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PubMed1962

Influence of guinea pig plasma factors on phagocytosis of Pasteurella pestis. I. Plasma from normal guinea pigs.

Stanziale, W. G. (Fort Detrick, Frederick, Md.) and J. D. White. Influence of guinea pig plasma factors on phagocytosis of Pasteurella pestis. I. Plasma from normal guinea pigs. J. Bacteriol. 83:175-181. 1962.-The existence of an enhancing effect of normal guinea pig plasma on the phagocytosis of Pasteurella pestis has been reaffirmed, using a test system consisting of neutrophils from normal nonimmune guinea pigs as the phagocytes. This enhancing influence was found to be attributable to the interplay of a thermolabile and a thermostable plasma component. The data suggest a consideration of complement as the thermolabile component, since the enhancing activity of plasma is destroyed by heating to 56 C and restored by addition of fresh plasma. The possibility that the thermolabile plasma component is opsonin was discounted. Surface alterations which did occur resulted in preserving the integrity of the envelope substance of the plague bacillus. Treatment of neutrophils with plasma prior to phagocytosis did not increase their phagocytic capacity. The thermostable enhancing component was not demonstrated to be present in any of three purified serum protein fractions tested. In fact, all of these fractions inhibited phagocytosis of P. pestis. An enhancing effect of purified fibrinogen could not be demonstrated.

Animals↗
PubMed1962

Influence of guinea pig plasma factors on phagocytosis of Pasteurella pestis. II. Plasma from plague-infected guinea pigs.

Stanziale, W. G. (Fort Detrick, Frederick, Md.) and J. D. White. Influence of guinea pig plasma factors on phagocytosis of Pasteurella pestis. II. Plasma from plague-infected guinea pigs. J. Bacteriol. 83:182-186. 1962.-The phagocytosis enhancing property of normal guinea pig plasma was altered during experimental plague infection. The most notable changes occurred in the plasma from afebrile, moribund animals and from those convalescing from acute infection. The plasma of the moribund guinea pigs, which was obtained 7 to 8 days after exposure to Pasteurella pestis, inhibited phagocytosis to a considerable degree. Plasma from convalescent guinea pigs enhanced phagocytosis to a higher degree than the plasma of normal animals. In contrast, plasma from recovered guinea pigs in which cultural or serological evidence of infection was not demonstrated enhanced phagocytosis to a degree equivalent to that of normal plasma. Plasma taken from guinea pigs during the febrile phase of plague infection enhanced phagocytosis to a lower degree than did normal plasma.

Animals↗
PubMed1962

MECHANISM OF ACTION OF THE TOXIN OF BACILLUS ANTHRACIS II. : Alkaline Phosphatasemia Produced by Culture Filtrates of Various Bacilli.

Slein, Milton W. (Fort Detrick, Frederick, Md.) and Gerald F. Logan, Jr. Mechanism of action of the toxin of Bacillus anthracis. II. Alkaline phosphatasemia produced by culture filtrates of various bacilli. J. Bacteriol. 83:359-369. 1962.-A factor which produces hyperphosphatasemia after intravenous injection into animals has been found in culture filtrates of several bacilli. The factor appears not to be lecithinase, although it has been found only in culture filtrates of microorganisms, such as Bacillus cereus variants and B. thuringiensis, which are known to produce lecithinase. The factor has been shown to be different from the protective antigen fraction of the toxin of B. anthracis. The phosphatasemic response can be prevented by mixing the factor with antiserum before injection. Immunological tests indicate that the centers of ossification may be a principal source of the excess alkaline phosphatase which is liberated into the bloodstream. However, moderate changes in bone alkaline phosphatase activity levels, brought about by the development of scurvy in guinea pigs or by treatment of scorbutic guinea pigs with ascorbic acid, did not seem to affect the phosphatasemic response of the animals. Marked release of alkaline phosphatase occurs when slices of rabbit epiphyseal bone or kidney cortex, or suspensions of leukocytes, are incubated with small amounts of the phosphatasemia factor.

