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Sample records for actinobacillus pleuropneumoniae haptoglobin

  1. Survival of Actinobacillus pleuropneumoniae outside the pig.

    PubMed

    Assavacheep, P; Rycroft, A N

    2013-02-01

    Transmission of Actinobacillus pleuropneumoniae is primarily thought to be via direct transfer of mucus from pig to pig. For transfer between farms, the organism may need to persist in the wet or dried state to be carried on an inanimate surface. The survival of A. pleuropneumoniae was investigated under controlled laboratory conditions. In aqueous suspension, survival was improved by the presence of NaCl and mucin; it was prolonged at lower temperature. In dry state, it survived best on a hydrophobic surface either under desiccated conditions or saturated humidity. Detectable viability was maintained for 3-4 days. When frozen, A. pleuropneumoniae survived for more than 17 weeks at -20 °C, but the viability declined to 0.01% during that time. Survival at -70 °C was effective for long term storage. Results obtained from this investigation would be applicable for sampling method, transport techniques, epidemiological study, and biosecurity implementation. PMID:22892250

  2. Lipopolysaccharides of Actinobacillus pleuropneumoniae bind pig hemoglobin.

    PubMed Central

    Bélanger, M; Bégin, C; Jacques, M

    1995-01-01

    A previous study indicated that lipopolysaccharides (LPS) extracted from Actinobacillus pleuropneumoniae bind two low-molecular-mass proteins, of approximately 10 and 11 kDa, present in porcine respiratory tract secretions (M. Bélanger, D. Dubreuil, and M. Jacques, Infect. Immun. 62:868-873, 1994). In the present study, we determined the N-terminal amino acid sequences of these two proteins, which revealed high homology with the alpha and beta chains of pig hemoglobin. Some isolates of A. pleuropneumoniae were able to use hemoglobin from various animal species as well as other heme compounds as sole sources of iron for growth, while other isolates were unable to use them. Immunoelectron microscopy showed binding of pig hemoglobin at the surface of all A. pleuropneumoniae isolates as well as labeling of outer membrane blebs. We observed, using Western blotting (immunoblotting), that the lipid A-core region of LPS of all isolates was binding pig hemoglobin. Furthermore, lipid A obtained after acid hydrolysis of LPS extracted from A. pleuropneumoniae was able to bind pig hemoglobin and this binding was completely abolished by preincubation of lipid A with polymyxin B but was not inhibited by preincubation with glucosamines. Fatty acids constituting the lipid A of A. pleuropneumoniae, namely, dodecanoic acid, tetradecanoic acid, 3-hydroxytetradecanoic acid, hexadecanoic acid, and octadecanoic acid, were also binding pig hemoglobin. Our results indicate that LPS of all A. pleuropneumoniae isolates tested bind pig hemoglobin and that lipid A is involved in this binding. Our results also indicate that some A. pleuropneumoniae isolates are, in addition, able to use hemoglobin for growth. Binding of hemoglobin to LPS might represent an important means by which A. pleuropneumoniae acquires iron in vivo from hemoglobin released from erythrocytes lysed by the action of its hemolysins. PMID:7822035

  3. Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs.

    PubMed Central

    Jobert, J L; Savoye, C; Cariolet, R; Kobisch, M; Madec, F

    2000-01-01

    In order to demonstrate the possible role of aerosol in the transmission of Actinobacillus pleuropneumoniae, an experiment including 18 specific pathogen-free (SPF), 10-week-old piglets, randomly distributed into 2 adjacent units, was carried out. In these facilities, air was forced through absolute filters to prevent any contact with infectious agents. During the first 6 d post inoculation, the 2 units were connected by a rectangular opening and the air circulation was forced by the ventilation system from unit A (inoculated pigs) to unit B (non-inoculated pigs). The A. pleuropneumoniae strain (biovar 1 serovar 9) was isolated in France from an outbreak of porcine pleuropneumonia. Two different infecting doses, 10(7) cfu/animal and 10(8) cfu/animal, were inoculated by intranasal route in 6 pigs of unit A. The infection spread quickly from the inoculated pigs to the non-inoculated pigs. Clinical signs were acute during the 4 d post inoculation: hyperthermia, respiratory distress and, sometimes, death (6 pigs of the unit A and 2 pigs of the unit B). All pigs seroconverted against A. pleuropneumoniae serovar 9 within 2 weeks. Lung lesions were severe: fibrinous pleurisy and lung hemorrhages in the acute stage, pleural adherences and focal pulmonary necrosis in the chronic stage. Actinobacillus pleuropneumoniae was isolated from the tonsils and/or lungs in 16 animals. It could be also isolated from the air of the experimental unit. This study showed that A. pleuropneumoniae was readily transmitted through aerosol over a distance of at least 2.5 m. Images Figure 1. Figure 2. PMID:10680652

  4. Antimicrobial resistance of Actinobacillus pleuropneumoniae isolated from swine.

    PubMed

    Vanni, Michele; Merenda, Marianna; Barigazzi, Giuseppe; Garbarino, Chiara; Luppi, Andrea; Tognetti, Rosalba; Intorre, Luigi

    2012-04-23

    The aim of this retrospective study was to evaluate the antimicrobial resistance rates and the trend in resistance of Actinobacillus pleuropneumoniae isolated from pigs in Italy from 1994 to 2009. A total of 992 A. pleuropneumoniae isolates were tested for their susceptibility to a panel of antimicrobial agents in a disk diffusion method. Resistance to 7 drugs (amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, cotrimoxazole, penicillin G and tilmicosin) showed a significant increasing trend over the time, while for 2 drugs (gentamycin and marbofloxacin) a significant decrease was observed. Resistance to the remaining 14 antimicrobial agents tested did not change significantly over the study period. Most of the isolates retained high susceptibility to antimicrobials usually effective against A. pleuropneumoniae such as amphenicols, fluoroquinolones and ceftiofur. However, high rates of resistance were observed for potentiated sulfa drugs, tetracyclines and penicillins which are currently recommended antimicrobials for pig pleuropneumonia therapy. Our results suggest the importance of continued monitoring of A. pleuropneumoniae clinical isolates in order to choose the most appropriate treatment of infections and to control the increase of resistance to currently used antimicrobials. PMID:22104584

  5. Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo.

    PubMed Central

    Huang, H; Potter, A A; Campos, M; Leighton, F A; Willson, P J; Yates, W D

    1998-01-01

    To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions. PMID:9553707

  6. A cohort study on Actinobacillus pleuropneumoniae colonisation in suckling piglets.

    PubMed

    Tobias, T J; Klinkenberg, D; Bouma, A; van den Broek, J; Daemen, A J J M; Wagenaar, J A; Stegeman, J A

    2014-06-01

    Actinobacillus pleuropneumoniae causes respiratory disease in pigs and despite the use of preventive measures such as vaccination and antimicrobials clinical outbreaks still occur. At weaning often many piglets are not colonised. If differences in prevalence between litters are large and if factors were known that could explain these differences, this may provide an opportunity to raise groups of A. pleuropneumoniae free piglets. To this end, a cohort study was performed on two endemically infected farrow-to-finish farms. Seventy-six of 133 sows were selected using stratified random selection by parity. Farmers complied with a strict hygiene and animal management protocol to prevent transmission between litters. Tonsil brush and serum samples taken three weeks before parturition were tested for antigen with an apxIVA qPCR and antibodies with Apx and Omp ELISAs, respectively. Three days before weaning tonsil brush samples from all piglets (n=871) were collected and tested for antigen. Whereas all sows tested positive both in serology tests as well as qPCR, 0.41 of the litters tested fully negative and 0.73 of all piglets tested negative. The proportion of positively tested piglets in positive litters ranged from 0.08-1.0 (median=0.36). A grouped logistic regression model with a beta binomial distribution of the probability for piglets to become infected was fitted to the data and associations with explanatory variables were explored. To test the possibility that alternatively the clustering was caused by onwards transmission among the piglets, a transmission model was fitted to the data incorporating sow-piglet and piglet-piglet transmission, but this model did not fit better. The results of this study showed that the number of colonised suckling piglets was highly clustered and mainly attributable to the variability of infectiousness of the dam, but no dam related risk factor for colonisation status of litter or piglets within litters could be identified. PMID

  7. Preparation, characterization, and immunogenicity of conjugate vaccines directed against Actinobacillus pleuropneumoniae virulence determinants.

    PubMed

    Byrd, W; Kadis, S

    1992-08-01

    Conjugate vaccines were prepared in an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP). Two subunit conjugates were prepared by coupling the A. pleuropneumoniae 4074 serotype 1 capsular polysaccharide (CP) to the hemolysin protein (HP) and the lipopolysaccharide (LPS) to the HP. Adipic acid dihydrazide was used as a spacer to facilitate the conjugation in a carbodiimide-mediated reaction. The CP and the LPS were found to be covalently coupled to the HP in the conjugates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detergent gel chromatography analyses. Following a booster vaccination, pigs exhibited significantly high (P less than 0.05) immunoglobulin G antibodies against CP, LPS, and HP. The anti-CP and anti-LPS immunoglobulin G antibodies were found to function as opsonins in the phagocytosis of A. pleuropneumoniae by polymorphonuclear leukocytes, whereas antibodies to the HP neutralized the cytotoxic effect of the HP on polymorphonuclear leukocytes. No killing of A. pleuropneumoniae was observed when the effects of the antibodies were tested in the presence of complement. Thus, polysaccharide-protein A. pleuropneumoniae conjugates elicit significant antibody responses against each component of each conjugate, which could be instrumental in protecting swine against SPAP. PMID:1639471

  8. High-molecular-mass lipopolysaccharides are involved in Actinobacillus pleuropneumoniae adherence to porcine respiratory tract cells.

    PubMed Central

    Paradis, S E; Dubreuil, D; Rioux, S; Gottschalk, M; Jacques, M

    1994-01-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The major adhesin of A. pleuropneumoniae has been identified as the lipopolysaccharides (LPSs) (M. Bélanger, D. Dubreuil, J. Harel, C. Girard, and M. Jacques, Infect. Immun. 58:3523-3530, 1990). Using immunoelectron microscopy and flow cytometry, we showed in the present study that LPSs were well exposed at the surface of this encapsulated microorganism. Immunolocalization with porcine lung and tracheal frozen sections showed that extracted LPS bound to the lung mesenchyme and vascular endothelium and to the tracheal epithelium, respectively. Inhibition of adherence of A. pleuropneumoniae with extracted LPS was also performed with lung and tracheal frozen sections. Acid hydrolysis of LPS revealed that the active component of LPS was not lipid A but the polysaccharides. LPSs from A. pleuropneumoniae serotypes 1 and 2 were separated by chromatography on Sephacryl S-300 SF, in the presence of sodium deoxycholate, according to their molecular masses. The adherence-inhibitory activity was found in the high-molecular-mass fractions. These high-molecular-mass fractions contained 2-keto-3-deoxyoctulosonic acid and neutral sugars, and they were recognized by a monoclonal antibody directed against A. pleuropneumoniae O antigen but not recognized by a monoclonal antibody against capsular antigen. Images PMID:8039902

  9. Overexpression of Porcine Beta-Defensin 2 Enhances Resistance to Actinobacillus pleuropneumoniae Infection in Pigs

    PubMed Central

    Yang, Xi; Cheng, Yu-Ting; Tan, Mei-Fang; Zhang, Hua-Wei; Liu, Wan-Quan; Zou, Geng; Zhang, Liang-Sheng; Zhang, Chun-Yan; Deng, Si-Min; Yu, Lei; Hu, Xue-Ying; Li, Lu

    2015-01-01

    To reduce the need for antibiotics in animal production, alternative approaches are needed to control infection. We hypothesized that overexpression of native defensin genes will provide food animals with enhanced resistance to bacterial infections. In this study, recombinant porcine beta-defensin 2 (PBD-2) was overexpressed in stably transfected PK-15 porcine kidney cells. PBD-2 antibacterial activities against Actinobacillus pleuropneumoniae, an important respiratory pathogen causing porcine contagious pleuropneumonia, were evaluated on agar plates. Transgenic pigs constitutively overexpressing PBD-2 were produced by a somatic cell cloning method, and their resistance to bacterial infection was evaluated by direct or cohabitation infection with A. pleuropneumoniae. Recombinant PBD-2 peptide that was overexpressed in the PK-15 cells showed antibacterial activity against A. pleuropneumoniae. PBD-2 was overexpressed in the heart, liver, spleen, lungs, kidneys, and jejunum of the transgenic pigs, which showed significantly lower bacterial loads in the lungs and reduced lung lesions after direct or cohabitation infection with A. pleuropneumoniae. The results demonstrate that transgenic overexpression of PBD-2 in pigs confers enhanced resistance against A. pleuropneumoniae infection. PMID:25916992

  10. Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8.

    PubMed

    Li, Gang; Xie, Fang; Zhang, Yanhe; Bossé, Janine T; Langford, Paul R; Wang, Chunlai

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae. PMID:26509499

  11. Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8

    PubMed Central

    Li, Gang; Xie, Fang; Zhang, Yanhe; Bossé, Janine T.; Langford, Paul R.; Wang, Chunlai

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5’-diphosphate 3’-diphosphate (ppGpp) and guanosine 5’-triphosphate 3’-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae. PMID:26509499

  12. PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II.

    PubMed

    Hüssy, Daniela; Schlatter, Yvonne; Miserez, Raymond; Inzana, Thomas; Frey, Joachim

    2004-04-19

    A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae. PMID:15066734

  13. New plasmid tools for genetic analysis of Actinobacillus pleuropneumoniae and other pasteurellaceae.

    PubMed

    Bossé, Janine T; Durham, Andrew L; Rycroft, Andrew N; Kroll, J Simon; Langford, Paul R

    2009-10-01

    We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae. A tandem reporter plasmid, pMC-Tandem, carrying promoterless xylE and gfpmut3 genes downstream of a multiple-cloning site (MCS), can be used for identification of transcriptional regulators and conditions which favor gene expression from different cloned promoters. The ability to detect transcriptional regulators using the tandem reporter system was validated in A. pleuropneumoniae using the cloned rpoE (sigma(E)) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of sigma(E), among a bank of random transposon mutants, as well as to detect induction of sigma(E) following exposure of A. pleuropneumoniae to ethanol or heat shock. pMCsodCP, carrying the cloned sodC promoter of A. pleuropneumoniae, was functional in A. pleuropneumoniae, Haemophilus influenzae, Haemophilus parasuis, Mannheimia haemolytica, and Pasteurella multocida. Two general expression vectors, pMK-Express and pMC-Express, which differ in their antibiotic resistance markers (kanamycin and chloramphenicol, respectively), were constructed for the Pasteurellaceae. Both plasmids have the A. pleuropneumoniae sodC promoter upstream of the gfpmut3 gene and an extended MCS. Replacement of gfpmut3 with a gene of interest allows complementation and heterologous gene expression, as evidenced by expression of the Haemophilus ducreyi nadV gene in A. pleuropneumoniae, rendering the latter NAD independent. PMID:19666733

  14. Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catechol siderophores.

    PubMed Central

    Diarra, M S; Dolence, J A; Dolence, E K; Darwish, I; Miller, M J; Malouin, F; Jacques, M

    1996-01-01

    Siderophores bind ferric ions and are involved in receptor-specific iron transport into bacteria. Six types of siderophores were tested against strains representing the 12 different serotypes of Actinobacillus pleuropneumoniae. Ferrichrome and bis-catechol-based siderophores showed strong growth-promoting activities for A. pleuropneumoniae in a disk diffusion assay. Most strains of A. pleuropneumoniae tested were able to use ferrichrome (21 of 22 or 95%), ferrichrome A (20 of 22 or 90%), and lysine-based bis-catechol (20 of 22 or 90%), while growth of 36% (8 of 22) was promoted by a synthetic hydroxamate, N5-acetyl-N5-hydroxy-L-ornithine tripeptide. A. pleuropneumoniae serotype 1 (strain FMV 87-682) and serotype 5 (strain 2245) exhibited a distinct yellow halo around colonies on Chrome Azurol S agar plates, suggesting that both strains can produce an iron chelator (siderophore) in response to iron stress. The siderophore was found to be neither a phenolate nor a hydroxamate by the chemical tests of Arnow and Csaky, respectively. This is the first report demonstrating the production of an iron chelator and the use of exogenous siderophores by A. pleuropneumoniae. A spermidine-based bis-catechol siderophore conjugated to a carbacephalosporin was shown to inhibit growth of A. pleuropneumoniae. A siderophore-antibiotic-resistant strain was isolated and shown to have lost the ability to use ferrichrome, synthetic hydroxamate, or catechol-based siderophores when grown under conditions of iron restriction. This observation indicated that a common iron uptake pathway, or a common intermediate, for hydroxamate- and catechol-based siderophores may exist in A. pleuropneumoniae. PMID:8975614

  15. Proteins found within porcine respiratory tract secretions bind lipopolysaccharides of Actinobacillus pleuropneumoniae.

    PubMed Central

    Bélanger, M; Dubreuil, D; Jacques, M

    1994-01-01

    Affinity for porcine respiratory tract secretions was found in some isolates of Actinobacillus pleuropneumoniae and involved lipopolysaccharides (LPS) (M. Bélanger, S. Rioux, B. Foiry, and M. Jacques, FEMS Microbiol. Lett. 97:119-126, 1992). In the present study, the affinity for a crude preparation of porcine respiratory tract mucus of isolates of the Pasteurellaceae family, i.e., Actinobacillus, Haemophilus, and Pasteurella spp., and of some unrelated gram-negative bacteria was examined. Affinity for crude porcine respiratory tract mucus was not a property shared by all Pasteurellaceae isolates tested. Furthermore, affinity for the porcine crude mucus preparation was not unique to the Pasteurellaceae group and did not seem to be restricted to bacteria originating from pigs. Different surface properties of A. pleuropneumoniae isolates in relation to their adherence to crude mucus were examined. The capsular layer seemed to mask the adhesin and interfered with adherence to crude mucus. Two poorly capsulated isolates, which had a more hydrophobic surface and bound Congo red, were also heavily labeled by gold particles coated with polymyxin, which is known to interact with the lipid A-core region of LPS, and adhered strongly to respiratory tract secretions. Tetramethylurea, charged polymers, divalent cations, chelators, monosaccharides and amino sugars, or lectins were unable to inhibit adherence of A. pleuropneumoniae to the crude mucus preparation. To identify the receptor(s) recognized by the lipopolysaccharidic adhesin of A. pleuropneumoniae, affinity chromatography was used. Two bands, which were proteinaceous in nature, of 10 and 11 kDa were recovered. Our results suggest that two low-molecular-mass proteins present in porcine respiratory tract secretions bind A. pleuropneumoniae LPS. Images PMID:8112857

  16. Capsular polysaccharide antigens for detection of serotype-specific antibodies to Actinobacillus pleuropneumoniae.

    PubMed Central

    Bossé, J T; Johnson, R P; Rosendal, S

    1990-01-01

    Capsular polysaccharides (CPS) of serotypes 1, 2, 5 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae were obtained from 18 h culture supernatants by precipitation with hexadecyltrimethyl-ammonium bromide (Cetavlon) followed by extraction with sodium chloride and reprecipitation in ethanol. These crude extracts, and portions purified further by phenol extraction to remove contaminating proteins, were evaluated as antigens for the detection of serotype-specific antibodies to A. pleuropneumoniae in sera from immunized rabbits and swine by an enzyme-linked immunosorbent assay. The crude extracts reacted strongly with homologous antisera, but except for serotype 1 showed considerable cross-reactivity with antisera to other serotypes. Phenol extraction greatly improved the serospecificity of the antigens from serotypes 1, 7 and, to a lesser extent, 5. The serotype 2 CPS antigen showed poor reactivity following phenol extraction, and did not appear as useful for detection of serotype-specific antibodies. PMID:2379111

  17. Construction of Actinobacillus pleuropneumoniae-Escherichia coli shuttle vectors: expression of antibiotic-resistance genes.

    PubMed

    West, S E; Romero, M J; Regassa, L B; Zielinski, N A; Welch, R A

    1995-07-01

    We constructed several cloning vectors, designated pGZRS-18/19 and pGZRS-38/39, which were based on an endogenous Actinobacillus pleuropneumoniae (Apl) 4.3-kb plasmid. They carry the lacZ alpha-complementation fragment and MCS from pUC18/19, and either the bla gene under the control of a putative Apl promoter or the KmR gene from Tn903. These vectors replicate in representative strains of Apl serotypes 1 and 7, Escherichia coli, Pasteurella haemolytica (Ph) and Haemophilus (Actinobacillus) actinomycetemcomitans. We also found that Apl and Ph did not express genes under the control of the lacZ or bla promoters, suggesting that their RNA polymerases may not utilize these promoters. PMID:7628722

  18. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

    PubMed Central

    Hathroubi, S.; Hancock, M. A.; Langford, P. R.; Tremblay, Y. D. N.; Labrie, J.

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

  19. A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae.

    PubMed

    Rossi, Ciro C; Bossé, Janine T; Li, Yanwen; Witney, Adam A; Gould, Kate A; Langford, Paul R; Bazzolli, Denise M S

    2016-09-01

    Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies. PMID:27402897

  20. A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

    PubMed Central

    Rossi, Ciro C.; Bossé, Janine T.; Li, Yanwen; Witney, Adam A.; Gould, Kate A.; Langford, Paul R.; Bazzolli, Denise M.S.

    2016-01-01

    Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies. PMID:27402897

  1. Identification of Actinobacillus pleuropneumoniae Genes Preferentially Expressed During Infection Using In Vivo-Induced Antigen Technology (IVIAT).

    PubMed

    Zhang, Fei; Zhang, Yangyi; Wen, Xintian; Huang, Xiaobo; Wen, Yiping; Wu, Rui; Yan, Qigui; Huang, Yong; Ma, Xiaoping; Zhao, Qin; Cao, Sanjie

    2015-10-01

    Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivoinduced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae. PMID:26059519

  2. A Transcriptome Map of Actinobacillus pleuropneumoniae at Single-Nucleotide Resolution Using Deep RNA-Seq

    PubMed Central

    Su, Zhipeng; Zhu, Jiawen; Xu, Zhuofei; Xiao, Ran; Zhou, Rui; Li, Lu; Chen, Huanchun

    2016-01-01

    Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumoniae, a highly contagious respiratory disease of swine. Although the genome of A. pleuropneumoniae was sequenced several years ago, limited information is available on the genome-wide transcriptional analysis to accurately annotate the gene structures and regulatory elements. High-throughput RNA sequencing (RNA-seq) has been applied to study the transcriptional landscape of bacteria, which can efficiently and accurately identify gene expression regions and unknown transcriptional units, especially small non-coding RNAs (sRNAs), UTRs and regulatory regions. The aim of this study is to comprehensively analyze the transcriptome of A. pleuropneumoniae by RNA-seq in order to improve the existing genome annotation and promote our understanding of A. pleuropneumoniae gene structures and RNA-based regulation. In this study, we utilized RNA-seq to construct a single nucleotide resolution transcriptome map of A. pleuropneumoniae. More than 3.8 million high-quality reads (average length ~90 bp) from a cDNA library were generated and aligned to the reference genome. We identified 32 open reading frames encoding novel proteins that were mis-annotated in the previous genome annotations. The start sites for 35 genes based on the current genome annotation were corrected. Furthermore, 51 sRNAs in the A. pleuropneumoniae genome were discovered, of which 40 sRNAs were never reported in previous studies. The transcriptome map also enabled visualization of 5'- and 3'-UTR regions, in which contained 11 sRNAs. In addition, 351 operons covering 1230 genes throughout the whole genome were identified. The RNA-Seq based transcriptome map validated annotated genes and corrected annotations of open reading frames in the genome, and led to the identification of many functional elements (e.g. regions encoding novel proteins, non-coding sRNAs and operon structures). The transcriptional units described in this study

  3. Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae.

    PubMed Central

    Gerlach, G F; Anderson, C; Potter, A A; Klashinsky, S; Willson, P J

    1992-01-01

    An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4. Images PMID:1541562

  4. Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

    PubMed Central

    Bossé, Janine T.; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M.; Rogers, Jon; Chaudhuri, Roy R.; Weinert, Lucy A.; Oshota, Olusegun; Holden, Matt T. G.; Maskell, Duncan J.; Tucker, Alexander W.; Wren, Brendan W.; Rycroft, Andrew N.; Langford, Paul R.

    2015-01-01

    Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. PMID:25957382

  5. Modulation of Gene Expression in Actinobacillus pleuropneumoniae Exposed to Bronchoalveolar Fluid

    PubMed Central

    Lone, Abdul G.; Deslandes, Vincent; Nash, John H. E.; Jacques, Mario; MacInnes, Janet I.

    2009-01-01

    Background Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially expressed in a medium that mimics the lung environment early in the infection process. Methods and Principal Findings Since bronchoalveolar lavage fluid (BALF) contains innate immune and other components found in the lungs, we examined gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after a 30 min exposure to BALF, using DNA microarrays and real-time PCR. The functional classes of genes found to be up-regulated most often in BALF were those encoding proteins involved in energy metabolism, especially anaerobic metabolism, and in cell envelope, DNA, and protein biosynthesis. Transcription of a number of known virulence genes including apxIVA and the gene for SapF, a protein which is involved in resistance to antimicrobial peptides, was also up-regulated in BALF. Seventy-nine percent of the genes that were up-regulated in BALF encoded a known protein product, and of these, 44% had been reported to be either expressed in vivo and/or involved in virulence. Conclusions The results of this study suggest that in early stages of infection, A. pleuropneumoniae may modulate expression of genes involved in anaerobic energy generation and in the synthesis of proteins involved in cell wall biogenesis, as well as established virulence factors. Given that many of these genes are thought to be expressed in vivo or involved in virulence, incubation in BALF appears, at least partially, to simulate in vivo conditions and may provide a useful medium for the discovery of novel vaccine or therapeutic targets. PMID:19578537

  6. Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI) operon.

    PubMed Central

    Jansen, R; Briaire, J; Kamp, E M; Gielkens, A L; Smits, M A

    1993-01-01

    Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed. Images PMID:8359891

  7. Sub-inhibitory concentrations of penicillin G induce biofilm formation by field isolates of Actinobacillus pleuropneumoniae.

    PubMed

    Hathroubi, S; Fontaine-Gosselin, S-È; Tremblay, Y D N; Labrie, J; Jacques, M

    2015-09-30

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium and causative agent of porcine pleuropneumonia. This is a highly contagious disease that causes important economic losses to the swine industry worldwide. Penicillins are extensively used in swine production and these antibiotics are associated with high systemic clearance and low oral bioavailability. This may expose A. pleuropneumoniae to sub-inhibitory concentrations of penicillin G when the antibiotic is administered orally. Our goal was to evaluate the effect of sub-minimum inhibitory concentration (MIC) of penicillin G on the biofilm formation of A. pleuropneumoniae. Biofilm production of 13 field isolates from serotypes 1, 5a, 7 and 15 was tested in the presence of sub-MIC of penicillin G using a polystyrene microtiter plate assay. Using microscopy techniques and enzymatic digestion, biofilm architecture and composition were also characterized after exposure to sub-MIC of penicillin G. Sub-MIC of penicillin G significantly induced biofilm formation of nine isolates. The penicillin G-induced biofilms contained more poly-N-acetyl-D-glucosamine (PGA), extracellular DNA and proteins when compared to control biofilms grown without penicillin G. Additionally, penicillin G-induced biofilms were sensitive to DNase which was not observed with the untreated controls. Furthermore, sub-MIC of penicillin G up-regulated the expression of pgaA, which encodes a protein involved in PGA synthesis, and the genes encoding the envelope-stress sensing two-component regulatory system CpxRA. In conclusion, sub-MICs of penicillin G significantly induce biofilm formation and this is likely the result of a cell envelope stress sensed by the CpxRA system resulting in an increased production of PGA and other matrix components. PMID:26130517

  8. Evaluation and Field Validation of PCR Tests for Detection of Actinobacillus pleuropneumoniae in Subclinically Infected Pigs

    PubMed Central

    Fittipaldi, Nahuel; Broes, André; Harel, Josée; Kobisch, Marylène; Gottschalk, Marcelo

    2003-01-01

    Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (109 to 102 CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (102 CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at −20°C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (κ = 0.65) was found between the presence of A

  9. Serodiagnosis of pleuropneumonia using enzyme-linked immunosorbent assay with capsular polysaccharide antigens of Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7.

    PubMed Central

    Bossé, J T; Johnson, R P; Rosendal, S

    1990-01-01

    Capsular polysaccharide antigens of serotypes 1, 2, 5 and 7 of Actinobacillus pleuropneumoniae were used in enzyme-linked immunosorbent assays (ELISAs) to test sera from experimentally infected and field pigs. Specific reactions were found in sera of experimental pigs with antigens of serotypes 1, 5 and 7 whereas the serotype 2 antigen was cross-reactive. A 1:200 serum dilution was used for testing of 300 sera from 21 swine herds in southern Ontario. Cases of pleuropneumonia had occurred in 11 of these herds, but not in the others. The negative cut-off value was the mean optical density at 405 nm (OD405) + three standard deviations (SD) for 16 negative reference sera. Sera from four pigs naturally infected with Actinobacillus suis were tested and found to react to varying degrees with each of the antigens. Therefore a second cut-off value was determined as the mean OD405 + 2 SD for the A. suis sera. Sera which, in the ELISA produced OD readings above the latter cut-off were considered positive for antibodies to A. pleuropneumoniae; those which were lower than the former cut-off were considered negative. Readings between the two cut-off values may have been due to low positive titers or cross-reactivity, possibly with A. suis, and could not be used to predict pleuropneumonia. Of the pleuropneumonia-free herds, none had positive reactors to serotypes 5 or 7, whereas one and two herds had positive reactors to serotypes 1 and 2, respectively. Of the pleuropneumonia positive herds, six had positive reactors to serotype 1, one to serotype 2, four to serotype 5, and eight to serotype 7. PMID:2249177

  10. Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.

    PubMed

    Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

    2016-07-01

    Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production. PMID:26996628

  11. Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test.

    PubMed

    Morioka, Ayako; Shimazaki, Yoko; Uchiyama, Mariko; Suzuki, Shoko

    2016-05-01

    We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2. PMID:26726101

  12. Treatment of pigs experimentally infected with Mycoplasma hyopneumoniae, Pasteurella multocida, and Actinobacillus pleuropneumoniae with various antibiotics.

    PubMed Central

    Stipkovits, L; Miller, D; Glavits, R; Fodor, L; Burch, D

    2001-01-01

    The authors have performed a comparative study of the efficacy of various in-feed medications for the treatment of 5- to 6-week-old specific pathogen-free (SPF) piglets experimentally infected on day 1 with Mycoplasma hyopneumoniae, on day 8 with Pasteurella multocida (serotype A), and on day 15 with Actinobacillus pleuropneumoniae (serotype 2). The treatment started on day 9 and continued for 12 consecutive days, then the piglets were euthanized for examination of macroscopic, histologic, and pathologic lesions and for the presence of mycoplasmas and bacteria in the lungs. Based on the results of clinical observations (respiratory signs, rectal temperature, body weight gain, and feed conversion efficiency), macroscopic and histologic lesions of the lungs, and microbiologic findings, the best results were obtained by treatment of pigs with Econor + chlortetracycline, followed by Tetramutin, Pulmotil, Cyfac, and lincomycin + chlortetracycline. PMID:11768127

  13. Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test

    PubMed Central

    MORIOKA, Ayako; SHIMAZAKI, Yoko; UCHIYAMA, Mariko; SUZUKI, Shoko

    2016-01-01

    We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2. PMID:26726101

  14. Characterization of an Actinobacillus pleuropneumoniae seeder pig challenge-exposure model.

    PubMed

    Lechtenberg, K F; Shryock, T R; Moore, G

    1994-12-01

    Five strains of Actinobacillus pleuropneumoniae serotype 1 were used to intranasally infect 5 groups of pigs. Using each bacterial strain, infected pigs (termed seeder pigs) were commingled for 48 hours with 5 groups of noninfected test pigs, then were removed. Seeder and test pigs were maintained in isolation and were observed for 14 days. Seeder pigs had mortality that was threefold greater than that of test pigs (24% vs 8%). Rectal temperature in excess of 40.3 C was achieved for 84% of test pigs and 88% of seeder pigs. Neither of these 2 variables was statistically different between the 2 groups of pigs. Clinical impression scores > or = 2 (on a 0 to 3 scale) were three-fold (64% vs 20%) greater for seeder than for test pigs (P < 0.05). The total number of bacterial isolations or nonrecoverable isolates was tabulated for test and seeder pigs' lungs at necropsy, irrespective of the amount of lesions. The number of A pleuropneumoniae isolations was not statistically different between test and seeder pig populations. Recovery of Pasteurella multocida or other bacteria was greater from the seeder pigs (P < 0.05), whereas the number of non-recoverable isolates was greater from test pigs than from seeder pigs (P < 0.05). Assessment of lung lesions at necropsy by either visual estimation or on a weight basis were in agreement.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7887514

  15. Experimental Actinobacillus pleuropneumoniae challenge in swine: Comparison of computed tomographic and radiographic findings during disease

    PubMed Central

    2012-01-01

    Background In pigs, diseases of the respiratory tract like pleuropneumonia due to Actinobacillus pleuropneumoniae (App) infection have led to high economic losses for decades. Further research on disease pathogenesis, pathogen-host-interactions and new prophylactic and therapeutic approaches are needed. In most studies, a large number of experimental animals are required to assess lung alterations at different stages of the disease. In order to reduce the required number of animals but nevertheless gather information on the nature and extent of lung alterations in living pigs, a computed tomographic scoring system for quantifying gross pathological findings was developed. In this study, five healthy pigs served as control animals while 24 pigs were infected with App, the causative agent of pleuropneumonia in pigs, in an established model for respiratory tract disease. Results Computed tomographic (CT) findings during the course of App challenge were verified by radiological imaging, clinical, serological, gross pathology and histological examinations. Findings from clinical examinations and both CT and radiological imaging, were recorded on day 7 and day 21 after challenge. Clinical signs after experimental App challenge were indicative of acute to chronic disease. Lung CT findings of infected pigs comprised ground-glass opacities and consolidation. On day 7 and 21 the clinical scores significantly correlated with the scores of both imaging techniques. At day 21, significant correlations were found between clinical scores, CT scores and lung lesion scores. In 19 out of 22 challenged pigs the determined disease grades (not affected, slightly affected, moderately affected, severely affected) from CT and gross pathological examination were in accordance. Disease classification by radiography and gross pathology agreed in 11 out of 24 pigs. Conclusions High-resolution, high-contrast CT examination with no overlapping of organs is superior to radiography in the

  16. ICEApl1, an Integrative Conjugative Element Related to ICEHin1056, Identified in the Pig Pathogen Actinobacillus pleuropneumoniae

    PubMed Central

    Bossé, Janine T.; Li, Yanwen; Fernandez Crespo, Roberto; Chaudhuri, Roy R.; Rogers, Jon; Holden, Matthew T. G.; Maskell, Duncan J.; Tucker, Alexander W.; Wren, Brendan W.; Rycroft, Andrew N.; Langford, Paul R.; Consortium, the BRaDP1T

    2016-01-01

    ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia. PMID:27379024

  17. Comparison of conventional and long-acting oxytetracyclines in prevention of induced Actinobacillus (Haemophilus) pleuropneumoniae infection of growing swine.

