Active and passive mouse-protecting capacity of Pseudomonas aeruginosa protein vaccines

Six different vaccines were prepared, each containing the soluble, practically lipopolysaccharide-free protein extract of 2 or 3 Pseudomonas aeruginosa strains. In active mouse protection tests the vaccines were shown to give protection against both homologous and heterologous serotype strains, and against strain PA-103 producing exotoxin A. In rabbits the vaccines were found to stimulate the production of protective antibodies demonstrable in a passive mouse protection test. The immune serum had a protective effect against the exotoxin A-producing strain PA-103, too. Toxicity of the vaccines was studied in mice (mouse weight gain test) and in rabbits (intracutaneous skin test and pyrogenicity). The vaccines were not or only slightly toxic.     

Screening of Nonfilamentous Bacteria for Production of Cutin-Degrading Enzymes

Two hundred thirty-two nonfilamentous bacterial strains, including saprophytes, plant pathogens, and opportunistic plant and human pathogens, were screened for the ability to produce cutinases (cutin-degrading esterases). Initially, esterase activity of culture filtrates of strains grown in nutrient broth-yeast extract medium supplemented with 0.4% apple or tomato cutin was determined by a spectrophotometric assay utilizing the model substrate p-nitrophenyl butyrate. The culture filtrates of the 10 Pseudomonas aeruginosa strains tested exhibited the highest esterase activity, with values of >500 nmol/min/ml. Of these 10 strains, 3 (K799, 1499A, and DAR41352) demonstrated significant induction (10-fold or above) of esterase activity by addition of cutin to nutrient broth-yeast extract medium. The ability of culture filtrates of the three strains to cause release of apple cutin monomers was confirmed by a novel high-performance liquid chromatography technique. Monomer identification was confirmed by gas chromatography-mass spectroscopy analyses. Addition of the nonionic detergent n-octylglucoside stimulated cutinase activity of culture filtrates from strains K799 and DAR41352, but not that of filtrates from strain 1499A. Time course studies in nutrient broth-yeast extract medium supplemented with apple cutin indicated maximal levels of cutinase in the culture fluids after cultures entered stationary phase. Incubation temperatures below the optimal temperature for growth (37°C) led to maximal production of cutinase.     

Isolation of different variants of Pseudomonas aeruginosa anatoxin and their characteristics

The conditions for the detoxification of the crude preparations of P. aeruginosa exotoxin A, obtained by the cultivation of strain PA-7 in Martin's broth, have been studied, and the schemes for obtaining nontoxic, stable, specifically antigenic preparations of toxoid from exotoxins A with different degrees of purification have been developed. Toxoid obtained by formalin treatment on the level of a crude preparation with its subsequent purification and additional detoxification with formalin in the presence of lysin has been shown to possess high immunogenic potency. The preparation has been found to induce immune response and to ensure the protection of experimental animals challenged not only with the lethal dose of exotoxin A, but also with P. aeruginosa toxigenic and protease-producing strains.     

Antiviral effects of bacteria isolated from manure

The objectives of this study were to determine the role of microbial activity in inactivation of hepatitis A virus (HAV) and to learn how the virus is inactivated. Of 31 bacterial strains isolated from animal manure, 10 efficiently inactivated HAV in fluid thioglycollate medium, with D₁₀ values (time, in days, required for a 90% reduction of virus titer) of 10 at 30°C. The D₁₀ value of the control suspension without bacteria was 35.1. Most of the 10 strains raised the pH of the medium during growth; comparisons suggested that alkalinity was not a principal antiviral property of these cultures. Cell-free filtrates of nine of these strains caused net 90% inactivation of HAV within 6 days at 37°C; the other did not. The inactivation capacity of four of the nine culture filtrates was significantly reduced by incubation with selected protease inhibitors before the virus was added. These protease inhibitors did not affect the activities of the other five culture filtrates. Fractions prepared by ultrafiltration (nominal molecular weights <1,000) from two of these cultures inactivated HAV suggesting that their mode of action was not enzymatic. Correspondence to: D.O. Cliver. mis|Department of Animal Health and Biomedical Sciences     

Relation of the immunosuppressivity of virulent Shigella to the structure of O antigen

The capacity of filtrates of virulent Shigella cultures (VSC) and their different fractions to render avirulent strains, injected intraperitoneally into mice simultaneously with these filtrates, capable of suppressing immune response (delayed hypersensitivity) has been studied. Among the fractions of VSC filtrates, the lipoid fraction soluble in the mixture of ethanol and ether has proved to be active. Active factors can be extracted from VSC filtrates by means of immunosorbents obtained from antibodies of pertinent specificity. VSC filtrates are completely inactivated by treatment with phenol and trichloroacetic acid. These data suggest that the immunosuppressive factors of shigellae, responsible for their capacity to suppress immunological memory, are incorporated into the O-antigen of these bacteria.     