Journal Article↗
PubMed1962

Temperaturegradient plates for growth of microorganisms.

Landman, Otto E. (Fort Detrick, Frederick, Md.), Howard T. Bausum, and Thomas S. Matney. Temperature-gradient plates for growth of microorganisms. J. Bacteriol. 83:463-469. 1962.-Different temperature-gradient plates have been devised for the study of microbial growth on solid media through continuous temperature ranges or in liquid media at finely graded temperatures. All plates are made of heavy-gauge aluminum; heat supplied at one end is dissipated along the length of the metal so that a gradient is produced. The shape and range of the gradient depends on the amount of heat supplied, the insulation, the ambient temperature, and other factors. Differences of 0.2 C in temperature sensitivity between bacterial strains can be detected. The plates are simple to construct and operate. The dimensions of the aluminum, the mode of temperature measurement, and the method of heating may all be modified without diminishing the basic utility of the device.A sharp growth front develops at the maximal temperature of growth of bacteria. In most strains, all bacteria below the front form colonies and all bacteria above the front are killed, except for a few temperature-resistant mutants.

Bacteria↗
PubMed1962

Studies on immunity in anthrax. IX. Effect of variations in cultural conditions on elaboration of protective antigen by strains of Bacillus anthracis.

Wright, George G. (Fort Detrick, Frederick, Md.), Milton Puziss, and W. Brook Neely. Studies on immunity in anthrax. IX. Effect of variation in cultural conditions on elaboration of protective antigen by strains of Bacillus anthracis. J. Bacteriol. 83:515-522. 1962.-Nonproteolytic and nonencapsulated avirulent mutants were isolated from six virulent strains of Bacillus anthracis and tested for elaboration of protective antigen in the chemically defined medium developed previously (599 medium). Initially the strains grew rather slowly in 599 medium; serial transfer in the medium increased the rate of growth but reduced the elaboration of antigen. Two of the strains tended to revert to the encapsulated form during serial passage in 599 medium. Modifications in the medium and cultural conditions were studied in an attempt to obtain improved growth; the most significant alterations were the addition of adenosine and l-alanine, reduction in the concentration of ferrous sulfate, and growth under anaerobic conditions with agitation. Study of amino acid utilization in the medium revealed that the concentrations of certain amino acids could be reduced. These modifications, together with substitution of more productive strains, resulted in an approximately fivefold increase in elaboration of protective antigen, and greatly simplified production of the antigen on a practical scale. Omission of bicarbonate from the medium had no significant influence on utilization of amino acids.

Alanine↗
PubMed1962

Nutrition of Leptospira pomona. 1. A chemically defined substitute for rabbit serum ultrafiltrate.

Johnson, R. C. (Fort Detrick, Frederick, Md.) and N. D. Gary. Nutrition of Leptospira pomona. I. A chemically defined substitute for rabbit serum ultrafiltrate. J. Bacteriol. 83:668-672. 1962.-Poor growth of Leptospira pomona, strain Wickard, in a medium containing exhaustively dialyzed rabbit serum was corrected by addition of rabbit serum ultrafiltrate. A dialyzed serum medium was used to investigate the nutritional qualities of various compounds when tested as substitutes for the ultrafiltrate fraction. l-Asparagine was the only amino acid that markedly stimulated growth. l-Glutamine was active only when autoclaved (converted to the ammonium salt of pyrrolidone carboxylic acid), and NH(4)Cl and urea satisfactorily replaced l-asparagine. Results indicated that amino acids functioned primarily as a nitrogen source in this medium. Thiamine was the only vitamin required for growth. The lag phase was usually decreased by 1 day with NaHCO(3).A medium composed of 0.02 m phospate buffer (pH 7.4), 15% dialyzed rabbit serum, thiamine (5 mug/ml), and NH(4)Cl (10(-3)m) supported growth equivalent to that obtained with undialyzed rabbit serum medium. L. pomona was transferred six times, in this medium and seven other serotypes were transferred three times, without any decrease in amount of growth. Growth of L. pomona was initiated with 20 organisms per ml.