    PubMed Central

    Kiorpes, A L; Bäckström, L R; Collins, M T; Kruse, G O

    1989-01-01

    These experiments tested the hypothesis that long-acting oxytetracycline (oxytetracycline-LA) was more effective than regular oxytetracycline in preventing porcine pleuropneumonia when administered either 24 or 48 h prior to experimental challenge with virulent strains of Actinobacillus pleuropneumoniae. Two experiments (1 and 2) were conducted using growing pigs (average weight 12-15 kg). Antibiotic treatments were administered once intramuscularly at 20 mg/kg body weight; controls received an equivalent volume of saline. Clinical signs were recorded over seven days, and mortality rates and pathological lesions were analyzed using analysis of variance. Serum oxytetracycline levels were compared 48 and 72 h postinjection. All pigs developed clinical disease following experimental infection. Actinobacillus pleuropneumoniae was recovered from 42% of experiment 1 pigs and all of experiment 2 pigs. The data showed that both oxytetracycline and oxytetracycline-LA given at the same dose protected pigs against experimental infection when given 24 h prior to challenge, and there was no difference between the efficacy of the two drugs in this experiment. When administered 48 h prior to challenge, only oxytetracycline-LA reduced the clinical signs and pathological changes following A. pleuropneumoniae challenge. Between 48 and 72 h postinjection, oxytetracycline-LA blood levels were significantly greater compared to oxytetracycline-treated pigs. PMID:2531629

  18. [Construction and immunogenicity of an attenuated mutant of Actinobacillus pleuropneumoniae by insertional inactivation of apxIC].

    PubMed

    Xu, Fu-Zhou; Shi, Ai-Hua; Chen, Xiao-Ling; Yang, Bing; Wang, Jin-Luo

    2007-10-01

    Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia. Apx toxin, an exotoxin secreted by A. pleuropneumoniae, is one of the most important virulence factors. To construct an avirulent mutant strain by inactivation of ApxI toxin, the apxIC gene of A. pleuropneumoniae serovar 10 was inactivated by inserting a chloramphenicol resistance gene cassette into the downstream XhoI site of the apxIC gene for constructing the transfer plasmid. The transfer plasmid was introduced into the electrocompetent A. pleuropneumoniae serovar 10 for homologous recombination by electroporation. The mutant strain was obtained and identified by PCR and Southern blotting. The mutant strain was phenotypically identical to the parent strain except that it showed no haemolytic activity. The mutant strain was also able to secret the same ApxI toxin as the parent strain. In the intra-peritoneal mouse model, the virulence of the mutant strain decreased at least 100 fold compared with the parent strain. The mutant was evaluated as a potential vaccine using a vaccination-challenge trial in which pigs were given two intra-nasal doses of the mutant with 14 days' interval and then challenged 14 days after the last vaccination with A. pleuropneumoniae serovar 1 and serovar 10 reference strains respectively. The death number and lung lesion score in the vaccinated pigs given the serovar 1 challenge were obviously lower than those in the unvaccinated pigs. And the lower lung lesion score was also observed in the vaccinated pigs challenged with serovar 10. And the positive numbers of A. pleuropneumoniae re-isolation and PCR detection showed the same consistency. The vaccination-challenge trial suggested that the mutant strain could offer partial cross-protection as a live attenuated vaccine against A . pleuropneumoniae infection. PMID:18062275

  19. Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae.

    PubMed

    Luna-Castro, Sarahí; Aguilar-Romero, Francisco; Samaniego-Barrón, Luisa; Godínez-Vargas, Delfino; de la Garza, Mireya

    2014-10-01

    Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24-42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60-70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App. PMID:24878848

  20. Secreted proteases from Actinobacillus pleuropneumoniae serotype 1 degrade porcine gelatin, hemoglobin and immunoglobulin A.

    PubMed Central

    Negrete-Abascal, E; Tenorio, V R; Serrano, J J; Garcia, C; de la Garza, M

    1994-01-01

    It was found that 48 hour cultures of Actinobacillus pleuropneumoniae secreted proteases into the medium. Electrophoresis in polyacrylamide gels (10%) copolymerized with porcine gelatin (0.1%), of the 70% (NH4)2SO4 precipitate from the culture supernatants, displayed protease activities of different molecular weights: > 200, 200, 90, 80, 70 and 50 kDa. They had activity over a broad range of pHs (4-8), with an optimal pH of 6-7. All were inhibited by 10 mM EDTA, and reactivated by 10 mM calcium. They were stable at -20 degrees C for more than a month. The proteases also degraded porcine IgA and porcine, human, and bovine hemoglobin, although they appeared to be less active against the hemoglobins. The IgA was totally cleaved in 48 h, using supernatants concentrated with polyvinyl pyrrolidone or the 70% (NH4)2SO4. Extracellular proteases could play a role in virulence. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:8004545

  1. [Generation of nalidixic acid-resistant strains and signature-tagged mutants of Actinobacillus pleuropneumoniae].

    PubMed

    Shang, Lin; Li, Wei; Li, Liangjun; Li, Lu; Zhang, Sihua; Li, Tingting; Li, Yaokun; Liu, Lei; Guo, Zhiwei; Zhou, Rui; Chen, Huanchun

    2008-01-01

    Actinobacillus pleuropneumoniae is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Signature-tagged mutagenesis (STM) is an effective method to identify virulence genes in bacteria. In this study, we selected nalidixic acid-resistant strains of APP serotypes 1 and 3 by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CC 118 lambdapir or S17-1 lambdapir containing mini-Tn10 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5alpha (pRK2073). We screened mutant strains by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains could easily be induced in vitro and the resistance was due to the mutation in the DNA gyrase A subunit gene gyrA. In the mating experiments, the bi-parental mating was more effective and easier than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data were helpful for the construction of STM mutants and pickup of virulence genes of APP. PMID:18338580

  2. Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA.

    PubMed

    Dreyfus, A; Schaller, A; Nivollet, S; Segers, R P A M; Kobisch, M; Mieli, L; Soerensen, V; Hüssy, D; Miserez, R; Zimmermann, W; Inderbitzin, F; Frey, J

    2004-04-19

    Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N'-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N'-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App were serologically negative in the ApxIV-ELISA. PMID:15066725

  3. Transcriptional Portrait of Actinobacillus pleuropneumoniae during Acute Disease - Potential Strategies for Survival and Persistence in the Host

    PubMed Central

    Klitgaard, Kirstine; Friis, Carsten; Jensen, Tim K.; Angen, Øystein; Boye, Mette

    2012-01-01

    Background Gene expression profiles of bacteria in their natural hosts can provide novel insight into the host-pathogen interactions and molecular determinants of bacterial infections. In the present study, the transcriptional profile of the porcine lung pathogen Actinobacillus pleuropneumoniae was monitored during the acute phase of infection in its natural host. Methodology/Principal Findings Bacterial expression profiles of A. pleuropneumoniae isolated from lung lesions of 25 infected pigs were compared in samples taken 6, 12, 24 and 48 hours post experimental challenge. Within 6 hours, focal, fibrino hemorrhagic lesions could be observed in the pig lungs, indicating that A. pleuropneumoniae had managed to establish itself successfully in the host. We identified 237 differentially regulated genes likely to encode functions required by the bacteria for colonization and survival in the host. This group was dominated by genes involved in various aspects of energy metabolism, especially anaerobic respiration and carbohydrate metabolism. Remodeling of the bacterial envelope and modifications of posttranslational processing of proteins also appeared to be of importance during early infection. The results suggested that A. pleuropneumoniae is using various strategies to increase its fitness, such as applying Na+ pumps as an alternative way of gaining energy. Furthermore, the transcriptional data provided potential clues as to how A. pleuropneumoniae is able to circumvent host immune factors and survive within the hostile environment of host macrophages. This persistence within macrophages may be related to urease activity, mobilization of various stress responses and active evasion of the host defenses by cell surface sialylation. Conclusions/Significance The data presented here highlight the importance of metabolic adjustments to host conditions as virulence factors of infecting microorganisms and help to provide insight into the mechanisms behind the efficient

  4. Effect of tulathromycin on the carrier status of Actinobacillus pleuropneumoniae serotype 2 in the tonsils of pigs.

    PubMed

    Angen, Ø; Andreasen, M; Nielsen, E O; Stockmarr, A; Baekbo, P

    2008-10-11

    The effect of a single or double dose of tulathromycin was evaluated in pigs carrying Actinobacillus pleuropneumoniae serotype 2 in their tonsils. Twenty-nine pigs from a reinfected specific pathogen-free-herd were selected from animals testing positive in an A pleuropneumoniae serotype 2-specific pcr test on tonsil scrapings and they were divided into three groups. The pigs in group 1 were treated subcutaneously with 2.5 mg/kg tulathromycin on day 0, the pigs in group 2 were treated with 2.5 mg/kg tulathromycin on days 0 and 4, and the pigs in group 3 were left untreated as controls. The pigs were tested by pcr on tonsil scrapings on days 0, 4, 11 and 33, and on day 33 all the animals were euthanased. There were no significant differences between the numbers of PCR-positive animals in the three groups on any of the sampling dates. PMID:18849576

  5. Construction of a broad host range shuttle vector for gene cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae.

    PubMed

    Frey, J

    1992-01-01

    We have constructed a pair of broad host range expression vectors, pJFF224-NX and pJFF224-XN, based on plasmid RSF1010, which enable cloning and efficient expression of genes in Actinobacillus pleuropneumoniae and Pasteurella haemolytica and in Escherichia coli. The vectors consist of the minimal autonomous replicon of the broad host range plasmid RSF1010 and a type II chloramphenicol acetyl transferase gene for chloramphenicol resistance selection. In addition, they contain a gene expression cassette based on the E. coli bacteriophage T4 gene 32 promoter region and a transcription stop signal, which are separated by a segment of multiple cloning sites in both orientations. Electroporation and subsequent selection for chloramphenicol resistance was used for the introduction of the vectors in A. pleuropneumoniae and P. haemolytica. A promoterless xy/E gene from the Pseudomonas putida TOL plasmid was cloned onto pJFF224-NX. This plasmid enabled efficient expression of active catechol2,3oxygenase in A. pleuropneumoniae and P. haemolytica. It was stably maintained in A. pleuropneumoniae without antibiotic selection, showing less than 0.1% loss after 100 generations, while native RSF1010 and other RSF1010-based vectors were unstable in this host. PMID:1448612

  6. Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2.

    PubMed

    MacDonald, J; Rycroft, A N

    1992-07-01

    The genetic determinants of the 120-kDa cytotoxin of Actinobacillus pleuropneumoniae serotype 2 were isolated from a lambda DNA library by a plaque immunoblot technique. Expression of the 120-kDa polypeptide was confirmed by Western immunoblot analysis of infected Escherichia coli cell lysates, which were shown to be toxic for porcine alveolar macrophages in vitro. The genetic determinants of the toxin were subcloned into the plasmid vector pUC18. This plasmid (pPTX1) directed the synthesis and secretion of the active 120-kDa cytotoxin in E. coli. The recombinant toxin was indistinguishable from native cytotoxin from A. pleuropneumoniae serotype 2 with respect to molecular size, reaction in Western blot analysis, heat lability, cytotoxic activity, and neutralization by serum antibody. A restriction endonuclease cleavage map of pPTX1 was prepared, and deletion mutants were used to locate the minimal region of DNA required for production of intracellular toxin; this gene was termed ptxA. Southern hybridization analysis with a 1.7-kb PvuII fragment located within the ptxA gene revealed sequences with a high degree of homology in serotype reference strains 2, 3, 4, 6, and 8. Other reference strains did not contain sequences that were recognized by this probe. However, related sequences (greater than 71% homology) were detected in Actinobacillus actinomycetemcomitans and A. equuli. Weak hybridization was observed between the ptxA probe and pLKT5, which carries the lktAC genes of Pasteurella haemolytica, and between the ptxA probe and pAPH1, which carries the structural gene for type II hemolysin from A. pleuropneumoniae. The isolation of the genetic determinants of this cytotoxin will enable investigations of the structure and organization of the ptx DNA region and further analysis of its role in the pathogenesis of pleuropneumonia. PMID:1612740

  7. The ClpP Protease Is Required for the Stress Tolerance and Biofilm Formation in Actinobacillus pleuropneumoniae

    PubMed Central

    Xie, Fang; Zhang, Yanhe; Li, Gang; Zhou, Long; Liu, Siguo; Wang, Chunlai

    2013-01-01

    In the respiratory tract and lung tissue, a balanced physiological response is essential for Actinobacillus pleuropneumoniae to survive various types of challenges. ClpP, the catalytic core of the Clp proteolytic complex, is involved in various stresses response and regulation of biofilm formation in many pathogenic bacteria. To investigate the role of ClpP in the virulence of A. pleuropneumoniae, the clpP gene was deleted by homologous recombination, resulting in the mutant strain S8ΔclpP. The reduced growth of S8ΔclpP mutant at high temperatures and under several other stress conditions suggests that the ClpP protein is required for the stress tolerance of A. pleuropneumoniae. Interestingly, we observed that the S8ΔclpP mutant exhibited an increased ability to take up iron in vitro compared to the wild-type strain. We also found that the cells without ClpP displayed rough and irregular surfaces and increased cell volume relative to the wild-type strain using scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) revealed that the S8ΔclpP mutant showed decreased biofilm formation compared to the wild-type strain. We examined the transcriptional profiles of the wild type S8 and the S8ΔclpP mutant strains of A. pleuropneumoniae using RNA sequencing. Our analysis revealed that the expression of 16 genes was changed by the deletion of the clpP gene. The data presented in this study illustrate the important role of ClpP protease in the stress response, iron acquisition, cell morphology and biofilm formation related to A. pleuropneumoniae and further suggest a putative role of ClpP protease in virulence regulation. PMID:23326465

  8. Pharmacokinetics of tulathromycin in edible tissues of healthy and experimentally infected pigs with Actinobacillus pleuropneumoniae.

    PubMed

    Bladek, Tomasz; Posyniak, Andrzej; Jablonski, Artur; Gajda, Anna

    2015-01-01

    The aim of this study was the comparison of the tissue pharmacokinetics of tulathromycin in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (App). Tulathromycin was given to 24 healthy and 24 infected pigs by intramuscular injection at a single dosage of 2.5 mg kg(-1) body weight (b.w.). Pigs were euthanised at each group and then samples of liver, kidney, muscle, injection site and skin with fat were taken at scheduled time points. Drug concentrations were determined by LC-MS/MS. In this study, higher values of the area under the concentration-time curves (AUC) were calculated in all tissue samples taken from infected than healthy pigs. In pigs with App the AUCs of liver, kidney, muscle, skin with fat and injection site were 1111, 1973, 235, 181 and 2931 mg kg(-1) h, while in pigs without inflammation they were 509, 1295, 151, 111 and 1587 mg kg(-1) h, respectively. Maximum drug tissue concentrations (Cmax) in infected animals were 2370, 6650, 2016, 666 and 83,870 µg kg(-1), while in healthy pigs they were 1483, 6677, 1733, 509 and 55,006 µg kg(-1), respectively. The eliminations half-times (T1/2) were respectively longer in all tissue samples taken from infected animals (from 157.3 to 187.3 h) than in healthy ones (from 138.6 to 161.2 h). The tulathromycin tissue concentrations were significantly higher (p < 0.05) in all tissue samples of the infected pigs compared with the healthy animals at 360 h (from 0.0014 to 0.0280) and at 792 h (from 0.0007 to 0.0242) after drug administration. The results suggest that the tissue pharmacokinetic properties and residue depletion of tulathromycin can be influenced by the disease state of animals. PMID:26247868

  9. Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

    PubMed Central

    Labrie, Josée; Hernandez Reyes, Yenney; Burciaga Nava, Jorge A.; Gagnon, Carl A.; Jacques, Mario

    2014-01-01

    Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools. PMID:24878741

  10. malT knockout mutation invokes a stringent type gene-expression profile in Actinobacillus pleuropneumoniae in bronchoalveolar fluid

    PubMed Central

    2009-01-01

    Background Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min. Results In reverse transcription PCR differential display (RT-PCR DD) experiments, A. pleuropneumoniae CM5 exposed to BALF up-regulated, among other genes, a gene predicted to encode LamB, an outer-membrane transport protein of the maltose regulon. To determine the role of the lamB and other genes of the maltose regulon in the pathogenesis of A. pleuropneumoniae, knockout mutations were created in the lamB and malT genes, the latter being the positive transcriptional regulator of the maltose regulon. Relative to the lamB mutant and the wild type, the malT mutant had a significant (P < 0.05) decrease in growth rate and an increased sensitivity to fresh porcine serum and high concentrations (more than 0.5 M) of sodium chloride. In DNA microarray experiments, the BALF-exposed malT mutant exhibited a gene-expression profile resembling that of a stringent type gene-expression profile seen in bacteria facing amino acid or carbon starvation. Genes encoding proteins for protein synthesis, energy metabolism, and DNA replication were down-regulated, while genes involved in stringent response (e.g., relA), amino acid and nucleotide biosynthesis, biofilm formation, DNA transformation, and stress response were up-regulated. Conclusion These results suggest that MalT may be involved in protection against some stressors and in the transport of one or more essential nutrients in BALF. Moreover, if MalT is directly or indirectly linked to the stringent response, an important

  11. Actinobacillus pleuropneumoniae genes expression in biofilms cultured under static conditions and in a drip-flow apparatus

    PubMed Central

    2013-01-01

    Background Actinobacillus pleuropneumoniae is the Gram-negative bacterium responsible for porcine pleuropneumonia. This respiratory infection is highly contagious and characterized by high morbidity and mortality. The objectives of our study were to study the transcriptome of A. pleuropneumoniae biofilms at different stages and to develop a protocol to grow an A. pleuropneumoniae biofilm in a drip-flow apparatus. This biofilm reactor is a system with an air-liquid interface modeling lung-like environment. Bacteria attached to a surface (biofilm) and free floating bacteria (plankton) were harvested for RNA isolation. Labelled cDNA was hybridized to a microarray to compare the expression profiles of planktonic cells and biofilm cells. Results It was observed that 47 genes were differentially expressed (22 up, 25 down) in a 4 h-static growing/maturing biofilm and 117 genes were differentially expressed (49 up, 68 down) in a 6h-static dispersing biofilm. The transcriptomes of a 4 h biofilm and a 6 h biofilm were also compared and 456 genes (235 up, 221 down) were identified as differently expressed. Among the genes identified in the 4 h vs 6h biofilm experiment, several regulators of stress response were down-regulated and energy metabolism associated genes were up-regulated. Biofilm bacteria cultured using the drip-flow apparatus differentially expressed 161 genes (68 up, 93 down) compared to the effluent bacteria. Cross-referencing of differentially transcribed genes in the different assays revealed that drip-flow biofilms shared few differentially expressed genes with static biofilms (4 h or 6 h) but shared several differentially expressed genes with natural or experimental infections in pigs. Conclusion The formation of a static biofilm by A. pleuropneumoniae strain S4074 is a rapid process and transcriptional analysis indicated that dispersal observed at 6 h is driven by nutritional stresses. Furthermore, A. pleuropneumoniae can form a biofilm under low

  12. Construction and immunogenicity of a ∆apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis.

    PubMed

    Liu, Qiong; Gong, Yuheng; Cao, Yuqin; Wen, Xintian; Huang, Xiaobo; Yan, Qigui; Huang, Yong; Cao, Sanjie

    2013-01-01

    The apxIC genes of the Actinobacillus pleuropneumoniae serovar 5 (SC-1), encoding the ApxIactivating proteins, was deleted by a method involving sucrose counter-selection. In this study, a mutant strain of A. pleuropneumoniae (SC-1) was constructed and named DapxIC/ ompP2. The mutant strain contained foreign DNA in the deletion site of ompP2 gene of Haemophilus parasuis. It showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice. The LD50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (LD50 of the mutant strain and parent strain in mice were determined to be 1.0 × 10(7) CFU and 3.5 × 10(5) CFU respectively). The mutant strain that was attenuated could secrete inactivated ApxIA RTX toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of A. pleuropneumoniae and H. parasuis. PMID:23718128

  13. Viability of Actinobacillus pleuropneumoniae in frozen pig lung samples and comparison of different methods of direct diagnosis in fresh samples.

    PubMed

    Gutierrez, C B; Rodriguez Barbosa, J I; Gonzalez, O R; Tascon, R I; Rodriguez Ferri, E F

    1992-04-01

    A comparative study on different methods of diagnosis of Actinobacillus pleuropneumoniae from both fresh and frozen pig lungs is described. A total of 196 lung tissues with pneumonic lesions were examined for culture isolation on chocolate blood agar, as well as for antigen detection by means of the coagglutination test, the immunodiffusion test and the indirect ELISA. These samples were subsequently frozen for 1 yr and then they were recultured. A. pleuropneumoniae was recovered from fresh lung specimens in 30 cases (15.3%) and from frozen samples in only two cases (0.9%). Such a different degree of isolation demonstrates that long freezing had an adverse effect on the viability of this organism in lung samples. A pleuropneumoniae detection was positive in 134 samples (68.4%) by at least one of the immunological techniques examined. The indirect ELISA was the most sensitive and specific test, with antigen detected in 125 lungs (63.8%). In comparison with the coagglutination and immunodiffusion tests, the sensitivities of the indirect ELISA were 95.8 and 93.7%, and the specificities were 67.0 and 63.4%, respectively. PMID:1563263

  14. Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

    PubMed Central

    Negrete-Abascal, E; Tenorio, V R; Guerrero, A L; García, R M; Reyes, M E; de la Garza, M

    1998-01-01

    A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae. Images Figure 2A. Figure 2B. Figure 3. Figure 4. Figure 5A. Figure 5B. Figure 6A. Figure 6B. PMID:9684047

  15. Comparative in vitro activity of 16 antimicrobial agents against Actinobacillus pleuropneumoniae.

    PubMed

    Yoshimura, H; Takagi, M; Ishimura, M; Endoh, Y S

    2002-01-01

    Sixteen antimicrobial agents were tested for their activity against 68 isolates of Actinobacillus pleuropneumoniae by determining the minimum inhibitory concentrations (MICs). Ceftiofur and the fluoroquinolones danofloxacin and enrofloxacin were the most active compounds, with a MIC for 90% of the isolates (MIC90) of (0.05 microg/ml. The MIC90 values of benzylpenicillin, amoxicillin and aspoxicillin were 0.78 units/ml, 0.39 microg/ml and < or = 0.05 microg/ml, respectively. Three isolates (4.4%) were resistant to penicillins, but aspoxicillin was as active as ceftiofur against the susceptible isolates, with MICs of < or = 0.05 microg/ml for all isolates. Resistance to oxytetracycline, chloramphenicol and thiamphenicol occurred in 22 (32.4%), 14 (20.6%) and 15 (22.1%) of the isolates, respectively. Doxycycline was more active than oxytetracycline, with a MIC90 of 1.56 microg/ml as against 25 microg/ml. Florfenicol was not only as active as thiamphenicol, with a MIC for 50% of the isolates (MIC50) of 0.39 microg/ml, but also active against thiamphenicol-resistant isolates. All the isolates were susceptible to florfenicol. All the isolates were also susceptible to gentamicin, spectinomycin, tilmicosin, colistin and tiamulin. Of these, spectinomycin was the least active, with a MIC50 of 25 microg/ml, followed by tiamulin, with a MIC50 of 6.25 microg/ml. Of the 68 isolates tested, 49 (72.0%) were of serotype 2; 14 (20.5%) were of serotype 1; 2 each (3.0%) were of serotypes 5 and 6; and one was of serotype 7. Of the isolates, 23 (33.8%) were resistant to one or more of the major antibiotics. Antibiotic resistance was found only infrequently among serotype 2, with 5 (10.2%) of 49 isolates being resistant to chloramphenicol and/or oxytetracycline, while it occurred in 18 (94.7%) of the 19 isolates of other serotypes. PMID:11860083

  16. Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli*

    PubMed Central

    Naegeli, Andreas; Neupert, Christine; Fan, Yao-Yun; Lin, Chia-Wei; Poljak, Kristina; Papini, Anna Maria; Schwarz, Flavio; Aebi, Markus

    2014-01-01

    N-Linked protein glycosylation is a frequent post-translational modification that can be found in all three domains of life. In a canonical, highly conserved pathway, an oligosaccharide is transferred by a membrane-bound oligosaccharyltransferase from a lipid donor to asparagines in the sequon NX(S/T) of secreted polypeptides. The δ-proteobacterium Actinobacillus pleuropneumoniae encodes an unusual pathway for N-linked protein glycosylation. This pathway takes place in the cytoplasm and is mediated by a soluble N-glycosyltransferase (NGT) that uses nucleotide-activated monosaccharides to glycosylate asparagine residues. To characterize the process of cytoplasmic N-glycosylation in more detail, we studied the glycosylation in A. pleuropneumoniae and functionally transferred the glycosylation system to Escherichia coli. N-Linked glucose specific human sera were used for the analysis of the glycosylation process. We identified autotransporter adhesins as the preferred protein substrate of NGT in vivo, and in depth analysis of the modified sites in E. coli revealed a surprisingly relaxed peptide substrate specificity. Although NX(S/T) is the preferred acceptor sequon, we detected glycosylation of alternative sequons, including modification of glutamine and serine residues. We also demonstrate the use of NGT to glycosylate heterologous proteins. Therefore, our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria. PMID:24275653

  17. Genomic relatedness among Actinobacillus pleuropneumoniae field strains of sterotypes 1 and 5 isolated from healthy and diseased pigs.

    PubMed Central

    Chatellier, S; Harel, J; Dugourd, D; Chevallier, B; Kobisch, M; Gottschalk, M

    1999-01-01

    Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the molecular epidemiology of swine pleuropneumonia. Images Figure 1. Figure 3. PMID:10480458

  18. Impact of Actinobacillus pleuropneumoniae biofilm mode of growth on the lipid A structures and stimulation of immune cells.

    PubMed

    Hathroubi, Skander; Beaudry, Francis; Provost, Chantale; Martelet, Léa; Segura, Mariela; Gagnon, Carl A; Jacques, Mario

    2016-07-01

    Actinobacillus pleuropneumoniae (APP), the etiologic agent of porcine pleuropneumonia, forms biofilms on biotic and abiotic surfaces. APP biofilms confers resistance to antibiotics. To our knowledge, no studies have examined the role of APP biofilm in immune evasion and infection persistence. This study was undertaken to (i) investigate biofilm-associated LPS modifications occurring during the switch to biofilm mode of growth; and (ii) characterize pro-inflammatory cytokines expression in porcine pulmonary alveolar macrophages (PAMs) and proliferation in porcine PBMCs challenged with planktonic or biofilm APP cells. Extracted lipid A samples from biofilm and planktonic cultures were analyzed by HPLC high-resolution, accurate mass spectrometry. Biofilm cells displayed significant changes in lipid A profiles when compared with their planktonic counterparts. Furthermore, in vitro experiments were conducted to examine the inflammatory response of PAMs exposed to UV-inactivated APP grown in biofilm or in suspension. Relative mRNA expression of pro-inflammatory genes IL1, IL6, IL8 and MCP1 decreased in PAMs when exposed to biofilm cells compared to planktonic cells. Additionally, the biofilm state reduced PBMCs proliferation. Taken together, APP biofilm cells show a weaker ability to stimulate innate immune cells, which could be due, in part, to lipid A structure modifications. PMID:27226465

  19. Susceptibility testing of Actinobacillus pleuropneumoniae in Denmark. Evaluation of three different media of MIC-determinations and tablet diffusion tests.

    PubMed

    Aarestrup, F M; Jensen, N E

    1999-02-12

    This study was conducted to compare the applicability of three different media in sensitivity testing of Actinobacillus pleuropneumoniae by means of MIC and tablet diffusion tests. The media used were: modified PPLO agar, chocolatized Mueller-Hinton-II and Columbia agar supplemented with NAD. Seven antimicrobial agents were tested: ceftiofur, enrofloxacin, penicillin, spectinomycin, tiamulin, trimethoprim + sulfadiazine and tylosin, against 40 randomly selected A. pleuropneumoniae isolates. In general, good agreement was found between results obtained with all combinations of media, most antimicrobials tested and the two-test systems. Some variations between media were observed for spectinomycin, tiamulin and tylosin. For ceftiofur and trimethoprim + sulfadiazine some isolates with low MIC-values were classified as resistant using tablet diffusion, indicating that the break points of resistance for these antimicrobials using the tablet diffusion tests need adjustment. Using current break points for resistance with MIC-determinations, all isolates tested susceptible to ceftiofur, enrofloxacin, penicillin, tiamulin and trimethoprim + sulfadiazine. A larger number of isolates tested resistant to spectinomycin and tylosin on all three media using both MIC determinations and tablet diffusion. PMID:10063535

  20. Economic impacts of reduced pork production associated with the diagnosis of Actinobacillus pleuropneumoniae on grower/finisher swine operations in the United States.

    PubMed

    Losinger, Willard C

    2005-05-10

    An examination of the economic impacts of the diagnosis of Actinobacillus pleuropneumoniae on grower/finisher swine operations indicated that reduced pork production, associated with the diagnosis of A. pleuropneumoniae on the operation, diminished consumer surplus by $53+/-52 million, and resulted in a total loss of $32+/-30 million to the US economy in 1995. Most of the economic surplus lost by consumers was transferred to producers, whose economic surplus increased by $21+/-25 million (which was not significantly different from zero). Uncertainty analysis showed that an estimate of the decline in production associated with the diagnosis of A. pleuropneumoniae accounted for most of the uncertainty of the change in consumer surplus and of the total loss to the economy. The estimate of the price elasticity of demand for pork also contributed towards a lot of the uncertainty in the estimated change in producer surplus. PMID:15820115

  1. Identification of a hemolysin from Actinobacillus pleuropneumoniae and characterization of its channel properties in planar phospholipid bilayers.

    PubMed

    Lalonde, G; McDonald, T V; Gardner, P; O'Hanley, P D

    1989-08-15

    A proteinaceous hemolysin secreted by strain 4074 of serotype 1 of Actinobacillus pleuropneumoniae was purified by diafiltration and ion exchange chromatographic techniques. The hemolytic activity is associated with a 107-kDa band as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and confirmed by Western blotting and immunoprecipitation. This hemolysin produces pores in membranes as demonstrated by osmotic protection studies using red blood cells and carbohydrate compounds of various molecular weights. These assays suggest a pore diameter in the order of 2 nm. Phospholipid bilayers composed of 1:1 w/w phosphotidylserine:phosphotidylethanolamine exposed to this toxin display discrete current flow events typical of transmembrane channels and consistent with the interpretation that this toxin acts by forming pores in phospholipid membranes. The linear relationship of current amplitude to holding potential when examined over the -60 to +60 mV range indicates that this pore has a constant mean single channel conductance level of 350-400 pS. PMID:2474533

  2. Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release

    PubMed Central

    Wang, Lei; Qin, Wanhai; Zhang, Jing; Bao, Chuntong; Zhang, Hu; Che, Yanyi; Sun, Changjiang; Gu, Jingmin; Feng, Xin; Du, Chongtao; Han, Wenyu; Richard, Paul Langford; Lei, Liancheng

    2016-01-01

    Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs. PMID:27046446

  3. Conservation and antigenic cross-reactivity of the transferrin-binding proteins of Haemophilus influenzae, Actinobacillus pleuropneumoniae and Neisseria meningitidis.

    PubMed

    Holland, J; Parsons, T R; Hasan, A A; Cook, S M; Stevenson, P; Griffiths, E; Williams, P

    1996-12-01

    Haemophilus influenzae acquires iron from the iron-transporting glycoprotein transferrin via a receptor-mediated process. This involves two outer-membrane transferrin-binding proteins (Tbps) termed Tbp1 and Tbp2 which show considerable preference for the human form of transferrin. Since the Tbps are attracting considerable attention as potential vaccine components, we used transferrin affinity chromatography to examine their conservation amongst 28 H. influenzae type b strains belonging to different outer-membrane-protein subtypes as well as six non-typable strains. Whole cells of all type b and non-typable strains examined bound human transferrin; whilst most strains possessed a Tbp1 of approximately 105 kDa, the molecular mass of Tbp2 varied from 79 to 94 kDa. Antisera raised against affinity-purified native H. influenzae Tbp1/Tbp2 receptor complex cross-reacted on Western blots with the respective Tbps of all the Haemophilus strains examined. When used to probe Neisseria meningitidis Tbps, sera from each of four mice immunized with the Haemophilus Tbp1/2 complex recognized the 68 kDa Tbp2 of N. meningitidis strain B16B6 but not the 78 kDa Tbp2 of N. meningitidis strain 70942. Serum from one mouse also reacted weakly with Tbp1 of strain B16B6. Apart from a weak reaction with the Tbp2 of a serotype 5 strain, this mouse antiserum failed to recognize the Tbps of the porcine pathogen A. pleuropneumoniae. However, a monospecific polyclonal antiserum raised against the denatured Tbp2 of Neisseria meningitidis B16B6 recognized the Tbps of all Haemophilus and Actinobacillus strains examined. Since H. influenzae forms part of the natural flora of the upper respiratory tract, human sera were screened for the presence of antibodies to the Tbps. Sera from healthy adults contained antibodies which recognized both Tbp1 and Tbp2 from H. influenzae but not N. meningitidis. Convalescent sera from meningococcal meningitis patients contained antibodies which, on Western blots

  4. Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

    PubMed Central

    Trottier, Y L; Wright, P F; Larivière, S

    1992-01-01

    An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5. PMID:1734068

  5. Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1.

