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Fowl cholera is caused by Pasteurella multocida serovars A:1, A:3, and A:4. The 39-kDa cross-protective factor protein and four other membrane proteins of the membrane proteome of P. multocida were identified. We determined that the 39-kDa cross-protective protein was Pasteurella lipoprotein B, or PlpB.
Marchette, Nyven J. (University of Utah, Salt Lake City), and Paul S. Nicholes. Virulence and citrulline ureidase activity of Pasteurella tularensis. J. Bacteriol. 82:26-32. 1961.-The presence of a citrulline ureidase system in Pasteurella tularensis strains of high virulence, and its absence in avirulent strains and strains of low virulence was confirmed. The presence of this system, however, was shown to be not directly related to virulence. The only wild strain of P. tularensis tested that lacked a citrulline ureidase system was isolated from a rodent. All the strains, isolated from rabbits, rabbit ticks, a human being, and a horse, that were tested possessed this system. The existence of two North American varieties of P. tularensis was postulated on the basis of virulence and citrulline ureidase activity.
Gaspar, Andrew J. (Fort Detrick, Frederick, Md.), Hugh B. Tresselt, and Martha K. Ward. New solid medium for enhanced growth of Pasteurella tularensis. J. Bacteriol. 82:564-569. 1961.-The purpose of this work was to develop a solid medium for more rapid growth of Pasteurella tularensis, especially from small inocula comparable to those generally encountered in clinical materials. AFTER TITRATION OF VARIOUS INGREDIENTS, SEPARATELY AND IN COMBINATION, THE OPTIMAL BASE WAS FOUND TO BE: 2.6% tryptose broth (Difco) with thiamine, 0.5% cysteine-HCl, 0.2% sodium thioglycolate, 1.0% glucose, dissolved by mixing without heat, and adjusted to pH 7.2 +/- 0.03. To this base 1.0% agar (Difco) is added and dissolved by heating in flowing steam for about 5 min prior to autoclaving at 121 C for 20 min. After sterilization and cooling, 5.0% defibrinated rabbit blood is added aseptically. Plates of the completed medium are incubated at 37 C for 24 hr prior to use. Colonies of P. tularensis approximately 1.0 mm in diameter are obtained on this medium after 26 to 29 hr incubation if conditions of high relative humidity (90 to 100% saturation) are maintained.The mechanisms involved in the growth enhancement obtained on this medium are under study. The role of thioglycolate appears to be that of keeping in solution the relatively high concentration of cysteine-HCl required. The specific methods of preparation described, the incubation of plates prior to use, and final incubation under conditions of high humidity all are important for optimal results. This developmental work has employed only pure cultures. Attempts are being made to develop a selective medium, using this new preparation as base, for the direct isolation of P. tularensis from a variety of clinical materials.
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.
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.
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.
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.
Bichowsky-Slomnicki, Leah (The Weizmann Institute of Science, Rehovoth, Israel) and Shlomo Ben-Efraim. Biological activities in extracts of Pasteurella pestis and their relation to the "pH 6 antigen." J. Bacteriol. 86:101-111. 1963.-Three kinds of biological activities could be found in crude extracts prepared from all the tested strains of Pasteurella pestis: cytotoxicity for monocytes, ability to agglutinate red blood cells, and induction of primary inflammatory reactions of the skin. All these biological activities were related to the presence of the "pH 6 antigen." In addition, fractions of the pH 6 antigen devoid of agglutinating activity against red blood cells, but possessing the other biological activities, could be isolated. The active biological principles appear to be protein in nature. The possible role of the described biological activities in the pathogenesis of plague is discussed.
Surgalla, M. J. (Fort Detrick, Frederick, Md.), A. W. Andrews, and C. L. Baugh. Effects of bicarbonate on growth of Pasteurella pestis. I. Differential response of virulent and avirulent cells. J. Bacteriol. 88:269-272. 1964.-Virulent Pasteurella pestis and the avirulent mutants always present in a virulent inoculum have been demonstrated to give growth responses in opposite directions upon addition of bicarbonate to broth cultures under certain conditions. The effect of supplemental bicarbonate on initiation of growth from a virulent inoculum was found to be either stimulation of both virulent and avirulent cells at low levels of added bicarbonate, depression of both virulent and avirulent cells at higher initial concentrations, or depression of avirulent mutants with simultaneous stimulation of virulent cells at a narrow range of intermediate levels.