Animals↗
PubMed1962

Selection of thermostable Serratia marcescens from logarithmic-phase cultures as a means for inducing synchronous growth.

Hess, George E. (U. S. Army Chemical Corps, Fort Detrick, Maryland) and Milton Shon. Selection of thermostable Serratia marcescens from logarithmic-phase cultures as a means for inducing synchronous growth. J. Bacteriol. 83:781-784. 1962.-A relatively thermostable, apparently nonmultiplying, population of Serratia marcescens was found in cultures in the exponential phase of growth. Thermal treatment of the whole culture resulted in the death of the multiplying cell fraction, although the nonmultiplying fraction remained viable. Subcultivation of the thermally treated suspensions yielded cultures in which most of the viable organisms multiplied synchronously for at least two generations.

Serratia marcescens↗
PubMed1962

Pathogenesis of Coccidioides immitis in monkeys.

Converse, J. L. (U. S. Army Chemical Corps, Fort Detrick, Frederick, Md.), E. P. Lowe, M. W. Castleberry, G. P. Blundell, and A. R. Besemer. Pathogenesis of Coccidioides immitis in monkeys. J. Bacteriol. 83:871-878. 1962.-Respiratory exposure to arthrospores from the submerged growth of Coccidioides immitis, strain Cash, in liquid medium resulted in similar pathogenesis in monkeys to that of strain Silveira arthrospores harvested from solid medium. Infectivity of 100% was noted with doses of 50 to 10,000 arthrospores. The disease was characterized by loss of appetite and weight, malaise, and extreme respiratory distress accompanied by coughing, with the immediate cause of death being acute coccidioidal pneumonia. The pathological picture was one of extensive, progressive, destructive pulmonary disease in the higher dose levels and few, small, self-contained, fibrous lesions, with little destruction of lung tissue, in the low doses. This was correlated in general with the findings of serial X rays and serological tests. The presence of the parasitic phase (spherule and endospore) of the organism was noted in large numbers within the pulmonary lesions and bronchial exudates and was substantiated by cultural methods. Occasionally, hyphal elements of the saprophytic growth phase were noted around the periphery of residual cavitated areas of the lungs.

Animals↗
PubMed1962

Pyruvate oxidation by Pasteurella tularensis strains of graded virulence.

Weinstein, I. (U.S. Army Chemical Corps, Fort Detrick, Frederick, Md.), M. L. Guss, and R. A. Altenbern. Pyruvate oxidation by Pasteurella tularensis strains of graded virulence. J. Bacteriol. 83:1010-1016. 1962.-The rate of oxidation of sodium pyruvate by Pasteurella tularensis was shown to be correlated with the virulence of the strain studied. No qualitative differences in cofactor requirements for or products of pyruvate oxidation were found among strains of graded virulence. The rate of pyruvate oxidation was directly related to strain virulence, whereas the endogenous respiration rate was inversely correlated with virulence. For all tested strains possessing some degree of virulence, maximal rates of pyruvate oxidation were obtained with small concentrations of pyruvate (10 to 20 mumoles), above which the oxidation rate was depressed. Small concentrations of glutamate or other dicarboxylic acids effected nonadditive stimulation (sparking) of the pyruvate oxidation rate only at pyruvate concentrations above optimal (greater than 20 mumoles). The avirulent strain 38A was unique, being characterized by a low endogenous respiration rate, a maximal rate of pyruvate oxidation at all concentrations above 20 mumoles, and the absence of glutamate sparking. Studies of cell-free extracts indicated that the differences in pyruvate oxidation among the strains may be attributed in part to variation in the properties of enzymes of the tricarboxylic acid cycle.

Citric Acid Cycle↗
PubMed1962

Separation of the toxin of Bacillus cereus into two components and nonidentity of the toxin with phospholipase.