    PubMed

    Archambault, Marie; Labrie, Josée; Rioux, Clément R; Dumas, France; Thibault, Pierre; Elkins, Christopher; Jacques, Mario

    2003-10-01

    The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm. PMID:14620863

  6. Nasal immunization with M cell-targeting ligand-conjugated ApxIIA toxin fragment induces protective immunity against Actinobacillus pleuropneumoniae infection in a murine model.

    PubMed

    Park, Jisang; Seo, Ki-Weon; Kim, Sae-Hae; Lee, Ha-Yan; Kim, Bumseok; Lim, Chae Woong; Kim, Jin-Hee; Yoo, Han Sang; Jang, Yong-Suk

    2015-05-15

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and severe economic loss in the swine industry has been caused by the infection. Therefore, the development of an effective vaccine against the bacteria is necessary. ApxII toxin, among several virulence factors expressed by the bacteria, is considered to be a promising vaccine candidate because ApxII toxin not only accompanies cytotoxic and hemolytic activities, but is also expressed in all 15 serotypes of bacteria except serotypes 10 and 14. In this study, we identified the peptide ligand capable of targeting the ligand-conjugated ApxIIA #5 fragment antigen to nasopharynx-associated lymphoid tissue. It was found that nasal immunization with ligand-conjugated ApxIIA #5 induced efficient mucosal and systemic immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. More importantly, the nasal immunization induced protective immunity against nasal challenge infection of the bacteria, which was confirmed by histopathological studies and bacterial clearance after challenge infection. Collectively, we confirmed that the ligand capable of targeting the ligand-conjugated antigen to nasopharynx-associated lymphoid tissue can be used as an effective nasal vaccine adjuvant to induce protective immunity against A. pleuropneumoniae infection. PMID:25818577

  7. Minimum inhibitory concentration breakpoints and disk diffusion inhibitory zone interpretive criteria for tilmicosin susceptibility testing against Pasteurella multocida and Actinobacillus pleuropneumoniae associated with porcine respiratory disease.

    PubMed

    Shryock, Thomas R; Staples, J Mitchell; DeRosa, David C

    2002-09-01

    Tilmicosin is a novel macrolide antibiotic developed for exclusive use in veterinary medicine. Tilmicosin has been approved as a feed premix to control porcine respiratory disease associated with Pasteurella multocida and Actinobacillus pleuropneumoniae. The development of antimicrobial susceptibility testing guidelines for tilmicosin was predicated on the relationship of clinical efficacy studies that demonstrated a favorable therapeutic outcome, on pharmacokinetic data, and on in vitro test data, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The approved breakpoints for the minimum inhibitory concentration dilution testing for both species are resistant, > or = 32 microg/ml, and susceptible, < or = 16 microg/ml. The zone of inhibition interpretive criteria for disk diffusion testing with a 15-microg tilmicosin disk are resistant, < or = 10 mm, and susceptible, > or = 11 mm. PMID:12296390

  8. Comparative activities of selected fluoroquinolones against dynamic populations of Actinobacillus pleuropneumoniae in an in vitro model of time-kill continuous culture experiment.

    PubMed

    Damte, Dereje; Lee, Seung-Jin; Yohannes, Sileshi B; Hossain, Md Akil; Suh, Joo-Won; Park, Seung-Chun

    2013-12-01

    The aim of the current study was to demonstrate and compare the impact of different pharmacokinetics of marbofloxacin, enrofloxacin and difloxacin on their antimicrobial effects, their killing and re-growth kinetics, and the population dynamics of Actinobacillus pleuropneumoniae clinical isolates in an in vitro dynamic model. Selected clinical isolates of A. pleuropneumoniae and three fluoroquinolones at a range of simulated AUC(24)/MIC ratios of multiple doses were investigated. At the same simulated AUC(24)/MIC ratios of the three fluoroquinolones, the killing re-growth profile and I(E) values (intensity of the antimicrobial effect) revealed strain- and fluoroquinolone-specific effects. For example, a 31% lower I(E) of difloxacin was observed in AppK5 (biofilm-former) than in AppK2 (biofilm-non-former) at the same AUC(24)/MIC ratio of 120 h. In addition, losses in A. pleuropneumoniae susceptibility of both strains by the three fluoroquinolones were observed. AUC(24)/MPC ratios of 20.89 and 39.81 for marbofloxacin, 17.32 and 19.49 for enrofloxacin and 31.62 and 60.25 for difloxacin were estimated to be protective against the selection of AppK2 and AppK5 strain mutants, respectively. Integration of these in vitro data with published pharmacokinetics revealed the inadequacy of the conventional clinical doses of the three drugs to attain the above protective values for minimum biofilm eradication concentration (MBEC) and concentration to prevent growth of 90% of the mutant subpopulation (MPC(90)). In conclusion, the results suggest optimising doses could suffice for resistant mutants control, while for biofilm-forming strains combination with biofilm-disrupting agents to reduce the MBEC to achieve AUC/MBEC ratios within the possible dosing regimens is desired. PMID:24139884

  9. The Live Attenuated Actinobacillus pleuropneumoniae Triple-Deletion Mutant ΔapxIC ΔapxIIC ΔapxIV-ORF1 Strain, SLW05, Immunizes Pigs against Lethal Challenge with Haemophilus parasuis

    PubMed Central

    Fu, Shulin; Ou, Jiwen; Zhang, Minmin; Xu, Juan; Liu, Huazhen; Liu, Jinlin; Yuan, Fangyan; Chen, Huanchun

    2013-01-01

    Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (ΔapxIC ΔapxIIC ΔapxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-γ) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine. PMID:23220998

  10. Enriched Housing Reduces Disease Susceptibility to Co-Infection with Porcine Reproductive and Respiratory Virus (PRRSV) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae) in Young Pigs.

    PubMed

    van Dixhoorn, Ingrid D E; Reimert, Inonge; Middelkoop, Jenny; Bolhuis, J Elizabeth; Wisselink, Henk J; Groot Koerkamp, Peter W G; Kemp, Bas; Stockhofe-Zurwieden, Norbert

    2016-01-01

    Until today, anti-microbial drugs have been the therapy of choice to combat bacterial diseases. Resistance against antibiotics is of growing concern in man and animals. Stress, caused by demanding environmental conditions, can reduce immune protection in the host, influencing the onset and outcome of infectious diseases. Therefore psychoneuro-immunological intervention may prove to be a successful approach to diminish the impact of diseases and antibiotics use. This study was designed to investigate the effect of social and environmental enrichment on the impact of disease, referred to as "disease susceptibility", in pigs using a co-infection model of PRRSV and A. pleuropneumoniae. Twenty-eight pigs were raised in four pens under barren conditions and twenty-eight other pigs were raised in four pens under enriched conditions. In the enriched pens a combination of established social and environmental enrichment factors were introduced. Two pens of the barren (BH) and two pens of the enriched housed (EH) pigs were infected with PRRSV followed by A. pleuropneumoniae, the other two pens in each housing treatment served as control groups. We tested if differences in disease susceptibility in terms of pathological and clinical outcome were related to the different housing regimes and if this was reflected in differences in behavioural and immunological states of the animals. Enriched housed pigs showed a faster clearance of viral PRRSV RNA in blood serum (p = 0.014) and histologically 2.8 fold less interstitial pneumonia signs in the lungs (p = 0.014). More barren housed than enriched housed pigs developed lesions in the lungs (OR = 19.2, p = 0.048) and the lesions in the barren housed pigs showed a higher total pathologic tissue damage score (p<0.001) than those in enriched housed pigs. EH pigs showed less stress-related behaviour and differed immunologically and clinically from BH pigs. We conclude that enriched housing management reduces disease susceptibility to co

  11. Pharmacokinetic/pharmacodynamic evaluation of marbofloxacin in the treatment of Haemophilus parasuis and Actinobacillus pleuropneumoniae infections in nursery and fattener pigs using Monte Carlo simulations.

    PubMed

    Vilalta, C; Giboin, H; Schneider, M; El Garch, F; Fraile, L

    2014-12-01

    This study evaluated the theoretical clinical outcome of three marbofloxacin posology regimens in two groups of pigs (weaners and fatteners) for the treatment of Actinobacillus pleuropneumoniae (App) and Haemophilus parasuis (Hp) infection and the appearance of resistant bacteria due to the antibiotic treatment. The probability of target attainment (PTA) for pharmacokinetic/pharmacodynamics (PK/PD) ratios associated with clinical efficacy and with the appearance of antimicrobial resistance for fluoroquinolones at each minimum inhibitory concentration (MIC) or mutant prevention concentration (MPC) were calculated, respectively. The cumulative fraction of response (CFR) was calculated for the three posology regimens against App and they ranged from 91.12% to 96.37% in weaners and from 93% to 97.43% in fatteners, respectively. In the case of Hp, they ranged from 80.52% to 85.14% in weaners and from 82.01% to 88.49% in fatteners, respectively. Regarding the PTA of the PK/PD threshold associated with the appearance of antimicrobial resistance, results showed that marbofloxacin would prevent resistances in most of the animals up to the MPC value of 1 μg/mL. PMID:24903473

  12. [The seroprevalence of Actinobacillus pleuropneumoniae in Swiss pig breeding herds--a study with the ApxIV ELISA].

    PubMed

    Nussbaumer, I; Miserez, R; Hüssy, D; Doherr, M G; Frey, J; Zimmermann, W

    2008-03-01

    At the end of the national eradication program for Enzootic Pneumonia (EP) and Porcine Actinobacillosis (APP) in Switzerland (2003), A. pleuropneumoniae serotype 2 is considered to have been eradicated. There is no current information about the distribution of the other serotypes available. The ApxIV ELISA detects antibodies against all serotypes of A. pleuropneumoniae, without cross-reaction with other bacterial species. The aim of this study was to achieve actual data concerning the seroprevalence of A. pleuropneumoniae in breeding-herds and to validate the ApxIV ELISA under field conditions, especially for the diagnosis of latently infected breeding-herds without clinical signs, and to achieve more information about the role of herd book farms for the spread of the infectious agent. A total of 2068 serum samples from 96 pig herds in Switzerland were examinated. Over half of the examinated herd book farms showed positive results in this ELISA. 93% of the breeding herds were positive. On single animal level sensitivity was 96% and specifity 100%. Herd sensitivity ranged between 67% and 99%. Herd specifity was 100%. The results show that the ApxIV ELISA is a valuable tool for the detection of latently infected herds. PMID:18429500

  13. Mutation in the LPS outer core biosynthesis gene, galU, affects LPS interaction with the RTX toxins ApxI and ApxII and cytolytic activity of Actinobacillus pleuropneumoniae serotype 1.

    PubMed

    Ramjeet, Mahendrasingh; Cox, Andrew D; Hancock, Mark A; Mourez, Michael; Labrie, Josée; Gottschalk, Marcelo; Jacques, Mario

    2008-10-01

    Lipopolysaccharides (LPS) and Apx toxins are major virulence factors of Actinobacillus pleuropneumoniae, a pathogen of the respiratory tract of pigs. Here, we evaluated the effect of LPS core truncation in haemolytic and cytotoxic activities of this microorganism. We previously generated a highly attenuated galU mutant of A. pleuropneumoniae serotype 1 that has an LPS molecule lacking the GalNAc-Gal II-Gal I outer core residues. Our results demonstrate that this mutant exhibits wild-type haemolytic activity but is significantly less cytotoxic to porcine alveolar macrophages. However, no differences were found in gene expression and secretion of the haemolytic and cytotoxic toxins ApxI and ApxII, both secreted by A. pleuropneumoniae serotype 1. This suggests that the outer core truncation mediated by the galU mutation affects the toxins in their cytotoxic activities. Using both ELISA and surface plasmon resonance binding assays, we demonstrate a novel interaction between LPS and the ApxI and ApxII toxins via the core oligosaccharide. Our results indicate that the GalNAc-Gal II-Gal I trisaccharide of the outer core is fundamental to mediating LPS/Apx interactions. The present study suggests that a lack of binding between LPS and ApxI/II affects the cytotoxicity and virulence of A. pleuropneumoniae. PMID:18713318

  14. Pharmacokinetics of tildipirosin in porcine plasma, lung tissue, and bronchial fluid and effects of test conditions on in vitro activity against reference strains and field isolates of Actinobacillus pleuropneumoniae.

    PubMed

    Rose, M; Menge, M; Bohland, C; Zschiesche, E; Wilhelm, C; Kilp, S; Metz, W; Allan, M; Röpke, R; Nürnberger, M

    2013-04-01

    The pharmacokinetics of tildipirosin (Zuprevo(®) 40 mg/mL solution for injection for pigs), a novel 16-membered-ring macrolide for the treatment for swine respiratory disease (SRD), was investigated in studies collecting blood plasma and postmortem samples of lung tissue and bronchial fluid (BF) from swine. In view of factors influencing the in vitro activity of macrolides, and for the interpretation of tildipirosin pharmacokinetics in relation to minimum inhibitory concentrations (MIC), additional experiments were conducted to study the effects of pH, carbon dioxide-enriched atmosphere, buffers, and serum on tildipirosin MICs for various reference strains and Actinobacillus (A.) pleuropneumoniae field isolates. After single intramuscular (i.m.) injection at 4 mg/kg body weight, maximum plasma concentration (Cmax) was 0.9 μg/mL observed within 23 min (Tmax ). Mean residence time from the time of dosing to the time of last measurable concentration (MRTlast) and terminal half-life (T1/2) both were about 4 days. A dose-response relationship with no significant sex effect is observed for area under the plasma concentration-time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUClast) over the range of doses up to 6 mg/kg. However, linear dose proportionality could not be proven with statistical methods. The time-concentration profile of tildipirosin in BF and lung far exceeded that in blood plasma. In lung, tildipirosin concentrations reached 3.1 μg/g at 2 h, peaked at 4.3 μg/g at day 1, and slowly declined to 0.8 μg/g at day 17. In BF, tildipirosin levels were 14.3, 7.0, and 6.5 μg/g at days 5, 10, and 14. T1/2 in lung was ∼7 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination. Culture media pH and carbon dioxide-enriched atmosphere (CO2 -EA) had a marked impact on in vitro activity of tildipirosin in reference strains of various rapidly growing aerobic and

  15. Blood gas and hematological changes in experimental peracute porcine pleuropneumonia.

    PubMed Central

    Kiorpes, A L; MacWilliams, P S; Schenkman, D I; Bäckström, L R

    1990-01-01

    The effect of experimental, peracute, porcine pleuropneumonia on arterial blood gases, acid base status, the leukogram, and gross and microscopic lung structure was studied in nine growing pigs (mean weight +/- SD 10.6 +/- 2.0 kg). Pigs were inoculated intranasally with a virulent serotype 5 isolate of Actinobacillus pleuropneumoniae, and all showed signs typical of the disease within four hours. Death occurred in all pigs from 4.5 to 32 hours postinoculation (mean 14 hours). Gross and microscopic changes were typical of porcine pleuropneumonia in all pigs. Changes in the leukogram included a rapid decline in total white cells, segmented neutrophils, lymphocytes, monocytes, and eosinophils. Pigs maintained alveolar ventilation throughout the study as arterial CO2 tension was unchanged; however, arterial O2 tension and pH decreased from (mean +/- SD) 95.2 +/- 5.7 torr and 7.463 +/- 0.018 at baseline to 62.1 +/- 12.3 torr and 7.388 +/- 0.045, respectively, within 90 minutes prior to death. The data showed that in this model of peracute porcine pleuropneumonia, progressive ventilatory failure was not a feature of the disease, and the blood gas values and acid base status were maintained within physiological ranges. The histopathological hematological and physiological findings were consistent with the hypothesis that peracute porcine pleuropneumonia resembles septic shock. Images Fig. 2. Fig. 3. PMID:2106382

  16. Blood gas stability and hematological changes in experimentally-induced acute porcine pleuropneumonia.

    PubMed Central

    Kiorpes, A L; Mirsky, M L; MacWilliams, P S; Bäckström, L R; Collins, M T

    1989-01-01

    Blood gas and hematological responses to acute, mild Actinobacillus pleuropneumoniae infection of growing pigs was studied. Six pigs (average weight 10.1 kg) were experimentally infected intranasally with A. pleuropneumoniae serotype 5. Four pigs served as controls. Rectal temperatures and arterial blood for gas analysis and hematology were taken at 0, 8, 16, 24, 48 and 72 h postinfection. All infected pigs became febrile showing clinical signs typical of mild to moderate porcine pleuropneumonia; controls remained asymptomatic. Neutrophilia with bands and lymphopenia were observed only in infected pigs. Arterial partial pressures of O2 and CO2, and pH did not change in infected pigs. All pigs were killed after 72 h, and lungs were examined and cultured. Gross and microscopic lesions consistent with porcine pleuropneumonia were seen in 3/6 and 5/6 infected lungs, respectively. Control lungs were grossly normal with no histological evidence of pleuropneumonia. We conclude that in mild, acute porcine pleuropneumonia as established experimentally, a leukogram typical of acute inflammation and stress is seen; however, hypoxemia and alveolar hypoventilation are not features of this form of the disease. PMID:2914231

  17. Haptoglobin blood test

    MedlinePlus

    ... liver. It attaches to a certain type of hemoglobin in the blood. How the Test is Performed ... destroyed. When red blood cells die, they release hemoglobin. Haptoglobin attaches to this released hemoglobin, which is ...

  18. Hemoglobin binding to deglycosylated haptoglobin.

    PubMed

    Kaartinen, V; Mononen, I

    1988-04-14

    The carbohydrate portion of polymeric haptoglobin was gradually removed by exoglycosidases in order to investigate its role in complex formation between haptoglobin and hemoglobin. Total removal of sialic acid diminished the haptoglobin-hemoglobin complex formation 15%. Removal of about 25% of the galactose residues from asialohaptoglobin, i.e., about 40% of the total weight of the carbohydrate moiety, totally inhibited the ability of haptoglobin to form complex with hemoglobin and react with haptoglobin-specific antibodies. Liberation of further galactose residues resulted in slow precipitation of the protein. Removal of a similar part of the carbohydrate moiety from haptoglobin-hemoglobin complex did not liberate hemoglobin from it, and the complex reacted with haptoglobin antibodies. The combined data indicate that the carbohydrate portion is essential for the functionally active form of polymeric haptoglobin to complex with hemoglobin, but it hardly has any direct role in the binding event, and other factors are responsible for the stability of the complex. PMID:3128331

  19. Haptoglobin Enhances Cardiac Transplant Rejection

    PubMed Central

    Shen, Hua; Heuzey, Elizabeth; Mori, Daniel; Wong, Christine; Colangelo, Christopher; Chung, Lisa M.; Bruce, Can; Slizovskiy, Ilya B.; Booth, Carmen J.; Kreisel, Daniel; Goldstein, Daniel R.

    2015-01-01

    Rationale Early graft inflammation enhances both acute and chronic rejection of heart transplants, but it is unclear how this inflammation is initiated. Objective To identify specific inflammatory modulators and determine their underlying molecular mechanisms after cardiac transplantation. Methods and Results We used a murine heterotopic cardiac transplant model to identify inflammatory modulators of early graft inflammation. Unbiased mass spectrometric analysis of cardiac tissue before and up to 72 hours after transplantation revealed that 22 proteins including haptoglobin, a known anti-oxidant, are significantly upregulated in our grafts. Through the use of haptoglobin deficient mice, we show that 80% of haptoglobin deficient recipients treated with peri-operative administration of the costimulatory blocking agent CTLA4 immunoglobulin exhibited > 100 days survival of full major histocompatibility complex mismatched allografts, whereas all similarly treated wild type recipients rejected their transplants by 21 days post transplantation. We found that haptoglobin modifies the intra-allograft inflammatory milieu by enhancing levels of the inflammatory cytokine IL-6 and the chemokine MIP-2 but impair levels of the immunosuppressive cytokine IL-10. Haptoglobin also enhances dendritic cell graft recruitment and augments anti-donor T cell responses. Moreover, we confirmed that the protein is present in human cardiac allograft specimens undergoing acute graft rejection. Conclusions Our findings provide new insights into the mechanisms of inflammation after cardiac transplantation and suggest that, in contrast to its prior reported anti-oxidant function in vascular inflammation, haptoglobin is an enhancer of inflammation after cardiac transplantation. Haptoglobin may also be a key component in other sterile inflammatory conditions. PMID:25801896

  20. Microheterogeneity of mammalian haptoglobins in isoelectric focusing.

    PubMed

    Dobryszycka, W; Krawczyk, E

    1979-01-01

    1. Human haptoglobin type 1-1, porcine haptoglobin, and equine haptoglobin were isolated and purified. 2. These haptoglobins were similar in polyacrylamide gel electrophoresis and in subunit structure but showed microheterogeneity in isoelectric focusing. 3. Isoelectric points of human haptoglobin as determined with photopolymerized gels were found to be 4.03-4.24, of porcine haptoglobin 4.0-4.30, and of horse haptoglobin 3.80-4.15, respectively. 4. Results obtained with chemically polymerized gels were 0.08-0.3 pH units higher. 5. Examined haptoglobins differed also in the ability of complex formation with hemoglobin, in sialic acid content and in antigenic specificity. PMID:45546

  1. [Haptoglobins in gynecologic tumors (author's transl)].

    PubMed

    Tatra, G; Moser, C

    1978-05-01

    Serum concentrations of haptoglobins were checked in 100 women with benign or malign gynecologic tumors. Typification of haptoglobins was determined by starch gel electrophoresis, quantification by single radial immunodiffusion. Cases with malignancies showed significant increased levels in comparison to healthy women. In cases with cervical carcinoma, a significant increase of the haptoglobin level was evaluated according to advanced carcinoma. PMID:418536

  2. Structure of the haptoglobin-haemoglobin complex.

    PubMed

    Andersen, Christian Brix Folsted; Torvund-Jensen, Morten; Nielsen, Marianne Jensby; de Oliveira, Cristiano Luis Pinto; Hersleth, Hans-Petter; Andersen, Niels Højmark; Pedersen, Jan Skov; Andersen, Gregers Rom; Moestrup, Søren Kragh

    2012-09-20

    Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of

  3. Pleural effusion associated with acute and chronic pleuropneumonia and pleuritis secondary to thoracic wounds in horses: 43 cases (1982-1992).

    PubMed

    Collins, M B; Hodgson, D R; Hutchins, D R

    1994-12-15

    Case records of 43 horses with pleural effusion associated with acute pleuropneumonia, chronic pleuropneumonia, or pleuritis secondary to a penetrating thoracic wound were reviewed to determine the predisposing factors, diagnosis, and treatment of this condition. Acute pleuropneumonia was diagnosed in 36 horses, the majority of which were Thoroughbreds (89%). Of 22 (61%) horses that were in race training at the onset of illness, 11 (31%) had been recently transported a long distance and 4 (11%) had evidence of exercise-induced pulmonary hemorrhage. Physical examination findings and hematologic data were nonspecific. The most consistent abnormality was hyperfibrino-genemia. Affected horses were treated with antibiotics, thoracic drainage, nonsteroidal anti-inflammatory drugs, and supportive care. Twenty-two (61%) horses were discharged from the hospital, with the mean duration of hospitalization for those discharged being 23 days. Nine (25%) horses were euthanatized and 5 (14%) died. Bacterial culturing of thoracic fluid resulted in growth in 30 of the 36 (83%) horses. The finding of anaerobic bacteria in thoracic fluid was not associated with a lower survival rate (62%) than the overall survival rate (61%). Four horses with chronic pleuropneumonia had a history of lethargy and inappetence for > 2 weeks. Actinobacillus equuli was isolated, either alone or in combination with other bacteria, from thoracic fluid of these 4 horses. Each horse was treated with broad spectrum antibiotics and made a rapid recovery. Three horses with acute pleuritis secondary to penetrating thoracic wounds also had nonspecific clinical signs, apart from the wound and a large volume of pleural effusion. Bacteriologic isolates from these horses differed slightly from those of horses with acute pleuropneumonia. PMID:7744650

  4. 21 CFR 866.5460 - Haptoglobin immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Haptoglobin immunological test system. (a) Identification. A haptoglobin immunological test system is a device... release hemoglobin) related to the formation of hemoglobin-haptoglobin complexes and certain kidney diseases. (b) Classification. Class II (special controls). The device is exempt from the...

  5. 21 CFR 866.5460 - Haptoglobin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Haptoglobin immunological test system. (a) Identification. A haptoglobin immunological test system is a device... release hemoglobin) related to the formation of hemoglobin-haptoglobin complexes and certain kidney diseases. (b) Classification. Class II (special controls). The device is exempt from the...

  6. Structure of haptoglobin and the haptoglobin-hemoglobin complex by electron microscopy.

    PubMed

    Wejman, J C; Hovsepian, D; Wall, J S; Hainfeld, J F; Greer, J

    1984-04-01

    The human serum protein, haptoglobin, forms a stable, irreversible complex with hemoglobin. Haptoglobin is composed of two H chains, which are connected via two smaller L chains to give a protein of 85,000 Mr. In the complex, each H chain binds an alpha beta dimer of hemoglobin for a total molecular weight of 150,000. The scanning transmission electron microscope has been used to derive new information about the shape and structure of haptoglobin and hemoglobin, and about their relative orientation in the complex. The micrographs of negatively stained images show that haptoglobin has the shape of a barbell with two spherical head groups, which are the H chains. These are connected by a thin filament with a central knob, which corresponds to the L chains. The overall length of the molecule is about 124(+/- 8) A and the interhead distance is 87 (+/- 7) A. In the haptoglobin-hemoglobin complex, the head groups are ellipsoidal and under optimal staining conditions bilobal . Thus, the alpha beta dimers are binding to the H chains, but off the long axis of the barbell by 127 degrees in a trans configuration. This angle considerably restricts the region on the surface of the H chain structure that can contain the hemoglobin binding site. The interhead group distance for complex is 116.5(+/- 6.3) A or 30 A greater than for haptoglobin. The N terminus of the beta chain was located on the trans off-axis configured barbell structure of complex by using a hemoglobin that was crosslinked between the alpha beta dimers in the region of the beta N terminus. The distances and angles that are measured on the micrographs for the native and crosslinked complex molecules permit the directions of two of the alpha beta dimer ellipsoid axes to be assigned. Taken together, these data provide an approximate relative orientation for the binding of the alpha beta dimer to the H chain of haptoglobin. PMID:6716481

  7. Rate of Nitric Oxide Scavenging by hemoglobin bound to haptoglobin

    PubMed Central

    Azarov, Ivan; He, Xiaojun; Jeffers, Anne; Basu, Swati; Ucer, Burak; Hantgan, Roy R.; Levy, Andrew; Kim-Shapiro, Daniel B.

    2008-01-01

    Cell-free hemoglobin, released from the red cell, may play a major role in regulating the bioavailability of nitric oxide. The abundant serum protein haptoglobin, rapidly binds to free hemoglobin forming a stable complex accelerating its clearance. The haptoglobin gene is polymorphic with two classes of alleles denoted 1 and 2. We have previously demonstrated that the haptoglobin 1 protein-hemoglobin complex is cleared twice as fast as the haptoglobin 2 protein-hemoglobin complex. In this report we explored whether haptoglobin binding to hemoglobin reduces the rate of nitric oxide scavenging using time-resolved absorption spectroscopy. We found that both the haptoglobin 1 and haptoglobin 2 protein complexes react with nitric oxide at the same rate as unbound cell-free hemoglobin. To confirm these results we developed a novel assay where free hemoglobin and hemoglobin bound to haptoglobin competed in the reaction with NO. The relative rate of the NO reaction was then determined by examining the amount of reacted species using analytical ultracentrifugation. Since complexation of hemoglobin with haptoglobin does not reduce NO scavenging, we propose that the haptoglobin genotype may influence nitric oxide bioavailability by determining the clearance rate of the haptoglobin-hemoglobin complex. We provide computer simulations showing that a two-fold difference in the rate of uptake of the haptoglobin hemoglobin complex by macrophages significantly affects nitric oxide bioavailability thereby providing a plausible explanation for why there is more vasospasm after subarachnoid hemorrhage in individuals and transgenic mice homozygous for the Hp 2 allele. PMID:18364244

  8. Serum haptoglobin concentrations in dogs with liver disease.

    PubMed

    Crawford, K; Warman, S M; Marques, A I; Yool, D A; Eckersall, P D; McCulloch, E; Lynn, K; Mellanby, R J; Gow, A G

    2013-12-14

    Dogs with liver disease have been shown to have increased serum C-reactive protein (CRP) concentrations. However, it is unclear whether dogs with liver disease also have increased serum haptoglobin concentrations. The aim of the study was to measure serum haptoglobin concentrations in healthy dogs, hospitalised dogs and dogs with liver diseases. Haptoglobin concentrations were measured in 30 healthy dogs, 47 hospitalised dogs with non-hepatic illness, 46 dogs with congenital portosystemic shunt (cPSS) and 11 dogs with primary hepatopathy. Haptoglobin concentrations were not significantly different between cPSS dogs with and without hepatic encephalopathy (HE), thus all cPSS dogs were considered as one group. Haptoglobin concentrations were significantly different between the remaining groups (P<0.0001). Hospitalised ill dogs had significantly higher haptoglobin concentrations than healthy dogs (P<0.001), dogs with cPSS (P<0.001) and dogs with primary hepatopathy (P<0.05). There was no significant difference between haptoglobin concentrations in healthy dogs, dogs with cPSS and dogs with primary hepatopathy. Haptoglobin concentrations were not significantly increased in dogs with liver diseases or in dogs with cPSS and HE. This is in contrast with the previously reported CRP results. This study demonstrates that liver function should be considered when interpreting haptoglobin concentrations in dogs. PMID:24158322

  9. Covalent structure of human haptoglobin: a serine protease homolog.

    PubMed Central

    Kurosky, A; Barnett, D R; Lee, T H; Touchstone, B; Hay, R E; Arnott, M S; Bowman, B H; Fitch, W M

    1980-01-01

    The complete amino acid sequences and the disulfide arrangements of the two chains of human haptoglobin 1-1 were established. The alpha 1 and beta chains of haptoglobin contain 83 and 245 residues, respectively. Comparison of the primary structure of haptoglobin with that of the chymotrypsinogen family of serine proteases revealed a significant degree of chemical similarity. The probability was less than 10(-5) that the chemical similarity of the beta chain of haptoglobin to the proteases was due to chance. The amino acid sequence of the beta chain of haptoglobin is 29--33% identical to bovine trypsin, bovine chymotrypsin, porcine elastase, human thrombin, or human plasmin. Comparison of haptoglobin alpha 1 chain to activation peptide regions of the zymogens revealed an identity of 25% to the fifth "kringle" region of the activation peptide of plasminogen. The probability was less than 0.014 that this similarity was due to chance. These results strongly indicate haptoglobin to be a homolog of the chymotrypsinogen family of serine proteases. Alignment of the beta-chain sequence of haptoglobin to the serine proteases is remarkably consistent except for an insertion of 16 residues in the region corresponding to the methionyl loop of the serine proteases. The active-site residues typical of the serine proteases, histidine-57 and serine-195, are replaced in haptoglobin by lysine and alanine, respectively; however, aspartic acid-102 and the trypsin specificity, residue, aspartic acid-189, do occur in haptoglobin. Haptoglobin and the serine proteases represent a striking example of homologous proteins with different biological functions. PMID:6997877

  10. Actinobacillus suis septicaemia in two foals.

    PubMed

    Nelson, K M; Darien, B J; Konkle, D M; Hartmann, F A

    1996-01-13

    A 24-hour-old Hackney ony filly developed signs of weakness, depression and a poor suck reflex, with harsh lung sounds over both fields, and a 48-hour-old Arabian colt from a normal birth which had sucked vigorously developed loose stools and became depressed, weak and anorectic. Both foals had serum IgG concentrations greater than 800 mg/dl, but each had a severe neutropenia with a left shift, and blood cultures from both of them yielded Actinobacillus suis. The A suis isolates had different antimicrobial susceptibility patterns and, in the case of the Arabian, the isolate was resistant to commonly used broad spectrum antimicrobial agents. PMID:8629322

  11. Haptoglobin genotype and its role in diabetic cardiovascular disease.

    PubMed

    Costacou, Tina; Levy, Andrew P

    2012-08-01

    Over the past decade, several longitudinal epidemiological studies have brought attention to the haptoglobin genotype and its importance in determining diabetic vascular disease risk. This manuscript presents an overview of the biology of the haptoglobin genotype and reviews the literature concerning its role in the development of cardiovascular disease among individuals with diabetes mellitus. PMID:22447230

  12. 21 CFR 866.5460 - Haptoglobin immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Haptoglobin immunological test system. 866.5460 Section 866.5460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5460 Haptoglobin immunological test system....

  13. A unique loop extension in the serine protease domain of haptoglobin is essential for CD163 recognition of the haptoglobin-hemoglobin complex.

