Toxicity of Pasteurella tularensis killed by ionizing radiation

Approximately 2 x 10(11) viable Pasteurella tularensis cells per ml, contained in suspensions, were killed by exposure to 10(6) r of gamma-radiation. When injected intraperitoneally into mice, the irradiated suspensions initially contained about 10 ld(50) per ml, and immunized mice against challenge with fully virulent strains of P. tularensis. Toxicity and immunizing activity of the suspensions decreased significantly within a few days at 5 C. Mice were protected against the toxin by immune serum or by prior injection of endotoxin of Escherichia coli. Cortisone did not protect against the newly prepared suspension, but was effective against the aged suspension. Lethal doses of newly prepared suspension for guinea pigs and rabbits were approximately 0.5 ml and 2 ml, respectively. Cortisone protected rabbits, but not guinea pigs, against lethal challenge. Pyrogenic effects resembling those shown by endotoxin-containing suspensions were demonstrated in rabbits. The results suggested that two toxins are responsible for the toxicity of irradiated suspensions of P. tularensis: one labile and associated with the immunizing activity of the suspension, the other more stable and resembling classical endotoxin.     

Enhancement activity of anti-brucella sera in experimental Brucella abortus infection in mice.

The passive transfer of rabbit anti-brucella sera promotes the multiplication of Brucella abortus in the spleen of mice infected intravenously with a low dose of this bacteria. The enhancement activity of the anti-brucella sera is related to their modes of preparation in rabbits. The agglutinin titers of all these antisera were relatively high, while there was some discrepancies in their content in haemagglutinating, immune adherence and complement fixing antibodies.     

Differential affinity of pathogenic species of microorganisms for a set of lectins detectable by the sandwich method using fluorescein isothiocyanate (FITC)

The qualitative differences in the affinity of concanavalin A (Con A), wheat-germ agglutinin (WGA) and Phaseolus vulgaris lectin to the surface of 10 microbial strains inducing various diseases in humans and agricultural animals have been demonstrated by means of the indirect immunofluorescence tests. Enterobacteria, Coxiella burnetii and Bacillus anthracis have been found to possess pronounced affinity to Con A and WGA, while Rickettsia prowazekii, Francisella tularensis and Brucella abortus, as well as Treponema pallidum, have proved to be resistant to lectins. WGA has been found to bind specifically to Brucella abortus and Treponema pallidum. Con A and WGA are seemingly suitable for use in the preliminary test for the total capacity of lectin receptors to come in contact with biological macromolecules.     

In vitro interactions between rabbit alveolar macrophages and Pasteurella tularensis

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.     

Antigens of Pasteurella tularensis: Preparative Procedures

Ether-water (EW) extraction of Pasteurella tularensis produced better antigens than five other chemical procedures. EW extracts produced from stationary-phase, liquid-grown, saline suspensions of strain SCHU S4 cells regularly induced agglutinin and precipitin formation in rabbits. Mice, guinea pigs, and monkeys also responded to EW extracts but with lower antibody levels. The use of strains of lower virulence, acetone-dried cells, organisms grown on a solid medium, and abbreviated extraction conditions all resulted in extracts with a diminished antigenicity, but logarithmic-phase and stationary-phase cells yielded equivalent EW extracts. The use of adjuvant, hyperimmunization, and large doses of antigen increased the precipitin responses of rabbits without appreciably altering the agglutinin response. By the appropriate combination of centrifugal fractionation of EW extracts, use of adjuvant, and vaccination schedule, rabbit antisera with either predominantly agglutinating or precipitating activities were obtained.     

