bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunological activity of a 14-kilodalton recombinant protein of Mycobacterium tuberculosis H37Rv
Authors: Kingston A E , Salgame P R , Mitchison N A , Colston M J
Published: January 2020

A 14-kilodalton peptide antigen from Mycobacterium tuberculosis was isolated from an Escherichia coli lambda gt 11 recombinant DNA clone and was identified by Western blotting (immunoblotting) with monoclonal antibody TB68. Immunization of mice and guinea pigs with the recombinant peptide (rTB68) induced in vitro lymphoproliferative responses in draining lymph node lymphocyte cultures as well as in vivo delayed-type hypersensitivity reactions. Moreover, rTB68 was found both to induce and to cross-react with Mycobacterium leprae immune lymphocytes, but did not generate protective effects against live M. leprae challenge in mice. These findings showed that a 14-kilodalton peptide which has been characterized as specific for M. tuberculosis on the basis of B-cell recognition was capable of generating cell-mediated immune responses and moreover contained T-cell epitopes which were cross-reactive with M. leprae antigens.
Relationship of bacterial growth phase to killing of Listeria monocytogenes by oxidative agents generated by neutrophils and enzyme systems
Authors: Bortolussi R , Vandenbroucke-Grauls C M , van Asbeck B S , Verhoef J
Published: January 2020

Listeria monocytogenes, a gram-positive motile bacterium which can cause severe bacterial infection in humans, is considered to be pathogenic by virtue of its ability to resist intracellular killing. Since the mechanism of intracellular survival is poorly understood, we assessed the sensitivity of L. monocytogenes to several potent antibacterial products. Phorbol myristate acetate (PMA)-stimulated polymorphonuclear cells (PMNs) produced extracellular antibacterial products which were inhibited completely by catalase, suggesting a role for oxidative agents in this process. L. monocytogenes in logarithmic (log) growth phase resisted PMA-stimulated PMN extracellular products significantly more than L. monocytogenes in stationary (stat) growth phase or Escherichia coli (three strains) in either phase of growth. The role of oxidative agents was studied further by using xanthine oxidase-xanthine, glucose oxidase-glucose, and myeloperoxidase enzyme systems to generate hydroxyl radical (.OH), hydrogen peroxide (H2O2), and hypochlorous acid (OCl-), respectively. L. monocytogenes in log phase resisted the antibacterial products of these enzyme systems under conditions which produced superoxide (O2-) and H2O2 at concentrations similar to those produced extracellularly by PMA-stimulated PMNs, while stat-growth-phase L. monocytogenes and E. coli in either phase of growth were susceptible. Antibacterial activity could be blocked or inhibited by exogenous catalase (for all oxygen radical-generating systems), mannitol, or desferoxamine (for xanthine oxidase-xanthine) and alanine (for myeloperoxidase), suggesting that .OH and OCl- were responsible for this activity. Log-phase L. monocytogenes had 2.5-fold higher bacteria-associated catalase activity, as compared with stat-phase L. monocytogenes. These experiments, therefore, suggest that log-phase L. monocytogenes resists oxidative antibacterial agents by producing sufficient catalase to inactivate these products. This may contribute to the ability of L. monocytogenes to survive intracellularly.
Fungicidal mechanisms of activated macrophages: evidence for nonoxidative mechanisms for killing of Blastomyces dermatitidis
Authors: Brummer E , Stevens D A
Published: January 2020

The mechanism(s) by which lymphokine-activated peritoneal macrophages kill Blastomyces dermatitidis was studied. Resident peritoneal macrophages from BALB/cByJ mice, when treated overnight with lymph node cells plus concanavalin A, supernatants from concanavalin A-stimulated spleen cells, or recombinant gamma interferon, were then able to kill a virulent B. dermatitidis isolate (ATCC 26199) (at levels of 25% +/- 4%, 28% +/- 8%, and 21% +/- 5%, respectively). Killing was not significantly decreased or enhanced in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethyl sulfoxide (300 mM), or azide (1 mM). Viable B. dermatitidis elicited a brisk oxidative burst and superoxide anion production in activated macrophages as measured by lucigenin-enhanced chemiluminescence, e.g., 10(4) cpm. However, these responses were not significantly different from those of control macrophages. Luminol-enhanced chemiluminescence responses by activated or control macrophages were meager (less than or equal to 10(2) cpm). These results indicate that activated macrophages kill B. dermatitidis by a mechanism(s) independent of products of the oxidative burst.
Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin
Authors: Mengaud J , Chenevert J , Geoffroy C , Gaillard J L , Cossart P
Published: January 2020

By immunoblotting with an antiserum raised against purified listeriolysin O, we have detected the presence of a truncated protein of 52 kilodaltons in culture supernatants of a Tn1545-induced nonhemolytic mutant of Listeria monocytogenes (J.L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986). The region of insertion of the transposon has been cloned and sequenced. The transposon had inserted in an open reading frame the listeriolysin O gene. The deduced amino acid sequence of this open reading frame revealed that listeriolysin O is homologous to streptolysin O and pneumolysin, although homologies were not detectable at the DNA level.
A highly conserved region present in transcripts encoding heterologous M proteins of group A streptococci
Authors: Hollingshead S K , Fischetti V A , Scott J R
Published: January 2020