    PubMed

    Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian; Thirup, Søren; Enghild, Jan Johannes; Graversen, Jonas Heilskov; Moestrup, Søren Kragh

    2007-01-12

    Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (alpha-chain) and a serine protease domain (beta-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the beta-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin beta-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin. PMID:17102136

  14. Epidemiology of Haemophilus pleuropneumoniae infection in pigs: a survey of Ontario Pork Producers, 1981.

    PubMed Central

    Rosendal, S; Mitchell, W R

    1983-01-01

    Information about factors associated with the spread and the effect of pleuropneumonia was obtained from 418 pork producers in Ontario, who returned a mailed questionnaire. The overall herd prevalence of pleuropneumonia was 23.2%. The prevalence among herds with feeder pigs only was 34.3% and 16% among sow herds. The chance of pleuropneumonia breaking out in a herd was increased with increased traffic of pigs into the herd. The source of supplementary stock had an important effect on the chance of pleuropneumonia occurring. The highest risk resulted from introducing stock from salesbarns and the lowest from stock of health status known to the purchaser and supplied by one breeder only. Mortality, primarily among feeder pigs, and unthriftiness were the major effects of Haemophilus pleuropneumoniae infection. Stress, such as crowding or inclement climatic conditions, was associated with outbreaks of pleuropneumonia. This would suggest that the infection with H. pleuropneumoniae can be subclinical until stress precipitates the disease. PMID:6831302

  15. Quantitative determination of haptoglobin glycoform variants in psoriasis.

    PubMed

    Maresca, Bernardetta; Cigliano, Luisa; Corsaro, Maria M; Pieretti, Giuseppina; Natale, Massimo; Bucci, Enrico M; Dal Piaz, Fabrizio; Balato, Nicola; Nino, Massimiliano; Ayala, Fabio; Abrescia, Paolo

    2010-12-01

    Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of this study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQ-VDIGLIK in either case. The number of peptides with distinct triantennary and tetraantennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and are different or missing in normal haptoglobin, might be associated with disease activity. PMID:21087091

  16. Release of toxic microvesicles by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Nowotny, A; Behling, U H; Hammond, B; Lai, C H; Listgarten, M; Pham, P H; Sanavi, F

    1982-01-01

    Oral isolates of Actinobacillus actinomycetemcomitans (strain Y4) release spherical microvesicles in large numbers during normal growth. The biological activities of these products were studied, and it was estimated that approximately 1/10 of their dry weight was made up of heat- and proteolysis-resistant endotoxin. The chicken embryo lethality and bone-resorbing activity of the microvesicles were heat stable but proteolysis sensitive. Other laboratories have reported the presence of a heat- and proteolysis-sensitive leukotoxin in similar preparations. Accordingly, the microvesicles released by strain Y4 may contain, in addition to endotoxin, several potent substances which are highly toxic and active in bone resorption, and these may be significant factors in the pathogenesis of periodontal diseases. PMID:7049947

  17. Release of toxic microvesicles by Actinobacillus actinomycetemcomitans.

    PubMed

    Nowotny, A; Behling, U H; Hammond, B; Lai, C H; Listgarten, M; Pham, P H; Sanavi, F

    1982-07-01

    Oral isolates of Actinobacillus actinomycetemcomitans (strain Y4) release spherical microvesicles in large numbers during normal growth. The biological activities of these products were studied, and it was estimated that approximately 1/10 of their dry weight was made up of heat- and proteolysis-resistant endotoxin. The chicken embryo lethality and bone-resorbing activity of the microvesicles were heat stable but proteolysis sensitive. Other laboratories have reported the presence of a heat- and proteolysis-sensitive leukotoxin in similar preparations. Accordingly, the microvesicles released by strain Y4 may contain, in addition to endotoxin, several potent substances which are highly toxic and active in bone resorption, and these may be significant factors in the pathogenesis of periodontal diseases. PMID:7049947

  18. Haptoglobin: the hemoglobin detoxifier in plasma.

    PubMed

    Alayash, Abdu I; Andersen, Christian Brix Folsted; Moestrup, Søren Kragh; Bülow, Leif

    2013-01-01

    Hemoglobin (Hb) is one of the most studied proteins. However, oxidative toxicity associated with free Hb in circulation and its contribution to inflammation and complications of transfusion have only recently become active areas of research. New insights into the protective mechanisms of haptoglobin (Hp), a plasma protein, and a timely resolution of the crystal structure of the Hb-Hp complex made it possible to definitively link the functional and structural interplay between the two proteins. Here, we summarize current knowledge of the interactions between Hb and Hp under oxidative stress conditions, and how Hb's own damaging radicals are harnessed by complex formation. Potential therapeutic benefits of using Hp for inactivation and clearance of free Hb under a number of clinical settings are considered. PMID:23140673

  19. Nasal immunization with mannan-decorated mucoadhesive HPMCP microspheres containing ApxIIA toxin induces protective immunity against challenge infection with Actinobacillus pleuropneumoiae in mice.

    PubMed

    Li, Hui-Shan; Shin, Min-Kyoung; Singh, Bijay; Maharjan, Sushila; Park, Tae-Eun; Kang, Sang-Kee; Yoo, Han-Sang; Hong, Zhong-Shan; Cho, Chong-Su; Choi, Yun-Jaie

    2016-07-10

    The development of subunit mucosal vaccines requires an appropriate delivery system or an immune modulator such as an adjuvant to improve antigen immunogenicity. The nasal route for vaccine delivery by microparticles has attracted considerable interest, although challenges such as the rapid mucociliary clearance in the respiratory mucosa and the low immunogenicity of subunit vaccine still remain. Here, we aimed to develop mannan-decorated mucoadhesive thiolated hydroxypropylmethyl cellulose phthalate (HPMCP) microspheres (Man-THM) that contain ApxIIA subunit vaccine - an exotoxin fragment as a candidate for a subunit nasal vaccine against Actinobacillus pleuropneumoniae. For adjuvant activity, mucoadhesive thiolated HPMCP microspheres decorated with mannan could be targeted to the PRRs (pathogen recognition receptors) and mannose receptors (MR) of antigen presenting cells (APCs) in the respiratory immune system. The potential adjuvant ability of Man-THM for intranasal immunization was confirmed by in vitro and in vivo experiments. In a mechanistic study using APCs in vitro, it was found that Man-THM enhanced receptor-mediated endocytosis by stimulating the MR of APCs. In vivo, the nasal vaccination of ApxIIA-loaded Man-THM in mice resulted in higher levels of mucosal sIgA and serum IgG than mice in the ApxIIA and ApxIIA-loaded THM groups due to the specific recognition of the mannan in the Man-THM by the MRs of the APCs. Moreover, ApxIIA-containing Man-THM protected immunized mice when challenged with strains of A. pleuropneumoniae serotype 5. These results suggest that mucoadhesive Man-THM may be a promising candidate for a nasal vaccine delivery system to elicit systemic and mucosal immunity that can protect from pathogenic bacteria infection. PMID:27189136

  20. Inhibition of fibroblast proliferation by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Shenker, B J; Kushner, M E; Tsai, C C

    1982-01-01

    We have examined soluble sonic extracts of Actinobacillus actinomycetemcomitans for their ability to alter human and murine fibroblast proliferation. We found that extracts of all A. actinomycetemcomitans strains examined (both leukotoxic and nonleukotoxic) caused a dose-dependent inhibition of both murine and human fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation). Addition of sonic extract simultaneously with [3H]thymidine had no effect on incorporation, indicating that suppression was not due to the presence of excessive amounts of cold thymidine. Inhibition of DNA synthesis was also paralleled by decreased RNA synthesis ([3H]uridine incorporation) and by a decrease in cell growth as assessed by direct cell counts; there was no effect on cell viability. The suppressive factor(s) is heat labile; preliminary purification and characterization studies indicate that it is a distinct and separate moiety from other A. actinomycetemcomitans mediators previously reported, including leukotoxin, immune suppressive factor, and endotoxin. Although it is not clear how A. actinomycetemcomitans acts to cause disease, we propose that one aspect of the pathogenicity of this organism rests in its ability to inhibit fibroblast growth, which in turn could contribute to the collagen loss associated with certain forms of periodontal disease, in particular juvenile periodontitis. PMID:7152684

  1. Killing of Actinobacillus actinomycetemcomitans by human lactoferrin.

    PubMed Central

    Kalmar, J R; Arnold, R R

    1988-01-01

    Actinobacillus actinomycetemcomitans is a fastidious, facultative gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human neutrophils have demonstrated bactericidal activity against A. actinomycetemcomitans, and much of the oxygen-dependent killing has been attributed to the myeloperoxidase-H2O2-halide system. However, the contribution of other neutrophil components to killing activity is obscure. Lactoferrin, an iron-binding glycoprotein, is a major constituent of neutrophil-specific granules and is also found in mucosal secretions. In this report, we show that human lactoferrin is bactericidal for A. actinomycetemcomitans. Killing activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log decrease in viability within 120 min at 37 degrees C at a concentration of 1.9 microM. Besides exhibiting concentration dependence, killing kinetics were affected by minor variations in temperature and pH. Magnesium, a divalent cation thought to stabilize lipopolysaccharide interactions on the surface of gram-negative organisms, enhanced lactoferrin killing of A. actinomycetemcomitans, while other cations, such as potassium and calcium, had no effect. Our data suggest that lactoferrin contributes to killing of A. actinomycetemcomitans by human neutrophils and that it may also play a significant role in innate secretory defense against this potential periodontopathogen. PMID:3417349

  2. Immunosuppressive properties of Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed Central

    Rabie, G; Lally, E T; Shenker, B J

    1988-01-01

    Actinobacillus actinomycetemcomitans produces a leukotoxin that kills human polymorphonuclear cells (PMNs) and monocytes but not lymphocytes. In this study, we examined A. actinomycetemcomitans leukotoxin for its ability to alter human peripheral blood lymphocyte (HPBL) responsiveness. After a 90-min exposure to the leukotoxin, all monocytes were killed and HPBL responsiveness to mitogens and antigens was significantly inhibited. The ability of the leukotoxin to inhibit HPBL responses was not surprising, since monocytes and macrophages are required for many lymphocyte functions. However, we were unable to totally restore HPBL responsiveness when adherent autologous monocytes were added back to cultures of leukotoxin-treated lymphocytes. These studies demonstrate that A. actinomycetemcomitans leukotoxin may also exert nonlethal effects directly on lymphocytes. Furthermore, impaired lymphocyte function did not appear to be the result of indirect effects of products released by dying monocytes. Although it is not clear how A. actinomycetemcomitans acts to cause disease, several investigators have proposed that impaired host defenses may play a pivotal role. Several studies have demonstrated defects in PMN, monocyte, and lymphocyte function in patients with periodontal disease. These findings, along with the data presented in this paper, support the hypothesis that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. The effects of this suppression may be to enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism. PMID:3335399

  3. Preparation of Monoclonal Antibodies Against Bovine Haptoglobin

    PubMed Central

    Wang, Caihong; Gu, Cheng; Guo, Donghua; Gao, Jing; Li, Chunqiu; Liu, Na; Geng, Yufei; Su, Mingjun; Wang, Xinyu

    2014-01-01

    Female, 8-week-old BALB/c mice were immunized with purified recombinant proteins of the predicted immunodominant region of bovine haptoglobin (pirBoHp). Two monoclonal antibodies (MAbs), named 1B3 and 6D6, were prepared by conventional B lymphocyte hybridoma technique. Titers of ascitic fluid and cell culture supernatant of MAb 1B3 were 1:9.6×108 and 1:8.2×104, respectively, and that of MAb 6D6 were 1:4.4×105 and 1:1.0×104, respectively. The subtype of MAbs 1B3 and 6D6 was IgG1κ. In Western blot analysis, MAbs 1B3 and 6D6 could recognize the α-chain of native BoHp from plasma of dairy cows. These data indicated that MAbs 1B3 and 6D6 have a potential use for developing diagnostic reagents of BoHp. PMID:25358005

  4. Isolation of Actinobacillus suis from a cat's lung.

    PubMed Central

    Daignault, D; Chouinard, L; Møller, K; Ahrens, P; Messier, S; Higgins, R

    1999-01-01

    Actinobacillus suis has been isolated from the lungs of 9-month-old cat. The bacterium was characterized biochemically as well as genetically, and its sensitivity profile to different antimicrobial agents was established. The role of this isolate in the cat's condition is discussed. PMID:9919368

  5. Human haptoglobin structure and function--a molecular modelling study.

    PubMed

    Polticelli, F; Bocedi, A; Minervini, G; Ascenzi, P

    2008-11-01

    Hemoglobin is the most prominent protein in blood, transporting O(2) and facilitating reactive oxygen and nitrogen species detoxification. Hemoglobin metabolism leads to the release of extra-erythrocytic hemoglobin, with potentially severe consequences for health. Extra-erythrocytic hemoglobin is complexed to haptoglobin for clearance by tissue macrophages. The human gene for haptoglobin consists of three structural alleles: Hp1F, Hp1S and Hp2. The products of the Hp1F and Hp1S alleles differ by only one amino acid, whereas the Hp2 allele is the result of a fusion of the Hp1F and Hp1S alleles, is present only in humans and gives rise to a longer alpha-chain. Haptoglobin consists of a dimer of alphabeta-chains covalently linked by a disulphide bond between the Cys15 residue of each alpha-chain. However, the presence of the Hp1 and Hp2 alleles in humans gives rise to HPT1-1 dimers (covalently linked by Cys15 residues), HPT1-2 hetero-oligomers and HPT2-2 oligomers. In fact, the HPT2 variant displays two free Cys residues (Cys15 and Cys74) whose participation in intermolecular disulphide bonds gives rise to higher-order covalent multimers. Here, the complete modelling of both haptoglobin variants, together with their basic quaternary structure arrangements (i.e. HPT1 dimer and HPT2 trimer), is reported. The structural details of the models, which represent the first complete view of the molecular details of human haptoglobin variants, are discussed in relation to the known haptoglobin function(s). PMID:18959750

  6. Biofilm Growth and Detachment of Actinobacillus actinomycetemcomitans

    PubMed Central

    Kaplan, Jeffrey B.; Meyenhofer, Markus F.; Fine, Daniel H.

    2003-01-01

    The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony. PMID:12562811

  7. First isolation of Actinobacillus genomospecies 2 in Japan

    PubMed Central

    MURAKAMI, Miyuki; SHIMONISHI, Yoshimasa; HOBO, Seiji; NIWA, Hidekazu; ITO, Hiroya

    2015-01-01

    We describe here the first isolation of Actinobacillus genomospecies 2 in Japan. The isolate was found in a septicemic foal and characterized by phenotypic and genetic analyses, with the latter consisting of 16S rDNA nucleotide sequence analysis plus multilocus sequence analysis using three housekeeping genes, recN, rpoA and thdF, that have been proposed for use as a genomic tool in place of DNA-DNA hybridization. PMID:26668165

  8. First Human Case of Meningitis and Sepsis in a Child Caused by Actinobacillus suis or Actinobacillus equuli

    PubMed Central

    Montagnani, Carlotta; Pecile, Patrizia; Moriondo, Maria; Petricci, Patrizia; Becciani, Sabrina; Chiappini, Elena; Indolfi, Giuseppe; Rossolini, Gian Maria; de Martino, Maurizio

    2015-01-01

    We report the first human case of meningitis and sepsis caused in a child by Actinobacillus suis or A. equuli, a common opportunistic pathogen of swine or horses, respectively. Identification was performed by matrix-assisted laser desorption ionization–time of flight mass spectrometry and real-time PCR assay. A previous visit to a farm was suspected as the source of infection. PMID:25878346

  9. Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes

    PubMed Central

    Yang, Huan; Wang, Haichao; Levine, Yaakov A.; Gunasekaran, Manoj K.; Wang, Yongjun; Addorisio, Meghan; Zhu, Shu; Li, Wei; Li, Jianhua; de Kleijn, Dominique P.V.; Olofsson, Peder S.; Warren, H. Shaw; He, Mingzhu; Al-Abed, Yousef; Roth, Jesse; Antoine, Daniel J.; Chavan, Sangeeta S.; Andersson, Ulf; Tracey, Kevin J.

    2016-01-01

    Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases. PMID:27294203

  10. Structure and assembly of haptoglobin polymers by electron microscopy.

    PubMed

    Wejman, J C; Hovsepian, D; Wall, J S; Hainfeld, J F; Greer, J

    1984-04-01

    Haptoglobin (Hp) consists of light (L) and heavy (H) chains, the latter of which combine with hemoglobin alpha beta dimers to form a highly stable complex. Human haptoglobin assembles as HL units that occur in two allelic forms; HL1 , which is monovalent, and HL2 , which is divalent. As a result, three phenotypic forms exist in the human population: Hp1-1, the homozygous form in which the monovalent HL1 unit occurs as a dimer; Hp2-2, the homozygous form of the divalent HL2 unit, which gives a series of polymers; and the heterozygous Hp2-1 form, which gives a different series of polymers. We have investigated the structures and assembly properties of these two haptoglobin polymeric series in their complexes with hemoglobin using high-resolution scanning transmission electron microscopy. Polymers of complex are composed of ellipsoidal or bilobal head groups, which are the H alpha beta subunits connected by thin filament-like structures, which are the L chains. Polymers of size up to pentamers can be identified easily by counting the number of head groups in the molecule. Complex 2-1 and complex 2-2 trimers were studied extensively. The differences in detailed morphology show that while the 2-1 trimer is a linear polymer, the 2-2 trimer is a closed circular molecule. The micrograph images suggest that complex 2-2 tetramers and pentamers, and perhaps higher forms may also be cyclic. The structure of the L2 subunit of haptoglobin is shown to be composed of two domains, which may be similar in structure to the single domain of the monovalent L1 chain. The two L2 domains are connected by a hinge that has quite limited flexibility. Using these structural models, assembly characteristics and structural properties of the trimers and tetramers of complex 2-1 and complex 2-2 are described. PMID:6716482

  11. Measurement of serum haptoglobin as an indicator of the efficacy of malaria intervention trials.

    PubMed

    Sisay, F; Byass, P; Snow, R W; Greenwood, B M; Perrin, L H; Yerly, S

    1992-01-01

    Serum haptoglobin levels were measured by an enzyme-linked immunosorbent assay in Gambian children who participated in 3 malaria intervention trials with untreated or impregnated bed nets. In one study, in which a significant effect on clinical malaria was observed, the mean serum haptoglobin level was significantly higher in the intervention than in the control group. In the other 2 studies, in which no significant protection was observed, mean haptoglobin levels were similar in intervention and control groups. Measurement of serum haptoglobin may provide a useful indirect measure of the effectiveness of malaria control programmes. PMID:1566291

  12. 21 CFR 522.313b - Ceftiofur hydrochloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... bacterial pneumonia) associated with Actinobacillus pleuropneumoniae, Pasteurella multocida, Salmonella... treatment of bovine respiratory disease (BRD, shipping fever, pneumonia) associated with...

  13. Uses of serology for the diagnosis of contagious bovine pleuropneumonia.

    PubMed

    Rurangirwa, F R

    1995-09-01

    A serological test involves detection of specific changes, induced by a pathogen, in the properties or actions of serum of an infected host. The test may detect the presence in serum of either antibodies to the pathogen (produced by the host) or antigens (i.e. the infecting agent itself and/or its components). The many serological tests which have been developed for the diagnosis of contagious bovine pleuropneumonia (CBPP) are classified into two groups on the basis of this distinction. To date, no single serological test is able to detect all stages of the disease. Thus the choice of serological test (or combination of tests) will depend on the specific aim of the investigation. Meanwhile, a sensitive, specific and simple 'pen-side' test for the diagnosis of all forms of CBPP is still lacking. PMID:8593394

  14. [Initial action of synthetic sexual steroids on serum-haptoglobin concentration (author's transl)].

    PubMed

    Klinger, G; Günther, E; Günther, M; Gerlach, D

    1981-01-01

    Sera of 21 clinically intact women, who had been on Non-ovlon, Gravistat or Deposiston, were tested for their haptoglobin levels, during two control cycles and in the first three cycles on contraceptives. Serum-haptoglobin concentrations were reduced. A primary role is played, in that context, by oestrogens. PMID:7223167

  15. Serum haptoglobin concentrations in dairy cattle with lameness due to claw disorders.

    PubMed

    Smith, Billy I; Kauffold, Johannes; Sherman, Lisa

    2010-11-01

    In cattle, elevated blood serum concentrations of haptoglobin, an acute phase protein, have been demonstrated in association with several diseases, but not with lameness. Serum haptoglobin was measured in 60 Holstein dairy cattle diagnosed with lameness due to four claw disorders, pododermatitis septica (PS; n=41), pododermatitis circumscripta (PC; n=8), interdigital necrobacillosis (IN; n=7), papillomatous digital dermatitis (PDD; n=4). Haptoglobin was measured on day 1 (0-3 days after lameness was observed but before treatment) and on days 3 and 5. A total of 10 healthy cows served as controls (haptoglobin values <1.0 mg/dL). Each of the claw disorders was associated with elevated haptoglobin on day 1 (PS, PC, IN and PDD: 65.9%, 37.5%, 71.4% and 25.0%, respectively). Trimming and antibiotic treatment led to a reduction in the number of PS and IN cows with increased haptoglobin concentrations, respectively (P<0.05), but trimming did not lead to any reduction in cows with PC. The study showed that lameness due to claw disorders can be associated with a systemic acute phase response and elevated serum haptoglobin in dairy cattle. Based on the course of haptoglobin, treatments seemed effective for all claw disorders except for PC. PMID:19751983

  16. N-linked glycan changes of serum haptoglobin β chain in liver disease patients.

    PubMed

    Zhang, Shu; Shu, Hong; Luo, Kaixuan; Kang, Xiaonan; Zhang, Ying; Lu, Haojie; Liu, Yinkun

    2011-05-01

    Human haptoglobin is a serum glycoprotein secreted by the liver with four potential N-glycosylation sites on its β chain. Many studies have reported glycan changes of haptoglobin in diseases such as breast cancer and pancreatic cancer. The objective of our study is to analyze N-linked glycan alterations of serum haptoglobin β chain obtained from patients with the hepatitis B virus (HBV), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). MALDI-QIT-TOF mass spectrometry revealed the intensity of m/z 1809.6, identified as a fucosylated glycan, was much higher in samples from patients with LC and HCC relative to the patients with HBV and healthy controls. Compared with LC patients, triantennary glycan was elevated and the biantennary structure was decreased in the haptoglobin β chain of HCC patients. Thus, alterations in the glycan structure of the haptoglobin β chain may constitute significant spectral signatures of cirrhosis and HCC disease. PMID:21380457

  17. Serum-haptoglobin concentration in Danish slaughter pigs of different health status.

    PubMed

    Petersen, Henrik Hagbard; Ersbøll, Annette Kjaer; Jensen, Charlotte Sonne; Nielsen, Jens Peter

    2002-08-30

    A cross-sectional study was conducted with 617 finishing pigs aged 10-25 weeks in 11 commercial herds of different health statuses as defined by the Danish monitoring program for specific-pathogen-free (SPF) herds. A standard clinical examination was performed and a blood sample was obtained from each pig for determination of haptoglobin concentration in serum. Pigs aged 10-14, 15-19 and 20-25 weeks in conventional herds had higher haptoglobin concentrations than high-health SPF (SPF-x) pigs of the same age. There was no significant difference between SPF-x pigs of different ages. Conventional pigs aged 15-19 and 20-25 weeks had higher haptoglobin concentrations than conventional pigs aged 10-14 weeks. Herd influenced the haptoglobin concentration. Lame pigs and pigs with tail or ear bite had elevated haptoglobin concentrations. No significant effect of respiratory symptoms or umbilical hernia was found. PMID:12163249

  18. Evidence that extracellular components function in adherence of Actinobacillus actinomycetemcomitans to epithelial cells.

    PubMed Central

    Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Extracellular microvesicles and a highly proteinaceous polymer associated with a leukotoxin-producing strain, Actinobacillus actinomycetemcomitans SUNY 75, were shown to increase adherence of other weakly adherent A. actinomycetemcomitans strains to KB epithelial cells. Images PMID:8406899

  19. Actinobacillus spp. and related bacteria in infected wounds of humans bitten by horses and sheep.

    PubMed Central

    Peel, M M; Hornidge, K A; Luppino, M; Stacpoole, A M; Weaver, R E

    1991-01-01

    We describe the isolation of Actinobacillus lignieresii and an A. equuli-like bacterium from an infected horse-bite wound in a 22-year-old stable foreman and A. suis from a bite injury in a 35-year-old man who had been attacked by a horse. A. lignieresii was also isolated in pure culture from an infected sheep-bite wound in a rural worker. These species of the genus Actinobacillus are primarily associated with animals and animal diseases and are rarely isolated from humans. The purpose of this report is to raise awareness of the possible occurrence of Actinobacillus spp. in bite wounds inflicted by farm animals and to discuss the difficulties encountered in the identification of species of Actinobacillus and related bacteria. PMID:1774260

  20. Studies on the structure of haptoglobin and the haptoglobin-haemoglobin complex by spin and fluorescence labelling.

    PubMed

    Osada, J; Sawaryn, A; Dobryszycka, W

    1978-01-01

    Human haptoglobin (Hp) of the 1-1 type incorporated one spin or fluorescence marker per molecule; the markers were found in the beta chain. Formation of the complex between spin-labelled Hp and haemoglobin or antibody caused conformational changes in the Hp molecular, evidenced by increased participation in the electron paramagnetic resonance spectrum of the component bound with the slowly rotating marker. From fluorescence-labelled Hp, the beta chain was isolated and cleaved by CNBr; only in one of the obtained peptides, one out of 4 histidine residues was modified with the marker. PMID:220830

  1. Serum haptoglobin in clinical biochemistry: change of a paradigm.

    PubMed

    Robert, L

    2013-12-01

    Serum haptoglobin (Hp) was discovered by Max Fernand Jayle as a young assistant professor in the Department of Biochemistry of the Paris Medical Faculty, headed by Professor Michel Polonovski. Jayle showed that Hp was an acute phase glycoprotein and worked out its routine determination in the blood-serum, based on its complex formation with hemoglobin, using the increased peroxidase activity of the hemoglobin-haptoglobin (Hb-Hp) complex, for routine determination in clinical biochemistry for the characterisation of inflammatory processes, together with other acute phase glycoproteins as orosomucoide. Later Smithies described the genetic control of human Hp-isoforms and quite recently Andersen et al. reported the elucidation of the crystal structure of the porcine Hb-Hp complex. In that article there was no mention of the discovery of Hp, neither of its determination in clinical biochemistry as an inflammatory marker. The only biologically significant role assigned to Hp by Andersen et al. is its (hypothetical) role to prevent or minimize the harmful effects of Hb during intravascular hemolysis, by the generation of reactive oxygen species (ROS) and complexing it. This shift of paradigm, not at all exceptional in medical biochemistry, will be described and discussed with its pitfalls and consequences. PMID:24011966

  2. Haptoglobin gene polymorphisms and interleukin-6 and -8 levels in patients with sickle cell anemia

    PubMed Central

    Pierrot-Gallo, Bruna Spinella; Vicari, Perla; Matsuda, Sandra Satiko; Adegoke, Samuel Ademola; Mecabo, Grazielle; Figueiredo, Maria Stella

    2015-01-01

    Background Haptoglobin genotypes, and interleukin-6 and -8 participate in the pathophysiology of sickle cell anemia. The expression of cytokines is regulated by genetic mechanisms however the effect of haptoglobin polymorphisms on these cytokines is not fully understood. This study aimed to compare the frequency of haptoglobin genotypes and the interleukin-6 and -8 concentrations in sickle cell anemia patients and controls to investigate the association between haptoglobin genotypes and cytokine levels. Methods Sixty sickle cell anemia patients and 74 healthy individuals were analyzed. Haptoglobin genotypes were determined by multiplex polymerase chain reaction, and the interleukin-6 and -8 levels by enzyme linked immunosorbent assay. The association between haptoglobin genotypes and cytokines was investigated by statistical tests. Results Hp2-1 was the most common genotype in both the cases and controls while Hp1-1 was less frequent among sickle cell anemia patients. Interleukin-6 and -8 levels were higher in patients than controls (p-value <0.0001). There was no significant difference in interleukin-6 and -8 concentrations between the genotypes (p-value >0.05). A similar trend was observed among the controls. Conclusion Although, levels of interleukin-6 and -8 were higher in the sickle cell anemia patients, they appeared not to be related to the haptoglobin genotypes. Further investigations are necessary to identify factors responsible for increased secretion of the interleukin-6 and -8 pro-inflammatory cytokines in patients with sickle cell anemia. PMID:26408368

  3. Studies on hemoglobin tryptophanyl contact residues in the haptoglobin-hemoglobin complex.

    PubMed

    Rogard, M; Waks, M

    1977-07-15

    Hemoglobin and apohemoglobin bind heptoglobin in the same molar ratio. Structural studies on haptoglobin-hemoglobin complex do not suggest any important structural changes in either protein upon binding. However, when apohemoglobin is bound to haptoglobin, a marked reduction in secondary structure, attributed to unfolding of globin chains, has been observed. Here we describe some properties of the haptoglobin-apohemoglobin (Hp-apoHb) complex, prepared by isoelectric focusing in the presence of an excess of haptoglobin. This complex does not exhibit the irreversibility of complexes obtained with hemoglobin in identical experimental conditions. The 'freezing' of the conformation of apohemoglobin upon binding to haptoglobin has been studied by fluorescence quenching experiments carried out in the presence of 8 M acrylamide. Changes in conformation of haptoglobin upon binding to apohemoglobin have been detected by titration of the exposed tryptophans using N-bromosuccinimide. Comparison of the additivity of exposed tryptophans in the complexes reveal that two tryptophans become inaccessible in the complex formation of haptoglobin with hemoglobin but not with apohemoglobin. These tryptophans, probably located on the alpha1beta2 contact interface of hemoglobin, have been tentatively identified as Trp-C3(37)beta. PMID:891540

  4. Actinobacillus actinomycetemcomitans contamination of toothbrushes from patients harbouring the organism.

    PubMed

    Müller, H P; Lange, D E; Müller, R F

    1989-07-01

    The main ecological niche of Actinobacillus actinomycetemcomitans (A.a.) seems to be the periodontal pocket, but it can also be isolated from supragingival plaque, buccal and tongue mucosa, or saliva. We examined toothbrushes from 21 patients, all identified as harbouring moderate to large numbers of A.a. in subgingival plaque, for contamination with this organism. 29% of the toothbrushes presented by our patients yielded detectable numbers of A.a. Immediately after toothbrushing this figure rose to 62%, but dropped to 50% after 1 h. Numbers of isolated A.a. on toothbrushes were weakly correlated with the degree of periodontal destruction, and significantly more numbers of A.a. on toothbrushes could be detected if the organism was found on mucous membranes or in saliva. There was no association with gingival inflammation, supragingival plaque nor mean numbers of isolated subgingival A.a. PMID:2760252

  5. Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the beta-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain.

    PubMed

    Yoshioka, N; Atassi, M Z

    1986-03-01

    A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146. PMID:3718478

  6. Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes.

    PubMed

    Madsen, M; Graversen, J H; Moestrup, S K

    2001-01-01

    The plasma protein haptoglobin and the endocytic hemoglobin receptor HbSR/CD163 are key molecules in the process of removing hemoglobin released from ruptured erythrocytes. Hemoglobin in plasma is instantly bound with high affinity to haptoglobin--an interaction leading to the recognition of the complex by HbSR/CD163 and endocytosis in macrophages. The haptoglobin-dependent HbSR/CD163 scavenging system for hemoglobin clearance prevents toxic effects of hemoglobin in plasma and kidney and explains the decrease in the haptoglobin plasma concentration in patients with accelerated hemolysis. The HbSR/CD163 activity may be of quantitative importance for iron uptake in macrophages in general and for some iron-associated pathological processes, e.g. the atherogenesis-promoting oxidation of LDL leading to foam cell formation and apoptosis in the vessel wall. PMID:11865982

  7. Combination of medical and surgical therapy for pleuropneumonia in a horse.

    PubMed Central

    Dechan, J

    1997-01-01

    Medical management was unable to prevent the development of an extrapulmonary abscess in a 10-year-old Thoroughbred gelding with anaerobic pleuropneumonia. Intercostal thoracostomy achieved drainage of the abscess. Resolution of the abscess and subsequent bronchopleural fistulas was monitored by ultrasonography and video-endoscopy. The horse returned to training 4 mo after discharge. PMID:9262860

  8. A model aerosol exposure system for induction of porcine Haemophilus pleuropneumonia.

    PubMed Central

    Sebunya, T N; Saunders, J R; Osborne, A D

    1983-01-01

    One group of six pigs and another group of three pigs were separately exposed in a polyethylene enclosed chamber for ten minutes, respectively, to Haemophilus pleuropneumoniae serotype 1 and Bacillus subtilis aerosols generated by an ultrasonic nebulizer. Haemophilus pleuropneumoniae and B. subtilis were deposited throughout the lungs immediately following aerosol exposure. The number of H. pleuropneumoniae and B. subtilis deposited varied within and between lungs in each group. The mean numbers of both organisms deposited in the posterior (caudal and accessory) lobes were significantly greater than those in the anterior (cranial and middle) lobes (P less than 0.001). The four principals that received H. pleuropneumoniae aerosols and the two contact controls developed fatal fibrinous pneumonia which simulated that seen in natural infections. Since this exposure system consistently resulted in clinical disease it has good potential as a model for the study of pathogenesis of the disease and more specifically for the evaluation of vaccines. Images Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:6403208

  9. Complete Genome Sequence of Mycoplasma mycoides subsp. mycoides T1/44, a Vaccine Strain against Contagious Bovine Pleuropneumonia.