Molnar, Dorothy M. (U.S. Army Chemical Corps, Fort Detrick, Frederick, Md.). Separation of the toxin of Bacillus cereus into two components and nonidentity of the toxin with phospholipase. J. Bacteriol. 84:147-153. 1962-Bacillus cereus produced toxin in a Casamino acids medium without added serum or other protein. The toxin was separated into two components by adsorption on columns of calcium phosphate gel followed by elution with phosphate buffer (pH 7.5). The component eluted first has been called factor I and the component eluted later factor II. When tested alone each component was relatively nontoxic, but when combined they formed a toxic mixture as evidenced by skin reactions in guinea pigs and tests for lethal effects in mice. The phospholipase activity, determined by the egg-yolk reaction, was found in the fraction containing factor I. However, using columns of alumina-C(gamma) gel the phospholipase activity was found in the fraction containing factor II. This suggests that the phospholipase is not the same chemical entity as either factor I or II. The following are further evidence for the non-identity of the toxin and phospholipase: (i) differential precipitation of the two activities by (NH(4))(2)SO(4); (ii) differential neutralization by various antisera; and (iii) differential inhibition with ethanol.

Animals↗
PubMed1962

Pesticins. II. Production of pesticin I and II.

Brubaker, Robert R. (Fort Detrick, Frederick, Md.) and Michael J. Surgalla. Pesticins. II. Production of pesticin I and II. J. Bacteriol. 84:539-545. 1962.-Pesticin I was separated from pesticin I inhibitor by ion-exchange chromatography of cell-free culture supernatant fluids and by acid precipitation of soluble preparations obtained from mechanically disrupted cells. The latter procedure resulted in formation of an insoluble pesticin I complex which, upon removal by centrifugation and subsequent dissolution in neutral buffer, exhibited a 100- to 1,000-fold increase in antibacterial activity over that originally observed. However, activity returned to the former level upon addition of the acid-soluble fraction, which contained pesticin I inhibitor. Since the presence of pesticin I inhibitor leads to serious errors in the determination of pesticin I, an assay medium containing ethylenediaminetetraacetic acid in excess Ca(++) was developed; this medium eliminated the effect of the inhibitor. By use of the above medium, sufficient pesticin I was found to be contained within 500 nonirradiated cells to inhibit growth of a suitable indicator strain; at least 10(7) cells were required to effect a corresponding inhibition by pesticin II. Although both pesticins are located primarily within the cell during growth, pesticin I may arise extracellularly during storage of static cells. Slightly higher activity of pesticin I inhibitor was found in culture supernatant fluids than occurred in corresponding cell extracts of equal volume. The differences and similarities between pesticin I and some known bacteriocins are discussed.

Anti-Bacterial Agents↗
PubMed1962

Genotypic alterations associated with avirulence in streptomycin-resistant Pasteurella pestis.

Brubaker, Robert R. (Fort Detrick, Frederick, Md.) and Michael J. Surgalla. Genotypic alterations associated with avirulence in streptomycin-resistant Pasteurella pestis. J. Bacteriol. 84:615-624. 1962.-Avirulence in Pasteurella pestis, as indicated by the existence of a new genotype, was associated with the genetic loss of Ca(++) dependence rather than with the usual concomitant loss of ability to produce virulence antigens. Strains of the new type exhibited all four of the known virulence factors of P. pestis but were avirulent for mice and guinea pigs (ld(50) > 10(8) cells). In addition to producing virulence antigens, the new strains formed rough colonies at 37 C when grown on Ca(++)-deficient medium, as opposed to typical avirulent strains which formed smooth colonies on this medium and failed to produce virulence antigens. Growth of the new strains in a corresponding Ca(++)-deficient broth medium was slow, as compared with that of typical avirulent strains, but was more rapid than that of virulent strains. The new strains were obtained (i) at 26 C by selecting for resistance to streptomycin or (ii) at 37 C by isolation from rough colonies growing on a Ca(++)-deficient agar medium plated with either a virulent streptomycin-sensitive or -resistant culture. Usually, isolates obtained at 26 C exhibited a marked reduction in ability to form colonies upon primary plating at 37 C. Of 108 streptomycin-resistant isolates obtained from six virulent strains, 20% were of the new type. In addition, five isolates were of reduced virulence (mouse ld(50) approximately 10(4) cells) but differed in being Ca(++)-dependent; they possessed all of the known virulence properties. Certain streptomycin-resistant strains failed to give rise to avirulent colonies when plated on a Ca(++)-deficient agar medium containing streptomycin. The significance of this phenomenon, which was not exhibited by the five strains of reduced virulence, is discussed, as are some apparent differences between isolates from a glycerol-fermenting strain and similar isolates obtained from five nonglycerol-fermenting strains.