Behavior and Pathogenicity of Tritrichomonas foetus in Chick Liver Cell Cultures*

SYNOPSIS. Clones of Tritrichomonas foetus, referred to as strains KV-1, DK-2, and UT-1, which were judged by subcutaneous mouse assay to differ in pathogenicity, had different effects on trypsin-dispersed liver cell cultures prepared from 3 lines (Light Brown Leghorn, Massachusetts Brown, Massachusetts Low Growth) of chicks. The general pattern of parasite-cell interaction was similar in all cell cultures, but the intensity of certain responses of the cultures to trichomonads, reflected best in the levels of macrophage activity, depended on the line of chicks. The mild UT-1 strain was readily engulfed and digested by the macrophages, caused very little damage to the epithelial cells and fibroblasts, and had a minimal inhibitory effect on the division rate of the latter. Many abnormal changes were seen, however, in the cytoplasm and nucleil of fibroblasts and epithelial cells in cultures exposed to strains of high (KV-1) and intermediate (DK-2) pathogenicity or to cell-free filtrates of rich active cultures (henceforth referred to as filtrates) of these strains. The changes included retraction of cytoplasm which caused cell-free spaces to appear in the sheets of fibroblasts and around the epithelial islands; the islands became detached from the surrounding fibroblast sheets and tended to form multilayered cell mounds. The flagellates and filtrates of KV-1 strain greatly inhibited fibroblast division, and similar, but less pronounced, inhibitory effects were exerted by DK-2 strain and its filtrates. Trichomonads of all 3 strains did not attach themselves to fibroblasts and epithelial cells. Thus the abnormal changes in cultures infected with KV-1 and DK-2 parasites apparently were caused by some toxic substances produced by the trichomonads, which had to be responsible also for the identical changes in cultures exposed to filtrates of these strains. KV-1 and DK-2 strains inhibited the phagocytic activity of the macrophages in the early stages of infection, the former causing stronger and longer lasting inhibition. Trichomonads of KV-1 strain multiplied actively within the phagocytes which developed degenerative changes and ultimately burst, releasing healthy parasites into the medium. DK-2 flagellates, altho incapable of dividing inside the macrophages, usually were not digested and caused degeneration of the cells which contained them.     

Immunogenicity of the recombinant Bacillus strains with cloned gene of biosynthesis of protective antigen against Bacillus anthracis

Microbe Russian Anti-Plague Research Institute, Saratov A hybrid plasmid pUB110PA-1 demonstrating stable functioning in the cells of Bacillus strains and containing the gene of biosynthesis of Bacillus anthracis protective antigen was constructed. The recombinant strains surpassing the anthrax vaccinal cultures in the secreted synthesis of the protective antigen were obtained and their immunological efficacy was assessed. A single inoculation of Guinea pigs with the dose of 5 x 107 spores of the recombinant strains imparted efficient protection against B. anthracis challenge. Immune responses were characterized by high indices of immunity and titers of antibodies to the protective antigen. In contrast to the anthrax vaccinal preparations, the gene-engineering strains imposed no residual virulence for BALB/n mice and Guinea pigs.     

A New Screening Method of Viral-neuraminidase Inhibitors

A new and practical method for the screening of neuraminidase inhibitors (NI) by means of the viral hemagglutination (HA)-dehemagglutination(deHA) reactions was suggested. The best conditions for the HA and deHA reactions were investigated. Existence of strong inhibition activity on the viral deHA has been recognized in the culture filtrates of some strains of actinomycetes. All of these deHA inhibitors showed NI activity that is not specified to the strain of the test viruses. About 0.25 mg/ml of the preparation obtained from the culture filtrate of the strongest actinomycetes, No. 289, inhibited the liberation of neuraminic acid from bovine submaxillary mucin by 80 HA units/ml of influenza A Fukuoka/1/70 (H₃N₂) virus up to 80%.     

Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material

Expression of the pedA gene from Pediococcus acidilactici, coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of pediocin to support additional studies, including structure-function research. Following various cloning strategies, a KM71H (Mut(s)) strain was selected. A significant concentration (74 microg/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA, inhibited the biological activity of natural pediocin PA-1. The recombinant pediocin appeared as a mixture of three main fractions (7-8, 11, 20 kDa vs. 4.6 kDa for natural pediocin PA-1). The recombinant pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some "collagen-like" material was tightly associated, most probably via covalent binding, to the recombinant pediocin. The "collagen-like" material was most probably responsible for the lack of biological activity of the recombinant pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that pediocin PA-1 may possess a somewhat "collagen-like" nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into fibrils.     

The expression of proteinases and haemolysins by Aeromonas hydrophila under modified atmospheres

The study estimated the proteolytic activity (against Hide Powder Azure) and haemolytic activity (against horse erythrocytes) in cell-free filtrates (CFF) from four strains of Aeromonas hydrophila growing under a range of commercially relevant modified atmospheres (2% O2, 78% N2, 20% CO2; 10% O2, 80% N2, 10% CO2; 50% N2, 50% CO2; 100% CO2). The examined strains exhibited significant qualitative and quantitative differences in the extent and times of onset of expression of these enzymes under aerobic and modified atmospheres. No proteolytic or haemolytic activities were detected in any Aer. hydrophila cultures grown at sub-optimal temperatures under modified atmospheres containing high concentrations of CO2 (i.e. 50% CO2 or 100% CO2). Although Aer. hydrophila can grow rapidly in modified atmospheres, the overall spoilage and pathogenic potential is grossly affected. Implications of these findings are discussed.     

In Vitro Production of Choleragen and Vascular Permeability Factor by Vibrio cholerae

The in vitro production of significant amounts of extracellular choleragen and vascular permeability factor (PF) by Vibrio cholerae strain VC-12 (Ogawa) in a basal peptone medium required forced aeration, low incubation temperature, and a low initial pH. Filtrates of alkaline peptone cultures of VC-12 grown at 37 C contained an ion translocase inhibitory activity but neither choleragen nor PF activity, Sterile filtrates of pH 6.5 peptone cultures of VC-12 grown at 29 C contained no ion translocase inhibitory activity but possessed choleragen activity (lethality for the suckling rabbit) and PF activity to the extent that the intradermal inoculation of 0.1 ml of a 1:12,288 dilution of such a filtrate gave rise to a vascular permeability reaction (8 by 8 mm in diameter) in the guinea pig. PF excretion occurred during the late logarithmic phase of growth but did not appear to be the consequence of cell lysis. The PF activities of strains VC-12 and 569B (Inaba) were neutralized to the same extent by anticholeragen antiserum prepared against crude 569B choleragen.     

Effect of various microorganisms on the activity of levorin

Levorin added to nutrient media with growing cultures of aerobic gram-positive bacilli, Escherichia, enterococci and filamentous fungi was partially inactivated. The antibiotic activity decrease depended on the strain characteristics, incubation period and temperature. Fermentation broth filtrates of the experimental strains also inactivated levorin while to a lesser extent than the growing organisms. In contaminated levorin pastes, the antibiotic activity was lower. The fermentative nature of inactivation was not proved.     

Simple cultural test for relative cellulolytic activity of fungi

A simple method is described for determining the relative cellulolytic activity of fungi. Opaque columns of an agar medium containing a partially crystalline cellulose preparation were inoculated with the fungi. Depth of the clear zone that developed beneath the growing cultures provided a visual measure of cellulolytic activity on a continuous, cumulative basis. Depth of clearing (DC) was determined for 25 species of fungi differing widely in cellulolytic activity, and compared by correlation analysis with results of three other methods for measuring cellulolytic activity. Relatively high coefficients of correlation (greater than 0.6) were obtained between DC and weight loss of cotton sliver, loss in tensile strength of cotton duck, and carboxymethyl cellulase activity in culture filtrates. In comparison with conventional assay procedures, the clearing method offered several advantages: (i) results were at least as well correlated with the capacity to utilize native cellulose as a substrate; (ii) the method measured activity of growing cultures rather than culture filtrates, thus involving less risk of losses due to product inhibition, binding, or denaturation of enzymes; (iii) repeated measurements were made on the same experimental set up, so that errors due to arbitrarily selected times of harvest were avoided conveniently; and (iv) the method required less working time and very simple equipment, making it convenient for large-scale screening tests.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO THIRAM, ZIRAM, FERBAM, NABAM AND ZINEB