    PubMed

    Gourgues, Géraldine; Barré, Aurélien; Beaudoing, Emmanuel; Weber, Johann; Magdelenat, Ghislaine; Barbe, Valérie; Schieck, Elise; Jores, Joerg; Vashee, Sanjay; Blanchard, Alain; Lartigue, Carole; Sirand-Pugnet, Pascal

    2016-01-01

    Mycoplasma mycoidessubsp.mycoidesis the etiologic agent of contagious bovine pleuropneumonia. We report here the complete genome sequence of the strain T1/44, which is widely used as a live vaccine in Africa. PMID:27081135

  10. Complete Genome Sequence of Mycoplasma mycoides subsp. mycoides T1/44, a Vaccine Strain against Contagious Bovine Pleuropneumonia

    PubMed Central

    Gourgues, Géraldine; Barré, Aurélien; Beaudoing, Emmanuel; Weber, Johann; Magdelenat, Ghislaine; Barbe, Valérie; Schieck, Elise; Jores, Joerg; Vashee, Sanjay; Blanchard, Alain; Lartigue, Carole

    2016-01-01

    Mycoplasma mycoides subsp. mycoides is the etiologic agent of contagious bovine pleuropneumonia. We report here the complete genome sequence of the strain T1/44, which is widely used as a live vaccine in Africa. PMID:27081135

  11. Study of chaperone-like activity of human haptoglobin: conformational changes under heat shock conditions and localization of interaction sites.

    PubMed

    Ettrich, Rüdiger; Brandt, Wolfgang; Kopecký, Vladimír; Baumruk, Vladimír; Hofbauerová, Katerina; Pavlícek, Zdenek

    2002-10-01

    With respect to the mechanism of chaperone-like activity, we examined the behavior of haptoglobin under heat shock conditions. Secondary structure changes during heat treatment were followed by circular dichroism, Raman and infrared spectroscopy. A model of the haptoglobin tetramer, based on its sequence homology with serine proteases and the CCP modules, has been proposed. Sequence regions responsible for the chaperone-like activity were not fully identical with the region that takes part in formation of the hemoglobin-haptoglobin complex. We can postulate the presence of at least two different chaperone-binding sites on each haptoglobin heavy chain. PMID:12452443

  12. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.

    PubMed

    Lally, E T; Kieba, I R; Demuth, D R; Rosenbloom, J; Golub, E E; Taichman, N S; Gibson, C W

    1989-02-28

    The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity. PMID:2647082

  13. Economical succinic acid production from cane molasses by Actinobacillus succinogenes.

    PubMed

    Liu, Yu-Peng; Zheng, Pu; Sun, Zhi-Hao; Ni, Ye; Dong, Jin-Jun; Zhu, Lei-Lei

    2008-04-01

    In this work, production of succinic acid by Actinobacillus succinogenes CGMCC1593 using cane molasses as a low cost carbon source was developed. In anaerobic bottles fermentation, succinic acid concentration of 50.6+/-0.9 g l(-1) was attained at 60 h using an optimum medium containing molasses pretreated with sulfuric acid, resulting in a succinic acid yield of 79.5+/-1.1% and sugar utilization of 97.1+/-0.6%. When batch fermentation was carried out in a 5-l stirred bioreactor with pretreated molasses, 46.4 g l(-1) of succinic acid was attained at 48 h and faster cells growth was also observed. Fed batch fermentation was performed to minimize the substrate (sugar) inhibition effect, giving 55.2 g l(-1) of succinic acid and 1.15 g l(-1)h(-1) of productivity at 48 h. The present study suggests that the inexpensive cane molasses could be utilized for the economical and efficient production of succinic acid by A. succinogenes. PMID:17532626

  14. Haptoglobin Is Required to Prevent Oxidative Stress and Muscle Atrophy

    PubMed Central

    Lo Verso, Francesca; Santini, Ferruccio; Vitti, Paolo; Chisari, Carmelo; Sandri, Marco; Maffei, Margherita

    2014-01-01

    Background Oxidative stress (OS) plays a major role on tissue function. Several catabolic or stress conditions exacerbate OS, inducing organ deterioration. Haptoglobin (Hp) is a circulating acute phase protein, produced by liver and adipose tissue, and has an important anti-oxidant function. Hp is induced in pro-oxidative conditions such as systemic inflammation or obesity. The role of systemic factors that modulate oxidative stress inside muscle cells is still poorly investigated. Results We used Hp knockout mice (Hp-/-) to determine the role of this protein and therefore, of systemic OS in maintenance of muscle mass and function. Absence of Hp caused muscle atrophy and weakness due to activation of an atrophy program. When animals were stressed by acute exercise or by high fat diet (HFD), OS, muscle atrophy and force drop were exacerbated in Hp-/-. Depending from the stress condition, autophagy-lysosome and ubiquitin-proteasome systems were differently induced. Conclusions Hp is required to prevent OS and the activation of pathways leading to muscle atrophy and weakness in normal condition and upon metabolic challenges. PMID:24959824

  15. Role of heme compounds and haptoglobin in Vibrio vulnificus pathogenicity.

    PubMed

    Helms, S D; Oliver, J D; Travis, J C

    1984-08-01

    An induced peritonitis model was employed in mice to determine whether heme-containing molecules enhance the lethality of infections by Vibrio vulnificus. The lethality of intraperitoneal (ip) inocula of the bacteria was increased by concurrent injections (ip) of hemoglobin, methemoglobin, or hematin, but not by myoglobin. Similar results were obtained in mice with phenylhydrazine-induced hemoglobinemia, in which after ip injections of V. vulnificus, a direct correlation between lethality and levels of plasma hemoglobin was observed. In vitro studies indicated that the growth of V. vulnificus, which was limited in an iron-poor medium, was enhanced by the addition of hemoglobin in a manner similar to an inorganic iron source, ferric ammonium citrate. These results suggest that V. vulnificus is capable of extracting iron from hemoglobin for use as a nutrilite, thereby promoting growth and increased lethality in the in vivo models. Further studies with human serum cultures demonstrated that the growth of V. vulnificus was not decreased when hemoglobin added to the serum was completely complexed with haptoglobin; these results are in opposition to those with cultures of Escherichia coli. These results are discussed relative to the capacity of V. vulnificus to produce fatal human infections. PMID:6746093

  16. Role of heme compounds and haptoglobin in Vibrio vulnificus pathogenicity.

    PubMed Central

    Helms, S D; Oliver, J D; Travis, J C

    1984-01-01

    An induced peritonitis model was employed in mice to determine whether heme-containing molecules enhance the lethality of infections by Vibrio vulnificus. The lethality of intraperitoneal (ip) inocula of the bacteria was increased by concurrent injections (ip) of hemoglobin, methemoglobin, or hematin, but not by myoglobin. Similar results were obtained in mice with phenylhydrazine-induced hemoglobinemia, in which after ip injections of V. vulnificus, a direct correlation between lethality and levels of plasma hemoglobin was observed. In vitro studies indicated that the growth of V. vulnificus, which was limited in an iron-poor medium, was enhanced by the addition of hemoglobin in a manner similar to an inorganic iron source, ferric ammonium citrate. These results suggest that V. vulnificus is capable of extracting iron from hemoglobin for use as a nutrilite, thereby promoting growth and increased lethality in the in vivo models. Further studies with human serum cultures demonstrated that the growth of V. vulnificus was not decreased when hemoglobin added to the serum was completely complexed with haptoglobin; these results are in opposition to those with cultures of Escherichia coli. These results are discussed relative to the capacity of V. vulnificus to produce fatal human infections. PMID:6746093

  17. The acute phase protein haptoglobin regulates host immunity

    PubMed Central

    Huntoon, Kristin M.; Wang, Yanping; Eppolito, Cheryl A.; Barbour, Karen W.; Berger, Franklin G.; Shrikant, Protul A.; Baumann, Heinz

    2008-01-01

    The contribution of acute phase plasma proteins to host immune responses remains poorly characterized. To better understand the role of the acute phase reactant and major hemoglobin-binding protein haptoglobin (Hp) on the function of immune cells, we generated Hp-deficient C57BL/6J mice. These mice exhibit stunted development of lymphoid organs associated with lower counts of mature T and B cells in the blood and secondary lymphoid compartments. Moreover, these mice show markedly reduced adaptive immune responses as represented by reduced accumulation of IgG antibody after immunization with adjuvant and nominal antigen, abrogation of Th1-dominated delayed-type hypersensitivity reaction, loss of mitogenic responses mounted by T cells, and reduced T cell responses conveyed by APCs. Collectively, these defects are in agreement with the observations that Hp-deficient mice are not capable of generating a recall response or deterring a Salmonella infection as well as failing to generate tumor antigen-specific responses. The administration of Hp to lymphocytes in tissue culture partially ameliorates these functional defects, lending further support to our contention that the acute phase response protein Hp has the ability to regulate immune cell responses and host immunity. The phenotype of Hp-deficient mice suggests a major regulatory activity for Hp in supporting proliferation and functional differentiation of B and T cells as part of homeostasis and in response to antigen stimulation. PMID:18436583

  18. Haptoglobin activates innate immunity to enhance acute transplant rejection in mice

    PubMed Central

    Shen, Hua; Song, Yang; Colangelo, Christopher M.; Wu, Terence; Bruce, Can; Scabia, Gaia; Galan, Anjela; Maffei, Margherita; Goldstein, Daniel R.

    2011-01-01

    Immune tolerance to transplanted organs is impaired when the innate immune system is activated in response to the tissue necrosis that occurs during harvesting and implantation procedures. A key molecule in this immune pathway is the intracellular TLR signal adaptor known as myeloid differentiation primary response gene 88 (MyD88). After transplantation, MyD88 induces DC maturation as well as the production of inflammatory mediators, such as IL-6 and TNF-α. However, upstream activators of MyD88 function in response to transplantation have not been identified. Here, we show that haptoglobin, an acute phase protein, is an initiator of this MyD88-dependent inflammatory process in a mouse model of skin transplantation. Necrotic lysates from transplanted skin elicited higher inflammatory responses in DCs than did nontransplanted lysates, suggesting DC-mediated responses are triggered by factors released during transplantation. Analysis of transplanted lysates identified haptoglobin as one of the proteins upregulated during transplantation. Expression of donor haptoglobin enhanced the onset of acute skin transplant rejection, whereas haptoglobin-deficient skin grafts showed delayed acute rejection and antidonor T cell priming in a MyD88-dependent graft rejection model. Thus, our results show that haptoglobin release following skin necrosis contributes to accelerated transplant rejection, with potential implications for the development of localized immunosuppressive therapies. PMID:22156194

  19. Haptoglobin genotype- and diabetes-dependent differences in iron-mediated oxidative stress in vitro and in vivo.

    PubMed

    Asleh, Rabea; Guetta, Julia; Kalet-Litman, Shiri; Miller-Lotan, Rachel; Levy, Andrew P

    2005-03-01

    We have recently demonstrated in multiple independent population-based longitudinal and cross sectional analyses that the haptoglobin 2-2 genotype is associated with an increased risk for diabetic cardiovascular disease. The chief function of haptoglobin (Hp) is to bind to hemoglobin and thereby prevent hemoglobin-induced oxidative tissue damage. This antioxidant function of haptoglobin is mediated in part by the ability of haptoglobin to prevent the release of iron from hemoglobin on its binding. We hypothesized that there may be diabetes- and haptoglobin genotype-dependent differences in the amount of catalytically active redox active iron derived from hemoglobin. We tested this hypothesis using several complementary approaches both in vitro and in vivo. First, measuring redox active iron associated with haptoglobin-hemoglobin complexes in vitro, we demonstrate a marked increase in redox active iron associated with Hp 2-2-glycohemoglobin complexes. Second, we demonstrate increased oxidative stress in tissue culture cells exposed to haptoglobin 2-2-hemoglobin complexes as opposed to haptoglobin 1-1-hemoglobin complexes, which is inhibitable by desferrioxamine by either a chelation or reduction mechanism. Third, we demonstrate marked diabetes-dependent differences in the amount of redox active iron present in the plasma of mice genetically modified expressing the Hp 2 allele as compared with the Hp 1 allele. Taken together these data implicate redox active iron in the increased susceptibility of individuals with the Hp 2 allele to diabetic vascular disease. PMID:15662028

  20. Molecular modeling of the human hemoglobin-haptoglobin complex sheds light on the protective mechanisms of haptoglobin.

    PubMed

    Nantasenamat, Chanin; Prachayasittikul, Virapong; Bulow, Leif

    2013-01-01

    Hemoglobin (Hb) plays a critical role in human physiological function by transporting O2. Hb is safe and inert within the confinement of the red blood cell but becomes reactive and toxic upon hemolysis. Haptoglobin (Hp) is an acute-phase serum protein that scavenges Hb and the resulting Hb-Hp complex is subjected to CD163-mediated endocytosis by macrophages. The interaction between Hb and Hp is extraordinarily strong and largely irreversible. As the structural details of the human Hb-Hp complex are not yet available, this study reports for the first time on insights of the binding modalities and molecular details of the human Hb-Hp interaction by means of protein-protein docking. Furthermore, residues that are pertinent for complex formation were identified by computational alanine scanning mutagenesis. Results revealed that the surface of the binding interface of Hb-Hp is not flat and protrudes into each binding partner. It was also observed that the secondary structures at the Hb-Hp interface are oriented as coils and α-helices. When dissecting the interface in more detail, it is obvious that several tyrosine residues of Hb, particularly β145Tyr, α42Tyr and α140Tyr, are buried in the complex and protected from further oxidative reactions. Such finding opens up new avenues for the design of Hp mimics which may be used as alternative clinical Hb scavengers. PMID:23638175

  1. The gingival immune response to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

    PubMed

    Hall, E R; Falkler, W A; Martin, S A; Suzuki, J B

    1991-12-01

    The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients. PMID:1765941

  2. Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells.

    PubMed

    Kato, Satsuki; Sugimura, Norihiko; Nakashima, Keisuke; Nishihara, Tatsuji; Kowashi, Yusuke

    2005-03-01

    It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans

  3. The haptoglobin 2-2 genotype is associated with inflammation and carotid artery intima-media thickness

    PubMed Central

    Dalan, Rinkoo; Liew, Huiling; Goh, Liuh Ling; Gao, Xiao; Chew, Daniel EK; Boehm, Bernhard O; Leow, Melvin Khee Shing

    2016-01-01

    The haptoglobin 2-2 genotype is associated with atherosclerosis in type 2 diabetes mellitus. We examined the associations of the haptoglobin 2-2 genotype with C-reactive protein (high-sensitivity C-reactive protein) and carotid artery intima-media thickness, adjusting for age, gender, ethnicity, type 2 diabetes mellitus, smoking status, body mass index, blood pressure, glycated haemoglobin, non-high-density lipoprotein cholesterol and medications via logistic multivariate regression in 200 subjects (160 type 2 diabetes mellitus versus 40 healthy individuals). The prevalence of the haptoglobin 2-2 genotype was 58% (115/200), higher in the Indians than in Chinese (72% versus 45%, p = 0.001). Multivariate analysis showed that the haptoglobin 2-2 genotype was associated with high-sensitivity C-reactive protein [mean: 3.5 ± 3.9 versus 2.2 ± 2.6 mg/L (non-haptoglobin 2-2), p < 0.001], haptoglobin concentration [mean: 116.9 ± 54.4.0 versus 147.2 ± 54.5 mg/dL (non-haptoglobin 2-2), p < 0.001] and average carotid artery intima-media thickness (multiplied by 10) [6.15 ± 1.22 versus 5.98 ± 1.20 mm (non-haptoglobin 2-2), p = 0.013]. This pilot study shows an association of the haptoglobin 2-2 genotype with low-grade inflammation, haptoglobin concentration and carotid artery intima-media thickness in multi-ethnic Singapore. PMID:27190085

  4. The haptoglobin 2-2 genotype is associated with inflammation and carotid artery intima-media thickness.

    PubMed

    Dalan, Rinkoo; Liew, Huiling; Goh, Liuh Ling; Gao, Xiao; Chew, Daniel Ek; Boehm, Bernhard O; Leow, Melvin Khee Shing

    2016-09-01

    The haptoglobin 2-2 genotype is associated with atherosclerosis in type 2 diabetes mellitus. We examined the associations of the haptoglobin 2-2 genotype with C-reactive protein (high-sensitivity C-reactive protein) and carotid artery intima-media thickness, adjusting for age, gender, ethnicity, type 2 diabetes mellitus, smoking status, body mass index, blood pressure, glycated haemoglobin, non-high-density lipoprotein cholesterol and medications via logistic multivariate regression in 200 subjects (160 type 2 diabetes mellitus versus 40 healthy individuals). The prevalence of the haptoglobin 2-2 genotype was 58% (115/200), higher in the Indians than in Chinese (72% versus 45%, p = 0.001). Multivariate analysis showed that the haptoglobin 2-2 genotype was associated with high-sensitivity C-reactive protein [mean: 3.5 ± 3.9 versus 2.2 ± 2.6 mg/L (non-haptoglobin 2-2), p < 0.001], haptoglobin concentration [mean: 116.9 ± 54.4.0 versus 147.2 ± 54.5 mg/dL (non-haptoglobin 2-2), p < 0.001] and average carotid artery intima-media thickness (multiplied by 10) [6.15 ± 1.22 versus 5.98 ± 1.20 mm (non-haptoglobin 2-2), p = 0.013]. This pilot study shows an association of the haptoglobin 2-2 genotype with low-grade inflammation, haptoglobin concentration and carotid artery intima-media thickness in multi-ethnic Singapore. PMID:27190085

  5. Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury*

    PubMed Central

    Asleh, Rabea; Ward, John; Levy, Nina S.; Safuri, Shady; Aronson, Doron; Levy, Andrew P.

    2014-01-01

    The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb. PMID:24778180

  6. Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR.

    PubMed Central

    Flemmig, T F; Rüdiger, S; Hofmann, U; Schmidt, H; Plaschke, B; Strätz, A; Klaiber, B; Karch, H

    1995-01-01

    The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the

  7. Biogenesis of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Holotoxin

    PubMed Central

    Ueno, Yoko; Ohara, Masaru; Kawamoto, Toru; Fujiwara, Tamaki; Komatsuzawa, Hitoshi; Oswald, Eric; Sugai, Motoyuki

    2006-01-01

    The cell cycle G2/M specific inhibitor cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is composed of CdtA, CdtB, and CdtC coded on the cdtA, cdtB, and cdtC genes that are tandem on the chromosomal cdt locus. A. actinomycetemcomitans CdtA has the lipid binding consensus domain, the so-called “lipobox”, at the N-terminal signal sequence. Using Escherichia coli carrying plasmid pTK3022, we show that the 16th residue, cysteine, of CdtA bound [3H]palmitate or [3H]glycerol. Further, posttranslational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblotting show the lipid-modified CdtA is present in the outer membrane. Immunoprecipitation and the pull-down assay of the CDT complex from E. coli carrying a plasmid containing cdtABC demonstrated that the CDT complex in the periplasm is composed of CdtA, CdtB, and CdtC and that the CDT complex in culture supernatant is an N-terminally truncated (36 to 43 amino acids) form of CdtA (CdtA′), CdtB, and CdtC. This suggests that CDT is present as a complex both in the periplasm and the supernatant where CdtA undergoes posttranslation processing to CdtA′ in the process of biogenesis and secretion of CDT holotoxin into the culture supernatant. Site-directed mutagenesis of the 16th cysteine residue to glycine in CdtA altered localization of CdtA in the cell and reduced the amount of CDT activity in the culture supernatant. This suggests that CDT forms a complex inside the periplasm for lipid modification where posttranslational processing of CdtA plays an important role for the efficient production of CDT holotoxin into the culture supernatant. PMID:16714579

  8. Haptoglobin Duplicon, Hemoglobin, and Vitamin C: Analyses in the British Women's Heart and Health Study and Caerphilly Prospective Study

    PubMed Central

    Guthrie, Philip A. I.; Abdollahi, Mohammad R.; Gaunt, Tom; Lawlor, Debbie A.; Ben-Shlomo, Yoav; Gallacher, John; Davey Smith, George; Day, Ian N. M.

    2014-01-01

    Background. Haptoglobin acts as an antioxidant by limiting peroxidative tissue damage by free hemoglobin. The haptoglobin gene allele Hp2 comprises a 1.7 kb partial duplication. Relative to allele Hp1, Hp2 carriers form protein multimers, suboptimal for hemoglobin scavenging. Objective. To examine the association of haptoglobin genotype with a range of phenotypes, with emphasis on vitamin C and hemoglobin levels. Methods. We applied a quantitative PCR assay for the duplication junction to two population cohorts including 2747 British women and 1198 British men. We examined the association of haptoglobin duplicon copy number with hemoglobin and vitamin C and used the copy number to complete a phenome scan. Results. Hemoglobin concentrations were greater in those with Hp2,2 genotype, in women only (Hp1,1 13.45 g/dL, Hp1,2 13.49 g/dL, Hp2,2 13.61 g/dL; P = 0.002), though statistically there was no evidence of a difference between the sexes (z value = 1.2, P = 0.24). Haptoglobin genotype was not associated with vitamin C or any other phenotype in either cohort. Conclusions. Our results do not support association of haptoglobin genotype with vitamin C or with other phenotypes measured in two population cohorts. The apparent association between haptoglobin genotype and hemoglobin in the women's cohort merits further investigation. PMID:25525287

  9. Biosensor assay for determination of haptoglobin in bovine milk.

    PubMed

    Akerstedt, Maria; Björck, Lennart; Persson Waller, Karin; Sternesjö, Ase

    2006-08-01

    Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples

  10. Mass spectrometric assay for analysis of haptoglobin fucosylation in pancreatic cancer.

    PubMed

    Lin, Zhenxin; Simeone, Diane M; Anderson, Michelle A; Brand, Randall E; Xie, Xiaolei; Shedden, Kerby A; Ruffin, Mack T; Lubman, David M

    2011-05-01

    A mass spectrometric method was developed to elucidate the N-glycan structures of serum glycoproteins and utilize fucosylated glycans as potential markers for pancreatic cancer. This assay was applied to haptoglobin in human serum where N-glycans derived from the serum of 16 pancreatic cancer patients were compared with those from 15 individuals with benign conditions (5 normals, 5 chronic pancreatitis, and 5 type II diabetes). This assay used only 10 μL of serum where haptoglobin was extracted using a monoclonal antibody and quantitative permethylation was performed on desialylated N-glycans followed by MALDI-QIT-TOF MS analysis. Eight desialylated N-glycan structures of haptoglobin were identified where a bifucosylated triantennary structure was reported for the first time in pancreatic cancer samples. Both core and antennary fucosylation were elevated in pancreatic cancer samples compared to samples from benign conditions. Fucosylation degree indices were calculated and show a significant difference between pancreatic cancer patients of all stages and the benign conditions analyzed. This study demonstrates that a serum assay based on haptoglobin fucosylation patterns using mass spectrometric analysis may serve as a novel method for the diagnosis of pancreatic cancer. PMID:21417406

  11. HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation.

    PubMed

    He, Zhicong; Aristoteli, Lina P; Kritharides, Leonard; Garner, Brett

    2006-05-01

    Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples. PMID:16546121

  12. A Mass Spectrometric Assay for Analysis of Haptoglobin Fucosylation in Pancreatic Cancer

    PubMed Central

    Lin, Zhenxin; Simeone, Diane M.; Anderson, Michelle A.; Brand, Randall E.; Xie, Xiaolei; Shedden, Kerby A.; Ruffin, Mack T.; Lubman, David M.

    2011-01-01

    A mass spectrometric method was developed to elucidate the N-glycan structures of serum glycoproteins and utilize fucosylated glycans as potential markers for pancreatic cancer. This assay was applied to haptoglobin in human serum where N-glycans derived from the serum of 16 pancreatic cancer patients were compared with those from 15 individuals with benign conditions (5 normals, 5 chronic pancreatitis, and 5 type II diabetes). This assay used only 10uL of serum where haptoglobin was extracted using a monoclonal antibody and quantitative permethylation was performed on desialylated N-glycans followed by MALDI-QIT-TOF MS analysis. Eight desialylated N-glycan structures of haptoglobin were identified where a bifucosylated tri-antennary structure was reported for the first time in pancreatic cancer samples. Both core and antennary fucosylation were elevated in pancreatic cancer samples compared to samples from benign conditions. Fucosylation degree indices were calculated and show a significant difference between pancreatic cancer patients of all stages and the benign conditions analyzed. This study demonstrates that a serum assay based on haptoglobin fucosylation patterns using mass spectrometric analysis may serve as a novel method for the diagnosis of pancreatic cancer. PMID:21417406

  13. Site-specific Glycoforms of Haptoglobin in Liver Cirrhosis and Hepatocellular Carcinoma*

    PubMed Central

    Pompach, Petr; Brnakova, Zuzana; Sanda, Miloslav; Wu, Jing; Edwards, Nathan; Goldman, Radoslav

    2013-01-01

    Haptoglobin is a liver-secreted glycoprotein with four N-glycosylation sites. Its glycosylation was reported to change in several cancer diseases, which prompted us to examine site-specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma. To this end, we have used two-dimensional separation composed of hydrophilic interaction and nano-reverse phase chromatography coupled to QTOF mass spectrometry of the enriched glycopeptides. Our results show increased fucosylation of haptoglobin in liver disease with up to six fucoses associated with specific glycoforms of one glycopeptide. Structural analysis using exoglycosidase treatment and MALDI-MS/MS of detached permethylated glycans led to the identification of Lewis Y-type structures observed particularly in the pooled hepatocellular carcinoma sample. To confirm the increase of the Lewis Y structures observed by LC-MS, we have used immunoaffinity detection with Lewis Y-specific antibodies. The presence of multiply fucosylated Lewis Y glycoforms of haptoglobin in the disease context could have important functional implications. PMID:23389049

  14. Parallel evolutionary events in the haptoglobin gene clusters of rhesus monkey and human

    SciTech Connect

    Erickson, L.M.; Maeda, N.

    1994-08-01

    Parallel occurrences of evolutionary events in the haptoglobin gene clusters of rhesus monkeys and humans were studied. We found six different haplotypes among 11 individuals from two rhesus monkey families. The six haplotypes include two types of haptoglobin gene clusters: one type with a single gene and the other with two genes. DNA sequence analysis indicates that the one-gene and the two-gene clusters were both formed by unequal homologous crossovers between two genes of an ancestral three-gene cluster, near exon 5, the longest exon of the gene. This exon is also the location where a separate unequal homologous crossover occured in the human lineage, forming the human two-gene haptoglobin gene cluster from an ancestral three-gene cluster. The occurrence of independent homologous unequal crossovers in rhesus monkey and in human within the same region of DNA suggests that the evolutionary history of the haptoglobin gene cluster in primates is the consequence of frequent homologous pairings facilitated by the longest and most conserved exon of the gene. 27 refs., 7 figs., 1 tab.

  15. Combination fibrinolytic therapy in the treatment of chronic septic pleuropneumonia in a Thoroughbred gelding.

    PubMed

    Rendle, D I; Armstrong, S K; Hughes, K J

    2012-09-01

    This report documents the treatment of a case of chronic pleuropneumonia in a 3-year-old Thoroughbred gelding. A recombinant tissue plasminogen activator (tenecteplase) and a recombinant deoxyribonucleic acidase (alphadornase) were infused into the pleural cavity as adjunctive therapy in the early stages of treatment. Instillation of fibrinolytic drugs was associated with a subjective reduction in the amount of fibrin deposition and decreased fluid accumulation within the pleural cavities. Fibrinolytic therapy may be a useful adjunctive therapy in selected cases of intrapleural disease in horses. PMID:22928684

  16. Proteomic identification of fucosylated haptoglobin alpha isoforms in ascitic fluids and its localization in ovarian carcinoma tissues from Mexican patients

    PubMed Central

    2014-01-01

    Background Ovarian cancer is the most lethal gynecologic disease due to delayed diagnosis, and ascites production is a characteristic of patients in advanced stages. The aim of this study was to perform the proteomic analysis of ascitic fluids of Mexican patients with ovarian carcinoma, in order to detect proteins with a differential expression pattern in the continuing search to identify biomarkers for this disease. Methods Samples were collected from 50 patients from the Instituto Nacional de Cancerología of México under informed consent and with approval of the bioethics and scientific committees. After elimination of abundant proteins (Albumin/IgGs) samples were processed for 2D electrophoresis and further protein identification by Mass Spectrometry (MALDI-TOF). Molecules of interest were followed by western blot and lectin binding assays, and their tissue location by histo-immunofluorescence and confocal analysis. Results and discussion An area with a differential expression pattern among samples was located in the 2D gels. Identified proteins were 6 alpha 1 isoforms and 1 alpha 2 isoform of Haptoglobin, and 2 isoforms of Transthyretin. While Transthyretin isoforms were constitutively expressed in all samples, clear differences in the expression pattern of Haptoglobin alpha isoforms were found. Moreover, increased levels of fucosylation of Haptoglobin alpha isoforms analyzed in 40 samples by Aleuria aurantia lectin binding by 1D overlay assay showed a positive correlation with advanced stages of the disease. Tissue detection of Haptoglobin and its fucosylated form, by histo-immunofluorescence in biopsies of ovarian cancer, also showed a correlation with ovarian cancer progression. Moreover, results show that fucosylated Haptoglobin is produced by tumor cells. Conclusions Increased numbers of highly fucosylated Haptoglobin alpha isoforms in ascitic fluids and the presence of fucosylated Haptoglobin in tumor tissues of ovarian cancer Mexican patients

  17. A Genome-Wide Association Study Identifies rs2000999 as a Strong Genetic Determinant of Circulating Haptoglobin Levels

    PubMed Central

    Bonnefond, Amélie; Bouatia-Naji, Nabila; Dechaume, Aurélie; Siest, Gérard; Herbeth, Bernard; Falchi, Mario; Bottolo, Leonardo; Guéant-Rodriguez, Rosa-Maria; Lecoeur, Cécile; Langlois, Michel R.; Labrune, Yann; Ruokonen, Aimo; El Shamieh, Said; Stathopoulou, Maria G.; Morandi, Anita; Maffeis, Claudio; Meyre, David; Delanghe, Joris R.; Jacobson, Peter; Sjöström, Lars; Carlsson, Lena M. S.; Walley, Andrew; Elliott, Paul; Jarvelin, Marjo-Riita; Dedoussis, George V.; Visvikis-Siest, Sophie

    2012-01-01

    Haptoglobin is an acute phase inflammatory marker. Its main function is to bind hemoglobin released from erythrocytes to aid its elimination, and thereby haptoglobin prevents the generation of reactive oxygen species in the blood. Haptoglobin levels have been repeatedly associated with a variety of inflammation-linked infectious and non-infectious diseases, including malaria, tuberculosis, human immunodeficiency virus, hepatitis C, diabetes, carotid atherosclerosis, and acute myocardial infarction. However, a comprehensive genetic assessment of the inter-individual variability of circulating haptoglobin levels has not been conducted so far. We used a genome-wide association study initially conducted in 631 French children followed by a replication in three additional European sample sets and we identified a common single nucleotide polymorphism (SNP), rs2000999 located in the Haptoglobin gene (HP) as a strong genetic predictor of circulating Haptoglobin levels (Poverall = 8.1×10−59), explaining 45.4% of its genetic variability (11.8% of Hp global variance). The functional relevance of rs2000999 was further demonstrated by its specific association with HP mRNA levels (β = 0.23±0.08, P = 0.007). Finally, SNP rs2000999 was associated with decreased total and low-density lipoprotein cholesterol in 8,789 European children (Ptotal cholesterol = 0.002 and PLDL = 0.0008). Given the central position of haptoglobin in many inflammation-related metabolic pathways, the relevance of rs2000999 genotyping when evaluating haptoglobin concentration should be further investigated in order to improve its diagnostic/therapeutic and/or prevention impact. PMID:22403646

  18. 21 CFR 558.618 - Tilmicosin.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... respiratory disease associated with Actinobacillus pleuropneumoniae and Pasteurella multocida. (3) Limitations... licensed veterinarian before reinitiating a further course of therapy with an appropriate...

  19. 21 CFR 558.618 - Tilmicosin.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... respiratory disease associated with Actinobacillus pleuropneumoniae and Pasteurella multocida. (3) Limitations... licensed veterinarian before reinitiating a further course of therapy with an appropriate...

  20. Baseline haptoglobin concentrations are repeatable and predictive of certain aspects of a subsequent experimentally-induced inflammatory response.

    PubMed

    Matson, Kevin D; Horrocks, Nicholas P C; Versteegh, Maaike A; Tieleman, B Irene

    2012-05-01

    Ecologists sometimes assume immunological indices reflect fundamental attributes of individuals-an important assumption if an index is to be interpreted in an evolutionary context since among-individual variation drives natural selection. Yet the extent to which individuals vary over different timescales is poorly understood. Haptoglobin, an acute phase protein, is an interesting parameter for studying variability as it is easily quantified and concentrations vary widely due to the molecule's role in inflammation, infection and trauma. We quantified haptoglobin in pigeon plasma samples collected over fourteen months and calculated repeatability to evaluate if haptoglobin concentration is a distinctive trait of individuals. We also explored the capacity of baseline haptoglobin concentrations to predict an array of physiological changes associated with a subsequent experimentally-induced inflammatory response. Maximum repeatability, which occurred over a short mid-winter interval, equaled 0.57. Baseline haptoglobin concentrations predicted response haptoglobin concentrations better than any other endotoxin-induced change. Overall, we identified several strengths and limitations of baseline [Hp] quantification. Acknowledging these qualities should lead to more refined conclusions in studies of the ecology and evolution of immune function. PMID:22316629

  1. Recurring exon deletions in the HP (haptoglobin) gene contribute to lower blood cholesterol levels.

    PubMed

    Boettger, Linda M; Salem, Rany M; Handsaker, Robert E; Peloso, Gina M; Kathiresan, Sekar; Hirschhorn, Joel N; McCarroll, Steven A

    2016-04-01

    One of the first protein polymorphisms identified in humans involves the abundant blood protein haptoglobin. Two exons of the HP gene (encoding haptoglobin) exhibit copy number variation that affects HP protein structure and multimerization. The evolutionary origins and medical relevance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from many recurring deletions, more specifically, reversions of an ancient hominin-specific duplication of these exons. Although this polymorphism has been largely invisible to genome-wide genetic studies thus far, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We further show that these deletions, and a SNP that affects HP expression, appear to drive the strong association of cholesterol levels with SNPs near HP. Recurring exonic deletions in HP likely enhance human health by lowering cholesterol levels in the blood. PMID:26901066

  2. Recurring exon deletions in the haptoglobin (HP) gene associate with lower blood cholesterol levels

    PubMed Central

    Boettger, Linda M.; Salem, Rany M.; Handsaker, Robert E.; Peloso, Gina; Kathiresan, Sekar; Hirschhorn, Joel; McCarroll, Steven A.

