bluuebunny/crossref_metadata_2025 huggingface dataset:
Swine immunity to an attenuated Salmonella typhimurium mutant containing a recombinant plasmid which codes for production of a 31-kilodalton protein of Brucella abortus
Authors: Stabel T J , Mayfield J E , Tabatabai L B , Wannemuehler M J
Published: January 2020

Salmonella typhimurium chi 4064, an attenuated delta cya delta crp mutant of S. typhimurium SR-11, was shown to be avirulent in swine. S. typhimurium chi 4064 was used as a carrier for plasmid pBA31-R7, which codes for the expression of a 31-kDa protein from Brucella abortus (BCSP31). Given orally, S. typhimurium chi 4064(pBA31-R7) colonized the intestine and mesenteric lymph nodes of 5- to 6-week-old crossbred swine. Orally immunized animals developed serum and intestinal antibody responses to the B. abortus 31-kDa protein and to salmonella endotoxin as measured by enzyme-linked immunosorbent assay. Similarly immunized swine did not develop delayed-type hypersensitivity following a subcutaneous injection of recombinant BCSP31. However, swine parenterally immunized with recombinant BCSP31 incorporated in Freund incomplete adjuvant did develop a delayed-type hypersensitivity response to the homologous antigen. The data indicated that oral presentation of antigen to swine in the context of recombinant S. typhimurium effective stimulated mucosal and systemic antibody-mediated immunity but failed to sensitize swine for either an antigen-specific delayed-type hypersensitivity or a blastogenic response to the cloned BCSP31.
Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by use of monoclonal antibodies
Authors: Kamp E M , Popma J K , Anakotta J , Smits M A
Published: January 2020

Monoclonal antibodies (MAbs) were raised against extracellular hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae serotypes 2 and 9. MAbs were tested for inhibition of hemolytic and cytotoxic activities of the reference strains of A. pleuropneumoniae serotypes 1 to 12. Five MAbs inhibited hemolytic or cytotoxic activities of one or more serotypes and were used to probe Western blots (immunoblots) of cytotoxic and hemolytic culture filtrates of serotypes 1 to 12. The MAbs recognized three different proteins: (i) a protein of approximately 103 kDa that was associated with a weak hemolytic activity and a moderate cytotoxic activity, (ii) a protein of approximately 105 kDa that was associated with a strong hemolytic activity and a strong cytotoxic activity, and (iii) a protein of approximately 120 kDa that was associated with a strong cytotoxic activity but not with hemolytic activity. Serotypes 6, 7, and 12 produced only the 103-kDa protein, and serotype 10 produced only the 105-kDa protein. The other serotypes produced two proteins: serotypes 1, 5, 9, and 11 produced the 103- and 105-kDa proteins, and serotypes 2, 3, 4, and 8 produced the 103- and 120-kDa proteins.
Activation of bovine neutrophils by partially purified Pasteurella haemolytica leukotoxin
Authors: Czuprynski C J , Noel E J , Ortiz-Carranza O , Srikumaran S
Published: January 2020

In this study we developed a new method for the partial purification of Pasteurella haemolytica leukotoxin by size-exclusion high-performance liquid chromatography. The partially purified leukotoxin had a molecular weight of 104,000, as estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reacted on an immunoblot with an antileukotoxin monoclonal antibody. As expected, high concentrations of the leukotoxin were inhibitory or lethal to bovine neutrophils. Incubation of bovine neutrophils with diluted leukotoxin, however, resulted in significant neutrophil activation that was comparable in magnitude to that obtained with standard activating agents such as opsonized zymosan or zymosan-activated serum. Dilute leukotoxin (1:128 to 1:8,192 dilutions) stimulated an oxidative burst (luminol-dependent chemiluminescence) by bovine neutrophils that was comparable in magnitude to that obtained with opsonized zymosan. Preincubation with leukotoxin did not significantly prime the neutrophils for an enhanced oxidative burst when they were then exposed to opsonized zymosan as a second stimulus. Dilute leukotoxin (1:100 to 1:1,000 dilutions) also stimulated cytoskeletal alterations in bovine neutrophils, as measured by a significant shape change response. Preferential release of secondary granule constituents (lactoferrin) occurred when neutrophils were incubated with 1:100 to 1:500 dilutions of leukotoxin. Significant release of primary granules, as measured by beta-glucosaminidase activity, was not observed except at low dilutions (1:20) of leukotoxin that resulted in significant release of cytosolic constituents (i.e., lactate dehydrogenase activity). The neutrophil-activating activity of the leukotoxin was heat labile, unaffected by polymyxin B, and abrogated by a leukotoxin-neutralizing monoclonal antibody. These data indicate that P. haemolytica leukotoxin, like the closely related Escherichia coli hemolysin, is a potent neutrophil-activating agent. Leukotoxin-stimulated release of neutrophil oxygen intermediates and granule constituents may contribute to the intense inflammation that characterizes bovine pulmonary pasteurellosis.
Killing of Plasmodium falciparum in vitro by nitric oxide derivatives
Authors: Rockett K A , Awburn M M , Cowden W B , Clark I A
Published: January 2020