The concentrations of thiram preventing germination of spores of Botrytis cinerea in drops of a 1% solution of sucrose, and on the surface of a sucrose-nitrate agar have been determined. Thiram had much less effect on germination in the agar medium, even when a purified agar was used. There was no growth on sucrose-nitrate agar if the concentration of thiram exceeded 31 p.p.m. Attempts to obtain strains able to grow at higher concentrations were unsuccessful.
Similar results were obtained with ziram, nabam and zineb.
Ferbam also was more effective in preventing spore germination in spore drops than on agar media; this effect was obtained with ordinary and with purified agar.
On a sucrose-nitrate agar generally there was no growth if the concentration of ferbam exceeded 125 p.p.m., but in one of forty-eight plates containing 250 p.p.m. ferbam, five slowly growing colonies were produced, and from these colonies arose mycelium which grew and sporulated rapidly on 500 p.p.m. ferbam agar. Agar disk inocula were transferred from these cultures to agar containing higher concentrations of ferbam and in this way, and by repeating the process, a strain was obtained which grew slowly but continuously, and sporulated on agar containing 5000 p.p.m. ferbam. However, the poor solubility of this fungicide made it difficult to assess quantitatively the degree of adaptation.
A proportion of the spores from this strain germinated in drops containing about twice the concentration of ferbam which prevented germination of parent spores.
The resistance of the mycelium of the resistant strain was not lost after repeated subculture on fungicide-free agar. The resistant strain was as susceptible as the parent strain to thiram, ziram, nabam and zineb.
Attempts to obtain strains of Venturia inaequalis resistant to thiram, ferbam, ziram and zineb were unsuccessful.     

Determination of the biological activity of Clostridium difficile toxins in in vivo and in vitro experiments

The biological activity of the filtrates of 29 C. difficile strains was studied in vivo (suckling white mice) and in vitro (cell cultures of different species and origin). The action of the filtrates on the experimental models in vivo was evaluated from the cytotoxic effect index, while in vitro the intensity of the cytotoxic effect was evaluated from the percentage of dead cells in the monolayer. The results of the comparative determination of toxicity characteristics in vivo and in vitro demonstrated that cell cultures were more sensitive experimental models than suckling white mice. The use of cell cultures permitted the quantitative evaluation of the cytotoxic activity of the filtrates under study, as well as the detection of their cell-directed action at minimal concentrations.     

Enteropathogenicity of Vibrio parahaemolyticus in the ligated rabbit ileum.

The enteropathogenicity of Vibrio parahaemolyticus was investigated by contrasting the effects of whole cells, cell fragments, cell-free preparations, and media constituents injected into rabbit ileal loops. Three of 20 cultures utilized were Kanagawa-negative strains from seawater and sea fish. The remaining 17 cultures included both Kanagawa-positive and -negative strains from Japanese victims of gastroenteritis. Broth culture filtrates concentrated 10-fold by dialysis against 30% Carbowax were unreactive, whereas lyophilized filtrates, regardless of Kanagawa type, as well as all sterile broth preparations containing greater than or equal to 5% NaCl gave positive reactions in the rabbit gut. In contrast, crude lysates derived from broth cultures of Kanagawa-positive strains caused loop dilatation; lysate supernatants were unreactive. Lysates of cells washed from brain heart infusion agar were more reactive than lysates from Trypticase soy agar-grown cells. When agar-grown cell lysates prepared by disruption in saline were dialyzed against distilled water, they were devoid of gut reactivity. Reactivity was restored in dialysands resuspended in saline and in dialysates concentrated 10-fold. The agar-grown cell lysates exhibited Kanagawa-type hemolysis. Our data support the conclusion that the rabbit loop reactivity observed with lyophilized, cell-free culture filtrates may result from excessively elevated NaCl concentrations, and that a toxic factor associated with large-cell particles may be dialyzable, depends on saline for expression, and resembles the Kanagawa hemolysin.     