In group A streptococcal strains of 10 different serotypes, the sequence previously identified as homologous to the structural gene for type 6 M protein (emm6) was found to be transcribed. The conserved sequence, which shows greater than 95% homology among heterologous M proteins, was identified as the 3' third of the gene.
Effects of diet and genetics on Mycobacterium bovis BCG vaccine efficacy in inbred guinea pigs
Authors: Cohen M K , Bartow R A , Mintzer C L , McMurray D N
Published: January 2020

Strain 2 and strain 13 guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on low-protein or protein-adequate diets. Five weeks later all animals were infected by the respiratory route with virulent Mycobacterium tuberculosis H37Rv organisms. Four weeks postchallenge, guinea pigs were skin tested with purified protein derivative and sacrificed. Protein deficiency resulted in significant reductions in body weight and thymus weight and in an impairment in the ability to control the M. bovis BCG vaccine organisms and to mount delayed hypersensitivity reactions. Protein deficiency also adversely affected the efficacy of the BCG vaccine as demonstrated by the numbers of virulent organisms recovered in spleens and lungs. Strain differences were observed in the number of leukocytes, thymus weight, and the responsiveness of blood lymphocytes to purified protein derivative stimulation. In general, strain 13 guinea pigs responded more dramatically to dietary insult than did their strain 2 counterparts. Protein deprivation completely abolished BCG vaccine protection in the lungs and spleens of strain 13 animals and significantly reduced the protection afforded to strain 2 animals. In both strains, the BCG vaccine protected normally nourished guinea pigs. There was no significant difference between strains with respect to susceptibility to pulmonary infection with virulent mycobacteria. Thus, diet and genetic pedigree each had a significant influence on BCG vaccine efficacy.
Rapid parasite multiplication rate, rather than immunosuppression, causes the death of mice infected with virulent Plasmodium yoelii
Authors: Fahey J R , Spitalny G L
Published: January 2020

Protective immunity against a lethal malaria challenge infection was passively transferred to naive recipient mice with spleen cells from donor mice bearing a lethal infection with the virulent YM strain of Plasmodium yoelii. Successful transfer of protection was contingent upon the elimination of residual, viable parasites from donor spleen cell suspensions prior to the infusion of cells. Passive transfer experiments failed to detect suppressor cells in the spleens of lethally infected mice because unfractionated spleen cells or T-cell-enriched spleen cells from mice infected with P. yoelii YM did not enhance parasitemias upon infusion into mice infected with cross-reactive nonvirulent P. yoelii 17X. We concluded that a form of protective immunity was generated during the course of virulent infection but that its expression was inconsequential because parasite growth apparently exceeded the capacity of the immune system to clear the infection.
Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci
Authors: Jones K F , Fischetti V A
Published: January 2020

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.
Antibody- and complement-dependent cell injury assayed by 51Cr release from human peripheral blood mononuclear cells pretreated with lipopolysaccharide
Authors: Repo H , Leirisalo-Repo M , Nurminen M , Mäkelä P H
Published: January 2020

Exposure of human peripheral blood mononuclear (MN) cells to deesterified (alkali-treated) lipopolysaccharide (LPS-OH) and then to 51Cr rendered the cells susceptible to 51Cr release in the presence of specific antibody and complement. The assay was optimized by using rough (Rb2 or Re) LPS. 51Cr release did not occur from cells preexposed to untreated or electrodialyzed LPS. Studies of isolated monocytes and lymphocytes revealed that the majority of the 51Cr released was derived from monocytes. The optimum concentration of LPS-OH was 10 micrograms/ml. Antiyersinia agglutinin-positive serum, but not a negative serum, obtained from patients with reactive yersinia arthritis caused 51Cr release from MN cells pretreated with yersinia LPS-OH. This implies that during yersinia infection antibodies are generated that can attack the cell membrane--LPS-OH complex. We conclude that the method provides a tool to demonstrate binding of LPS to MN cells in a manner that leads to cell injury in an immune host.
Impairment of the oxidative metabolism of mouse peritoneal macrophages by intracellular Leishmania spp
Authors: Buchmüller-Rouiller Y , Mauël J
Published: January 2020

When stimulated in vitro with macrophage-activating factor or lipopolysaccharide, mouse peritoneal macrophages acquire the capacity to develop a strong respiratory burst when they are triggered by membrane-active agents. The presence of intracellular parasites of the genus Leishmania (L. enriettii, L. major) significantly inhibited such activity, as measured by chemiluminescence, reduction of cytochrome c and Nitro Blue Tetrazolium, and hexose monophosphate shunt levels. On the contrary, inert intracellular particles such as latex beads strongly increased the macrophage respiratory burst, suggesting that the Leishmania-linked inhibition resulted from a specific parasite effect. Impairment of macrophage oxidative metabolism by intracellular Leishmania spp. was a function of the number of infecting microorganisms and was more pronounced in macrophages infected with living than with dead parasites. Moreover, the metabolic inhibition was less apparent in L. enriettii-infected macrophages that were exposed to both macrophage-activating factor and lipopolysaccharide, i.e., conditions leading to complete parasite destruction. The mechanisms of respiratory burst inhibition by intracellular Leishmania spp. are unclear, but these observations suggest that such effects may contribute significantly to intracellular survival of the microorganisms.
Enzymatic and antigenic characterization of immunoglobulin A1 proteases from Bacteroides and Capnocytophaga spp
Authors: Frandsen E V , Reinholdt J , Kilian M
Published: January 2020