We have investigated the in vitro susceptibility of the human malaria parasite Plasmodium falciparum to killing by nitric oxide and related molecules. A saturated solution of nitric oxide did not inhibit parasite growth, but two oxidation products of nitric oxide (nitrite and nitrate ions) were toxic to the parasite in millimolar concentrations. Nitrosothiol derivatives of cysteine and glutathione were found to be about a thousand times more active (50% growth inhibitory concentration, approximately 40 microM) than nitrite.
Complete nucleotide sequence of the gene for a surface protein antigen of Streptococcus sobrinus
Authors: Tokuda M , Okahashi N , Takahashi I , Nakai M , Nagaoka S , Kawagoe M , Koga T
Published: January 2020

The complete nucleotide sequence of the gene for a cell surface protein antigen (SpaA) of Streptococcus sobrinus MT3791 (serotype g) was determined. The spaA gene consisted of 4,698 bp and coded for a protein of 170,202 Da. A putative signal peptide was found in the amino-terminal end of the protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in SpaA. One repeating region, located in the amino-terminal region, was rich in alanine, and the other, located in the central region, was rich in proline. The molecular structure of SpaA was very similar to that of the surface protein antigen of Streptococcus mutans.
Experimental
Shigella
Infections in Laboratory Animals I. Antagonism by Human Normal Flora Components in Gnotobiotic Mice
Authors: Maier Bruce R. , Hentges David J.
Published: January 2020

Germfree mice were associated with selected species of human intestinal bacteria and then challenged with a streptomycin-resistant
Shigella flexneri
strain. Antagonism against
Shigella
was most pronounced in mice associated with
Escherichia coli
and least pronounced in mice associated with
Bacteroides fragilis.
A moderate degree of antagonism could be demonstrated in mice associated with either
Streptococcus faecalis
or
Bifidobacterium adolescentis. Shigella
persisted in the cecal contents of
E. coli
-associated mice at very low, stable levels.
Shigella
populations were reduced to levels below detection in the ceca of mice diassociated with
E. coli
and
Bacteroides.
Upon subsequent administration of streptomycin,
Bacteroides
disappeared from the ceca. The
E. coli
population was greatly reduced, and
Shigella
reappeared at very high population levels as an apparent recombinant which resembled
E. coli
biochemically. A streptomycin-resistant
E. coli
population subsequently emerged and became dominant in the ceca.
Shigella
concomitantly declined to levels below detection.
Growth of Venezuelan Encephalitis Virus in Embryonic Cell Cultures of Wild Birds
Authors: Dickerman R. W. , Bonacorsa C. M.
Published: January 2020

Primary embryonic cell cultures made from nine species of wild and domestic birds were infected with Venezuelan encephalitis virus and reproduced each strain to moderately high levels.
Comparative Immunogenicity of Heat-Killed and Living Oral
Salmonella
Vaccines
Authors: Collins Frank M. , Carter Philip B.
Published: January 2020

CD-1 mice were vaccinated intragastrically or intramuscularly with one or two doses of 200 μg of heat-killed
Salmonella enteritidis
5694. Control mice were vaccinated with sublethal doses of living
S. enteritidis
Se795. The mice were challenged intragastrically with approximately 10
6
S. enteritidis
5694 SM
R
7 to 14 days later, and the growth of the challenge population in the liver, spleen, mesenteric lymph nodes, lungs, and intestine was measured quantitatively. Mice receiving two doses of heat-killed vaccine by mouth were able to delay the systemic emergence of a gastrically introduced salmonella infection by 1 to 2 days. The corresponding liver and spleen populations were slightly lower than those seen in the normal controls. On the other hand, mice receiving the living, attenuated vaccine (either intravenously or intragastrically) developed an effective anti-salmonella immunity against subsequent reinfection.
Oxygen-Stable Hemolysins of Group A Streptococci VIII. Leukotoxic and Antiphagocytic Effects of Streptolysins S and O
Authors: Ofek I. , Bergner-Rabinowitz S. , Ginsburg I.
Published: January 2020

Streptolysin S exists in a cell-bound form and as an extracellular complex between a nonspecific carrier (serum, serum albumin, ribonucleic acid [RNA], Triton, Tween) and a hemolytic moiety (probably a peptide) synthesized by streptococci. Although all the forms of streptolysin S, at 100 hemolytic units, killed mouse leukocyte monolayers, the time needed to kill 100% of the cells varied with the different streptolysin S preparations. Whereas 30 min was sufficient for the cell-bound hemolysin to kill all of the cells, 60 and 180 min were required when RNA streptolysin S and serum streptolysin S, respectively, were employed. Addition of 10% mouse serum to RNA streptolysin S or to cell-bound hemolysin delayed the killing of the leukocytes. The delayed killing observed with serum and albumin hemolysins is probably due to competition for the hemolytic moiety between the carrier molecules and target sites (phospholipids) upon the leukocyte membrane. Serum streptolysin S must be constantly incubated with the cells for 90 min for 100% of the cells to undergo cytopathic changes upon subsequent incubation for an additional 90 min. Streptolysin S inhibitor (trypan blue) added to the system after 30 or 60 min of incubation resulted in the killing of 50 and 100% of the leukocytes, respectively, when the cells were further incubated for 120 min. It is suggested that 30 min of incubation was not sufficient for the transfer of enough streptolysin S molecules upon the cell surface to allow killing of all of the cells. Sublethal amounts of streptolysin S, streptolysin O, and saponin suppressed phagocytosis of streptococci by mouse peritoneal macrophages. This effect was abolished by inhibitors of streptolysin S (trypan blue) and of streptolysin O and saponin (cholesterol). With sublethal amounts of streptolysin S, no inhibition of the reduction of nitro blue tetrazolium by nonphagocytosing cells was observed, but these amounts of streptolysin S caused a 50% inhibition of the reduction of nitro blue tetrazolium by phagocytosing leukocytes. It is suggested that some metabolic systems, which are normally enhanced during phagocytosis, have been affected by sublethal doses of streptolysin S. The results indicate that the in vivo production of small amounts of streptolysins S and O by group A streptococci may inhibit phagocytosis and may thus contribute to the invasiveness and pathogenicity of this microorganism.
Immunization of Mice Against Encephalomyocarditis Virus II. Intraperitoneal and Respiratory Immunization with Ultraviolet-Inactivated Vaccine: Effect of
Bordetella pertussis
Extract on the Immune Response
Authors: Bogaerts W. J. C. , Durville-van der Oord B. J.
Published: January 2020