Animals↗
PubMed1962

LIVE TULAREMIA VACCINE I. : Host-Parasite Relationship in Monkeys Vaccinated Intracutaneously or Aerogenically.

Eigelsbach, H. T. (Fort Detrick, Frederick, Md.), J. J. Tulis, M. H. McGavran and J. D. White. Live tularemia vaccine. I. Host-parasite relationship in monkeys vaccinated intracutaneously or aerogenically. J. Bacteriol. 84:1020-1027. 1962.-Bacteriological, histological, immunohistochemical, and serological studies were made on monkeys administered live tularemia vaccine strain LVS by either of two routes. Comparative data are presented on nonvaccinated monkeys exposed via the respiratory route to a highly virulent strain of Pasteurella tularensis. Tissue changes resulting from either aerogenic or intracutaneous vaccination were mild, and consisted primarily of the proliferation of histiocytes without the formation of granulomas. The vaccine strain was isolated from the site of vaccination of animals inoculated dermally, from the lungs of animals vaccinated aerogenically, and from the regional lymph nodes, liver, and spleen of both groups; it was not isolated from the blood or bone marrow. Proliferation of the vaccine strain at the site of dermal inoculation and in the lungs of animals exposed aerogenically was observed within 24 hr; in both groups, the maximal viable population was reached within 3 days and maintained through the 10th day. A reduction in the number of viable vaccine organisms had begun by the 14th day; isolations were obtained only from the regional lymph nodes on the 28th day, and the vaccine strain was not isolated from any of the tissues cultured on the 90th day. Because the monkey is less resistant to tularemia than is man, the benign response of this animal to live tularemia vaccine indicates that the vaccine might also be safe for man when administered by either the dermal or respiratory route.

Journal Article↗
PubMed1962

Survival of Serratia marcescens after freeze-drying or aerosolization at unfavorable humidity. I. Effects of sugars.

Zimmerman, Leonard (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.). Survival of Serratia marcescens after freeze-drying or aerosolization at unfavorable humidity. I. Effects of sugars. J. Bacteriol. 84:1297-1302. 1962.-Suspensions of Serratia marcescens were subjected to freeze-drying or to aerosolization at unfavorable humidity levels. The survival of the cells during one or the other of these treatments was markedly improved in the presence of common sugars, but no one sugar stabilized the cells against both stresses. The protective effects of the sugars were correlated with their penetrability into cells; minimally penetrable sugars stabilized cells against aerosolization, and freely penetrable sugars stabilized cells during freeze-drying. These results were attributed to the modifications of intracellular water content induced by the presence of the sugars in the cell suspensions.

Carbohydrates↗
PubMed1963

GENETIC TRANSFER OF THE ABILITY TO GROW AT 55 C IN BACILLUS SUBTILIS.

McDonald, William C. (U.S. Army Chemical Corps, Fort Detrick, Frederick, Md.) and Thomas S. Matney. Genetic transfer of the ability to grow at 55 C in Bacillus subtilis. J. Bacteriol. 85:218-220. 1962.-Two strains of Bacillus subtilis, 168 and P1, were found to grow at 55 C (55(+)) on complete media; strain 168S(r) failed to grow at temperatures above 50 C (55(-)). When 168S(r) bacteria were grown in the presence of deoxyribonucleic acid extracted from 168, the ability to grow at 55 C was transformed at a frequency of 10(-4). An incubation period of 3 to 4 hr at 37 C was necessary for phenotypic expression of the 55(+) character. Only 10 to 20% of the 55(+) transformants retained the high-level streptomycin resistance (S(r)) of the recipient, indicating close linkage between the S(s) and 55(+) loci.