Knisely, Ralph F. (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.), Lois M. Swaney, and Harold Friedlander. Selective media for the isolation of Pasteurella pestis. J. Bacteriol. 88:491-496. 1964.-Several selective media are described that were successfully used to isolate virulent and avirulent strains of Pasteurella pestis from material heavily contaminated with other organisms. These media are comparatively easy to prepare, consist of readily available ingredients, and usually require no adjustment of the pH. One of the selective media described permits excellent recovery and the growth of large, easily distinguishable colonies of P. pestis in 48 hr at 26 C, and also allows the detection of fewer numbers of P. pestis organisms in soil than a previously recommended selective medium. The inhibition of other organisms frequently present in clinical specimens is described.
Baugh, C. L. (Fort Detrick, Frederick, Md.), J. W. Lanham, and M. J. Surgalla. Effects of bicarbonate on growth of Pasteurella pestis. II. Carbon dioxide fixation into oxalacetate by cell-free extracts. J. Bacteriol. 88:553-558. 1964.-Enzyme preparations from Pasteurella pestis will carboxylate phosphoenolpyruvate to form oxalacetate by two distinct reactions. The reactions are similar to those catalyzed by the enzymes, phosphoenolpyruvic carboxylase and phosphoenolpyruvate carboxykinase. No significant differences in enzyme characteristics or enzyme content were found when virulent cells were compared with avirulent under the conditions of our experiments. The carboxykinase of P. pestis differs from that of animal origin, because it is dependent upon adenine derivatives rather than inosine or guanosine nucleotides. The latter two nucleotides can act indirectly by way of adenosine nucleotides, because nucleoside diphosphokinase and myokinase are present in the extract.
Hatch, Melvin T. (University of Utah, Salt Lake City), and Paul S. Nicholes. Immunogenic substances in culture filtrates and lysates of Pasteurella tularensis. J. Bacteriol. 88:566-573. 1964.-Culture filtrates and lysates of Pasteurella tularensis were tested for immunogenicity in mice subsequently infected with either strain 425 or 425 F4G. The efficacy of the vaccines varied with dosage and was significantly dependent upon methods of preparation. The optimal procedures for the production of an immunologically potent vaccine included: (i) aging the cultures after growth under partial anaerobiosis at 37 C, and (ii) inactivating the cells with phenol or formaldehyde. An unusual "survival phenomenon" was suggested when mice were administered large doses of cell-free vaccines and subsequently large doses of moderately virulent P. tularensis. The data indicated that the filtrates and lysates elicited an immune response sufficient to protect against an active infection with strains 425 or 425 F4G, and that the challenge dose per se stimulated an enhanced immunity. Furthermore, this survival phenomenon was demonstrable when immunized mice were subsequently given massive doses of strain 425 and challenged with approximately 1,000 ld(50) of the fully virulent strain Schu. On the basis of our data, we have hypothesized that the protective antigens were released into the suspending medium as a result of alterations in the permeability of cells undergoing either complete or partial enzymatic degradation. We believe that the envelope antigens were released from the cell by mechanisms analogous to those causing leakage of intracellular constituents in cells maintained at an incubation temperature in an unfavorable growth environment.
Hertman, I. (Israel Institute for Biological Research, Ness Ziona, Israel). Bacteriophage common to Pasteurella pestis and Escherichia coli. J. Bacteriol. 88:1002-1005. 1964.-Phage Y of Pasteurella pestis and phage T(3) of Escherichia coli are serologically related and have common bacterial hosts. Anti-Y serum neutralizes the homologous phage and phage T(3) to a similar extent. Anti-Y serum, absorbed with T(3), still neutralizes the activity of phage Y for P. pestis, but not the activity for E. coli B/r. A mutant of phage Y was isolated from turbid plaques formed on P. pestis at 27 C. This mutant infected P. pestis only if this organism had been grown at 27 C; it lost its infectivity towards E. coli B/r and was not neutralized by anti-T(3) serum. From this phage, additional mutants were isolated on P. pestis, some of which were infective for both P. pestis and E. coli B/r and some of which remained infective for P. pestis only. The reacquired infectivity for E. coli B/r was neutralized by anti-T(3) serum. The name T(3)P is suggested for phage Y.