    2016-01-01

    Two exons of the human haptoglobin (HP) gene exhibit copy number variation that affects HP multimerization and underlies one of the first protein polymorphisms identified in humans. The evolutionary origins and medical significance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from the recurring reversion of an ancient hominin-specific duplication of these exons. Though this polymorphism has been largely invisible to genome-wide genetic studies to date, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We show that these deletions, and a SNP that affects HP expression, are the likely drivers of the strong but complex association of cholesterol levels to SNPs near HP. Recurring exonic deletions in the haptoglobin gene likely enhance human health by lowering cholesterol levels in the blood. PMID:26901066

  3. A Polyclonal Antibody Against Recombinant Bovine Haptoglobin Expressed in Escherichia coli

    PubMed Central

    Guo, Donghua; Zhang, Hong; Li, Chunqiu

    2013-01-01

    The nucleotide sequence of the predicted immunodominant region of bovine haptoglobin (pirBoHp), without the signal peptide sequence, was synthesized based on the codon usage bias of Escherichia coli. The synthesized pirBoHp gene was cloned into the prokaryotic expression vector pET-32a (+), which contains a His-tag. The recombinant pirBoHp protein was successfully expressed in E. coli BL21 (DE3) cells. Western blot analysis showed that the purified recombinant pirBoHp protein could be recognized by an anti-His-tag monoclonal antibody. Further investigations indicated that a polyclonal antibody against the recombinant pirBoHp protein could recognize the α and β chains of native bovine haptoglobin in a pooled plasma sample from dairy cattle suffering from foot rot. PMID:24328747

  4. Evolutionary diversification of the trypanosome haptoglobin-haemoglobin receptor from an ancestral haemoglobin receptor

    PubMed Central

    Lane-Serff, Harriet; MacGregor, Paula; Peacock, Lori; Macleod, Olivia JS; Kay, Christopher; Gibson, Wendy; Higgins, Matthew K; Carrington, Mark

    2016-01-01

    The haptoglobin-haemoglobin receptor of the African trypanosome species, Trypanosoma brucei, is expressed when the parasite is in the bloodstream of the mammalian host, allowing it to acquire haem through the uptake of haptoglobin-haemoglobin complexes. Here we show that in Trypanosoma congolense this receptor is instead expressed in the epimastigote developmental stage that occurs in the tsetse fly, where it acts as a haemoglobin receptor. We also present the structure of the T. congolense receptor in complex with haemoglobin. This allows us to propose an evolutionary history for this receptor, charting the structural and cellular changes that took place as it adapted from a role in the insect to a new role in the mammalian host. DOI: http://dx.doi.org/10.7554/eLife.13044.001 PMID:27083048

  5. Conventional transmission electron microscopy of 1-1 haptoglobin isolated molecules and paracrystalline aggregates.

    PubMed

    Blonda, C; Albergamo, A; Casale, A; Felluga, B; Bernini, L; Santulli, A; Annunziata, A; Mazza, A

    1986-04-01

    Data are presented relating conventional transmission electron microscope (CTEM) ultrastructural observations of the monomeric phenotypic variant (Hp 1-1) of the haptoglobin class of blood glycoproteins. Through comparison of these findings with homologous published data, obtained by means of scanning transmission electron microscopy (STEM), the validity of CTEM in molecule shape and fine structure determination is confirmed. An experimental procedure for Hp 1-1 crystallization is also reported. PMID:3712515

  6. Hemolysis in runners as evidenced by low serum haptoglobin: Implications for preflight monitoring of astronauts

    NASA Technical Reports Server (NTRS)

    Owens, Joyce; Spitler, Diane L.; Frey, Mary Anne Bassett

    1987-01-01

    Hematological parameters and serum haptoglobin were examined in 21 male employees of the Kennedy Space Center who were at 3 levels of physical activity: 7 subjects regularly ran more than 40 km (25 miles) per week (Group I); 7 ran 13 to 24 km (8 to 15 miles) per week (II), and 7 were sedentary (III). Blood was drawn on a different day of the week for five weeks. Differences between day of the week, visit number, and activity level were examined. No differences were observed for day of week or visit number; thus mean values for each variable were calculated for each subject. Variables did not differ among groups. However, trends with level of training were observed in some critical variables. Hemoglobin (Hb) and hematocrit (Hct) conformed to a staircase effect with Group I (14.5 gm/dl and 41.3 percent) lower than Group III (15.1 gm/dl and 42.9 percent). Reticulocyte count was higher and haptoglobin levels lower in Group I (1.35% and 75.7 gm/dl) than Group III (.99 percent and 132.9 gm/dl), with haptoglobin for the high mileage Group I in the clinically abnormal range. Since haptoglobin binds free Hb following RBC destruction, these results suggest that intravascular hemolysis occurs in trained male runners. These results may have special meaning for astronauts training before long-duration spaceflights, since the further reduction in red blood cells which is reported to occur during spaceflight could become detrimental to their health and performance.

  7. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

    PubMed Central

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-01-01

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. DOI: http://dx.doi.org/10.7554/eLife.05553.001 PMID:25497229

  8. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

    PubMed

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-01-01

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. PMID:25497229

  9. Determination of haptoglobin genotype in an Iranian population with idiopathic generalized epilepsy

    PubMed Central

    Al-balaghee, Sukaina; Al-balaghee, Zeinab; Shabani, Ashraf; Ghadam, Parinaz; Bandehpour, Mojgan; Askari Mehr, Ali; Kazemi, Bahram

    2015-01-01

    Background: Haptoglobin (Hp) is a plasma α2-sialoglycoprotein that contains alpha and beta chains. It displays in three common phenotypes, Hp1-1, Hp2-1, and Hp2-2. Proteins expressed by polymorphic genes have grossly different molecular sizes resulting in different diffusion rates in the brain. Haptoglobin expressed by the Hp2-2 genotype has lower hemoglobin-binding capacity than Hp1-1 or Hp2-1 and is associated with idiopathic generalized epilepsy. Methods: To determine polymorphism in haptoglobin genes in patients with idiopathic generalized tonic-clonic seizures, 42 men, 42 women, and 50 controls were selected for this study. Genomic DNA was extracted from blood and studied by polymerase chain reactions (PCR). Results: The amplified fragments for the Hp1-1 and Hp2-2 genotypes were 1757 and 3481 base pairs (bp) respectively, and the Hp2-1 genotype had both fragments, in addition to a 349-bp fragment. The distribution of the three major Hp phenotypes in epilepsy patients was 28.6 (1-1), 38.1 (2-1), and 33.3% (2-2) in the men, and 31 (1-1), 40.5 (2-1), and 28.6% (2-2) in the women. The distribution of Hp genotypes in controls was 22 (1-1), 40 (2-1), and 38% (2-2). Conclusion: We show that all Hp genotypes participate in idiopathic generalized epilepsy. PMID:26989737

  10. Actinobacillus actinomycetemcomitans strains Y4 and N27 adhere to hydroxyapatite by distinctive mechanisms.

    PubMed Central

    Kagermeier, A S; London, J

    1985-01-01

    Actinobacillus actinomycetemcomitans strains Y4 and N27 absorb to spheroidal hydroxyapatite in roughly the same numbers per milligram of substrate and with the same tenacity as two previously tested Cytophaga species. Although the two strains of A. actinomycetemcomitans exhibited similar affinities and number of binding sites for SHA, their response to enzyme treatment and heating were very different. The capacity of strain Y4 to attach to spheroidal hydroxyapatite was diminished by treatment with proteases and phospholipases and was unaffected by neuraminidase, while strain N27 was unaffected by proteases and phospholipases and lost its binding capabilities when treated with neuraminidase. Images PMID:3972445

  11. Fatal transmission of contagious caprine pleuropneumonia to an Arabian oryx (Oryx leucoryx).

    PubMed

    Chaber, A L; Lignereux, L; Al Qassimi, M; Saegerman, C; Manso-Silván, L; Dupuy, V; Thiaucourt, F

    2014-09-17

    Contagious caprine pleuropneumonia (CCPP) is an infectious respiratory disease mainly affecting domestic goats. As CCPP has never been documented in grazing antelopes (subfamily hippotraginae), they were not considered susceptible. Mycoplasma capricolum subspecies capripneumoniae (Mccp) was isolated from pleural liquid collected during the necropsy of a severely emaciated Arabian oryx with mild nasal discharge. The Mccp isolate was then genotyped using a multilocus sequence scheme; the sequence type was identical to the Mccp strain previously identified in a sand gazelle from a nearby enclosure. This case shows for the first time that members of the hippotraginae subfamily, here the Arabian oryx, can be affected by CCPP. In addition, genotyping shows that the oryx was most probably infected, at a distance, by sand gazelles. PMID:25069622

  12. Investigations into the role of carrier animals in the spread of contagious bovine pleuropneumonia.

    PubMed

    Windsor, R S; Masiga, W N

    1977-09-01

    An attempt was made to transmit contagious bovine pleuropneumonia (CBPP) from 22 animals recovered from artificial infection to healthy animals. Despite close contact and the imposition of a number of stresses no disease was transmitted. An unsuccessful attempt was made to reactivate old CBPP lesions by corticosteroid treatment, and by splenectomy. Four animals recovered from artificial infection did not become reinfected when put in contact with acute disease. Seven animals were reinoculated with Mycoplasmamycoides sub-species mycoides by endobronchial intubation. No clinical disease resulted. It is concluded that sequestra do not break down easily and that it is difficult to reinfect recovered animals. It is suggested that in field outbreaks of obscure origin, investigation should be thorough before it is concluded that an animal with an old sequestrum was responsible. PMID:337429

  13. Identification of fucosylated haptoglobin as a novel tumor marker for pancreatic cancer and its possible application for a clinical diagnostic test.

    PubMed

    Miyoshi, Eiji; Shinzaki, Shinichiro; Moriwaki, Kenta; Matsumoto, Hitoshi

    2010-01-01

    Fucosylation is one of the most important oligosaccharide modifications in cancer and inflammation. The fucosylation level is increased in total cellular proteins of cancer cells as well as in sera of patients with cancer. Recently, on AAL blot analysis, we found a fucosylated glycoprotein of 40 kDa in sera of patients with pancreatic cancer. Based on its N-terminal sequence, this protein was identified as haptoglobin. Fucosylated haptoglobin was increased in sera of patients with several kinds of cancer and the positive rate was higher in pancreatic cancer. The level of fucosylated haptoglobin was not correlated with total haptoglobin, suggesting that a factor other than inflammation could regulate the production of fucosylated haptoglobin. Mass spectrometry analysis revealed the detailed oligosaccharide structure of fucosylated haptoglobin purified from sera of patients with pancreatic cancer. For clinical applications, we developed a lectin-antibody ELISA system for quantifying fucosylated haptoglobin. In this review, we would like to summarize the history of the identification of fucosylated haptoglobin as a marker for pancreatic cancer and its possible application for a clinical diagnostic test. PMID:20816478

  14. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

    SciTech Connect

    Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul

    2008-05-01

    A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.

  15. Nucleic Acid Similarities Among Pseudomonas pseudomallei, Pseudomonas multivorans, and Actinobacillus mallei1

    PubMed Central

    Rogul, M.; Brendle, J. J.; Haapala, D. K.; Alexander, A. D.

    1970-01-01

    Annealing experiments on membrane filters were carried out with deoxyribonucleic acids (DNA) from selected strains of the nomen-species of Pseudomonas, Actinobacillus, Chromobacterium, and Micrococcus, with the use of DNA of Pseudomonas pseudomallei and Actinobacillus mallei as reference materials. Under the usual conditions employed in these experiments, the results were not quantitatively reproducible. Incorporation of dimethylsulfoxide (DMSO) into the incubation medium greatly increased differences in comparative binding. DNA binding in agar matrices was examined in the presence and absence of DMSO at various incubation temperatures. It was found that the greatest specificity, stability, and total binding for DNA containing high amounts of guanine and cytosine occurred in the presence of DMSO. Under the most stringent annealing conditions permitted in agar, DNA species from P. pseudomallei and A. mallei in the presence of DMSO demonstrated interspecific relative bindings of 76 to 86% when compared to the homologous reactions. The thermal elution midpoints (Em) of these duplexed interspecific DNA species were quite close to the homologous Em values. The relative bindings of P. multivorans DNA types to either reference DNA ranged between 6 to 27%, and the Em values were 4 to 7 C less than those for the homologous reactions. PMID:5438051

  16. Production of succinic acid from oil palm empty fruit bunch cellulose using Actinobacillus succinogenes

    NASA Astrophysics Data System (ADS)

    Pasma, Satriani Aga; Daik, Rusli; Maskat, Mohamad Yusof

    2013-11-01

    Succinic acid is a common metabolite in plants, animals and microorganisms. It has been used widely in agricultural, food and pharmaceutical industries. Enzymatic hydrolysate glucose from oil palm empty fruit bunch (OPEFB) cellulose was used as a substrate for succinic acid production using Actinobacillus succinogenes. Using cellulose extraction from OPEFB can enhance the production of glucose as a main substrate for succinic acid production. The highest concentration of glucose produced from enzymatic hydrolysis is 167 mg/mL and the sugar recovery is 0.73 g/g of OPEFB. By optimizing the culture medium for succinic acid fermentation with enzymatic hydrolysate of OPEFB cellulose, the nitrogen sources could be reduced to just only 2.5 g yeast extract and 2.5 g corn step liquor. Batch fermentation was carried out using enzymatic hydrolysate of OPEFB cellulose with yeast extract, corn steep liquor and the salts mixture, 23.5 g/L succinic acid was obtained with consumption of 72 g/L glucose in enzymatic hydrolysate of OPEFB cellulose at 38 hours and 37°C. This study suggests that enzymatic hydrolysate of OPEFB cellulose maybe an alternative substrate for the efficient production of succinic acid by Actinobacillus succinogenes.

  17. Evaluation of serum amyloid A and haptoglobin concentrations as prognostic indicators for horses with colic.

    PubMed

    Westerman, Trina L; Foster, Crystal M; Tornquist, Susan J; Poulsen, Keith P

    2016-04-15

    OBJECTIVE To evaluate the use of the acute-phase proteins serum amyloid A (SAA) and haptoglobin as prognostic indicators in horses with colic with regard to the need for surgical intervention, development of complications, and hospitalization cost and duration. DESIGN Prospective observational study. ANIMALS 20 clinically normal horses and 42 horses with colic. PROCEDURES Total WBC and neutrophil counts and plasma fibrinogen, SAA, and haptoglobin concentrations were compared between healthy (control) horses and horses admitted to a veterinary teaching hospital for colic. Clinicopathologic values were compared between medical and surgical colic cases to test the ability of acute-phase proteins to predict indication for surgical intervention, development of complications, and duration and cost of hospitalization. RESULTS Mean SAA concentration was significantly higher in the surgical group, compared with that for both the control and medical groups. Haptoglobin concentration did not differ significantly among groups. Horses with colic and an abnormally increased SAA concentration (> 5 μg/mL) were more likely to be managed surgically than medically (OR, 5.7; 95% confidence interval, 1.4 to 22.8). Horses with small intestinal lesions had significantly higher SAA concentrations than did control horses. Euthanasia due to a poor prognosis or the development of thrombophlebitis was more likely for horses with an SAA concentration > 5 μg/mL (OR, 7.6; 95% confidence interval, 1.1 to 52.4). A weak positive correlation (r = 0.30) was observed between cost of treatment and SAA concentration. CONCLUSIONS AND CLINICAL RELEVANCE Horses with colic that had an abnormally increased SAA concentration were more likely to require surgical intervention, develop thrombophlebitis, or be euthanized because of a poor prognosis despite treatment. PMID:27031421

  18. Galectin-1-binding glycoforms of haptoglobin with altered intracellular trafficking, and increase in metastatic breast cancer patients.

    PubMed

    Carlsson, Michael C; Cederfur, Cecilia; Schaar, Viveka; Balog, Crina I A; Lepur, Adriana; Touret, Franck; Salomonsson, Emma; Deelder, André M; Fernö, Mårten; Olsson, Håkan; Wuhrer, Manfred; Leffler, Hakon

    2011-01-01

    Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7-2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8-3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20-80) in cancer sera and about 30% (range 25-50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells. PMID:22028908

  19. Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation.

    PubMed

    Okuyama, Noriko; Ide, Yoshihito; Nakano, Miyako; Nakagawa, Tsutomu; Yamanaka, Kanako; Moriwaki, Kenta; Murata, Kohei; Ohigashi, Hiroaki; Yokoyama, Shigekazu; Eguchi, Hidetoshi; Ishikawa, Osamu; Ito, Toshifumi; Kato, Michio; Kasahara, Akinori; Kawano, Sunao; Gu, Jianguo; Taniguchi, Naoyuki; Miyoshi, Eiji

    2006-06-01

    Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An approximately 40 kD protein was found to be highly fucosylated in pancreatic cancer and an N-terminal analysis revealed that it was the beta chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN-1, showed that the alpha 1-3/alpha 1-4/alpha 1-6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver. PMID:16385567

  20. Haptoglobin binding to apolipoprotein A-I prevents damage from hydroxyl radicals on its stimulatory activity of the enzyme lecithin-cholesterol acyl-transferase.

    PubMed

    Salvatore, Alfonso; Cigliano, Luisa; Bucci, Enrico M; Corpillo, Davide; Velasco, Silvia; Carlucci, Alessandro; Pedone, Carlo; Abrescia, Paolo

    2007-10-01

    Apolipoprotein A-I (ApoA-I), a major component of HDL, binds haptoglobin, a plasma protein transporting to liver or macrophages free Hb for preventing hydroxyl radical production. This work aimed to assess whether haptoglobin protects ApoA-I against this radical. Human ApoA-I structure, as analyzed by electrophoresis and MS, was found severely altered by hydroxyl radicals in vitro. Lower alteration of ApoA-I was found when HDL was oxidized in the presence of haptoglobin. ApoA-I oxidation was limited also when the complex of haptoglobin with both high-density lipoprotein and Hb, immobilized on resin beads, was exposed to hydroxyl radicals. ApoA-I function to stimulate cholesterol esterification was assayed in vitro by using ApoA-I-containing liposomes. Decreased stimulation was observed when liposomes oxidized without haptoglobin were used. Conversely, after oxidative stress in the presence of haptoglobin (0.5 microM monomer), the liposome activity did not change. Plasma of carrageenan-treated mice was analyzed by ELISA for the levels of haptoglobin and ApoA-I, and used to isolate HDL for MS analysis. Hydroxyproline-containing fragments of ApoA-I were found associated with low levels of haptoglobin (18 microM monomer), whereas they were not detected when the haptoglobin level increased (34-70 microM monomer). Therefore haptoglobin, when circulating at enhanced levels with free Hb during the acute phase of inflammation, might protect ApoA-I structure and function against hydroxyl radicals. PMID:17824618

  1. The role of Haptoglobin and its related protein, Zonulin, in inflammatory bowel disease.

    PubMed

    Vanuytsel, Tim; Vermeire, Séverine; Cleynen, Isabelle

    2013-12-01

    Crohn's disease (CD) and ulcerative colitis (UC), collectively called inflammatory bowel disease (IBD), are immune-mediated conditions characterized by a chronic inflammation of the gut. Their precise etiology is unknown, although an increased intestinal permeability has been shown to play a central role in the pathogenesis of IBD. The intestinal epithelium provides the largest interface between the external environment and the host, and is thus a crucial regulation site of innate and adaptive immunity. Zonulin is one of the few known physiological mediators of paracellular intestinal permeability. It was found upregulated in different immune diseases like Celiac disease and Type 1 Diabetes (T1D). Recently, human zonulin was identified as prehaptoglobin-2 (pre-HP2) which before only had been regarded as the inactive precursor for HP2. Haptoglobin (HP) is a hemoglobin-binding protein with immunomodulatory properties. Its gene harbors a common polymorphism with 2 different alleles: HP1 and HP2. Allele HP2 and genotype HP22 has been shown to be overrepresented in different immune diseases like Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and T1D, and has also been found to be more frequent in patients with IBD (UC and CD) than in healthy controls.   In order to get some clues about the mechanism of action of HP(2) in IBD pathogenesis, we here review the current state of knowledge about zonulin and haptoglobin structure and function, and their plausible role in immune mediated diseases with an emphasis on IBD. PMID:24868498

  2. SERS-based quantitative detection of ovarian cancer prognostic factor haptoglobin

    PubMed Central

    Perumal, Jayakumar; Balasundaram, Ghayathri; Mahyuddin, Aniza P; Choolani, Mahesh; Olivo, Malini

    2015-01-01

    Surface-enhanced Raman spectroscopy (SERS) is increasingly being used for biosensing because of its high sensitivity and low detection limit, which are made possible by the unique Raman ‘fingerprint’ spectra from the biomolecules. Here we propose a novel SERS method for the fast, sensitive, and reliable quantitative analysis of haptoglobin (Hp), an acute phase plasma glycoprotein that is widely gaining application as a prognostic ovarian cancer biomarker. We exploited the peroxidase activity of the hemoglobin–haptoglobin (Hb–Hp) complex formed by the selective and specific binding of Hp to free Hb to catalyze the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and hydrogen peroxide to result in the final product of strongly SERS-active TMB2+. We observed a linear increase in the SERS signal of TMB2+ with increasing concentrations of Hb–Hp complex from 50 nM to 34 μM. Based on this concentration-dependent SERS spectrum, we quantified Hp in clinical samples. We observed that our inference about the prognosis of the disease coincided with the histology data and that our method was much more sensitive than the enzyme-linked immunosorbent assay method. PMID:25834423

  3. Identification of an inducible factor secreted by pancreatic cancer cell lines that stimulates the production of fucosylated haptoglobin in hepatoma cells.

    PubMed

    Narisada, Megumi; Kawamoto, Sayuri; Kuwamoto, Kana; Moriwaki, Kenta; Nakagawa, Tsutomu; Matsumoto, Hitoshi; Asahi, Michio; Koyama, Nobuto; Miyoshi, Eiji

    2008-12-19

    Fucosylation is one of the most important oligosaccharide modifications and is involved in cancer and inflammation. Recently, fucosylated haptoglobin was identified as a possible tumor marker for pancreatic cancer. The molecular mechanism underlying increases in fucosylated haptoglobin in sera of patients with pancreatic cancer seems to be complicated. Our previous study [N. Okuyama, Y. Ide, M. Nakano, T. Nakagawa, K. Yamanaka, K. Moriwaki, K. Murata, H. Ohigashi, S. Yokoyama, H. Eguchi, O. Ishikawa, T. Ito, M. Kato, A. Kasahara, S. Kawano, J. Gu, N. Taniguchi, E. Miyoshi, Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation, Int. J. Cancer 118 (11) (2006) 2803-2808] demonstrated that pancreatic cancer cells secrete a factor, which induces the production of haptoglobin in hepatoma cells. In the present study, we found that interleukin 6 (IL6) expressed in pancreatic cancer is a factor that induces the haptoglobin production, using a neutralizing antibody for IL6. Real-time PCR analyses revealed the up-regulation of fucosylation regulatory genes after IL6 treatment, resulting increases in fucosylated haptoglobin being revealed by a lectin ELISA. This pathway could be one of the possible mechanisms underlying increases in haptoglobin in sera of patients with pancreatic cancer. PMID:18951869

  4. Isolation of Actinobacillus seminis from a goat with clinical epididymo-orchitis in Brazil

    PubMed Central

    dos Santos, Fabrine Alexandre; de Azevedo, Edísio Oliveira; de Azevedo, Sérgio Santos; Júnior, Felício Garino; Mota, Rinaldo Aparecido; de Cássia Peixoto Kim, Pomy; Gomes, Ana Lisa Vale; Alves, Clebert José

    2014-01-01

    The present study reports the first isolation of Actinobacillus seminis from a goat in Brazil. A four-year-old Moxotó breeding goat in a flock of 70 goats and 65 sheep reared together in the county of Patos, semiarid region of Northeastern Brazil, showed clinical signs of unilateral orchitis and epididymitis. Diagnosis of A. seminis infection was confirmed by association of clinical findings, bacterial isolation and 16S rRNA gene sequencing. This result suggests that A. seminis may be an additional cause of infertility in goats, and that sheep may be the source of infection because the mixed farming system allows the contact between sheep and goats in the semiarid region of Northeastern Brazil. PMID:24948932

  5. Antibodies to Aqx toxin of Actinobacillus equuli in horses and foals.

    PubMed

    Berthoud, H; Frey, J; Sternberg, S; Straub, R; Kuhnert, P

    2004-08-21

    Actinobacillus equuli is found in the normal oral flora of horses, but has been associated with several diseases, and particularly with the usually fatal septicaemia in neonatal foals which is thought to be associated with a failure of the passive transfer of immunoglobulins via the colostrum. The Aqx protein of A equuli, belonging to the RTX family of pore-forming toxins, is also cytotoxic to horse lymphocytes. The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx. PMID:15384504

  6. Succinic acid production from duckweed (Landoltia punctata) hydrolysate by batch fermentation of Actinobacillus succinogenes GXAS137.

    PubMed

    Shen, Naikun; Wang, Qingyan; Zhu, Jing; Qin, Yan; Liao, Siming; Li, Yi; Zhu, Qixia; Jin, Yanling; Du, Liqin; Huang, Ribo

    2016-07-01

    Duckweed is potentially an ideal succinic acid (SA) feedstock due to its high proportion of starch and low lignin content. Pretreatment methods, substrate content and nitrogen source were investigated to enhance the bioconversion of duckweed to SA and to reduce the costs of production. Results showed that acid hydrolysis was an effective pretreatment method because of its high SA yield. The optimum substrate concentration was 140g/L. The optimum substrate concentration was 140g/L. Corn steep liquor powder could be considered a feasible and inexpensive alternative to yeast extract as a nitrogen source. Approximately 57.85g/L of SA was produced when batch fermentation was conducted in a 1.3L stirred bioreactor. Therefore, inexpensive duckweed can be a promising feedstock for the economical and efficient production of SA through fermentation by Actinobacillus succinogenes GXAS137. PMID:27023386

  7. Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Flanagan, J J; Arjomandi, A; Delanoy, M L; Du Paty, E; Galea, P; Laune, D; Rieunier, F; Walker, R P; Binder, S R

    2014-04-01

    Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5-335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum. PMID:24583194

  8. Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

    PubMed Central

    Garibay-Cerdenares, OL; Hernández-Ramírez, VI; Osorio-Trujillo, JC; Gallardo-Rincón, D; Talamás-Rohana, P

    2015-01-01

    Haptoglobin (Hp) is an acute-phase protein that is produced by the liver to capture the iron that is present in the blood circulation, thus avoiding its accumulation in the blood. Moreover, Hp has been detected in a wide variety of tissues, in which it performs various functions. In addition, this protein is considered a potential biomarker in many diseases, such as cancer, including ovarian carcinoma; however, its participation in the cancerous processes has not yet been determined. The objective of this work was to demonstrate the expression of Hp and its receptor CCR2 in the ovarian cancer cells and its possible involvement in the process of cell migration through changes in the rearrangement of the actin cytoskeleton using western blot and wound-healing assays and confirming by confocal microscopy. Ovarian cancer cells express both Hp and its receptor CCR2 but only after exposure to ascitic fluid, inducing moderated cell migration. However, when the cells are exposed to exogenous Hp, the expression of CCR2 is induced together with drastic changes in the actin cytoskeleton rearrangement. At the same time, Hp induced cell migration in a much more efficient manner than did ascitic fluid. These effects were blocked when the CCR2 synthetic antagonist RS102895 was used to pretreat the cells. These results suggest that Hp-induced changes in the cell morphology, actin cytoskeleton structure, and migration ability of tumor cells, is possibly “preparing” these cells for the potential induction of the metastatic phenotype. PMID:26211665

  9. Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: exploring a new role of haptoglobin in the tumoral microenvironment.

    PubMed

    Garibay-Cerdenares, O L; Hernández-Ramírez, V I; Osorio-Trujillo, J C; Gallardo-Rincón, D; Talamás-Rohana, P

    2015-01-01

    Haptoglobin (Hp) is an acute-phase protein that is produced by the liver to capture the iron that is present in the blood circulation, thus avoiding its accumulation in the blood. Moreover, Hp has been detected in a wide variety of tissues, in which it performs various functions. In addition, this protein is considered a potential biomarker in many diseases, such as cancer, including ovarian carcinoma; however, its participation in the cancerous processes has not yet been determined. The objective of this work was to demonstrate the expression of Hp and its receptor CCR2 in the ovarian cancer cells and its possible involvement in the process of cell migration through changes in the rearrangement of the actin cytoskeleton using western blot and wound-healing assays and confirming by confocal microscopy. Ovarian cancer cells express both Hp and its receptor CCR2 but only after exposure to ascitic fluid, inducing moderated cell migration. However, when the cells are exposed to exogenous Hp, the expression of CCR2 is induced together with drastic changes in the actin cytoskeleton rearrangement. At the same time, Hp induced cell migration in a much more efficient manner than did ascitic fluid. These effects were blocked when the CCR2 synthetic antagonist RS102895 was used to pretreat the cells. These results suggest that Hp-induced changes in the cell morphology, actin cytoskeleton structure, and migration ability of tumor cells, is possibly "preparing" these cells for the potential induction of the metastatic phenotype. PMID:26211665

  10. Capsular polysaccharide from Mycoplasma mycoides subsp. mycoides shows potential for protection against contagious bovine pleuropneumonia.

    PubMed

    Mwirigi, Martin; Nkando, Isabel; Olum, Moses; Attah-Poku, Samuel; Ochanda, Horace; Berberov, Emil; Potter, Andrew; Gerdts, Volker; Perez-Casal, Jose; Wesonga, Hezron; Soi, Reuben; Naessens, Jan

    2016-10-01

    Contagious Bovine Pleuropneumonia (CBPP) is a severe respiratory disease caused by Mycoplasma mycoides subsp. mycoides (Mmm) which is widespread in Africa. The capsule polysaccharide (CPS) of Mmm is one of the few identified virulence determinants. In a previous study, immunization of mice against CPS generated antibodies, but they were not able to prevent multiplication of Mmm in this model animal. However, mice cannot be considered as a suitable animal model, as Mmm does not induce pathology in this species. Our aim was to induce antibody responses to CPS in cattle, and challenge them when they had specific CPS antibody titres similar or higher than those from cattle vaccinated with the live vaccine. The CPS was linked to the carrier protein ovalbumin via a carbodiimide-mediated condensation with 1-ethyl-3(3-imethylaminopropyl) carbodiimide (EDC). Ten animals were immunized twice and challenged three weeks after the booster inoculation, and compared to a group of challenged non-immunized cattle. When administered subcutaneously to adult cattle, the vaccine elicited CPS-specific antibody responses with the same or a higher titre than animals vaccinated with the live vaccine. Pathology in the group of immunized animals was significantly reduced (57%) after challenge with Mmm strain Afadé compared to the non-immunized group, a figure in the range of the protection provided by the live vaccine. PMID:27496744

  11. Pyogranulomatous pleuropneumonia and mediastinitis in ferrets (Mustela putorius furo) associated with Pseudomonas luteola Infection.

    PubMed

    Martínez, J; Martorell, J; Abarca, M L; Olvera, A; Ramis, A; Woods, L; Cheville, N; Juan-Sallés, C; Moya, A; Riera, A; Soto, S

    2012-01-01

    Between 2008 and 2009, three pet ferrets from different sources presented with acute episode of dyspnoea. Cytological examination of pleural exudates revealed severe purulent inflammation with abundant clusters of rod-shaped microorganisms with a clear surrounding halo. Treatment was ineffective and the ferrets died 2-5 days later. Two ferrets were subjected to necropsy examination, which revealed pyothorax, mediastinal lymphadenopathy and multiple white nodules (1-2mm) in the lungs. Microscopical examination showed multifocal necrotizing-pyogranulomatous pleuropneumonia and lymphadenitis with aggregates of encapsulated microorganisms, some of which were positively stained by periodic acid-Schiff and alcian blue. In-situ hybridization for Pneumocystis spp., Ziehl-Neelsen staining and immunohistochemistry for distemper, coronavirus and influenza antigen were negative in all cases. Electron microscopically, the bacteria were 2-3 μm long with a thick electron-lucent capsule. Microbiology from one ferret yielded a pure culture of gram-negative bacteria identified phenotypically as Pseudomonas luteola. This speciation was later confirmed by 16S RNA gene amplification. PMID:21601873

  12. Challenges of controlling contagious bovine pleuropneumonia in sub-Saharan Africa: a Zambian perspective.

    PubMed

    Muuka, Geoffrey; Songolo, Nadi; Kabilika, Swithine; Hang'ombe, Bernard M; Nalubamba, King S; Muma, John B

    2013-01-01

    Contagious bovine pleuropneumonia (CBPP) is a disease of economic importance that is widely distributed in sub-Saharan African and contributes significantly to cattle morbidity and mortality. Control of CBPP offers a number of challenges as a result many developing countries in Africa are still struggling with this disease. In this study, we look at the challenges encountered in CBPP control in sub-Saharan Africa from the Zambian perspective. In conducting this study, we reviewed scientific literature and reports from the Ministry of Agriculture and Livestock and related animal institutions, and also made interviews with experts and key government officials involved in CBPP control in Zambia. Among the challenges identified for the successful control of CBPP were as follows: failure in the delivery of veterinary services, lack of a cattle identification system, natural phenomenon, livestock husbandry systems in the traditional sector, human movements, traditional practices among cattle farmers and cattle marketing systems. It was seen that the epidemiology of CBPP in Zambia is influenced by both ecological and anthological factors. Therefore, design and implementation of any control or eradication programme should be area/regional-dependent taking into account the different factors influencing disease transmission and maintenance. PMID:22843213

  13. Iron-Chelating Activity of Tetracyclines and Its Impact on the Susceptibility of Actinobacillus actinomycetemcomitans to These Antibiotics

    PubMed Central

    Grenier, Daniel; Huot, Marie-Pierre; Mayrand, Denis

    2000-01-01

    Three tetracyclines (tetracycline, doxycycline, and minocycline) were found to possess iron-chelating activity in a colorimetric siderophore assay. Determination of MICs indicated that the activity of doxycycline against the periodontopathogen Actinobacillus actinomycetemcomitans was only slightly influenced by the presence of an excess of iron that likely saturates the antibiotic. On the other hand, the MICs of doxycycline and minocycline were significantly lower for A. actinomycetemcomitans cultivated under iron-poor conditions than under iron-rich conditions. PMID:10681353

  14. [Effect of sheep haptoglobin on the hemoglobin molecule in the Hp-Hb complex].