Bacteroides and Capnocytophaga species have been implicated as periodontal pathogens. Some of these species possess immunoglobulin A1 (IgA1) proteases that are capable of cleaving the human IgA1 molecule in the hinge region, leaving intact Fc alpha and Fab alpha fragments. The purpose of this study was to characterize this activity. In addition to IgA1 protease activity in already known species, IgA1 protease activity was a feature of Bacteroides buccalis, Bacteroides oralis, Bacteroides veroralis, Bacteroides capillus, and Bacteroides pentosaceus. Results of immunoelectrophoretic and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses suggested that all species cleave the alpha-chain at the same peptide bond, i.e., the prolyl-seryl bond between residues 223 and 224 in the hinge region. The Bacteroides proteases could be classified as thiol proteases, which were at the same time dependent on metal ions, while the Capnocytophaga proteases were metallo enzymes. None of the proteases were inhibited by the physiologic proteases inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Investigations with enzyme-neutralizing antibodies raised in rabbits against protease preparations from the respective type strains revealed that, despite otherwise identical characteristics, the IgA1 protease of each Bacteroides species was antigenically distinct. Bacteroides buccae and the two later synonymous species B. capillus and B. pentosaceus produced identical proteases. In contrast, IgA1 proteases from Capnocytophaga ochracea and Capnocytophaga sputigena strains were apparently identical, while Capnocytophaga gingivalis had a protease that differed from those of the other Capnocytophaga species.
Prophylactic administration of interleukin-2 protects mice from lethal challenge with gram-negative bacteria
Authors: Chong K T
Published: January 2020

Prophylactic administration of recombinant human interleukin-2 (IL-2) in mice enhanced survival and produced complete recovery from an otherwise lethal acute bacterial infection. IL-2 was administered as a single intraperitoneal or intravenous bolus dose to CDI mice 18 h before challenge with a lethal dose of a clinical isolate of Escherichia coli type O2 (minimal 100% lethal dose, 6 X 10(7) CFU per mouse). At IL-2 dosages of 7 X 10(6) U/kg, 90% of treated CDI mice survived as compared to 0% for the excipient buffer control animals (P less than 0.001). This protective effect was also demonstrable in immune-deficient beige mice. The IL-2 effect was dose dependent; protection was consistently observed in mice pretreated with IL-2 at doses ranging from 1.8 X 10(6) to 7 X 10(6) U/kg. However, at 3.5 X 10(5) U/kg the protective effect was more variable. The route of administration of IL-2 was shown to play an important role; when IL-2 and challenge bacteria were given by the same route (either intravenously or intraperitoneally), protection was readily observable, but when IL-2 and challenge bacteria were given by different routes, little or no protective effect was observed. The protective effect was fully inducible as early as 1 h after IL-2 administration and was effective against various strains of gram-negative bacteria, indicating that the probable mode of action represents control of the establishment of infection by increased activity of the nonspecific host defense mechanisms. The IL-2 effect was abrogated by the administration of carrageenan, suggesting a possible role of macrophages. These data demonstrate that IL-2 may be a potentially useful adjunct for the prophylaxis of bacterial infections in both clinical and veterinary medicine.
Lipoarabinomannan and lipid-free arabinomannan antigens of Mycobacterium paratuberculosis
Authors: Sugden E A , Samagh B S , Bundle D R , Duncan J R
Published: January 2020

Lipoarabinomannan (LAM) and lipid-free arabinomannan (AM) were prepared from Mycobacterium paratuberculosis. Purification of LAM was done by ultracentrifugation of the phenol-water-extracted crude polysaccharide, followed by affinity and anion exchange chromatography. AM was purified from the supernatant of the ultracentrifuged polysaccharide or from alkaline-extracted material by gel filtration and anion exchange chromatography. Chemical analysis revealed arabinose and mannose in LAM (1.4:1) and AM (3.5:1) and the presence of palmitic, stearic, and tuberculostearic acids for a total of 7.8% lipid in LAM. Traces of phosphorus were found in the AMs, particularly LAM (0.05%). Nuclear magnetic resonance confirmed the presence of alpha-arabinosyl residues and the acylated nature of LAM. LAM exhibited lipid-dependent aggregation, as indicated by a Triton-induced decrease in molecular weight. By using bovine sera, LAM was found to be active in the complement fixation test, whereas AM was inactive and inhibited this activity. Thus, the presence of AM in crude polysaccharide could explain the variable complement fixation results. Triton-dissociated LAM exhibited a precipitin (Cl) in common with that of AM, confirming shared determinants. LAM in its lipid-dependent aggregated form, however, exhibited a second precipitin (C2), which may be due to the disparity in antigen size or a novel epitope. The lipid content of LAM rendered it 100 times more effective for coating plates in the enzyme immunoassay than lipid-free AM.
Purification and partial characterization of the multicomponent dextranase complex of Streptococcus sobrinus and cloning of the dextranase gene
Authors: Barrett J F , Barrett T A , Curtiss R
Published: January 2020