Baugh, C. L. (Fort Detrick, Frederick, Md.), A. W. Andrews, and M. J. Surgalla. Effects of bicarbonate on growth of Pasteurella pestis. III. Replacement of bicarbonate by pyrimidines. J. Bacteriol. 88:1394-1398. 1964.-The effect of carbon dioxide on the growth of virulent Pasteurella pestis cultures at 37 C with aeration was studied by substituting known products of carbon dioxide fixation for bicarbonate in the test system. The growth of the virulent cells in the inoculum is stimulated and the culture remains virulent, if bicarbonate is replaced by orotic acid. The addition of cytosine, uracil, or citrulline also results in the retention of virulence, but the effect on the growth of the virulent cells is not as pronounced as with bicarbonate or orotic acid. It is proposed that an impaired pyrimidine synthesis due to a deficiency in carbomyl phosphate is responsible for the loss of virulence by P. pestis in aerated broth cultures at 37 C. The carbamyl phosphate deficiency may be enhanced by the loss of metabolically produced carbon dioxide at 37 C.
Montie, Thomas C. (Albert Einstein Medical Center, Philadelphia, Pa.), and Samuel J. Ajl. Selective inhibition by tryptophan analogues of murine toxin synthesis in Pasteurella pestis. J. Bacteriol. 88:1467-1475. 1964.-Washed-cell suspensions of Pasteurella pestis, avirulent strain "Tjiwidej," exhibited a preferential inhibition of toxin synthesis relative to total protein formation, when grown in the presence of various tryptophan analogues. Growth was partially inhibited in the presence of methyl analogues. High concentrations of 5-fluorotryptophan induced slight growth-inhibitory effects. However, toxin production was more sensitive to these levels of the analogue. Growth inhibition appeared not to relate to toxin inhibition. Inhibition of toxin synthesis by analogues was reversed by l-tryptophan and indole. Shikimic acid but not anthranilic acid antagonized the action of 4-methyltryptophan on selective toxin synthesis. The formation of tryptophanless protein accounted for continued protein synthesis in tryptophan-depleted cells. Protein resolved by acrylamide gel electrophoresis from crude cell extracts exhibited two toxic protein bands. The synthesis of one toxin-protein band, the less-mobile of the two, appeared to be associated with the membrane fraction of the cell, and was selectively blocked in cells grown in the presence of tryptophan analogues. Cellular tryptophan levels may determine the quantity and quality of proteins made.
Sawyer, William D. (U.S. Army Medical Unit, Fort Detrick, Frederick, Md.), Joseph V. Jemski, Arthur L. Hogge, Jr., Henry T. Eigelsbach, Elwood K. Wolfe, Harry G. Dangerfield, William S. Gochenour, Jr., and Dan Crozier. Effect of aerosol age on the infectivity of airborne Pasteurella tularensis for Macaca mulatta and man. J. Bacteriol. 91:2180-2184. 1966.-In aging aerosols of Pasteurella tularensis SCHU-S4, the respiratory infectivity for man and Macaca mulatta decreased more rapidly than the viability of the organisms. Infectivity was diminished after 120 min, and was reduced 10-fold after 180 min. These findings confirmed previous observations made in mice and guinea pigs, and also revealed that smaller losses of infectivity were detectable in the primate hosts.
Nutter, J. E. (Fort Detrick, Frederick, Md.), and Q. N. Myrvik. In vitro interactions between rabbit alveolar macrophages and Pasteurella tularensis. J. Bacteriol. 92:645-651. 1966.-Rabbit alveolar macrophages were successfully employed in a study of host cell-Pasteurella tularensis interactions in vitro. Under cell culture conditions in which inhibitory antibiotics were not employed and small infection ratios were used, the relative in vivo virulence of two strains of P. tularensis was duplicated. As a consequence of intracellular multiplication, normal macrophages were killed in relation to the virulence of the strain employed. Alveolar macrophages were also collected from immune rabbits, and macrophage mortality and bacterial growth were significantly suppressed below levels observed with normal macrophage preparations. The effect of immune serum could only be ascribed a minor role in the observed reactions. A marked intravenous toxicity of P. tularensis for the rabbit was observed with both virulent and attenuated strains. The toxicity was possessed only by viable preparations and could be elicited in animals immune to virulent challenge.
Several oxidase reagents and commercial products were evaluated as to their efficacy in detecting oxidase activity in species of the genus Pasteurella. Recommendations are made concerning the reagent of choice for determining oxidase activity in the genus Pasteurella. Recommendations are made also concerning the use of commercial products and their efficacy in detecting oxidase activity in this genus.