Journal Article↗
PubMed1963

Studies on immunity in anthrax. X. Gel-adsorbed protective antigen for immunization of man.

Puziss, Milton (Fort Detrick, Frederick, Md.) and George G. Wright. Studies on immunity in anthrax. X. Gel-adsorbed protective antigen for immunization of man. J. Bacteriol. 85:230-236. 1963.-Protective antigen in filtrates of anaerobic cultures of Bacillus anthracis was not readily precipitable by alum or other aluminum salts, but was adsorbed onto a preformed aluminum hydroxide gel under specific conditions. The adsorbed product was highly effective in immunizing rabbits, guinea pigs, and mice against challenge with virulent B. anthracis spores. Gel-adsorbed anaerobic antigens preserved with 1:10,000 thimerosal were unstable on storage at 4 C; replacement of this preservative with 1:40,000 benzethonium chloride produced a more stable product. Addition of 0.0009% formalin further increased the stability during accelerated aging at 37 C. The final product had high protective activity in animals and was well tolerated in man.

Alum Compounds↗
PubMed1963

Studies on immunity in anthrax. XI. Control of cellular permeability by bicarbonate ion in relation to protective antigen elaboration.

Puziss, Milton (Fort Detrick, Frederick, Md.) and Mary B. Howard. Studies on immunity in anthrax. XI. Control of cellular permeability by bicarbonate ion in relation to protective antigen elaboration. J. Bacteriol. 85:237-243. 1963.-No elaboration of Bacillus anthracis protective antigen was demonstrated after addition of bicarbonate to cultures incubated 42 hr in a bicarbonate-free medium. Antigen accumulation in culture filtrates was greater if the bicarbonate was added to these cultures early in the growth period. Cell-free extracts of washed cell suspensions, prepared by hand grinding or lysozyme treatment, had no demonstrable intracellular protective antigen. Sonic treatment released antigen in measurable amounts, but tended to destroy or degrade the labile antigen. Freezing and thawing disrupted the cells and liberated the antigen; protective antigen was shown to be of internal rather than of extracellular origin. Maximal intracellular antigen concentration occurred before the peak concentration was reached in the culture filtrates. Antigen also was found in cell extracts from bicarbonate-free culture media when none was present in the corresponding culture filtrates. A high level of internal protective antigen was present in cells grown in a bicarbonate-free medium maintained at a constantly alkaline pH; only a trace of antigen was present in the corresponding pH-adjusted culture filtrate. The hypothesis is presented that bicarbonate functions to control cellular permeability and release of antigen from the cells into the ambient medium.

Anthrax↗
PubMed1963

Reversion of L forms and spheroplasts of Proteus mirabilis.

Altenbern, Robert A. (U.S. Army Chemical Corps, Fort Detrick, Frederick, Md.). Reversion of L forms and spheroplasts of Proteus mirabilis. J. Bacteriol. 85:269-272. 1963.-Spheroplasts or 3-B type L forms of Proteus mirabilis formed by growth in the presence of penicillin are able to revert to the bacillary form, as judged by regain of resistance to osmotic shock. Reversion takes place in the absence of exogenous nutrients, although at a lesser rate than that observed in a nitrogen-rich medium. Either chloramphenicol or 6-azauracil can completely inhibit reversion of spheroplasts in a penicillin-free medium. By direct measurement, there was no detectable change in total protein content throughout the period of reversion of a spheroplast suspension. Assay for acid-insoluble diaminopimelic acid (DAP) revealed that a large part of the DAP of the cell wall of normal cells was lost when the cells were converted to spheroplasts by exposure to penicillin. Upon transfer to penicillin-free medium, there was a rapid increase in acid-insoluble DAP in the wall of the reverting spheroplast. When spheroplasts were transferred to penicillin-free medium containing chloramphenicol, the acid-insoluble DAP in the wall of the spheroplast continued to decrease. The significance of these results in regard to sites of activity of the antibiotics involved is discussed.

Cell Wall↗