The presence of proteases in culture supernatant fluids and on the cell surface of Streptococcus sobrinus and the aggregation of multicomponent enzyme complexes make the isolation and characterization of cell surface proteins difficult. We report a simple purification procedure for dextranase and the cloning of the dextranase structural gene. S. sobrinus culture supernatant fluids were precipitated with 70% ammonium sulfate, and the precipitate was dialyzed against sodium acetate buffer and loaded onto a hemoglobin-Sepharose 4B column connected to a blue dextran-agarose column at 4 degrees C. After being washed with low concentrations of salt, the dextranase and the dextran-binding proteins were eluted with 5 M KI and further purified by gel filtration. Two dextranases (molecular weights, 175,000 and 160,000) were purified and partially characterized. The structural gene for the dextranase of S. sobrinus 6715 strain UAB66, serotype g, was cloned into the cosmid vector, pHC79. Clones were selected for expression of dextranase activity by detection of zones of enzyme-mediated hydrolysis of a blue dextran substrate incorporated into minimal medium agar plates. Release of dextranase was achieved by induction of thermoinducible, excision-defective Escherichia coli K-12 lysogens containing recombinant cosmid molecules of S. sobrinus DNA. Recombinant cosmid molecules were repackaged simultaneously into infectious lambdoid particles. Recombinant clones expressing dextranase activity which varied in size from the high-molecular-weight protein produced by S. sobrinus (i.e., 175,000) to lower-molecular-weight forms expressed by S. sobrinus have been identified and partially characterized.
Recognition of carbohydrate and protein epitopes by monoclonal antibodies to a cell wall antigen from Streptococcus mutans
Authors: Mitchell C G , Smith R , Lehner T
Published: January 2020

The nature of the determinants recognized by a panel of monoclonal antibodies (MAbs) raised against a cell wall antigen of Streptococcus mutans (SA I/II) was investigated. Mild periodate oxidation of SA I/II showed that MAbs Guy 1, 2, 3, and 5 recognized carbohydrate epitopes on the antigen. Glycosidases were used to identify the nature of the sugars involved in their binding. Treatment with beta-glucosidase inhibited the binding of Guy 1, 2, 3, and 5 by 90%. No competition was found for any of the MAbs between SA I/II and a series of carbohydrates, including the serotype c polysaccharide from S. mutans. The results show that MAbs Guy 1, 2, 3, and 5 recognize carbohydrate epitopes on SA I/II which are distinct from the serotype polysaccharide. The other MAbs recognized protein epitopes on SA I/II.
Anaphylaxis or so-called encephalopathy in mice sensitized to an antigen with the aid of pertussigen (pertussis toxin)
Authors: Munoz J J , Peacock M G , Hadlow W J
Published: January 2020

Sensitization of mice with 1 mg of bovine serum albumin (BSA) or chicken egg albumin (EA) given intraperitoneally and 300 to 400 ng of pertussigen (pertussis toxin [Ptx]) given intravenously (i.v.) induced a high degree of anaphylactic sensitivity when the mice were challenged i.v. with 1 mg of antigen 14 days later. Regardless of H-2 haplotype, all of the strains tested (CFW, BALB/cJ, DBA/2J, and C3H.SW/SnJ) were susceptible to anaphylaxis. Sensitization of mice by a multiple-dose procedure that has been reported to induce fatal encephalopathy in mice (L. Steinman, A. Weiss, N. Adelman, M. Lim, R. Zuniga, J. Oehlert, E. Hewlett, and S. Falkow, Proc. Natl. Acad. Sci. USA 82, 8733-8736, 1982) (1 mg of BSA on day -1, 100 to 400 ng of Ptx on day zero 1 mg of BSA on day +1, 100 to 400 ng of Ptx on day +2, and 1 mg of BSA on day +6) induced shock in BALB/cJ, DBA/2J, and C3H.SW/SnJ mice, but not in CFW mice. When EA was used instead of BSA, CFW, BALB/cJ, and C3H.SW/SnJ mice did not develop fatal shock, whereas DBA/2J mice did. When dose 3 of antigen (BSA or EA) was postponed to day +21, all mouse strains sensitized by the multiple-dose procedure were found to be susceptible to shock. The fatal shock induced by this procedure, as well as that induced by giving a single sensitizing dose of antigen and Ptx, could be prevented by one to three 1-ml doses of saline given i.v. at the time signs of severe shock appeared. Although only one dose of saline was often sufficient to save the mice, two or three doses were usually needed. Microscopic changes were not found in midsagittal sections of the brains of mice sensitized by either procedure. This was true of mice that died from shock or were saved from shock by injections of saline. From these results, we concluded that the proposed model for encephalopathy induced in mice by Ptx and BSA demonstrates only the well-known anaphylactogenic effect of Ptx or pertussis vaccine. Since there are many other more sensitive methods to detect Ptx, induction of anaphylaxis is not of much value for detection or quantitation of Ptx in pertussis vaccine.
Chemical composition and immunological characterization of polysaccharide antigen from Eubacterium saburreum T18
Authors: Nakazawa F , Ito T , Sato N , Sato M , Hoshino E
Published: January 2020

Two types of polysaccharide were obtained from the oral microorganism Eubacterium saburreum T18 by formamide extraction and subsequent gel filtration and ion-exchange chromatography. One polysaccharide, which was composed of D-glycero-D-galacto-heptose, had antigenic activity in an immunoprecipitation reaction with rabbit anti-T18 serum due to immunoglobulin M antibodies. The second polysaccharide was composed of D-glycero-D-manno-heptose and L-rhamnose, but it did not have immunoprecipitation activity. These polysaccharide antigens were not alkali labile and differed from E. saburreum L44 and T27 antigens, which were composed of D-glycero-D-galacto-heptose.
Killing of Plasmodium falciparum by eosinophil secretory products
Authors: Waters L S , Taverne J , Tai P C , Spry C J , Targett G A , Playfair J H
Published: January 2020