PECTIC ENZYMES SECRETED BY VERTICILLIUM DAHLIAE AND THEIR ROLE IN THE DEVELOPMENT OF THE WILT DISEASE OF COTTON

An isolate of Verticillium dahliae obtained from Uganda was highly virulent to young cotton plants under greenhouse conditions. A hyaline variant which often appeared in culture was as virulent as the parent isolate, but preliminary experiments indicated that it did not survive as long in unsterile soil. The parent isolate grew rapidly in cotton plants after root inoculation and was isolated from stems and leaves well before the appearance of disease symptoms visible to the naked eye.
Protopectinase was produced in the absence of pectic materials, but more active preparations were obtained when media contained pectic substances. In general, there was a close correspondence between the protopectinase activity of culture filtrates and the toxicity of these filtrates to parenchymatous cells. Some separation of the two activities was obtained by heating enzyme solutions or by plasmolysing the test tissue.
Protopectinase solutions had little pectinesterase activity but rapidly reduced the viscosity of solutions of pectic substances. In general, the properties of protopectinase and the viscosity-reducing enzyme were similar.
Young cotton shoots wilted rapidly when placed in cell-free filtrates from cultures of the pathogen. Wilting was delayed under conditions unfavourable for transpiration. Evidence was obtained which showed that wilting was caused by the uptake of thermostable compounds of high molecular weight which impeded the upward flow of the vascular sap. Pronounced vascular browning was obtained only when solutions containing protopectinase were used. Wilting and vascular browning were obtained with solutions having little pectinesterase activity; in contrast, a solution having high pectinesterase activity produced relatively little vascular browning.     

Temperature and carbon source affect the production and secretion of a thermostable β-xylosidase by Aspergillus fumigatus

The effect of the temperature of growth and carbon source on the production and secretion of β-xylosidase (EC 3.2.1.37) by the thermotolerant fungi Aspergillus fumigatus was studied in submerged cultures. In cultures developed at optimal temperature (30 °C), the enzyme was predominantly cell-bound, while in cultures developed at higher temperature (42 °C), the β-xylosidase activity was predominantly found in the cell-free filtrates. The use of corn cob powder instead of xylan as substrate increased considerably the secretion of enzyme. The highest level of extracellular β-xylosidase (45 U/ml or 360 U/mg protein) was obtained in 3% corn cob cultures grown at 42 °C for 72 h. The partially purified enzyme was active and stable at high temperatures. The presence of high titres of β-xylosidase activity in association with xylanase in the culture filtrates enhanced the efficiency of the pulp hydrolysis process.     

a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a.

Conjugation between haploid cells of Saccharomyces cerevisiae is mediated through the action of diffusible mating hormones, two of which have been designated as a-factor and alpha-factor. Partially purified fractions exhibiting a-factor activity have been obtained from culture filtrates of a cells by ultrafiltration, ion-exchange chromatography, and gel filtration. The a-factor preparations specifically caused both G1 arrest and morphological alterations in cells of alpha-mating type, whereas a cells, a/alpha diploids, and nonmating alpha mutants were not affected. The a-factor activity was found in the culture filtrates of all a strains tested, but not in filtrates of alpha or a/alpha cell cultures. The hormone is sensitive to various proteases, showing that it is associated with a peptide or protein. Gel filtration studies suggest an apparent molecular weight greater than 600,000; however, this result may be due to aggregation with carbohydrate present in the preparations. Although the biological activities of a-factor are analogous to those described previously for alpha-factor, the chemical properties of these two hormones appear to be quite different.     

The role of higher polythionates in the reduction of chromium(VI) by Acidithiobacillus and Thiobacillus cultures

In this paper, we report the chromium(VI) reduction by filtrates of Acidithiobacillus and Thiobacillus cultures. Chromium(VI) reduction by filtrates of A. ferrooxidans cultures under acidic conditions was higher than that observed for A. thiooxidans. However, at pH close to 7, chromium(VI) reduction by filtrates of T. thioparus cultures was as high as that by filtrates of A. thiooxidans cultures and much higher than that observed for A. ferrooxidans cultures at the same pH. The capability of these cultures to reduce chromium(VI) was associated specifically with the fraction of cultures (cells, sulphur and associated sulphur compounds) retained by filtration through a 0.45mum filter. In the fraction that comes from A. thiooxidans culture, polythionates (S(x)O(6)(2-)) with 3-7 sulphur atoms were detected and identified (by HPLC with MS as detector). The model of vesicles containing polythionates, sulphur and water agrees with our results.