Mice were immunized by intraperitoneal (ip) or respiratory administration of ultraviolet-inactivated virus alone or with
Bordetella pertussis
extract (BPE) as an adjuvant. The effect of immunization was tested by determination of antibody titers and by survival of a lethal challenge with 200 LD
50
of a virulent (large-plaque variant) strain of EMC virus. For plain vaccine the ip 50% effective dose (ED
50
) was 37 hemagglutination units (HAU; ca. 4 × 10
6
plaque-forming unit equivalents); with adjuvant the ip ED
50
was reduced to 20 HAU. After respiratory immunization by intratracheal injection, an ED
50
value of 100 HAU was found, which was not affected by BPE. After ip vaccination the primary immune response was enhanced by BPE, but the challenge response, measured 3 weeks after challenge, was unaffected. Respiratory immunization induced a primary response which was not influenced by BPE, but here the challenge response was enhanced by the adjuvant. After secondary treatment (challenge or booster vaccination) serum antibodies and protection against challenge persisted for at least 1 year.
Effect of specific antibody on adherence of Staphylococcus aureus to bovine mammary epithelial cells
Authors: Olmsted S B , Norcross N L
Published: January 2020

Staphylococcus aureus is a major pathogen in the bovine mammary gland. The ability of S. aureus to adhere to epithelial cells in the ductules and alveoli of the bovine mammary gland is believed to add greatly to its virulence and may be necessary for colonization. Two in vitro methods were developed for the purposes of quantifying adherence and of determining the effect which specific antibody may have in inhibiting the adherence of this organism. Both methods utilize bovine mammary epithelial primary cells as targets for labeled bacteria. In one assay, the bacteria are labeled with [methyl-3H]thymidine and incubated on the primary epithelial monolayers. The second assay involves labeling the bacteria with biotin. An enzyme-linked immunosorbent assay is then performed with streptavidin conjugated to horseradish peroxidase. Both methods have proven to be reliable and allow for the testing of many criteria in one assay. Cows were immunized with a whole-cell vaccine, and immune serum and milk were collected. The bacteria were then incubated in the presence of serum or milk as a test for antiadherent capability. By using the methods described, distinct antiadherent activity in both serum and milk was demonstrated.
Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria
Authors: Corfield A P , Wagner S A , Clamp J R , Kriaris M S , Hoskins L C
Published: January 2020

Oligosaccharide side chains of human colonic mucins contain O-acetylated sialic acids and glycosulfate esters. Although these substituents are considered to protect the chains against degradation by bacterial glycosidases, sialate O-acetylesterase, N-acetylneuraminate lyase, and glycosulfatase activities have been found in fecal extracts. To better define the source of these activities, we measured extracellular and cell-bound sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities produced by 23 isolates of human fecal bacteria grown anaerobically in a hog gastric mucin culture medium; these represented dominant populations of fecal anaerobes, facultative anaerobes, and the subset of mucin oligosaccharide-degrading bacteria. Every strain produced sialidase and high levels of arylesterase, and all but five facultative anaerobes produced sialate O-acetylesterase. Sialic acids containing 2 mol or more of O-acetyl ester per mol of sialic acid were cleaved from mucin glycoproteins more slowly by sialidases of mucin oligosaccharide-degrading stains than were sialic acids containing 1 or 0 mol, and only N-acetyl- and mono-O-acetylated sialic acids were recovered from enzyme digests of a mucin containing di-O-acetylated sialic acids. No detectable N-acetylneuraminate lyase activity was produced by any strain, but low activity was induced by increasing the glycoprotein-bound sialic acid concentration in the culture medium of six Escherichia coli strains. Using lactitol-6-sulfate as a substrate, we found weak glycosulfatase activity in the partially purified, concentrated enzyme mixture in the culture supernatants of four mucin oligosaccharide-degrading strains but in none of the unconcentrated culture fractions. We conclude that the presence of two or more O-acetyl groups on sialic acids inhibits enteric bacterial sialidases but that production of sialate O-acetylesterases by several populations of enteric bacteria lessens the likelihood that mucin oligosaccharide chains terminating in O-acetylated sialic acids are protected from degradation. Sialate O-acetylesterases have a role in bacterial degradation of mucin glycoproteins in the human colon.
Experimental verocytotoxemia in rabbits
Authors: Richardson S E , Rotman T A , Jay V , Smith C R , Becker L E , Petric M , Olivieri N F , Karmali M A
Published: January 2020

