated with similar biological functions or pathways. The use of gene sets in the analyses of high-throughput gene expression data has been intensively studied and applied in clinical research. However, the main interest remains in finding modules of biological knowledge, or corresponding gene sets, significantly associated with disease conditions. Risk prediction from censored survival times using gene sets hasn't been well studied. In this work, we propose a hybrid method that uses both single gene and gene set information together to predict patient survival risks from gene expression profiles. In the proposed method, gene sets provide context-level information that is poorly reflected by single genes. Complementarily, single genes help to supplement incomplete information of gene sets due to our imperfect biomedical knowledge. Through the tests over multiple data sets of cancer and trauma injury, the proposed method showed robust and improved performance compared with the conventional approaches with only single genes or gene sets solely. Additionally, we examined the prediction result in the trauma injury data, and showed that the modules of biological knowledge used in the prediction by the proposed method were highly interpretable in biology. A wide range of survival prediction problems in clinical genomics is expected to benefit from the use of biological knowledge.