    PubMed

    Beĭsembaeva, R U; Dzhusupova, R Zh

    1986-01-01

    Sheep haptoglobin (HpC) binding hemoglobin increases the stability of the latter to acid denaturation and oxidation by atmospheric O2. HpC is also capable of binding methemoglobin (MetHb) denaturated at pH 3.5 to form a stable complex. This process is accompanied by partial reconstitution of the structural integrity and peroxidase activity of MetHb. Consequently, the formation of a HpC-MetHb complex leads to changes in the tertiary structure of the MetHb molecule. The increase in the peroxidase activity of MetHb at pH less than or equal to 4.0 after its binding to HpC is due to the stabilizing and stimulating activity of HpC. PMID:3006800

  15. Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer.

    PubMed

    Tsai, Hsien-Yu; Boonyapranai, Kongsak; Sriyam, Supawadee; Yu, Chong-Jen; Wu, Sz-Wei; Khoo, Kay-Hooi; Phutrakul, Suree; Chen, Shui-Tein

    2011-06-01

    Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6-linked tri-sialylated triantennary glycan containing α1,3-linked fucose attached to the four-linked position of the three-arm mannose of N-linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression. PMID:21538882

  16. Role of Haptoglobin in Health and Disease: A Focus on Diabetes.

    PubMed

    MacKellar, Mark; Vigerust, David J

    2016-07-01

    In Brief Prospective identification of individuals with diabetes who are at greatest risk for developing complications would have considerable public health importance by allowing appropriate resources to be focused on those who would benefit most from aggressive intervention. Haptoglobin (Hp) is an acute-phase protein that is crucial for the elimination of free hemoglobin and the neutralization of oxidative damage. In the past two decades, associations have been made between polymorphisms in Hp and complications arising from diabetes. Individuals with polymorphism in Hp have been shown to have significantly higher risk of developing cardiovascular disease. This review summarizes the current literature on the role of Hp in health and disease, with a focus on diabetes. PMID:27621532

  17. Chimerism in humans after intragenic recombination at the haptoglobin locus during early embryogenesis.

    PubMed

    Asakawa, J; Kodaira, M; Nakamura, N; Satoh, C; Fujita, M

    1999-08-31

    The human haptoglobin (HP) HP*2 allele contains a 1.7-kilobase (kb) intragenic duplication that arose after a unique nonhomologous recombination between the prototype HP*1 alleles. During a genetic screening of 13,000 children of survivors exposed to atomic-bomb radiation and 10,000 children of unexposed persons, two children suspected of carrying de novo mutations at the haptoglobin locus were identified (one in each group). DNA analyses of single-cell-derived colonies of Epstein-Barr virus-transformed B cells revealed that the two children were mosaics comprising HP*2/HP*2 and HP*2/HP*1 cells at a ratio of approximately 3:1. We infer that the latter cells are caused by reversion of one HP*2 allele to HP*1 through an intramolecular homologous recombination between the duplicated segments of the Hp*2 allele that excised one of the segments. Because the mosaicism is substantial (approximately 25%), this recombination must have occurred in early embryogenesis. The frequency of finding these children and the extent of their mosaicisms corresponds to an HP*2 to HP*1 reversion rate of 8 x 10(-6) per cell during development. This leads to the prediction that the HP*1 allele also will be represented, although usually at a very low frequency, in any HP2-2 person. We tested this prediction by using PCR for a single individual and found the HP*1 allele at frequencies of 4 x 10(-6) and 3 x 10(-6) in somatic and sperm cells. The HP*1 allele was detected by PCR in all four other HP2-2 individuals, which supports the regular but rare occurrence somatically of homologous recombination within duplicated regions in humans, in agreement with previous observations in mouse and Drosophila. PMID:10468605

  18. Haptoglobin concentrations in free-range and temporarily captive juvenile steller sea lions.

    PubMed

    Thomton, Jamie D; Mellish, Jo-Ann E

    2007-04-01

    Haptoglobin (Hp) is an acute-phase protein synthesized in the liver that circulates at elevated concentrations in response to tissue damage caused by inflammation, infection, and trauma. As part of a larger study, sera Hp concentrations were measured in temporarily captive (n = 21) and free-range (n = 38) western stock juvenile Steller sea lions (Eumetopias jubatus) sampled from 2003 to 2006. Baseline Hp concentration at time of capture was 133.3 +/- 17.4 mg/dl. Temporarily captive animals exhibited a 3.2-fold increase in Hp concentrations during the first 4 wk of captivity, followed by a return to entry levels by week 5. Haptoglobin levels were not influenced by age, season, or parasite load. There was a significant positive correlation between Hp concentrations and white blood cell count (P < 0.001) and globulin levels (P < 0.001) and a negative correlation to red blood cell count and hematocrit (P < 0.001 for both). There was no correlation between Hp levels and platelet count (P = 0.095) or hemoglobin (P = 0.457). Routine blubber biopsies collected under gas anesthesia did not produce a measurable Hp response. One animal with a large abscess had an Hp spike of 1,006.0 mg/dl that returned to entry levels after treatment. In conclusion, serum Hp levels correlate to the stable clinical health status observed during captivity, with moderate Hp response during capture and initial acclimation to captivity and acute response to inflammation and infection. PMID:17495310

  19. Haptoglobin phenotype is not a predictor of recurrence free survival in high-risk primary breast cancer patients

    PubMed Central

    Gast, Marie-Christine W; van Tinteren, Harm; Bontenbal, Marijke; van Hoesel, René QGCM; Nooij, Marianne A; Rodenhuis, Sjoerd; Span, Paul N; Tjan-Heijnen, Vivianne CG; de Vries, Elisabeth GE; Harris, Nathan; Twisk, Jos WR; Schellens, Jan HM; Beijnen, Jos H

    2008-01-01

    Background Better breast cancer prognostication may improve selection of patients for adjuvant therapy. We conducted a retrospective follow-up study in which we investigated sera of high-risk primary breast cancer patients, to search for proteins predictive of recurrence free survival. Methods Two sample sets of high-risk primary breast cancer patients participating in a randomised national trial investigating the effectiveness of high-dose chemotherapy were analysed. Sera in set I (n = 63) were analysed by surface enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF MS) for biomarker finding. Initial results were validated by analysis of sample set II (n = 371), using one-dimensional gel-electrophoresis. Results In sample set I, the expression of a peak at mass-to-charge ratio 9198 (relative intensity ≤ 20 or > 20), identified as haptoglobin (Hp) alpha-1 chain, was strongly associated with recurrence free survival (global Log-rank test; p = 0.0014). Haptoglobin is present in three distinct phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2), of which only individuals with phenotype Hp 1-1 or Hp 2-1 express the haptoglobin alpha-1 chain. As the expression of the haptoglobin alpha-1 chain, determined by SELDI-TOF MS, corresponds to the phenotype, initial results were validated by haptoglobin phenotyping of the independent sample set II by native one-dimensional gel-electrophoresis. With the Hp 1-1 phenotype as the reference category, the univariate hazard ratio for recurrence was 0.87 (95% CI: 0.56 – 1.34, p = 0.5221) and 1.03 (95% CI: 0.65 – 1.64, p = 0.8966) for the Hp 2-1 and Hp 2-2 phenotypes, respectively, in sample set II. Conclusion In contrast to our initial results, the haptoglobin phenotype was not identified as a predictor of recurrence free survival in high-risk primary breast cancer in our validation set. Our initial observation in the discovery set was probably the result of a type I error (i.e. false positive). This study

  20. Pharmacodynamics of Antimicrobials against Mycoplasma mycoides mycoides Small Colony, the Causative Agent of Contagious Bovine Pleuropneumonia

    PubMed Central

    Mitchell, John D.; McKellar, Quintin A.; McKeever, Declan J.

    2012-01-01

    Background Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC) is the causative agent of Contagious Bovine Pleuropneumonia (CBPP), a disease of substantial economic importance in sub-Saharan Africa. Failure of vaccination to curtail spread of this disease has led to calls for evaluation of the role of antimicrobials in CBPP control. Three major classes of antimicrobial are effective against mycoplasmas, namely tetracyclines, fluoroquinolones and macrolides. Therefore, the objectives of this study were to determine the effector kinetics of oxytetracycline, danofloxacin and tulathromycin against two MmmSC field strains in artificial medium and adult bovine serum. Methods Minimum inhibitory concentrations (MIC) were determined for oxytetracycline, danofloxacin and tulathromycin against MmmSC strains B237 and Tan8 using a macrodilution technique, and time-kill curves were constructed for various multiples of the MIC over a 24 hour period in artificial medium and serum. Data were fitted to sigmoid Emax models to obtain 24 hour-area under curve/MIC ratios for mycoplasmastasis and, where appropriate, for mycoplasmacidal activity and virtual mycoplasmal elimination. Results Minimum inhibitory concentrations against B237 were 20-fold higher, 2-fold higher and approximately 330-fold lower in serum than in artificial medium for oxytetracycline, danofloxacin and tulathromycin, respectively. Such differences were mirrored in experiments using Tan8. Oxytetracycline was mycoplasmastatic against both strains in both matrices. Danofloxacin elicited mycoplasmacidal activity against B237 and virtual elimination of Tan8; similar maximum antimycoplasmal effects were observed in artificial medium and serum. Tulathromycin effected virtual elimination of B237 but was mycoplasmastatic against Tan8 in artificial medium. However, this drug was mycoplasmastatic against both strains in the more physiologically relevant matrix of serum. Conclusions Oxytetracycline, danofloxacin and

  1. Contagious bovine pleuropneumonia: Seroprevalence and risk factors in Western Oromia, Ethiopia.

    PubMed

    Daniel, Garuma; Abdurahaman, Mukarim; Tuli, Getachew; Deresa, Benti

    2016-01-01

    Contagious bovine pleuropneumonia (CBPP) is one of the most important threats to cattle health and production in Ethiopia. At the livestock farm of the Bako Agricultural Research Center, an outbreak of respiratory disease of cattle occurred in May 2011, and many animals were affected and died before the disease was diagnosed. Therefore, this study was designed to determine the seroprevalence of CBPP antibodies in selected districts of Western Oromia Region and to assess the potential risk factors for the occurrence of the disease. A crosssectional study was conducted from November 2013 to March 2014 in three selected districts of Western Oromia Region. A total of 386 sera were examined for the presence of specific antibodies against Mycoplasma mycoidesmycoides small colony (MmmSC), using a competitive enzyme-linked immunosorbent assay. The risk factors that were evaluated in this study were geographical location, age, sex, breed and body condition. The overall seroprevalence in this study was 28.5%. The seroprevalence of Mycoplasma mycoidesmycoides small colony antibodies at the district level was 40.3%, 19.0% and 5.7% in Gobbu Sayyo, BakoTibbe and Horro districts, respectively. There was a statistically significant variation ( p < 0.05) in the prevalence of antibodies amongst the districts. However, animal-related risk factors, such as age, sex, breed and body condition, were not significantly associated ( p > 0.05) with the serological status of the animal. This study showed that the overall prevalence of CBPP in Western Oromia Zones was high. This warrants the implementation of appropriate preventive and control measures to minimise the economic losses associated with the disease. PMID:27247066

  2. Isothermal loop-mediated amplification (lamp) for diagnosis of contagious bovine pleuro-pneumonia

    PubMed Central

    2013-01-01

    Background Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. Results We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis. Conclusion The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a

  3. A meta-analysis of contagious caprine pleuropneumonia (CCPP) in Ethiopia.

    PubMed

    Asmare, Kassahun; Abayneh, Takele; Mekuria, Solomon; Ayelet, Gelagay; Sibhat, Berhanu; Skjerve, Eystein; Szonyi, Barbara; Wieland, Barbara

    2016-06-01

    This systematic literature review was initiated due to lack of comprehensive information on the status and distribution of contagious caprine pleuropneumonia (CCPP) in Ethiopia. The objectives of the review were thus to provide a pooled prevalence estimate of CCPP in the country and asses the level of in between study variance among the available reports. Manual and electronic search was conducted between 8th of January and 25th of June 2015. A total of twelve published articles and one MSc thesis was retrieved from 19 initially identified studies. Twenty five animal level datasets were extracted at regional level considering some hypothesized predictors. The retrieved data were summarized in a meta-analytical approach. Accordingly, the pooled prevalence estimate of CCPP was 25.7% (95% CI:20.9,31.0). The inverse variance square (I(2)) that explains the variation in effect size attributed to reports true heterogeneity was 95.7%.The sub-group analysis was also computed for assumed predictors including, age, sex, type of study population, production systems and regional states. Among these predictors, study population type revealed statistically significant difference (P<0.05). Accordingly, the prevalence estimate for samples collected at abattoir was 39.2%, while that of samples collected at field level was 22.4%. In the final model, type of study population fitted the multivariable meta-regression model accounting for 22.87% of the explainable proportion of heterogeneity among the presumed predictors. Evidence on isolation and confirmation of Mycoplasma capricolum subspp. capripneumonie in the country was obtained from five regional states. In conclusion, it is recommended to further investigate facilities related with transportation and collection premises along with potential role of sheep in the epidemiology of CCPP. Finally, the review emphasizes the need for monitoring the ongoing CCPP control intervention and introduces amendments based on the findings

  4. Exploring site-specific N-glycosylation microheterogeneity of haptoglobin using glycopeptide CID tandem mass spectra and glycan database search.

    PubMed

    Chandler, Kevin Brown; Pompach, Petr; Goldman, Radoslav; Edwards, Nathan

    2013-08-01

    Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something that detached N-glycan and deglycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy that takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false discovery rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at a false discovery rate of 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at a false discovery rate of 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http

  5. Exploring Site-Specific N-Glycosylation Microheterogeneity of Haptoglobin using Glycopeptide CID Tandem Mass Spectra and Glycan Database Search

    PubMed Central

    Chandler, Kevin Brown; Pompach, Petr; Goldman, Radoslav

    2013-01-01

    Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something which detached N-glycan and de-glycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy which takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false-discovery-rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at false-discovery-rate 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at false-discovery-rate 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http

  6. The Influence of Cigarette Smoking on Gingival Bleeding and Serum Concentrations of Haptoglobin and Alpha 1-Antitrypsin

    PubMed Central

    Al-Bayaty, Fouad H.; Baharuddin, NorAdinar; Abdulla, Mahmood A.; Ali, Hapipah Mohd; Arkilla, Magaji B.; ALBayaty, Mustafa F.

    2013-01-01

    The objectives of this study were to evaluate the influence of cigarette smoking on gingival bleeding and serum concentrations of cotinine, haptoglobin, and alpha 1-antitrypsin in Malaysian smokers. A total of 197 male smokers and nonsmokers were recruited for this study. Plaque index, bleeding on probing (BOP), and levels of serum cotinine, haptoglobin, and alpha 1-antitrypsin were evaluated. The data were analyzed using SPSS version 20.0, with the significance level set at α ≤ 0.05. Linear regression analyses were performed. The mean cigarette consumption per day was 13.39 ± 5.75 cigarettes; the mean duration was 16.03 ± 8.78 years. Relatively low BOP values (26.05 ± 1.48) and moderate plaque indexes (51.35 ± 11.27) were found. The levels of serum cotinine (106.9 ± 30.71 ng/dL), haptoglobin (76.04 ± 52.48 mg/dL), and alpha 1-antitrypsin (141.90 ± 18.40 mg/dL) were significantly higher in smokers compared to non-smokers. Multiple logistic regression models for all variables and smokers demonstrated observed differences between BOP, the number of cigarettes per day, and duration of smoking, while serum cotinine, haptoglobin and alpha-1 antitrypsin levels showed no significant differences. Duration of smoking (years) and the cotinine level in serum showed a significant correlation with plaque index. The present analysis demonstrated that the duration of smoking in years, but not the number of cigarettes smoked per day, was associated with reduced gingival bleeding in smokers. PMID:24286083

  7. The influence of cigarette smoking on gingival bleeding and serum concentrations of haptoglobin and alpha 1-antitrypsin.

    PubMed

    Al-Bayaty, Fouad H; Baharuddin, Noradinar; Abdulla, Mahmood A; Ali, Hapipah Mohd; Arkilla, Magaji B; ALBayaty, Mustafa F

    2013-01-01

    The objectives of this study were to evaluate the influence of cigarette smoking on gingival bleeding and serum concentrations of cotinine, haptoglobin, and alpha 1-antitrypsin in Malaysian smokers. A total of 197 male smokers and nonsmokers were recruited for this study. Plaque index, bleeding on probing (BOP), and levels of serum cotinine, haptoglobin, and alpha 1-antitrypsin were evaluated. The data were analyzed using SPSS version 20.0, with the significance level set at α ≤ 0.05. Linear regression analyses were performed. The mean cigarette consumption per day was 13.39 ± 5.75 cigarettes; the mean duration was 16.03 ± 8.78 years. Relatively low BOP values (26.05 ± 1.48) and moderate plaque indexes (51.35 ± 11.27) were found. The levels of serum cotinine (106.9 ± 30.71 ng/dL), haptoglobin (76.04 ± 52.48 mg/dL), and alpha 1-antitrypsin (141.90 ± 18.40 mg/dL) were significantly higher in smokers compared to non-smokers. Multiple logistic regression models for all variables and smokers demonstrated observed differences between BOP, the number of cigarettes per day, and duration of smoking, while serum cotinine, haptoglobin and alpha-1 antitrypsin levels showed no significant differences. Duration of smoking (years) and the cotinine level in serum showed a significant correlation with plaque index. The present analysis demonstrated that the duration of smoking in years, but not the number of cigarettes smoked per day, was associated with reduced gingival bleeding in smokers. PMID:24286083

  8. Gene diversity for haptoglobin and transferrin classical markers among Hindu and Muslim populations of Aligarh City, India.

    PubMed

    Ara, G; Siddique, Y H; Afzal, M

    2011-06-01

    The present paper reports the distribution of serum protein markers viz. haptoglobin and transferrin in two major groups of Aligarh city of North India. In present study we have undertaken a survey of 538 individuals belonging to eight different populations, four from the Hindu community i.e. Brahmin, Bania, Rajput and Jatav, and the rest four among the Muslim community i.e. Syed, Sheikh, Pathan and Ansari. The heterozygosity ranged from 0.2939 (Ansari) to 0.4873 (Brahmin) for haptoglobin and from 0.000 (Rajput) to 0.1498 (Pathan) for transferrin. The values of D(ST) are 0.4122 and 0.4406, and that of G(ST) are 0.5059 and 0.9726 for haptoglobin and transferrin markers respectively. Through F(ST) test, it has been concluded that there is a high genetic differentiation of populations within Hindu and Muslim groups, though there is absence of any significant differences between these groups. PMID:21866866

  9. Serum haptoglobin as a novel molecular biomarker predicting colorectal cancer hepatic metastasis.

    PubMed

    Sun, Lichao; Hu, Shusheng; Yu, Long; Guo, Chunguang; Sun, Lixin; Yang, Zhihua; Qi, Jun; Ran, Yuliang

    2016-06-01

    Early detection of liver metastasis is important for improving colorectal cancer (CRC) patient survival. Our previous studies showed haptoglobin was highly expressed in primary CRC tissues, especially in heterochronous metastatic cases. Here, we assessed the potential of serum haptoglobin (sHP) as a biomarker for early detection of CRC liver metastasis by evaluating the sHP in 475 CRC patients and 152 healthy volunteers. In the training set (250 cases), sHP level in CRC-M1 (1773.18 ± 690.25 ng/mL) were significantly increased as compared to in CRC-M0 (1544.37 ± 1497.65 ng/mL) or healthy (917.76 ± 571.59 ng/mL). And the high sHP level was correlated with poor survival. Logistic regression analysis revealed that sHP, serum carcinoembryonic antigen (sCEA) and serum carbohydrate antigen 19.9 (sCA19.9) level were the significant parameters for detecting liver metastasis. In leave-one-out-cross-validation, these three markers resulted in 89.1% sensitivity and 85.8% specificity for hepatic metastasis detection. In an independent test set (225 cases), receiver operating characteristic curve analysis of sHP in CRC liver metastasis showed an area under the curve of 0.735, with a sensitivity of 87.2% and a specificity of 59.9%. Combination of sHP, sCEA and sCA19.9 improved diagnostic accuracy to 0.880, with a sensitivity of 88.5% and a specificity of 87.8%. Silencing of HP by specific shRNA significantly inhibited the LOVO and SW620 cell invasion, and suppressed xenograft tumor invasive growth. In summary, these results demonstrate that sHP is associated with poor prognosis of CRC patients and that HP promotes colorectal cancer cell invasion. sHP combining with sCA19.9 and sCEA may be used as accurate predictors of CRC liver metastasis. PMID:26756179

  10. Haplotype Association between Haptoglobin (Hp2) and Hp Promoter SNP (A-61C) May Explain Previous Controversy of Haptoglobin and Malaria Protection

    PubMed Central

    Cox, Sharon E.; Doherty, Conor; Atkinson, Sarah H.; Nweneka, Chidi V.; Fulford, Anthony J.C.; Ghattas, Hala; Rockett, Kirk A.; Kwiatkowski, Dominic P.; Prentice, Andrew M.

    2007-01-01

    Background Malaria is one of the strongest recent selective pressures on the human genome, as evidenced by the high levels of varying haemoglobinopathies in human populations–despite the increased risk of mortality in the homozygous states. Previously, functional polymorphisms of Hp, coded by the co-dominant alleles Hp1 and Hp2, have been variously associated with several infectious diseases, including malaria susceptibility. Methodology/Principal Findings Risk of a clinical malarial episode over the course of a malarial transmission season was assessed using active surveillance in a cohort of Gambian children aged 10–72 months. We report for the first time that the major haplotype for the A-61C mutant allele in the promoter of haptoglobin (Hp)–an acute phase protein that clears haemoglobin released from haemolysis of red cells–is associated with protection from malarial infection in older children, (children aged ≥36 months, >500 parasites/ul and temperature >37.5°C; OR = 0.42; [95% CI 0.24–0.73] p = 0.002) (lr test for interaction, <36 vs ≥36 months, p = 0.014). Protection was also observed using two other definitions, including temperature >37.5°C, dipstick positive, plus clinical judgement of malaria blinded to dipstick result (all ages, OR = 0.48, [95% CI 0.30–0.78] p = 0.003; ≥36 months, OR = 0.31, [95% CI 0.15–0.62] p = 0.001). A similar level of protection was observed for the known protective genetic variant, sickle cell trait (HbAS). Conclusions/Significance We propose that previous conflicting results between Hp phenotypes/genotypes and malaria susceptibility may be explained by differing prevalence of the A-61C SNP in the populations studied, which we found to be highly associated with the Hp2 allele. We report the -61C allele to be associated with decreased Hp protein levels (independent of Hp phenotype), confirming in vitro studies. Decreased Hp expression may lead to increased oxidant stress and

  11. Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis.

    PubMed Central

    Haubek, D; Dirienzo, J M; Tinoco, E M; Westergaard, J; López, N J; Chung, C P; Poulsen, K; Kilian, M

    1997-01-01

    Actinobacillus actinomycetemcomitans strains with enhanced levels of production of leukotoxin are characterized by a 530-bp deletion from the promoter region of the leukotoxin gene operon. Previous isolates with this deletion constituted a single clone belonging to serotype b, although they displayed minor differences among each other. We have analyzed the geographic dissemination of this clone by examining 326 A. actinomycetemcomitans isolates from healthy and periodontally diseased individuals as well as from patients with different types of extraoral infections originating from countries worldwide. A total of 38 isolates, all belonging to the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the promoter region of the leukotoxin gene operon from 10 of these strains revealed complete identity, which indicates that the deletion originates from a single mutational event. This particular clone was exclusively associated with localized juvenile periodontitis (LJP). In at least 12 of 28 families from which the clone was isolated, more than one family member had LJP. Notably, all the subjects carrying this clone had a genetic affiliation with the African population. These observations suggest that juvenile periodontitis in some adolescents with an African origin is associated with a disseminating clone of A. actinomycetemcomitans. PMID:9399490

  12. Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

    PubMed Central

    2006-01-01

    Abstract Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis. PMID:16548331

  13. Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production.

    PubMed

    Schindler, Bryan D; Joshi, Rajasi V; Vieille, Claire

    2014-09-01

    Actinobacillus succinogenes 130Z naturally produces among the highest levels of succinate from a variety of inexpensive carbon substrates. A few studies have demonstrated that A. succinogenes can anaerobically metabolize glycerol, a waste product of biodiesel manufacture and an inexpensive feedstock, to produce high yields of succinate. However, all these studies were performed in the presence of yeast extract, which largely removes the redox constraints associated with fermenting glycerol, a highly reduced molecule. We demonstrated that A. succinogenes cannot ferment glycerol in minimal medium, but that it can metabolize glycerol by aerobic or anaerobic respiration. These results were expected based on the A. succinogenes genome, which encodes respiratory enzymes, but no pathway for 1,3-propanediol production. We investigated A. succinogenes's glycerol metabolism in minimal medium in a variety of respiratory conditions by comparing growth, metabolite production, and in vitro activity of terminal oxidoreductases. Nitrate inhibited succinate production by inhibiting fumarate reductase expression. In contrast, growth in the presence of dimethylsulfoxide and in microaerobic conditions allowed high succinate yields. The highest succinate yield was 0.75 mol/mol glycerol (75 % of the maximum theoretical yield) in continuous microaerobic cultures. A. succinogenes could also grow and produce succinate on partially refined glycerols obtained directly from biodiesel manufacture. Finally, by expressing a heterologous 1,3-propanediol synthesis pathway in A. succinogenes, we provide the first proof of concept that A. succinogenes can be engineered to grow fermentatively on glycerol. PMID:25047181

  14. Improving succinic acid production by Actinobacillus succinogenes from raw industrial carob pods.

    PubMed

    Carvalho, Margarida; Roca, Christophe; Reis, Maria A M

    2016-10-01

    Carob pods are an inexpensive by-product of locust bean gum industry that can be used as renewable feedstock for bio-based succinic acid. Here, for the first time, unprocessed raw carob pods were used to extract a highly enriched sugar solution, afterwards used as substrate to produce succinic acid using Actinobacillus succinogenes. Batch fermentations containing 30g/L sugars resulted in a production rate of 1.67gSA/L.h and a yield of 0.39gSA/g sugars. Taking advantage of A. succinogenes' metabolism, uncoupling cell growth from succinic acid production, a fed-batch mode was implemented to increase succinic acid yield and reduce by-products formation. This strategy resulted in a succinic acid yield of 0.94gSA/g sugars, the highest yield reported in the literature for fed-batch and continuous experiments, while maintaining by-products at residual values. Results demonstrate that raw carob pods are a highly efficient feedstock for bio-based succinic acid production. PMID:27394995

  15. Carob pod water extracts as feedstock for succinic acid production by Actinobacillus succinogenes 130Z.

    PubMed

    Carvalho, Margarida; Roca, Christophe; Reis, Maria A M

    2014-10-01

    Carob pods are a by-product of locust bean gum industry containing more than 50% (w/w) sucrose, glucose and fructose. In this work, carob pod water extracts were used, for the first time, for succinic acid production by Actinobacillus succinogenes 130Z. Kinetic studies of glucose, fructose and sucrose consumption as individual carbon sources till 30g/L showed no inhibition on cell growth, sugar consumption and SA production rates. Sugar extraction from carob pods was optimized varying solid/liquid ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Batch fermentations containing 10-15g/L total sugars resulted in a maximum specific SA production rate of 0.61Cmol/Cmol X.h, with a yield of 0.55Cmol SA/Cmol sugar and a volumetric productivity of 1.61g SA/L.h. Results demonstrate that carob pods can be a promising low cost feedstock for bio-based SA production. PMID:25164341

  16. Immobilization of Actinobacillus succinogenes by adhesion or entrapment for the production of succinic acid.

    PubMed

    Corona-González, Rosa Isela; Miramontes-Murillo, Ricardo; Arriola-Guevara, Enrique; Guatemala-Morales, Guadalupe; Toriz, Guillermo; Pelayo-Ortiz, Carlos

    2014-07-01

    The production of succinic acid was studied with entrapped and adsorbed Actinobacillus succinogenes. The adsorption of fermentation products (organic acids in the concentration range of 1-20 g/L) on different supports was evaluated. It was found that succinic acid was adsorbed in small quantities on diatomite and zeolite (12.6 mg/g support). The highest production of succinic acid was achieved with A. succinogenes entrapped in agar beads. Batch fermentations with immobilized cells were carried out with glucose concentrations ranging from 20 to 80 g/L. Succinic acid (43.4 g/L) was obtained from 78.3g/L glucose, and a high productivity (2.83 g/Lh) was obtained with a glucose concentration of 37.6g/L. For repeated batch fermentations (5 cycles in 72 h) with immobilized cells in agar, the total glucose consumed was 147.55 g/L, while the production of succinic acid was 107 g/L. Immobilized cells reduced significantly the fermentation time, yield, productivity and final concentration of succinic acid. PMID:24844165

  17. Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli.

    PubMed

    Gröndahl, G; Sternberg, S; Jensen-Waern, M; Johannisson, A

    2001-11-01

    Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice. PMID:11770988

  18. A complete industrial system for economical succinic acid production by Actinobacillus succinogenes.

    PubMed

    Li, Jian; Zheng, Xiao-Yu; Fang, Xiao-Jiang; Liu, Shu-Wen; Chen, Ke-Quan; Jiang, Min; Wei, Ping; Ouyang, Ping-Kai

    2011-05-01

    An industrial fermentation system using lignocellulosic hydrolysate, waste yeast hydrolysate, and mixed alkali to achieve high-yield, economical succinic acid production by Actinobacillus succinogenes was developed. Lignocellulosic hydrolysate and waste yeast hydrolysate were used efficiently as carbon sources and nitrogen source instead of the expensive glucose and yeast extract. Moreover, as a novel method for regulating pH mixed alkalis (Mg(OH)(2) and NaOH) were first used to replace the expensive MgCO(3) for succinic acid production. Using the three aforementioned substitutions, the total fermentation cost decreased by 55.9%, and 56.4 g/L succinic acid with yield of 0.73 g/g was obtained, which are almost the same production level as fermentation with glucose, yeast extract and MgCO(3). Therefore, the cheap carbon and nitrogen sources, as well as the mixed alkaline neutralize could be efficiently used instead of expensive composition for industrial succinic acid production. PMID:21470857

  19. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss. PMID:26817919

  20. Succinic acid production from acid hydrolysate of corn fiber by Actinobacillus succinogenes.

    PubMed

    Chen, Kequan; Jiang, Min; Wei, Ping; Yao, Jiaming; Wu, Hao

    2010-01-01

    Dilute acid hydrolysate of corn fiber was used as carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. The optimized hydrolysis conditions were obtained by orthogonal experiments. When corn fiber particles were of 20 mesh in size and treated with 1.0% sulfuric acid at 121 degrees C for 2 h, the total sugar yield could reach 63.3%. It was found that CaCO(3) neutralization combined with activated carbon adsorption was an effective method to remove fermentation inhibitors especially furfural that presented in the acid hydrolysate of corn fiber. Only 5.2% of the total sugar was lost, while 91.9% of furfural was removed. The yield of succinic acid was higher than 72.0% with the detoxified corn fiber hydrolysate as the carbon source in anaerobic bottles or 7.5 L fermentor cultures. It was proved that the corn fiber hydrolysate could be an alternative to glucose for the production of succinic acid by A. succinogenes NJ113. PMID:18830824

  1. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

    PubMed

    García, L; Tercero, J C; Legido, B; Ramos, J A; Alemany, J; Sanz, M

    1998-01-01

    The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens. PMID:9524322

  2. CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma.

    PubMed

    Graversen, Jonas Heilskov; Madsen, Mette; Moestrup, Søren K

    2002-04-01

    CD163 is a highly expressed macrophage membrane protein belonging to the scavenger receptor cysteine rich (SRCR) domain family. The CD163 expression is induced by interleukin-6, interleukin-10 and glucocorticoids. Its function has remained unknown until recently when CD163 was identified as the endocytic receptor binding hemoglobin (Hb) in complex with the plasma protein haptoglobin (Hp). This specific receptor-ligand interaction leading to removal from plasma of the Hp-Hb complex-but not free Hp or Hb-now explains the depletion of circulating Hp in individuals with increased intravascular hemolysis. Besides having a detoxificating effect by removing Hb from plasma, the CD163-mediated endocytosis of the Hp-Hb complex may represent a major pathway for uptake of iron in the tissue macrophages. The novel functional linkage of CD163 and Hp, which both are induced during inflammation, also reveal some interesting perspectives relating to the suggested anti-inflammatory properties of the receptor and the Hp phenotypes. PMID:11854028

  3. Human haptoglobin phenotypes and concentration determination by nanogold-enhanced electrochemical impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Cheng, Tsai-Mu; Lee, Tzu-Cheng; Tseng, Shin-Hua; Chu, Hsueh-Liang; Pan, Ju-Pin; Chang, Chia-Ching

    2011-06-01

    Haptoglobin (Hp) is an acute phase protein that binds free hemoglobin (Hb), preventing Hb-induced oxidative damage in the vascular system. There are three phenotypes in human Hp, whose heterogeneous polymorphic structures and varying concentrations in plasma have been attributed to the cause of diseases and outcome of clinical treatments. Different phenotypes of Hp may be composed of the same subunits but different copy numbers, rendering their determination difficult by a single procedure. In this study, we have developed a simple, fast, reliable and sensitive method, using label-free nanogold-modified bioprobes coupled with self-development electrochemical impedance spectroscopy (EIS). By this method, probe surface charge transfer resistance is detected. The relative charge transfer resistance ratios for Hp 1-1, Hp 2-1 and Hp 2-2 were characterized. We were able to determine protein size difference within 3 nm, and the linear region of the calibration curve for Hp levels in the range of 90 pg ml - 1 and 90 µg ml - 1 (~1 fM to 1 pM). We surmise that similar approaches can be used to investigate protein polymorphism and altered protein-protein interaction associated with diseases.