The multiplication of two strains of Plasmodium falciparum in culture, as measured by [3H]hypoxanthine incorporation, was inhibited in a dose-dependent manner by granule proteins secreted by purified eosinophils obtained from patients with the hypereosinophilic syndrome. Morphological examination revealed the presence of abnormal parasites inside erythrocytes, indicating that they were killed in situ, and the later stages of the developmental cycle were found to be most susceptible to these toxic effects. A monoclonal antibody against eosinophil cationic protein partially blocked the inhibitory effect, suggesting that it was caused by more than one of the eosinophil granule proteins. Thus some of the antimalarial effects of molecules such as the tumor necrosis factor, which activates eosinophils, may be mediated through the enhanced production of eosinophil secretion products.
Purification and characterization of serotype 6 fimbriae from Bordetella pertussis and comparison of their properties with serotype 2 fimbriae
Authors: Cowell J L , Zhang J M , Urisu A , Suzuki A , Steven A C , Liu T , Liu T Y , Manclark C R
Published: January 2020

Fimbriae were removed from Bordetella pertussis (serotype 1.3.6) by mechanical shearing and purified by precipitation with ammonium sulfate, pH-dependent precipitation at pH 7.4, followed by two successive extractions of the precipitated fimbriae with 4 M urea. By electron microscopy, the precipitated fimbriae appeared as aggregated bundles of long, relatively straight filaments which were disaggregated to individual flexuous filaments at pH 10.5. These purified fimbriae were identified as serotype 6 agglutinogens, since antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.3.6, 1.2.3.6, or 1.2.3.4.6 but did not agglutinate strains of serotype 1.2.3.4, 1.2.3, or 1.3. In contrast, antibody to serotype 2 fimbriae only agglutinated B. pertussis strains containing serotype 2 agglutinogen. Purified type 6 and 2 fimbriae were found to be weakly cross-reactive by enzyme-linked immunosorbent assay, using polyclonal antibody to each type of fimbria. In an immunoblot assay, polyclonal antibodies to a 22,000-dalton subunit of fimbriae from B. bronchiseptica reacted strongly with the type 2 fimbrial subunit of B. pertussis, but only weakly with the type 6 subunit. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein subunit of the type 6 fimbriae migrated with a molecular weight of 21,500, whereas the type 2 fimbrial subunit had a molecular weight of 22,000. The two types of subunits had similar amino acid compositions and showed amino-terminal sequence homology in 15 of 21 amino acids. The amino-terminal amino acid sequences of the B. pertussis fimbriae were distinct from those reported for fimbriae from other gram-negative bacteria. Neither the type 6 nor the type 2 fimbriae caused hemagglutination when assayed with several types of erythrocytes.
Immunoglobulin A1 protease types of Neisseria gonorrhoeae and their relationship to auxotype and serovar
Authors: Mulks M H , Knapp J S
Published: January 2020

Immunoglobulin A1 (IgA1) proteases are extracellular bacterial proteolytic enzymes which correlate with virulence in several species of human pathogens. We report that Neisseria gonorrhoeae produced two distinct types of IgA1 protease, each of which cleaved a different peptide bond in the hinge region of human IgA1. The type of IgA1 protease produced correlated with both nutritional auxotype and outer membrane protein I serovar in this organism. Gonococcal type 1 IgA1 protease was produced primarily by N. gonorrhoeae strains which require arginine, hypoxanthine, and uracil (AHU) and which belong to the protein IA-1 or IA-2 serovar. Most isolates of other auxotypes and serovars produced type 2 IgA1 protease. Although both the AHU auxotype and protein IA serogroup were found to be associated with disseminated gonococcal infection, there was no direct correlation of IgA1 protease type with disseminated or with uncomplicated gonorrhea.
Test of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis
Authors: Nnalue N A , Stocker B A
Published: January 2020

Aromatic compound-dependent (aro) derivatives of three mouse-virulent strains of Salmonella choleraesuis (Salmonella cholerae-suis) were constructed and shown to be nonvirulent for mice (intraperitoneal [i.p.] 50% lethal dose [LD50], greater than 5 X 10(6) CFU). A pur derivative, and a thy derivative, each of a different virulent parent, remained moderately virulent (i.p. LD50S for BALB/c mice, ca. 10(5) and 5 X 10(4) CFU, respectively). Tested as live vaccines i.p., the aro strains were ineffective in salmonella-susceptible BALB/c and C57BL/6 mice but were somewhat effective in salmonella-resistant CBA/J mice and in outbred CD-1 mice. The pur and thy strains were effective as live vaccines in BALB/c mice when given in sublethal doses. Two previously isolated nonvirulent galE derivatives of S. choleraesuis (i.p. LD50 in BALB/c mice, greater than 10(6) CFU) were also ineffective as live vaccines in BALB/c and C57BL/6 mice. The main antigenic difference between S. choleraesuis (O-6,7) and S. typhimurium (O-4,12) is in O-antigen character, thought to largely determine the specificity of protection in salmonellosis. Paired, nearly isogenic O-6,7 and O-4,12 derivatives were constructed from an aro S. typhimurium strain of proven efficacy as a live vaccine. Used as live vaccines, the O-4,12 member protected BALB/c mice against challenge with virulent S. typhimurium, whereas the O-6,7 member did not protect against virulent S. choleraesuis. However, BALB/c mice vaccinated with the O-6,7 member and mice vaccinated with an aro S. choleraesuis strain were protected against challenge with a moderately virulent (LD50, 5 X 10(4) CFU) O-6,7 derivative of an S. typhimurium strain.
A rabbit model of toxic shock syndrome that uses a constant, subcutaneous infusion of toxic shock syndrome toxin 1
Authors: Parsonnet J , Gillis Z A , Richter A G , Pier G B
Published: January 2020