Endogenous gamma interferon and interleukin-10 in Brucella abortus 2308 infection in mice

Abstract CD-1 mice intravenously infected with the virulent Brucella abortus 2308 strain simultaneously produce significant levels of gamma interferon (IFN-γ) and interleukin-10 (IL-10) in their spleens between the second and eighth day post-infection with no production of interleukin-4 (IL-4). Endogenous synthesis of IL-10 does not affect the production of IFN-γ in this organ, while the production of both cytokines during this period of time is accompanied by a statistically significant increase ( P < 0.001) in the number of colony forming units (cfu) of B. abortus 2308 present in the organ. These findings suggest that although the endogenous synthesis of IL-10 apparently does not affect IFN-γ production, it may affect the effector functions of macrophages to control intracellular brucellae. Production of the Th1 cytokine IFN-γ during B. abortus 2308 infection is also associated with a specific IgG3 and IgG2a response against the B. abortus 2308 lipopolysaccharide (S-LPS) antigen.     

Survival of Airborne Pasteurella tularensis at Different Atmospheric Temperatures

The aerosol survival, recovery, and death rate of Pasteurella tularensis SCHU S5 disseminated in particle sizes of 1 to 5 mum were significantly affected by air temperature. The highest aerosol recovery of viable P. tularensis was observed within -7 and 3 C; the recovery decreased significantly below and above this temperature range. The death rate of airborne P. tularensis was not significantly influenced by an increase in temperature from -40 to 24 C. However, a progressive increase in atmospheric temperature from 24 to 35 C resulted in increased death rates; thus, a linear relationship appeared to be present between the temperature and death rates. At 49 C, the recoveries of viable airborne P. tularensis were significantly lower and the death rates were higher than at the other temperatures.     

Interaction of S- and R-lipopolysaccharides of Francisella tularensis with lypopolysaccharide-binding protein of human serum

Investigation of ability of Francisella tularensis S- and R-lypopolysaccharide (LPS) preparations as well as the live bacteria with different chemotypes to interact with human lypopolysaccharide-binding protein (LBP) was carried out. It was found that LPS preparations derived from virulent(S-LPS) or isogenic avirulent mutant (R-LPS) strains of F. tularensis had markedly lower affinity to LBP as compared with typical S-LPS of Salmonella abortus and R-LPS of Yersinia pestis. It was shown that R-LPS preparation from avirulent mutant binds LPB more effectively than S-LPS from F. tularensis virulent strain. Differences in S- and R-LPS affinity were also confirmed for LPS represented by the live cells. Thus, bacteria with S-chemotype of LPS (F. tularensis 15/10) bound only 20.3% of LBP, whereas cells with R-LPS (F. tularensis 543 cap(-)) bound 39.9%. Such pattern was observed in experiments with both normal non-immune human serum and sera from people immunized with live tularemia vaccine. The latter indicates that opsonization of LPS by specific antibodies does not change its affinity to LBP. The observed more efficient binding of avirulent strain R-LPS to LBP is likely determines the more intensive host response directed to destruction and rapid elimination of the causative agent. At the same time, weak affinity of the vaccine and virulent strains S-LPS to LBP probably allows the bacterium to avoid activation of host defense mechanisms thus contributing to its long-term persistence in microorganism and development of specific immunity against tularemia.     

The use if Francisella tularensis lipopolysaccharide in the dot solid phase enzyme immunoassay

To determine antitularemia antibodies in the sera of humans and animale, the possibility of using dot immunoassay with the use of F. tularensis lipopolysaccharide (LPS) as antigen-containing preparation was ascertained. Experiments demonstrated that this method made it possible to determine specific antitularemia antibodies in the sera of sick and immunized humans and animals. Investigetions carried out with the use of heterologous antisera to F. novicida, F. novicida-like and F. philomiragia, as well as Brucellf abortus, Vibrio cholerae and Yersinia enterocolitica, revealed that F. tularensis S-LPS was highly specific. The results obtained in this investigation are indicative of good prospects of using F. tularensis LPS in dot blotting for the laboratory diagnostics of tularemia in humans.     

Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens

Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.     