The clinicopathologic effects of intravenously administered purified verocytotoxin 1 (VT1; Shiga-like toxin 1) in 2-kg male rabbits was studied. The 50% lethal dose was 0.2 micrograms of protein per kg of body weight (2 x 10(4) 50% cytotoxic doses per kg). The clinical features included nonbloody diarrhea and a progressive flaccid paresis, usually culminating in death. The histopathology was characterized by edema and hemorrhage in the mucosa and submucosa of the cecum and edema, hemorrhage, and neuronal necrosis in the brain and gray matter of the spinal cord. Thrombotic microangiopathy, the characteristic histopathologic renal lesion in the hemolytic-uremic syndrome, was also found to be the underlying lesion in verocytotoxemic rabbits. To determine the specific distribution of VT1 in rabbit tissues, purified 125I-labelled VT1 was administered intravenously to 20 rabbits (both immunologically naive and VT1-immune rabbits). The highest specific uptake of 125I-VT1 was in the spinal cord, brain, cecum, colon, and small bowel in unimmunized animals but in the liver, spleen, and lungs in immune animals. Immunofluorescent staining of cecal and spinal cord tissues after intravenous administration of VT1 showed evidence of specific vascular endothelial cell binding of the toxin. The striking correlation of the central nervous system and gastrointestinal localization of 125I-VT1 with the sites of known histopathology is consistent with direct toxin-mediated injury to these tissues, initiated by the specific binding of VT1 to the vascular endothelium. We conclude that the vascular damage induced by VT1 in affected rabbit tissues is similar to that seen in the kidneys and other tissues in patients with verocytotoxin-producing Escherichia coli-associated hemolytic-uremic syndrome. This suggests that although the rabbit model fails to replicate human hemolytic-uremic syndrome, it is useful for studying the pathogenesis of the vascular lesions in verocytotoxin-producing E. coli-associated diseases.
Interleukin-6 antagonizes tumor necrosis factor-mediated mycobacteriostatic and mycobactericidal activities in macrophages
Authors: Bermudez L E , Wu M , Petrofsky M , Young L S
Published: January 2020

Interleukin-6 (IL-6) is a cytokine produced by a number of cells, including macrophages, and is directly involved in the inflammatory response. The production of IL-6 can be stimulated by monokines such as IL-1 and tumor necrosis factor (TNF). Mycobacterium avium complex organisms frequently cause disseminated disease in patients with AIDS. M. avium is an intracellular bacterium that that mainly infects macrophages. Treatment of M. avium-infected macrophage monolayers with recombinant IL-6 decreased the ability of TNF to activate cultured macrophages to inhibit growth of or kill intracellular M. avium (68% +/- 14% decrease in intracellular killing compared with that in monolayers not treated with IL-6). To further evaluate whether this effect was dependent on the down regulation of membrane receptors to TNF, we examined 125I-TNF binding to macrophages previously exposed to IL-6: the expression of TNF receptors was decreased by 78% +/- 9%. The effect of IL-6 on TNF receptors was observed after 4 h and was reversible. Infection of macrophages with different M. avium serovars was associated with release of IL-6, and IL-6 production peaked at 48 h after infection in concentrations ranging from 328 +/- 87 ng/10(5) cells to 907 +/- 224 ng/10(5) cells. IL-6 did not have any influence on the rate of growth of the tested strains of M. avium within or outside macrophages. These results suggest that release of IL-6 by M. avium-infected macrophages may influence the host's immune response and the outcome of the disease.
Role of Yersinia enterocolitica Yst toxin in experimental infection of young rabbits
Authors: Delor I , Cornelis G R
Published: January 2020

We constructed a Yst-negative mutant of Yersinia enterocolitica W1024 by reverse genetics, and we compared the virulence of the yst+ and yst isogenic strains in an experimental oral infection of the young rabbit. The rabbits infected with the yst+ strain suffered from the diarrhea and lost weight, and most of them died. By contrast, the occurrence of diarrhea, weight loss, and death in the group of rabbits infected with the yst mutant was as low as that in the group of uninfected rabbits. Bacteria from both strains were excreted in the feces and induced a serum antibody response against Yop proteins. The yst mutant disappeared more rapidly from the feces. We conclude that the enterotoxin Yst is a major factor involved in the Y. enterocolitica-associated diarrhea in the young rabbit. Given the similarity with the symptoms observed for children, this result suggests that Yst could also be an important factor in diarrhea in young children infected with Y. enterocolitica.
The 93-kilodalton protein of Borrelia burgdorferi: an immunodominant protoplasmic cylinder antigen
Authors: Luft B J , Mudri S , Jiang W , Dattwyler R J , Gorevic P D , Fischer T , Munoz P , Dunn J J , Schubach W H
Published: January 2020

Using immunoblots, we identified proteins of Borrelia burgdorferi recognized by sera from 62 patients with either acute or chronic Lyme disease. In all groups studied, the 41-kDa flagellar protein and a relatively minor 93-kDa protein (p93) were the most commonly recognized antigens in patients with acute and chronic disease due to B. burgdorferi. A murine monoclonal antibody (MAb 181.1) was developed against p93, and the antigen was detected by immunoblot analysis in four European and American strains of B. burgdorferi. On two-dimensional gel electrophoresis, p93 had an apparent pI of 6.8. Immunoelectronmicroscopy with MAb 181.1 demonstrated that p93 is located within the protoplasmic cylinder compartment of the organism. The gene encoding p93 was retrieved from a phage expression library. The derived amino acid sequence of p93 confirmed chemical characterization of the antigen, including its amino-terminal peptide sequence. The derived amino acid sequence predicted it to be predominantly alpha helical. A prominent antigenic domain located at the carboxy portion of the protein was recognized by human and rabbit polyclonal antisera and human (MAb D4) and mouse (MAb 181.1) MAbs.
Recombinant interleukin-6 protects mice against experimental bacterial infection
Authors: Liu Z , Simpson R J , Cheers C
Published: January 2020