We have developed a rabbit model of toxic shock syndrome that uses a subcutaneous infusion pump to administer toxic shock syndrome toxin 1 (TSST-1). A dose of 150 micrograms, infused at a constant rate over a period of 7 days, resulted in a characteristic illness highlighted by fever, conjunctival hyperemia, cachexia, and lethargy. The illness was uniformly fatal, with a mean interval until death of 3.2 +/- 0.4 days. Serial determinations of serum chemistries confirmed the multisystem nature of this illness. Rabbits developed profound hypocalcemia, with levels falling from 15.5 +/- 0.2 to 7.6 +/- 0.4 mg/dl under the influence of TSST-1. Blood urea nitrogen and creatinine rose dramatically, in the setting of oliguria or anuria. Serum glutamicpyruvic transaminase was the most reliable indicator of hepatic dysfunction, with the mean rising from 48 U/liter before administration of TSST-1 to 546 U/liter among rabbits surviving 2 days of the infusion. Creatine phosphokinase also rose dramatically in 10 of 16 rabbits. Rabbits demonstrated relative neutrophilia and lymphopenia as well as an increase in the partial thromboplastin time. Histopathologic examination demonstrated disease of multiple organs, particularly the liver, spleen, and lymph nodes, all of which demonstrated inflammation, thrombosis, hemorrhage, and erythrophagocytosis. The concurrent administration of prednisolone with TSST-1 prevented death in four of four rabbits and greatly lessened the morbidity. Rabbits were not protected from morbidity or mortality by the concurrent administration of polymyxin B. We believe that a constant, subcutaneous infusion of TSST-1 in rabbits provides a reproducible model for studying the pathogenesis of TSS.
Passive immunization with antibodies against iron-regulated outer membrane proteins protects turkeys from Escherichia coli septicemia
Authors: Bolin C A , Jensen A E
Published: January 2020

Escherichia coli septicemia is a common disease of young poultry and several species of mammals. Rabbit antiserum was prepared against iron-regulated outer membrane proteins of E. coli. Eighteen-day-old turkeys were passively immunized with antiserum and challenged by air sac inoculation of 1 X 10(6) to 2 X 10(6) CFU of E. coli O78:K80:H9. Turkeys injected with normal rabbit serum or saline solution before challenge served as controls. Fatalities (8 of 51 turkeys inoculated) occurred only in groups given saline solution or normal rabbit serum. The remaining turkeys were necropsied 96 h after challenge. Passive immunization with antiserum significantly (P less than 0.05) reduced the frequency of bacteremia at 96 h after challenge, the frequency of recovery of E. coli from air sacs, and the severity of gross lesions in inoculated birds as compared with birds given normal rabbit serum or saline solution.
Ultrastructural study of Chlamydia trachomatis surface antigens by immunogold staining with monoclonal antibodies
Authors: Kuo C C , Chi E Y
Published: January 2020

Surface antigens of Chlamydia trachomatis were studied by immunogold staining with monoclonal antibodies and by electron microscopy. The serovar- and subspecies-specific epitopes were the most surface accessible. The species- and genus-specific epitopes were the least surface exposed. Similar serological specificity as that in the microimmunofluorescence test was demonstrated by immunogold staining.
Differential growth of Legionella pneumophila in guinea pig versus mouse macrophage cultures
Authors: Yamamoto Y , Klein T W , Newton C A , Widen R , Friedman H
Published: January 2020

Legionella pneumophila is a facultative intracellular bacterium which replicated well in inbred guinea pig strain 2 peritoneal macrophages at a low infectivity ratio. In contrast, the growth of this organism was markedly restricted in mouse (BDF1) peritoneal macrophages, even at a relatively high infectivity ratio. The initial uptake of L. pneumophila organisms by macrophages was similar in both animal species, and both groups of macrophages supported the growth of Listeria monocytogenes. Treatment of L. pneumophila with immune guinea pig serum did not result in restriction of bacterial growth in macrophages, but guinea pig macrophages were readily induced to suppress the growth of L. pneumophila by preincubation with supernatants obtained from mitogen-activated normal guinea pig splenocyte cultures. Thus, lymphokines generated from mitogen-stimulated guinea pig lymphocytes induced a restriction of growth of these organisms similar to that observed naturally with macrophages from mice, which are considered highly resistant to these bacteria. Although guinea pigs are considered highly susceptible to L. pneumophila infections and mice are considered relatively resistant, the mechanism of this difference is not clear. The results of the present study suggest that the restriction of L. pneumophila growth by macrophages relates to host susceptibility to infection and that cell populations permissive for L. pneumophila can be transformed to nonpermissive by products from stimulated lymphocytes but not by opsonization with immune serum.
Congo red binding phenotype is associated with hemin binding and increased infectivity of Shigella flexneri in the HeLa cell model
Authors: Daskaleros P A , Payne S M
Published: January 2020