Immunosuppressive Activity of the 3a-1 Fraction of Papain

The maximal nonlethal dose of the 3a-1 fraction of papain, determined by use of 9-week-old white albino rabbits, was 10 mg per injection, administered intravenously. The immunosuppressive activity of the 3a-1 fraction of papain was studied by its inhibition of sheep red blood cell hemolysin, bacterial agglutinin, and horse serum precipitin. Immune suppression was observed when papain injections preceded antigen injections by 12 to 18 hr. The enzyme preparation was analyzed by use of cellulose acetate and starch-gel electrophoresis and by the micro-Kjeldahl method. Starch-gel electrophoresis of serum samples revealed qualitative alterations in the alpha(-1) region 7 hr after papain administration. Changes were also observed in the urinary aminopolysaccharide content.     

Species- and genus-specific antigenic epitopes of Francisella tularensis lipopolysaccharides

Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.     

Polynucleotide Homologies of Brucella Deoxyribonucleic Acids

Deoxyribonucleic acids (DNA's) extracted from organisms presently placed in the genus Brucella (B. abortus, B. melitensis, B. neotomae, and B. suis) possessed very similar polynucleotide sequences. Unlabeled, single-stranded DNA fragments from B. abortus, B. melitensis, B. neotomae, and B. suis were equally effective in competing with the interaction of corresponding radiolabeled, single-stranded DNA fragments with their homologous DNA-agars. Unlabeled fragments of B. ovis, however, did not compete as effectively as the homologous, unlabeled DNA's, and this organism, therefore, had a detectably different polynucleotide composition. The mole percentages of guanine plus cytosine in Brucella DNA's (56 to 58%) were also similar. DNA's from Francisella tularensis, Escherichia coli, and the slow loris did not compete.     

Optimization of production of Brucella abortus S19 culture in bioreactor using soyabean casein digest medium

A method of cultivating Brucella abortus S19 culture in bioreactor was attempted using three different media. Culture conditions in bioreactor were optimized by varying agitation and aeration parameters. Varying the aeration ranging from 0.5 vvm to 0.8 vvm and agitation rate ranging from 250 rpm to 400 rpm during bacterial growth was found to yield highest viable count within 48 hours of culture period. A count of > 1 x 10(11) CFU per ml within 48 to 60 hours post seeding was obtained consistently in all five consecutive batches (P > 0.05) with 6 x 10(11) CFU per ml being the maximum yield when the organism is grown in soyabean casein digest medium. B. abortus S19 maintained its smooth characteristics throughout its growth in bioreactor. The vaccine prepared with soyabean casein digest medium was found to be potent and safe with a protective index of 3.33 in mice. The vaccine was tested in 10 cattle calves of 3 to 13 months age and all the vaccinated animals were seropositive on 28, 60, 90, 120 and 150 days post-vaccination when analyzed by fluorescence polarization assay (FPA).     

Brucella abortus strain RB51 vaccination in elk. II. Failure of high dosage to prevent abortion

Brucella abortus strain RB51 is used as a vaccine because it induces antibodies that do not react on standard serologic tests for brucellosis allowing differentiation between vaccination and infection. Strain RB51 was evaluated in captive elk (Cervus elaphus) to determine if vaccination protected against abortion following experimental challenge. Thirty elk were vaccinated intramuscularly with 1.0 x 10(10) colony-forming units (CFU) of strain RB51 in March 1998. Fourteen of these were given a booster dose of 1.13 x 10(10) CFU exactly 1 yr later. All vaccinated elk seroconverted via a modified dot blot assay to strain RB51 with the booster group having higher titers (P < or = 0.001). Seventeen other elk served as unvaccinated controls. All elk were bred and determined pregnant using pregnancy-specific protein B analysis. Elk were challenged in March 2000 with 1.1 x 10(7) CFU of B. abortus strain 2308 administered intraconjunctivally and all elk seroconverted to strain 2308. Fifteen of 17 control elk aborted; 16 of 16 elk given a single vaccination aborted (P = 0.44); and 13 of 14 elk given a booster aborted (P = 0.86). There were two viable calves in the control group and one in the booster group. Strain 2308 was recovered from fetuses and nonviable calves in all groups. Based on the results of this and other studies, the use of strain RB51 to prevent abortion in elk cannot be recommended.     