  4. Structure of the trypanosome haptoglobin-hemoglobin receptor and implications for nutrient uptake and innate immunity.

    PubMed

    Higgins, Matthew K; Tkachenko, Olga; Brown, Alan; Reed, Jenny; Raper, Jayne; Carrington, Mark

    2013-01-29

    African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin-hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake. PMID:23319650

  5. Haptoglobin binds apolipoprotein E and influences cholesterol esterification in the cerebrospinal fluid.

    PubMed

    Salvatore, Alfonso; Cigliano, Luisa; Carlucci, Alessandro; Bucci, Enrico M; Abrescia, Paolo

    2009-07-01

    Haptoglobin (Hpt) binds the apolipoprotein (Apo) A-I domain, which is involved in stimulating the enzyme lecithin-cholesterol acyltransferase (LCAT) for cholesterol esterification. This binding was shown to protect ApoA-I against hydroxyl radicals, thus preventing loss of ApoA-I function in enzyme stimulation. In this study, we report that Hpt is also able to bind ApoE. The Hpt binding site on the ApoE structure was mapped by using synthetic peptides, and found homologous to the Hpt binding site of ApoA-I. Hydroxyl radicals promoted in vitro the formation of ApoE-containing adducts which were detected by immunoblotting. Hpt impaired this oxidative modification whereas albumin did not. CSF from patients with multiple sclerosis or subjects without neurodegeneration contains oxidized forms of ApoE and ApoA-I similar to those observed in vitro. CSF was analyzed for its level of ApoA-I, ApoE, Hpt, cholesteryl esters, and unesterified cholesterol. The ratio of esterified with unesterified cholesterol, assumed to reflect the LCAT activity ex vivo, did not correlate with either analyzed protein, but conversely correlated with the ratio [Hpt]/([ApoE]+[ApoA-I]). The results suggest that Hpt might save the function of ApoA-I and ApoE for cholesterol esterification, a process contributing to cholesterol elimination from the brain. PMID:19457062

  6. Cloning and expression of human haptoglobin subunits in Escherichia coli: delineation of a major antioxidant domain.

    PubMed

    Lai, I Hsiang; Tsai, Tsung I; Lin, Hong Huei; Lai, Wei Yen; Mao, Simon J T

    2007-04-01

    Human plasma haptoglobin (Hp) comprises alpha and beta subunits. The alpha subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that alpha1, alpha2, beta, and alpha2beta chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. Each cloned subunit retained its immunoreactivity as confirmed using antibodies specific to alpha or beta chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to the native Hp. The antioxidant activity was found to be associated with both alpha and beta chains when assessed by Cu(2+)-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of beta chain was extremely potent and markedly greater than that of native Hp (3.5x), alpha chain (10x) and probucol (15x). The latter is a clinically proved potent compound used for antioxidant therapy. The "unrestricted" structure of beta subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a "mini-Hp" in antioxidant therapy. It may also provide a new insight in understanding the mechanism involved in the antioxidant nature of Hp. PMID:17095249

  7. Convergent evolution of human and bovine haptoglobin: partial duplication of the genes.

    PubMed

    Wicher, Krzysztof B; Fries, Erik

    2007-10-01

    Haptoglobin (Hp) is a hemoglobin-binding plasma protein consisting of two types of chains, called alpha and beta, which originate from a common polypeptide. In humans, but not in other mammals, Hp has been shown to occur in two allelic forms, Hp1 and Hp2, which differ in the length of the alpha-chain. The longer alpha-chain (in Hp2) seems to have arisen by an internal duplication of a gene segment coding for almost the entire alpha-chain of Hp1. In this article we show that Hp of cow (Bos taurus) contains an alpha-chain, the structure of which is similar to that of the human Hp2 alpha-chain. Furthermore, comparison of the structure of bovine Hp and human Hp2 suggests that the bovine gene arose by a duplication of the gene segment homologous to that duplicated in human Hp2. However, a phylogenetic analysis indicates that the two genes were formed independently. The evolutionary pressure that has led to the fixation of the Hps with a longer alpha-chain is not known. PMID:17922076

  8. Role of porcine serum haptoglobin in the host-parasite relationship of Taenia solium cysticercosis.

    PubMed

    Navarrete-Perea, José; Toledano-Magaña, Yanis; De la Torre, Patricia; Sciutto, Edda; Bobes, Raúl José; Soberón, Xavier; Laclette, Juan Pedro

    2016-06-01

    Human and porcine cysticercosis is a parasitic disease caused by the larval stage (cysts) of the tapeworm Taenia solium. Cysts may live in several host tissues such as skeletal muscle or brain. We have previously described the presence of host haptoglobin (Hp) and hemoglobin (Hb) in different protein extracts of the T. solium cysts. Here, we report the binding of host Hp and Hb to a number of cyst proteins, evaluated through measuring electrophoretic and light absorbance changes. In the sera obtained from 18 cysticercotic pigs, Hp-Hb complexes were abundant, whereas free Hp was undetectable. In contrast, in the sera from non 18 cysticercotic pigs, Hp-Hb and free Hp were found. In the soluble protein fraction of cysts tissue, free Hp was detected showing a considerable Hb-binding ability, whereas in the vesicular fluid, Hp is mainly bound to Hb. Interestingly, assays carried out with the insoluble fraction of T. solium cysts tissue, showed binding of Hp and Hp-Hb in a saturable way, suggesting the existence of specific interactions. Our results suggested that the parasite can take advantage of the uptaken host Hp and Hb, either free or in complexes, as a source of iron or as a way to modulate the inflammatory response surrounding the T. solium cysts. PMID:27234210

  9. Measurement of porcine haptoglobin in meat juice using surface acoustic wave biosensor technology.

    PubMed

    Klauke, Thorsten N; Gronewold, Thomas M A; Perpeet, Markus; Plattes, Susanne; Petersen, Brigitte

    2013-11-01

    Aim of the study was the application of biosensor technique to measure the concentration of an acute phase protein (APP) within complex matrices from animal origin. For the first time, acute phase protein haptoglobin (Hp) was detected from unpurified meat juice of slaughter pigs by a label-free biosensor-system, the SAW-based sam®5 system. The system uses a sensor chip with specific antibodies to catch Hp while the mass-related phase shift is measured. The concentration is calculated as a function of these measured phase shifts. The results correlate very well with reference measurement results obtained by enzyme-linked immunosorbent assay (ELISA), R=0.98. The robust setup of the surface acoustic wave (SAW)-based system and its ability to measure within very short time periods qualifies it for large-scale analyses and is apt to identify rapidly pigs in the meat production process whose consumption would have an increased risk for consumers. PMID:23601789

  10. Rapid detection and quantification of free hemoglobin and haptoglobin by nanogold modified electrochemical impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Lu, Yu-Ning; Li, Hsing-Yuan; Chu, Hsueh-Liang; Cheng, Tsia-Mu; Tseng, Shin-Hua; Chang, Chia-Ching

    2013-03-01

    Free Hemoglobin (Hb) is a metabolic substance that damage tissue and vessel. It is released from destructed red blood cell and causes infection or inflammatory of human body. In blood plasma, haptoglobin (Hp) binds free Hb with high affinity and prevents the damage which is caused by cell free Hb. Hp has three phenotypes, that are Hp1-1, Hp 2-1, and Hp 2-2. Different phenotypes of Hp has been different affinities to Hb. It is known that electrochemical impedance spectroscopy (EIS) provide more information for detecting the small amount bio-molecules, include protein and DNA. In this study, we have developed a simple, fast, reliable and sensitive platform to quantify concentration of free Hb and Hp. In this platform, detection probe has been modified with nano gold and the surface charge transfer resistance of Hb and Hp binding could be detected and quantified within 18 min. This is a whole new platform to quantify free Hb in the serum of human to our knowledge.

  11. Pharmacogenomic interaction between the Haptoglobin genotype and vitamin E on atherosclerotic plaque progression and stability

    PubMed Central

    Veiner, Hilla-Lee; Gorbatov, Rostic; Vardi, Moshe; Doros, Gheorghe; Miller-Lotan, Rachel; Zohar, Yaniv; Sabo, Edmond; Asleh, Rabea; Levy, Nina S.; Goldfarb, Levi J.; Berk, Thomas A.; Haas, Tali; Shalom, Hadar; Suss-Toby, Edith; Kam, Adi; Kaplan, Marielle; Tamir, Ronit; Ziskind, Anna; Levy, Andrew P.

    2015-01-01

    Structured Abstract Objective Homozygosity for a 1.7kb intragenic duplication of the Haptoglobin (Hp) gene (Hp 2-2 genotype), present in 36% of the population, has been associated with a 2–3 fold increased incidence of atherothrombosis in individuals with Diabetes (DM) in 10 longitudinal studies compared to DM individuals not homozygous for this duplication (Hp 1-1/2-1). The increased CVD risk associated with the Hp 2-2 genotype has been shown to be prevented with vitamin E supplementation in man. We sought to determine if there was an interaction between the Hp genotype and vitamin E on atherosclerotic plaque growth and stability in a transgenic model of the Hp polymorphism. Methods and Results Brachiocephalic artery atherosclerotic plaque volume was serially assessed by high resolution ultrasound in 28 Hp 1-1 and 26 Hp 2-2 mice in a C57Bl/6 ApoE−/− background. Hp 2-2 mice had more rapid plaque growth and an increased incidence of plaque hemorrhage and rupture. Vitamin E significantly reduced plaque growth in Hp 2-2 but not in Hp 1-1 mice with a significant pharmacogenomic interaction between the Hp genotype and vitamin E on plaque growth. Conclusions These results may help explain why vitamin E supplementation in man can prevent CVD in Hp 2-2 DM but not in non Hp 2-2 DM individuals. PMID:25618031

  12. Molecular Characterization of Buffalo Haptoglobin: Sequence Based Structural Comparison Indicates Convergent Evolution Between Ruminants and Human.

    PubMed

    Niranjan, S K; Goyal, S; Dubey, P K; Vohra, V; Singh, S; Kathiravan, P; Kataria, R S

    2016-01-01

    Haptoglobin (Hp) protein has high affinity for hemoglobin (Hb) binding during intravascular hemolysis and scavenges the hemoglobin induced free radicals. Earlier reports indicate about uniqueness of Hp molecule in human and cattle, but in other animals, it is not much studied. In this paper, we characterized buffalo Hp molecule and determined its molecular structure, evolutionary importance, and tissue expression. Comparative analysis and predicted domain structure indicated that the buffalo Hp has an internal duplicated region in α-chain only similar to an alternate Hp2 allele in human. This duplicated part encoded for an extra complement control protein CCP domain. Phylogenetic analysis revealed that buffalo and other ruminants were found to group together separated from all other non-ruminants, including human. The key amino acid residues involved in Hp and Hb as well as Hp and macrophage scavenger receptor, CD163 interactions in buffalo, depicted a significant variation in comparison to other non-ruminant species. Constitutive expression of Hp was also confirmed across all the vital tissues of buffalo, for the first time. Results revealed that buffalo Hp is both structurally and functionally conserved, having internal duplication in α-chain similar to human Hp2 and other ruminant species, which might have evolved separately as a convergent evolutionary process. Furthermore, the presence of extra Hp CCP domain possibly in all ruminants may have an effect during dimerization of molecule in these species. PMID:26646629

  13. Molecular characterization and validation of commercially available methods for haptoglobin measurement in bottlenose dolphin☆

    PubMed Central

    Segawa, Takao; Amatsuji, Hazumu; Suzuki, Kento; Suzuki, Miwa; Yanagisawa, Makio; Itou, Takuya; Sakai, Takeo; Nakanishi, Teruyuki

    2013-01-01

    Haptoglobin (Hp) is a positive acute-phase protein and a valuable marker of inflammation in both human and veterinary medicine. The aim of this study was to validate the molecular characterization of Hp in dolphins and to validate commercially available Hp measurement methods such as Hp-ELISA (originally designed for pigs) and Hp–hemoglobin (Hb) binding assay. The dolphin Hp (dHp) amino acid sequence appeared most similar to pig Hp by sequence homology and phylogenetic clustering. Amino acid sequence analysis revealed that dHp comprises the Hp1 form of α1 and β chains. The anti-pig Hp antibody cross-reacted with both recombinant dHp, expressed by Escherichia coli, and dHp from serum. The intra- and inter-assay levels of imprecision of pig Hp-ELISA and the Hp–Hb binding assay were found to be tolerable for the determination of Hp in dolphin, and there was no significant discrepancy between the two determination methods. The ability of the assay to differentiate between healthy and inflammation groups was investigated, and a significant increase in Hp concentration was detected in inflammatory conditions. Thus, Hp is a useful inflammation marker for dolphin, and the Hp concentration in dolphin serum samples can be reliably measured using commercially available pig Hp-ELISA and Hp–Hb binding assay. PMID:24600559

  14. Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: Functional and antioxidative implications

    PubMed Central

    Roche, Camille J.; Dantsker, David; Alayash, Abdu I.; Friedman, Joel M.

    2012-01-01

    The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αβ dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb–Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αβ Hb dimer. The presented results show that the Hb dimer in the Hb–Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb–Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb–Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation. PMID:22521791

  15. Insights on N-glycosylation of human haptoglobin and its association with cancers.

    PubMed

    Zhang, Shu; Shang, Shuxin; Li, Wei; Qin, Xue; Liu, Yinkun

    2016-07-01

    Protein glycosylation is one of the most significant post-translation modifications and plays a critical role in various biological functions. Haptoglobin (Hp) is one of the acute-phase response proteins secreted by liver. Its glycosylation could be analyzed by many analytical techniques qualitatively and quantitatively. The glycosylation alterations of Hp are reported to be associated with different kinds of diseases. The main glycosylation alterations of Hp in cancer appear to be the presence of aberrantly fucosylated and sialylated structures as well as increased branching. In this mini review, we provided a brief overview of Hp structure and biological function, discussed its glycosylation alterations in different cancers, and described the existing technologies for analyzing glycosylation site and glycan of Hp. Given the importance of Hp glycosylation, its unknown and unclear biological complexity and significances, Hp glycosylation has become a major target in cancer research. Development of sensitive and specific detection of Hp glycosylation including large-scale validation may be significant steps forward to its clinical application. PMID:26873173

  16. Exploring farmer preferences for contagious bovine pleuropneumonia vaccination: a case study of Narok District of Kenya.

    PubMed

    Kairu-Wanyoike, Salome W; Kaitibie, Simeon; Taylor, Nick M; Gitau, George K; Heffernan, Claire; Schnier, Christian; Kiara, Henry; Taracha, Evans; McKeever, Declan

    2013-07-01

    Contagious bovine pleuropneumonia (CBPP) is an economically important disease in most of sub-Saharan Africa. A conjoint analysis and ordered probit regression models were used to measure the preferences of farmers for CBPP vaccine and vaccination attributes. This was with regard to inclusion or not of an indicator in the vaccine, vaccine safety, vaccine stability as well as frequency of vaccination, vaccine administration and the nature of vaccination. The analysis was carried out in 190 households in Narok District of Kenya between October and December 2006 using structured questionnaires, 16 attribute profiles and a five-point Likert scale. The factors affecting attribute valuation were shown through a two-way location interaction model. The study also demonstrated the relative importance (RI) of attributes and the compensation value of attribute levels. The attribute coefficient estimates showed that farmers prefer a vaccine that has an indicator, is 100% safe and is administered by the government (p<0.0001). The preferences for the vaccine attributes were consistent with expectations. Preferences for stability, frequency of vaccination and nature of vaccination differed amongst farmers (p>0.05). While inclusion of an indicator in the vaccine was the most important attribute (RI=43.6%), price was the least important (RI=0.5%). Of the 22 household factors considered, 15 affected attribute valuation. The compensation values for a change from non inclusion to inclusion of an indicator, 95-100% safety, 2h to greater than 2h stability and from compulsory to elective vaccination were positive while those for a change from annual to biannual vaccination and from government to private administration were negative. The study concluded that the farmers in Narok District had preferences for specific vaccine and vaccination attributes. These preferences were conditioned by various household characteristics and disease risk factors. On average the farmers would need to be

  17. Quantitative risk assessment of entry of contagious bovine pleuropneumonia through live cattle imported from northwestern Ethiopia.

    PubMed

    Woube, Yilkal Asfaw; Dibaba, Asseged Bogale; Tameru, Berhanu; Fite, Richard; Nganwa, David; Robnett, Vinaida; Demisse, Amsalu; Habtemariam, Tsegaye

    2015-11-01

    Contagious bovine pleuropneumonia (CBPP) is a highly contagious bacterial disease of cattle caused by Mycoplasma mycoides subspecies mycoides small colony (SC) bovine biotype (MmmSC). It has been eradicated from many countries; however, the disease persists in many parts of Africa and Asia. CBPP is one of the major trade-restricting diseases of cattle in Ethiopia. In this quantitative risk assessment the OIE concept of zoning was adopted to assess the entry of CBPP into an importing country when up to 280,000 live cattle are exported every year from the northwestern proposed disease free zone (DFZ) of Ethiopia. To estimate the level of risk, a six-tiered risk pathway (scenario tree) was developed, evidences collected and equations generated. The probability of occurrence of the hazard at each node was modelled as a probability distribution using Monte Carlo simulation (@RISK software) at 10,000 iterations to account for uncertainty and variability. The uncertainty and variability of data points surrounding the risk estimate were further quantified by sensitivity analysis. In this study a single animal destined for export from the northwestern DFZ of Ethiopia has a CBPP infection probability of 4.76×10(-6) (95% CI=7.25×10(-8) 1.92×10(-5)). The probability that at least one infected animal enters an importing country in one year is 0.53 (90% CI=0.042-0.97). The expected number of CBPP infected animals exported any given year is 1.28 (95% CI=0.021-5.42). According to the risk estimate, an average of 2.73×10(6) animals (90% CI=10,674-5.9×10(6)) must be exported to get the first infected case. By this account it would, on average, take 10.15 years (90% CI=0.24-23.18) for the first infected animal to be included in the consignment. Sensitivity analysis revealed that prevalence and vaccination had the highest impact on the uncertainty and variability of the overall risk. PMID:26427634

  18. Succinic acid production by Actinobacillus succinogenes from batch fermentation of mixed sugars.

    PubMed

    Almqvist, Henrik; Pateraki, Chrysanthi; Alexandri, Maria; Koutinas, Apostolis; Lidén, Gunnar

    2016-08-01

    Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8-26.8 g/L and a total yield of 0.59-0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14-0.20 g/g and formate 0.08-0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO2 sparging could replace carbonate supply in the form of MgCO3 without affecting the succinate yield. PMID:27255975

  19. Oral and systemic immunoglobulin G-subclass antibodies to Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed

    Engström, P E; George, M; Larsson, P; Lally, E T; Taichman, N S; Norhagen, G

    1999-04-01

    Salivary, gingival crevicular fluid and serum-specific immunoglobulin G (IgG)-subclass antibodies to Actinobacillus actinomycetemcomitans leuktoxin were quantified by enzyme-linked immunosorbent assay. Samples were taken from six patients with periodontal pockets > or = 5 mm, harboring A. actinomycetemcomitans in subgingival plaque and from six healthy, sex- and age-matched controls, who did not harbor A. actinomycetemcomitans. In individuals suffering from periodontitis, the median values of specific IgG1- and IgG2-subclass antibodies in saliva, gingival crevicular fluid and serum were, respectively IgG1 147 ng/ml, 5226 ng/ml and 7318 ng/ml and IgG2 4.8 ng/ml, 934 ng/ml and 860 ng/ml. In the patients, specific IgG3 antibodies were detected in one out of six individuals in saliva, in two individuals in gingival crevicular fluid and in five out of six patients in serum with a median value of 561 ng/ml. The median values of specific IgG4 antibodies in saliva, gingival crevicular fluid and serum were below detectable levels. The median values of the total IgG subclasses in saliva and serum were 14622 ng/ml and 10.3 g/l respectively. Individuals with periodontitis had, compared with controls, a higher ratio of specific IgG1 antibodies to total IgG1 in saliva (P < 0.05) and in serum (P < 0.05) and a higher ratio of specific IgG antibodies to total IgG in saliva (P < 0.05) and in serum (P < 0.01). The results show an elevation of both oral and systemic specific antibodies to A. actinomycetemcomitans leukotoxin. PMID:10219169

  20. Reclassification of Actinobacillus muris as Muribacter muris gen. nov., comb. nov.

    PubMed

    Nicklas, Werner; Bisgaard, Magne; Aalbæk, Bent; Kuhnert, Peter; Christensen, Henrik

    2015-10-01

    To reinvestigate the taxonomy of [Actinobacillus] muris, 474 strains, mainly from mice and rats, were characterized by phenotype and 130 strains selected for genotypic characterization by 16S rRNA and partial rpoB gene sequencing. The type strain was further investigated by whole-genome sequencing. Phylogenetic analysis of the DNA sequences showed one monophyletic group with intragroup similarities of 96.7 and 97.2 % for the 16S rRNA and rpoB genes, respectively. The highest 16S rRNA gene sequence similarity to a taxon with a validly published name outside the group was 95.9 %, to the type strain of [Pasteurella] pneumotropica. The closest related taxon based on rpoB sequence comparison was 'Haemophilus influenzae-murium', with 88.4 % similarity. A new genus and a new combination, Muribacter muris gen. nov., comb. nov., are proposed based on a distinct phylogenetic position based on 16S rRNA and rpoB gene sequence comparisons, with major divergence from the existing genera of the family Pasteurellaceae. The new genus has the characteristics of [A.] muris with the emendation that acid formation from ( - )-d-mannitol and hydrolysis of aesculin are variable, while the α-glucosidase test is positive. There is no requirement for exogenously supplied NAD (V factor) for the majority of strains investigated; however, one strain was found to require NAD. The major fatty acids of the type strain of Muribacter muris were C14 : 0, C14 : 0 3-OH/iso-C16 : 1 I, C16 : 1ω7c and C16 : 0, which is in line with most genera of the Pasteurellaceae. The type strain of Muribacter muris is CCUG 16938T ( = NCTC 12432T = ATCC 49577T). PMID:26296776

  1. Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

    PubMed

    Sosroseno, Wihaskoro; Herminajeng, Endang; Susilowati, Heni; Budiarti, Sri

    2002-12-01

    The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases. PMID:16887678

  2. Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams

    PubMed Central

    2013-01-01

    Background Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. Results The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. Conclusions The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis. PMID:23514236

  3. Molecular characterization of hpuAB, the haemoglobin-haptoglobin-utilization operon of Neisseria meningitidis.

    PubMed

    Lewis, L A; Gray, E; Wang, Y P; Roe, B A; Dyer, D W

    1997-02-01

    We previously identified HpuB, an 85 kDa Fe-repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin-haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high-affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5' to hpuB, and was designated hpuA. HpuA was identified in a total-membrane-protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [3H]-palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5' to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co-transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5' to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)-box sequences. The structure of this operon suggests that HpuA-HpuB is a two-component receptor analogous to the bipartite transferrin receptor TbpB-TbpA. PMID:9157245

  4. Assignment of the binding site for haptoglobin on apolipoprotein A-I.

    PubMed

    Spagnuolo, Maria Stefania; Cigliano, Luisa; D'Andrea, Luca D; Pedone, Carlo; Abrescia, Paolo

    2005-01-14

    Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function. PMID:15533931

  5. An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

    PubMed Central

    Levy, Nina S.; Vardi, Moshe; Blum, Shany; Miller-Lotan, Rachel; Afinbinder, Yefim; Cleary, Patricia A.; Paterson, Andrew D.; Bharaj, Bhupinder; Snell-Bergeon, Janet K.; Rewers, Marian J.; Lache, Orit; Levy, Andrew P.

    2013-01-01

    Background Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. Methods A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. Results Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. Conclusions The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies. PMID:23492570

  6. Study of Serum Haptoglobin Level and its Relation to Erythropoietic Activity in Beta Thalassemia Children

    PubMed Central

    Ragab, Seham M.; Safan, Manal A.; Badr, Eman A.

    2015-01-01

    Background Serum haptoglobin (Hp) is a reliable marker for hemolysis regardless the inflammatory state. Objective We investigated the possible relation between Hp depletion and hemolysis severity, hepatitis C virus (HCV) infection and iron load in β-thalassemia children. Methods Twenty two β-thalassemia major (TM),20 β-thalassemia intermedia (TI) children with 20 age and sex matched healthy controls were involved. Pre-transfusion hemoglobin level was considered. Serum ferritin, Hp and transferrin receptor levels (sTfR) (by ELISA ), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (by colorimetric method) were assayed. Markers of hepatitis C virus (HCV) were done by PCR. Results The mean Hp levels among the studied groups were as follows; 8.02 ± 0.93 (mg/dl), 8.6 ±0.72 (mg/dl) and 122 ± 18.5(mg/dl) for TM, TI and the controls respectively. Both patient groups had significantly lower Hp level compared to the controls (P<0.0001) with significant lower level in TM compared to TI children ( P= 0.034). Significant inverse correlations were found between serum Hp and sTfR levels ( reflecting the erythropoietic activity) in thalassemia children combined and in each group (TM and TI) as well as among HCV infected children. STfR was the only significant independent predictor for serum Hp level (t= −5.585, P<0.0001). Among HCV infected patients, no significant correlation was found between serum Hp and serum transaminases. Conclusion Serum Hp depletion in thalassemia had significant relation to disease severity and correlated well with their erythropoietic activity, as assessed by the measurement of sTfR without significant relation to HCV infection. Extensive multicenter studies are recommended. PMID:25745546

  7. Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors

    PubMed Central

    Bullard, Whitney; Kieft, Rudo; Capewell, Paul; Veitch, Nicola J.; Macleod, Annette; Hajduk, Stephen L.

    2012-01-01

    The haptoglobin-hemoglobin receptor (HpHbR) of African trypanosomes plays a critical role in human innate immunity against these parasites. Localized to the flagellar pocket of the veterinary pathogen Trypanosoma brucei brucei this receptor binds Trypanosome Lytic Factor-1 (TLF-1), a subclass of human high-density lipoprotein (HDL) facilitating endocytosis, lysosomal trafficking and subsequent killing. Recently, we found that group 1 Trypanosoma brucei gambiense does not express a functional HpHbR. We now show that loss of the TbbHpHbR reduces the susceptibility of T. b. brucei to human serum and TLF-1 by 100- and 10,000-fold, respectively. The relatively high concentrations of human serum and TLF-1 needed to kill trypanosomes lacking the HpHbR indicates that high affinity TbbHpHbR binding enhances the cytotoxicity; however, in the absence of TbbHpHbR, other receptors or fluid phase endocytosis are sufficient to provide some level of susceptibility. Human serum contains a second innate immune factor, TLF-2, that has been suggested to kill trypanosomes independently of the TbbHpHbR. We found that T. b. brucei killing by TLF-2 was reduced in TbbHpHbR-deficient cells but to a lesser extent than TLF-1. This suggests that both TLF-1 and TLF-2 can be taken up via the TbbHpHbR but that alternative pathways exist for the uptake of these toxins. Together the findings reported here extend our previously published studies and suggest that group 1 T. b. gambiense has evolved multiple mechanisms to avoid killing by trypanolytic human serum factors. PMID:22286709

  8. Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors.

    PubMed

    Bullard, Whitney; Kieft, Rudo; Capewell, Paul; Veitch, Nicola J; Macleod, Annette; Hajduk, Stephen L

    2012-01-01

    The haptoglobin-hemoglobin receptor (HpHbR) of African trypanosomes plays a critical role in human innate immunity against these parasites. Localized to the flagellar pocket of the veterinary pathogen Trypanosoma brucei brucei this receptor binds Trypanosome Lytic Factor-1 (TLF-1), a subclass of human high-density lipoprotein (HDL) facilitating endocytosis, lysosomal trafficking and subsequent killing. Recently, we found that group 1 Trypanosoma brucei gambiense does not express a functional HpHbR. We now show that loss of the TbbHpHbR reduces the susceptibility of T. b. brucei to human serum and TLF-1 by 100- and 10,000-fold, respectively. The relatively high concentrations of human serum and TLF-1 needed to kill trypanosomes lacking the HpHbR indicates that high affinity TbbHpHbR binding enhances the cytotoxicity; however, in the absence of TbbHpHbR, other receptors or fluid phase endocytosis are sufficient to provide some level of susceptibility. Human serum contains a second innate immune factor, TLF-2, that has been suggested to kill trypanosomes independently of the TbbHpHbR. We found that T. b. brucei killing by TLF-2 was reduced in TbbHpHbR-deficient cells but to a lesser extent than TLF-1. This suggests that both TLF-1 and TLF-2 can be taken up via the TbbHpHbR but that alternative pathways exist for the uptake of these toxins. Together the findings reported here extend our previously published studies and suggest that group 1 T. b. gambiense has evolved multiple mechanisms to avoid killing by trypanolytic human serum factors. PMID:22286709

  9. Age-Dependent Effects of Haptoglobin Deletion in Neurobehavioral and Anatomical Outcomes Following Traumatic Brain Injury

    PubMed Central

    Glushakov, Alexander V.; Arias, Rodrigo A.; Tolosano, Emanuela; Doré, Sylvain

    2016-01-01

    Cerebral hemorrhages are common features of traumatic brain injury (TBI) and their presence is associated with chronic disabilities. Recent clinical and experimental evidence suggests that haptoglobin (Hp), an endogenous hemoglobin-binding protein most abundant in blood plasma, is involved in the intrinsic molecular defensive mechanism, though its role in TBI is poorly understood. The aim of this study was to investigate the effects of Hp deletion on the anatomical and behavioral outcomes in the controlled cortical impact model using wildtype (WT) C57BL/6 mice and genetically modified mice lacking the Hp gene (Hp−∕−) in two age cohorts [2–4 mo-old (young adult) and 7–8 mo-old (older adult)]. The data obtained suggest age-dependent significant effects on behavioral and anatomical TBI outcomes and recovery from injury. Moreover, in the adult cohort, neurological deficits in Hp−∕− mice at 24 h were significantly improved compared to WT, whereas there were no significant differences in brain pathology between these genotypes. In contrast, in the older adult cohort, Hp−∕− mice had significantly larger lesion volumes compared to WT, but neurological deficits were not significantly different. Immunohistochemistry for ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) revealed significant differences in microglial and astrocytic reactivity between Hp−∕− and WT in selected brain regions of the adult but not the older adult-aged cohort. In conclusion, the data obtained in the study provide clarification on the age-dependent aspects of the intrinsic defensive mechanisms involving Hp that might be involved in complex pathways differentially affecting acute brain trauma outcomes. PMID:27486583

  10. Haptoglobin, hemopexin, and related defense pathways—basic science, clinical perspectives, and drug development

    PubMed Central

    Schaer, Dominik J.; Vinchi, Francesca; Ingoglia, Giada; Tolosano, Emanuela; Buehler, Paul W.

    2014-01-01

    Hemolysis, which occurs in many disease states, can trigger a diverse pathophysiologic cascade that is related to the specific biochemical activities of free Hb and its porphyrin component heme. Normal erythropoiesis and concomitant removal of senescent red blood cells (RBC) from the circulation occurs at rates of approximately 2 × 106 RBCs/second. Within this physiologic range of RBC turnover, a small fraction of hemoglobin (Hb) is released into plasma as free extracellular Hb. In humans, there is an efficient multicomponent system of Hb sequestration, oxidative neutralization and clearance. Haptoglobin (Hp) is the primary Hb-binding protein in human plasma, which attenuates the adverse biochemical and physiologic effects of extracellular Hb. The cellular receptor target of Hp is the monocyte/macrophage scavenger receptor, CD163. Following Hb-Hp binding to CD163, cellular internalization of the complex leads to globin and heme metabolism, which is followed by adaptive changes in antioxidant and iron metabolism pathways and macrophage phenotype polarization. When Hb is released from RBCs within the physiologic range of Hp, the potential deleterious effects of Hb are prevented. However, during hyper-hemolytic conditions or with chronic hemolysis, Hp is depleted and Hb readily distributes to tissues where it might be exposed to oxidative conditions. In such conditions, heme can be released from ferric Hb. The free heme can then accelerate tissue damage by promoting peroxidative reactions and activation of inflammatory cascades. Hemopexin (Hx) is another plasma glycoprotein able to bind heme with high affinity. Hx sequesters heme in an inert, non-toxic form and transports it to the liver for catabolism and excretion. In the present review we discuss the components of physiologic Hb/heme detoxification and their potential therapeutic application in a wide range of hemolytic conditions. PMID:25389409