Wild-type isolates of Shigella flexneri bind the dye Congo red from solid media, thus producing red (Crb+) colonies. Mutants which fail to bind the dye produce white colonies (Crb-) and are avirulent in a variety of systems. In S. flexneri the ability to bind Congo red correlates with the ability to bind hemin and protoporphyrin IX. Binding of hemin by Crb+ S. flexneri was observed both in solid media and in liquid assays. Results of competition experiments suggest that Congo red and hemin bind to the same site on the bacterial cell and are retained on the cell surface. Binding of hemin by Crb+ S. flexneri is independent of hemin transport since both Crb+ and Crb- cells can utilize hemin as a sole source of iron. Both Crb- and Crb- organisms were able to grow in HeLa cell lysates, indicating that the gene(s) that is responsible for Congo red binding does not play a role in the acquisition of intracellular heme iron. By using the HeLa cell invasion system, the effect of hemin prebinding on the invasiveness of Crb+ S. flexneri was determined. Crb+ cells which had prebound hemin exhibited increased invasiveness, indicating a possible role for the crb gene product in the initial stages of invasion by S. flexneri.
Intestinal mucus gel and secretory antibody are barriers to Campylobacter jejuni adherence to INT 407 cells
Authors: McSweegan E , Burr D H , Walker R I
Published: January 2020

An in vitro mucus assay was developed to study the role of mucus gel and secretory immunoglobulin A (sIgA) in preventing attachment of Campylobacter jejuni to INT 407 cells. An overlay of rabbit small intestinal mucus was found to impede the attachment of C. jejuni to a monolayer of INT 407 cells. Mucus from rabbits previously colonized with C. jejuni was found to completely inhibit bacterial adherence to the underlying cells. Anti-Campylobacter sIgA was readily detected in mucus samples from previously exposed rabbits and was responsible for eliminating bacterial adherence to the INT 407 cells. This was shown by loss of inhibition after mucus absorption with Campylobacter cells. sIgA-containing mucus caused aggregation of the C. jejuni cells within the mucus layer of the assay system. Nonimmune mucus and sIgA alone were unable to cause bacterial aggregation, suggesting a cooperative role for mucus and sIgA. Antibodies responsible for adhesion inhibition were cross-reactive among several Campylobacter strains and were not directed solely against flagellar antigens.
Cell-to-cell interaction of Streptococcus sanguis and Propionibacterium acnes on saliva-coated hydroxyapatite
Authors: Ciardi J E , McCray G F , Kolenbrander P E , Lau A
Published: January 2020

Cell-to-cell interaction (coaggregation) between Propionibacterium acnes PK93 and Streptococcus sanguis DL1 was measured on saliva-coated hydroxyapatite beads (SHA) at bacterial concentrations between 1.3 X 10(6) and 6.7 X 10(8) cells per ml. Four hundredfold more DL1 than PK93 cells adhered to the saliva-coated beads, and the adherence of S. sanguis was proportional to cell input. SHA precoated with 3 X 10(8) DL1 cells bound 75 to 80% of available PK93 cells at all input amounts tested, up to an input of 8 X 10(7) cells. Adherence of PK93 to DL1-coated SHA approached saturation at an input of approximately 10(9) PK93 cells, when 1.5 X 10(8) bound. The coaggregation on SHA occurred either in buffer or saliva and was inhibited by N-acetylgalactosamine and by lactose; the attachment of DL1 to SHA was not inhibited by these sugars. S. sanguis 34 and heat-treated DL1 cells, neither of which form coaggregates with PK93, attached to SHA, but such cells did not bind PK93 cells. The findings of this study indicate that bacteria unable to attach to saliva-coated hydroxyapatite can indeed adhere to such a surface by strong lectin-mediated cell-to-cell interactions with bacteria already attached to the surface.
Growth inhibition of Trypanosoma cruzi in cultured murine myocardial cells mediated by a specifically induced lymphokine
Authors: Reyes L , Chinchilla M
Published: January 2020

An in vitro model for studying the protection of mouse myocardial cells against Trypanosoma cruzi was developed. It was demonstrated that immune mouse lymphocytes stimulated with a specific antigen elicited a mediator which protected myocardial cells from newborn mice against T. cruzi infection. The substance responsible for this activity was sensitive to acid (pH 2) treatment.
Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms
Authors: MacInnes J I , Rosendal S
Published: January 2020

Outer membrane protein (OMP)-enriched extracts and whole-cell protein preparations of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms were examined by polyacrylamide gel electrophoresis and immunoblotting. Both the OMP-enriched and whole-cell protein profiles of Actinobacillus suis, A. pleuropneumoniae (NAD-independent biovar), A. lignieresii, and Pasteurella haemolytica were very similar to those of H. pleuropneumoniae serotypes 1 to 8. Antisera prepared against H. pleuropneumoniae typically recognized three major OMP antigens with approximate molecular weights of 17,000 (17K), 32K, and 42K in immunoblots of H. pleuropneumoniae serotypes 1 to 8, Actinobacillus spp., and P. haemolytica. Antisera prepared against Actinobacillus spp. and Haemophilus sp. "minor group" also recognized these 17K, 32K, and 42K antigens. Using absorbed sera, we demonstrated that the 17K antigen had an epitope (or epitopes) common to all the gram-negative organisms examined, including Escherichia coli. The 32K and 42K antigens had epitopes common to members of the family Pasteurellaceae but, in the case of the 32K antigen, also contained unique epitopes. These results provide a basis for understanding the lack of specificity of serodiagnostic tests for H. pleuropneumoniae infection and provide another line of evidence for the association of H. pleuropneumoniae with the genus Actinobacillus.
Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli
Authors: Barbieri J T , Collier R J
Published: January 2020