Because of reports of high levels of interleukin-6 (IL-6) in patients during infection, we studied the role of IL-6 in experimental infection. Mice infected with the facultative intracellular pathogen Listeria monocytogenes displayed high levels of IL-6 in their sera and tissues, particularly the spleen, 1 to 3 days after infection. At this time, the IL-6 titers correlated with bacterial numbers in individual mice and in groups of mice given graded doses of Listeria organisms. However, the presence of IL-6 in serum declined after 4 days, even when a large initial dose of bacteria meant that bacterial numbers were still increasing at this time. Recombinant mouse IL-6 injected intraperitoneally before infection protected mice in a dose-dependent manner. It was effective when given 4 h before infection but not when administration was delayed for 24 h postinfection. It is therefore believed that IL-6 plays a role in early priming of the immune response to infection. Its exact function in this model is being investigated.
Role of crl in avian pathogenic Escherichia coli: a knockout mutation of crl does not affect hemagglutination activity, fibronectin binding, or Curli production
Authors: Provence D L , Curtiss R
Published: January 2020

This study determined the role of crl in the production of curli by, the hemagglutination activity of, and fibronectin binding by avian pathogenic Escherichia coli chi 7122. Curli, an extracellular structure that binds fibronectin, was recently described (A. Olsén, A. Jonsson, and S. Normark, Nature [London] 338:652-655, 1989). The crl gene product was hypothesized to be the subunit monomer of curli and to bind fibronectin. E. coli HB101, which does not contain crl, binds fibronectin and produces curli when harboring a plasmid containing the crl gene. We show that HB101 hemagglutinates chicken erythrocytes when harboring the crl gene and that chi 7122 hemagglutinates chicken erythrocytes, binds fibronectin, and produces curli. Hemagglutination activity, fibronectin binding, and curli production are best expressed by chi 7122 and HB101 harboring the crl gene when the bacteria are grown on colonization factor antigen agar at 26 degrees C. The expression of hemagglutination activity, fibronectin binding, and curli production by both strains is decreased by growth on this agar at an increased temperature, of an increased osmolarity, or in an anaerobic atmosphere. This results indicates that the crl gene plays a role in the expression of the three phenotypes in HB101 and possibly in chi 7122 as well. We inactivated crl in chi 7122 by allele replacement in the expectation of abolishing hemagglutination activity, fibronectin binding, and curli production. The mutation was verified by Southern blot analysis and by a polymerase chain reaction, and there was no evidence of a second crl gene in chi 7122. However, the mutant of chi 7122 lacking crl hemagglutinated chicken erythrocytes, bound fibronectin, and produced curli at wild-type levels. This result indicates that crl plays a nonessential role in the expression of these three phenotypes in chi 7122.
Cytoadherence characteristics of rosette-forming Plasmodium falciparum
Authors: Udomsangpetch R , Webster H K , Pattanapanyasat K , Pitchayangkul S , Thaithong S
Published: January 2020

Sequestration of Plasmodium falciparum-infected erythrocytes to the capillary endothelium can cause obstruction and localized tissue damage. Occlusion of vessels in falciparum malaria infection has been related to two properties of the parasite: adhesion to endothelial cells and rosette formation. Our study on P. falciparum isolates from Thailand producing variable numbers of rosettes suggests the involvement of rosettes in capillary blockage caused by direct adhesion of the rosette-forming infected erythrocytes to various target cells, e.g., live human umbilical vein endothelial cells, monocytes, and platelets. These rosettes did not bind Formalin-fixed target cells, nor did they bind to live or fixed C32 or G361 melanoma cells. Classification of the receptors involved in cytoadherence of endothelial cells and monocytes by specific antibody blocking and flow cytometry indicated that CD36 was involved in the adherence of monocytes but that other receptors besides CD36 may be involved in parasite adherence to endothelial cells. The cytoadherence of infected erythrocytes to monocytes was also associated with CD54 (ICAM-1). Further, differentiation of adherent monocytes resulted in an inversion of CD36 and CD54 levels on the cell surface which correlated with a decrease in surface binding of infected erythrocytes. This observation suggests that the state of cell activation and differentiation may also contribute to sequestration of parasites and to the pathogenesis of malaria.
Uptake and intracellular survival of Bordetella pertussis in human macrophages
Authors: Friedman R L , Nordensson K , Wilson L , Akporiaye E T , Yocum D E
Published: January 2020