Evaluation of Media for Differentiating Nonfermenting Gram-negative Bacteria of Medical Significance

The response of the rabbit to viable or killed whole-cell Pasteurella tularensis vaccines was studied. The most practical preparation for the production of anti-P. tularensis antibodies was viable organisms of the live vaccine strain (LVS). The intravenous route of administration proved superior to either the subcutaneous or intradermal routes, and incorporation of LVS into Freund's adjuvants did not result in increased levels of antibody. Short-term hyperimmunization, three injections at weekly intervals, constituted the most efficient method for increasing levels of the antibodies.     

Serological prevalence of tularemia in cottontail rabbits of southern Illinois

Sera of cottontail rabbits (Sylvilagus floridanus) collected in southern Illinois in 1983 and 1984 were screened for the presence of antibodies against Francisella tularensis by rapid slide agglutination and enzyme linked immunosorbent assay techniques; 6% of 118 and 16% of 119 samples were positive by these methods, respectively. Rabbits gained, lost and maintained titers over at least an 8 mo period. Francisella tularensis tularensis was isolated from one serologically negative, clinically healthy rabbit.     

Isolation of a Francisella tularensis mutant that is sensitive to serum and oxidative killing and is avirulent in mice: Correlation with the loss of MinD homologue expression

Abstract We constructed mutant strains of Francisella tularensis biotype novicida by insertional mutagenesis with a kanamycin resistance (Km^R) cassette. One mutant, KEM7, was defective for survival in macrophages in comparison with the wild-type (WT) strain and a random insertion strain, KEM21. While all three strains exhibited intracellular growth, the number of viable KEM7 present after 24–48 h of infection was approximately 10 times less than that of WT or KEM21. This observation was apparently due to a reduced number of viable KEM7 associated with the macrophages one hour after phagocytosis. KEM7 was approximately 3 times more susceptible than WT or KEM21 to killing by the products of the xanthine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also found to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 1000- to 10 000-times less than that of either KEM21 or WT. DNA sequence analysis at the Km^R insertion site suggested that the F. tularensis homologue of min D had been interrupted. Western immunoblot analysis confirmed the presence of a MinD homologue in F. tularensis WT and KEM21, and demonstrated its absence in KEM7.     

Safety of Brucella abortus strain RB51 in bull elk

Some of the elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (Wyoming, Idaho, Montana; USA) are infected with Brucella abortus, the bacterium that causes bovine brucellosis. Brucella abortus strain RB51 vaccine is being considered as a means to control B. abortus induced abortions in cow elk. However, the most probable vaccination strategies for use in free-ranging elk might also result in some bull elk being inoculated, thus, it is important to insure that the vaccine is safe in these animals. In the winter of 1995, 10 free-ranging bull elk calves were captured, tested for B. abortus antibodies, and intramuscularly inoculated with 1.0 x 10(9) colony forming units (CFU) of B. abortus strain RB51. Blood was collected for hemoculture and serology every 2 wk after inoculation for 14 wk. Beginning 4 mo postinoculation and continuing until 10 mo postinoculation elk were serially euthanized, necropsied, and tissues collected for culture and histopathology. These elk cleared the organism from the blood within 6 wk and from all tissues within 10 mo. No lesions attributable to B. abortus were found grossly and only minimal to mild lymphoplasmacytic epididymitis was found in a few elk on histologic examination. In a separate study, six adult bull elk from Wind Cave National Park (South Dakota, USA) were taken to a ranch near Carrington (North Dakota, USA). Three were orally inoculated with approximately 1.0 x 10(10) CFU of RB51 and three were inoculated with corn syrup and saline. Ninety days post-inoculation semen was examined and cultured from these bulls. Strain RB51 was not cultured from their semen at that time. There were no palpable abnormalities in the genital tract and all elk produced viable sperm. Although they contain small sample sizes, these studies suggest that B. abortus strain RB51 is safe in bull elk.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.