A mutant, full-length form of diphtheria toxin was cloned into Escherichia coli K-12 and expressed under BL-1 + EK-1 containment. A gene fragment encoding the catalytic domain of the toxin was subjected to oligonucleotide-directed mutagenesis to produce a three-base mutation of an active site residue; Glu-148 was thereby replaced by Ser. Ser-148 fragment A had less than 1% of the ADP-ribosyltransferase activity of wild-type fragment A. Next, the complementary portion of the toxin structural gene was spliced with the mutated DNA fragment downstream of codon 148 to produce a construct that encoded mutant whole toxin with Ser at position 148. The mutant toxin was indistinguishable from authentic diphtheria toxin by Western blot analysis, but was about 800-fold less cytotoxic than wild-type toxin for BS-C-1 cells. Evidence from subunit exchange experiments indicated that a substantial fraction of the mutant toxin contained a fully functional B moiety, capable of mediating the entry of wild-type fragment A into sensitive mammalian cells. This combination of approaches provides a means of applying recombinant DNA methods in E. coli to study structure-function relationships in whole diphtheria toxin.
Antigenic mimicry of a human cellular polypeptide by Mycoplasma hyorhinis
Authors: Fernsten P D , Pekny K W , Harper J R , Walker L E
Published: January 2020

A 46-kilodalton (kDa) polypeptide was immunoprecipitated from radiolabeled extracts of human cell lines infected with Mycoplasma hyorhinis by murine monoclonal antibodies PF/2A and ML77. Both of these antibodies also reacted in an enzyme-linked immunosorbent assay (ELISA) with M. hyorhinis cells and with human and nonhuman cell lines infected with M. hyorhinis but failed to react with A7573 cells infected with any of 10 other species of the order Mycoplasmatales. PF/2A also reacted in the ELISA with certain human cell lines that were demonstrated to be free of mycoplasma infection. From extracts of these lines, a polypeptide antigen that appeared as a 24-kDa doublet on polyacrylamide gels was immunoprecipitated by PF/2A. When the PF/2A-reactive human cell lines were infected by M. hyorhinis, both the 46- and 24-kDa antigens were immunoprecipitated by PF/2A. ML77 did not react in the ELISA with any noninfected human cells tested and failed to immunoprecipitate a 24-kDa component from any human cells. In Western blotting analyses of extracts of M. hyorhinis cells, both PF/2A and ML77 stained a 46-kDa band. PF/2A also stained 24-kDa bands in Western blotting analyses of reactive human cells and M. hyorhinis cells, although a 24-kDa component was not precipitated from extracts of M. hyorhinis cells by PF/2A.
Role of volatile fatty acids in colonization resistance to Clostridium difficile in gnotobiotic mice
Authors: Su W J , Waechter M J , Bourlioux P , Dolegeal M , Fourniat J , Mahuzier G
Published: January 2020

Clostridium difficile is an agent involved in the development of antibiotic-associated pseudomembranous colitis. The purpose of this work was to investigate the role of volatile fatty acids (VFAs) in resistance to colonization by C. difficile by using a gnotobiotic animal model. Accordingly, germfree mice were associated with different hamster flora, and the VFAs in their cecal contents were measured by gas chromatography. The results showed that VFAs were produced mainly by the intestinal flora, especially by the strictly anaerobic bacteria. In these associated mice, the concentrations of acetic, propionic, and butyric acids were higher than those of other acids, but at pH 6.8 the MICs of these three acids in vitro for C. difficile were more than 200 mu eq/ml. In gnotobiotic mice monoassociated with C. difficile and in the isolated ceca of these mice, VFAs did not inhibit the growth of C. difficile. In gnotobiotic mice which were diassociated with C. difficile and C. butyricum and given drinking water with a lactose concentration of 20%, the cecal contents included about the same amount of butyric acid as did those of the monoassociated mice, although the population of C. difficile remained the same. Therefore, it is suggested that VFAs alone cannot inhibit intestinal colonization by C. difficile and that, consequently, other inhibitory mechanisms are also present.
Production, characterization, and protective effect of monoclonal antibodies to Clostridium chauvoei flagella
Authors: Tanaka M , Hirayama N , Tamura Y
Published: January 2020

Monoclonal antibodies to flagella of Clostridium chauvoei were obtained by the fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with partially purified flagella of strain Okinawa. Enzyme-linked immunosorbent assay and Western blot analysis with partially purified flagella, flagellated cells, and nonflagellated mutants were used to show that five monoclonal antibodies are specific for the flagella. In the Western blot analysis, all five antiflagellar antibodies reacted strongly with the 56,000-molecular-weight protein, which corresponds to the flagellin. By using the ELISA-derived reactivity of monoclonal antibodies to the various clostridia and the competitive binding assay, we showed that the flagella of C. chauvoei had at least three epitopes. The three antiflagellar monoclonal antibodies (one immunoglobulin G and two immunoglobulin M) demonstrated passive protective effects in mice. These results strongly suggest that the flagella of C. chauvoei are important for protective immunity in mice.