Recent reports have demonstrated that Bordetella pertussis has invasive behavior in vivo and in vitro. In this study, we investigated the ability of a virulent strain, avirulent mutants, and mutants deficient in specific virulence factors to enter and survive intracellularly in human macrophages in vitro. Uptake of virulent B. pertussis was dose dependent and occurred in the absence of serum or specific antibody, with entry occurring via a microfilament-dependent phagocytic process. The virulent wild-type parental strain was internalized and persisted intracellularly over the 3 days of experiments, as determined by transmission electron microscopy and by recovery of viable plate counts. This is the first report of long-term survival of B. pertussis in human macrophages. Avirulent mutants entered macrophages, but at only an average of 1.5% of virulent parental levels, and did not survive intracellularly. Mutants which did not express adenylate cyclase toxin, filamentous hemagglutinin, or pertussis toxin had decreased abilities to enter and to survive inside macrophages. The results suggest that the internalization process, as well as intracellular survival, is virulence dependent and that mutations which inactivate expression of virulence factors may affect both. The ability of B. pertussis to enter and persist inside macrophages may be important not only for survival of the bacteria but also in the pathogenesis of whooping cough.
Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis
Authors: Pal P G , Horwitz M A
Published: January 2020

We have studied the capacity of a selected fraction of Mycobacterium tuberculosis extracellular proteins (EP) released into broth culture by mid-logarithmic-growth-phase organisms to induce cell-mediated immune responses and protective immunity in a guinea pig model of pulmonary tuberculosis. Guinea pigs infected with M. tuberculosis by aerosol but not uninfected control guinea pigs exhibit strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. Guinea pigs immunized subcutaneously with EP but not sham-immunized control guinea pigs also develop strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. EP is nonlethal and nontoxic to guinea pigs upon subcutaneous immunization. Guinea pigs immunized with EP and then challenged with aerosolized M. tuberculosis exhibit protective immunity. In five independent experiments, EP-immunized guinea pigs were consistently protected against clinical illness, including weight loss. Compared with EP-immunized guinea pigs, sham-immunized control guinea pigs lost 12.9 +/- 2.0% (mean +/- SE) of their total weight. EP-immunized guinea pigs also had a 10-fold reduction in viable M. tuberculosis bacilli in their lungs and spleens (P = 0.004 and 0.001, respectively) compared with sham-immunized control animals. In the two experiments in which some guinea pigs died after aerosol challenge, EP-immunized animals were protected from death. Whereas all 12 (100%) EP-immunized guinea pigs survived challenge with aerosolized M. tuberculosis, only 6 of 12 (50%) sham-immunized control guinea pigs survived challenge (P = 0.007, Fisher exact test). This study demonstrates that actively growing M. tuberculosis cells release immunoprotective molecules extracellularly, that a subunit vaccine against tuberculosis is feasible, and that extracellular molecules of M. tuberculosis are potential candidates for a subunit vaccine.
Roles of peripheral leukocytes and tissue macrophages in antibacterial resistance induced by free or liposome-encapsulated muramyl tripeptide phosphatidylethanolamide
Authors: Melissen P M , van Vianen W , Bakker-Woudenberg I A
Published: January 2020

Administration of free muramyl tripeptide phosphatidylethanolamide (MTPPE) or liposome-encapsulated MTPPE (LE-MTPPE) in a twofold-lower dose at 24 h before bacterial inoculation resulted in clearance of intravenously inoculated Klebsiella pneumoniae by tissue macrophages, whereas in control mice, bacteria were not effectively cleared from the blood. In addition, MTPPE and LE-MTPPE led to increased numbers of leukocytes in the blood, which could compensate for the leukopenia in mice resulting from infection with K. pneumoniae. In an attempt to elucidate the relative contributions of the activation of tissue macrophages and the recruitment of leukocytes to the antibacterial resistance induced by MTPPE and LE-MTPPE, mice were infected intraperitoneally with K. pneumoniae. In these MTPPE- and LE-MTPPE treated mice, intraperitoneal influx of leukocytes and the phagocytic capacity of leukocytes were not higher than in untreated control mice. However, MTPPE- and LE-MTPPE-treated mice survived much longer; eventually 33% of the LE-MTPPE-treated mice survived, whereas all untreated control mice died as a result of bacterial septicemia. This prevention of early death appeared to be the result of an increased clearance of bacteria from the blood by activated tissue macrophages. It was observed that depletion of these tissue macrophages in liver and spleen abrogates the effect of LE-MTPPE treatment, indicating that tissue macrophages are of major importance in the LE-MTPPE-induced resistance against K. pneumoniae infection.
Gastrointestinal colonization and systemic dissemination by Candida albicans and Candida tropicalis in intact and immunocompromised mice
Authors: de Repentigny L , Phaneuf M , Mathieu L G
Published: January 2020

Gastrointestinal colonization and systemic dissemination by Candida albicans and Candida tropicalis were compared in intact and immunocompromised mice. Five-day-old CFW mice were inoculated by the oral-intragastric route with 1.0 x 10(7) CFU of two C. albicans and two C. tropicalis strains isolated from the blood of patients with acute leukemia and with C. albicans 4918 and its cerulenin-resistant mutant 4918-10. C. albicans and C. tropicalis spread to the lungs, liver, and kidneys within 30 min postinoculation, and organ CFU of the two species were comparable over the following 10 days. Close association of blastoconidia with the villous surface of the small intestine resulted in lysis of microvilli and then progressive invasion of villi. Blastoconidia within villi were surrounded by a conspicuous zone of clearing. Persistent colonization of the small and large intestines by C. albicans blood isolates and strains 4918 and 4918-10 was similar for 31 days after inoculation, but consistently exceeded that of C. tropicalis. In mice colonized with C. albicans, immunosuppression with cortisone acetate and cyclophosphamide on days 30 and 33 after inoculation increased stomach CFU 40- to 370-fold and intestinal CFU 30- to 80-fold. In contrast, persistent colonization by C. tropicalis was undetectable before immunosuppression and only became apparent after treatment. C. albicans disseminated more frequently and with higher organ CFU than C. tropicalis. Despite this fact, 20% of mice infected with C. tropicalis died, compared with 4% infected with C. albicans blood isolates. Indirect immunofluorescence revealed penetrative growth by Candida hyphae exclusively in the mucosa and submucosa of the stomach from immunosuppressed, persistently colonized mice. Taken together, the data indicate that C. tropicalis appears to be more virulent than C. albicans and that factors responsible for gastrointestinal colonization, systemic dissemination, and mortality in immunocompromised mice may not be identical.
eNAP-2, a novel cysteine-rich bactericidal peptide from equine leukocytes
Authors: Couto M A , Harwig S S , Cullor J S , Hughes J P , Lehrer R I
Published: January 2020

We purified a novel cysteine-rich antibiotic peptide, eNAP-2 (M(r), approximately 6,500), from acid extracts of equine neutrophils by sequential gel filtration and reversed-phase high-performance liquid chromatography and determined its partial N-terminal amino acid sequence. Although its cysteine motif distinguished eNAP-2 from all other currently known endogenous antibiotic peptides, including defensins and granulins, it showed substantial sequence similarity to WDNM1, a putative member of the four-disulfide-core protein family that also includes animal and human antiproteases, snake venom neurotoxins, and rat and mouse whey proteins. The antibacterial properties of eNAP-2 were tested against several equine uterine pathogens, namely, Streptococcus zooepidemicus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Killing of S. zooepidemicus was very efficient, as evidenced by a 94% decrease in numbers of CFU per milliliter after exposure to 100 micrograms of eNAP-2 per ml (approximately 15 microM) for 2 h. Exposure of E. coli and P. aeruginosa to 200 micrograms of eNAP-2 per ml for 2 h resulted in 90.2 and 77.6% reduction, respectively, in the numbers of CFU per milliliter. Bacteriostasis, without bactericidal activity, occurred after K. pneumoniae was incubated with 200 micrograms of eNAP-2 per ml. Additional studies will be required in other species and cell types to determine whether eNAP-2 is restricted to equine neutrophils or is the index member of a larger family of endogenous antibiotics.
Inhibition of Streptococcus mutans adherence to saliva-coated hydroxyapatite by human secretory immunoglobulin A (S-IgA) antibodies to cell surface protein antigen I/II: reversal by IgA1 protease cleavage
Authors: Hajishengallis G , Nikolova E , Russell M W
Published: January 2020

The effect of human secretory immunoglobulin A (S-IgA) and serum antibodies to surface protein antigen (Ag) I/II on the adherence of Ag I/II-bearing Streptococcus mutans and of free Ag I/II to saliva-coated hydroxyapatite (SHA) was investigated. The inhibition by S-IgA of binding of both S. mutans and free Ag I/II to SHA was dependent on antibody to Ag I/II. Essentially no difference was found between S-IgA1 and S-IgA2 with respect to antibody-dependent inhibition of Ag I/II binding to SHA, but S-IgA1 inhibited S. mutans adherence more effectively than did either serum immunoglobulin A1 (IgA1) or IgG antibodies. The antiadherence effect of S-IgA was abrogated after cleavage by IgA1 protease. Purified Fab alpha fragments containing Ag I/II-binding activity enhanced the binding of free Ag I/II to SHA and showed greater binding to SHA than did intact S-IgA1. Despite its relative inability to interact with precoated SHA, S-IgA1 containing antibody to Ag I/II was readily incorporated into the salivary pellicle during coating, but this did not promote Ag I/II binding. These data suggest that S-IgA antibodies can inhibit the initial adherence of S. mutans to salivary pellicle-coated tooth surfaces in an adhesin-specific fashion, but the presence in the oral cavity of bacterial IgA1 proteases would potentially interfere with this antiadherence mechanism.
Purification and characterization of the urease enzymes of Helicobacter species from humans and animals
Authors: Turbett G R , Høj P B , Horne R , Mee B J
Published: January 2020

The urease enzymes of Helicobacter pylori, H. mustelae, H. felis, and H. nemestrinae have been purified to homogeneity by affinity chromatography and characterized. The native urease enzymes of the four organisms were found to be almost identical, with a pI of 6.1 and molecular masses of 480 to 500 kDa, as determined by electrophoretic mobility in nondenaturing polyacrylamide gels. Transmission electron microscopy of the native urease showed it to be a molecule approximately 13 nm in diameter, with hexagonal symmetry. Denaturation studies indicated that each urease enzyme molecule was composed of two nonidentical subunits with molecular masses of approximately 64 and 30 kDa. The subunits were present in a 1:1 ratio, suggesting a hexameric stoichiometry for the native molecule. The predicted molecular mass of H. pylori urease, based on subunit molecular weight and stoichiometry, is 568 kDa. N-terminal amino acid sequencing of the enzyme subunits from the four species revealed high levels of homology. The large subunits (UreB) were found to be 92 to 100% homologous, and the small subunits (UreA) were 75 to 95% homologous over the first 12 to 20 residues. The high degree of homology suggests a common ancestral origin and an important role for the urease enzymes of these organisms.
Role of Vibrio cholerae neuraminidase in the function of cholera toxin
Authors: Galen J E , Ketley J M , Fasano A , Richardson S H , Wasserman S S , Kaper J B
Published: January 2020

Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase- V. cholerae 395 strains were then constructed by using suicide vector-mediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture filtrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ filtrates increased five- and eightfold, respectively, relative to that with NANase- filtrates. In addition, NANase+ filtrates increased the short-circuit current measured in Ussing chambers 65% relative to that with NANase- filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation ratios was seen at doses of 10(4) to 10(8) CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 10(9) CFU than NANase- strains when inoculated into nonfasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions.
Protective local and systemic antibody responses of swine exposed to an aerosol of Actinobacillus pleuropneumoniae serotype 1
Authors: Bossé J T , Johnson R P , Nemec M , Rosendal S
Published: January 2020

The isotype-specific antibody responses in serum and in nasal and pulmonary lavage fluids of swine following aerosol immunization with an attenuated strain of Actinobacillus pleuropneumoniae serotype 1, strain CM5A, was investigated. The presence of immunoglobulin G (IgG), IgA, and IgM with specificities for capsular polysaccharide, lipopolysaccharide, and hemolysin was determined by enzyme-linked immunosorbent assay by using purified antigens. Strain CM5A induced serum antibodies of each isotype to the three antigens. The serum antibody response was sustained and typical of persistent antigenic stimulation. The specific IgM response decreased and the specific IgG response increased after challenge with strain CM5. IgA specific for the three antigens was detected in nasal secretions from all immune pigs, whereas specific IgG could only be detected in samples contaminated with blood. Both IgA and IgG specific for each of the antigens were detected in pulmonary lavage samples. There was no significant increase in specific IgA in nasal secretions; however, levels of lipopolysaccharide-specific and hemolysin-specific IgG and IgA in pulmonary secretions rose after aerosol challenge with strain CM5. Passive transfer of immune swine serum resulted in protection against pleuropneumonia and in levels of specific serum IgG which were similar to those in actively immunized pigs. It is concluded that specific serum IgG antibodies are important in protection from porcine pleuropneumonia.
An enzymatic mutant of Shiga-like toxin II variant is a vaccine candidate for edema disease of swine
Authors: Gordon V M , Whipp S C , Moon H W , O'Brien A D , Samuel J E
Published: January 2020

Edema disease (ED) of weanling pigs is caused by an infection with Escherichia coli that produces Shiga-like toxin II variant (SLT-IIv). Pathology identical to that caused by ED can be duplicated in pigs that are injected with less than 10 ng of purified SLT-IIv per kg of body weight. Therefore, SLT-IIv was mutated to create an immunoreactive form of the toxin that was significantly reduced in enzymatic activity. Initially, purified SLT-IIv was treated with formaldehyde which abrogated cytotoxic activity. Pigs were vaccinated with the toxoid (100 micrograms) to determine whether a toxoid was a viable vaccine candidate and whether young pigs were capable of mounting an immune response. Although the pigs developed a neutralizing antibody titer (1:128 to 1:512) 28 days postinjection, they also lost weight and developed ED lesions. The deleterious effect of the toxoid appeared to result from residual enzymatic activity or a reversion to a toxic form. An alternative method, site-directed mutagenesis, was employed to consistently reduce the enzymatic activity of SLT-IIv. Glutamate at position 167 of the mature A subunit was replaced by aspartate (E167D), and arginine at position 170 was replaced by lysine (R170K). These mutations reduced cytotoxic activity 10(4)-fold and 10-fold, respectively, while the enzymatic activities were decreased 400-fold and 5-fold, respectively. The activity of a toxin that contained both mutations (SLT-IIvE167D/R170K) closely resembled that of SLT-IIvE167D. When position 167 was replaced by glutamine (E167Q), the cytotoxic activity decreased 10(6)-fold and the enzymatic activity decreased approximately 1,500-fold. Pigs that were vaccinated with purified, mutant toxin designated SLT-IIvE167Q developed a neutralizing antibody titer of 1:512 21 days postinjection, and their tissues were free of ED lesions. These data suggest that SLT-IIvE167Q may represent an effective vaccine against ED.
Topographic mapping of Helicobacter pylori colonization in long-term-infected pigs
Authors: Engstrand L , Rosberg K , Hübinette R , Berglindh T , Rolfsen W , Gustavsson S
Published: January 2020

Four barrier-born pigs were inoculated with Helicobacter pylori during gastroscopy. Infection in all pigs was established after 3 weeks, and the animals were kept isolated from other pigs in ordinary experimental sites. The pigs were sacrificed and examined 3, 5, 6, and 6.5 months postinoculation. A detailed urease mapping of the pig stomachs showed a patchy distribution of H. pylori. The bacteria colonized in all pigs, with a concentration of H. pylori-positive areas in the antrum and fundus. Furthermore, the number of colonized areas tended to increase with time, and some of these areas showed a strong urease reaction, indicating a heavy colonization with H. pylori. Biopsies from these areas contained 10(2) to 10(5) CFU per 2-mm-wide biopsy. We conclude that persistence of H. pylori infection in barrier-born pigs can be demonstrated for at least 6.5 months. The patchy distribution and the variability of viable bacteria were similar to those described for humans.