Effect of Specific Immune Mouse Serum on the Growth of Salmonella enteritidis in Mice Preimmunized With Living or Ethyl Alcohol-killed Vaccines

The effect of prior opsonization of virulent Salmonella enteritidis on the growth of this organism in blood, liver, spleen, peritoneal cavity, and inguinal lymph node of specific pathogen-free mice prevaccinated with ethyl alcohol-killed S. enteritidis or living S. gallinarum was determined by daily enumeration. Both the vaccines and the challenge inocula were injected by the intravenous, intraperitoneal, or subcutaneous routes to determine the effect of variations in the vaccinating procedure on the level of immunity induced. The survival percentage observed in mice vaccinated with killed organisms varied extensively, depending on the route of challenge. However, simultaneous organ enumeration studies revealed that vaccination with killed organisms failed to prevent the growth of the challenge organism in vivo. On the other hand, virulent S. enteritidis injected into mice vaccinated with living S: gallinarum failed to multiply and was subsequently eliminated. Immunity in these animals was so effective that a subcutaneously injected challenge did not spread beyond the regional node. Immunization with killed organisms slowed but was unable to prevent the spread of such a challenge beyond the draining node involved in the primary immune response. Neither the route of challenge nor the regimen used in the vaccination had any appreciable influence on the level of antibacterial immunity detected in the organs of the reticuloendothelial system at the time of challenge.     

Effect of Specific Immune Mouse Serum on the Growth of Salmonella enteritidis in Nonvaccinated Mice Challenged by Various Routes

Salmonella enteritidis was injected intravenously, intraperitoneally, or subcutaneously into specific pathogen-free mice. The number of organisms in the blood, liver, spleen, peritoneal cavity, and draining inguinal lymph node was determined by daily enumeration. Opsonization of the organism with hyperimmune serum increased the rate of phagocytosis, resulting in rapid blood clearance together with an alteration in the relative numbers of organisms accumulating in the liver and spleen. Serum treatment also brought about a substantial increase in the number of bacteria killed during the first 60 min of the infection. However, the survivors of this initial period of inactivation then multiplied rapidly in the liver and spleen, ultimately resulting in the death of the animal from a generalized infection. Attempts to passively protect mice with hyperimmune serum were uniformly negative. The effects of treatment of the virulent S. enteritidis with hyperimmune serum were consistent with the general thesis that cellular rather than humoral factors play the major role in the expression of an effective antibacterial immunity against salmonella infections.     

Cross-protection against Salmonella enteritidis infection in mice

Mice were vaccinated with six strains of Salmonella and two strains of Escherichia coli, as well as with Pseudomonas aeruginosa, Proteus vulgaris, and Serratia marcescens. The amount of in vivo growth of each organism was followed by viable counting techniques on organ homogenates. The vaccinated mice, along with unvaccinated controls, were challenged intravenously with 1,000 ld(50) of a streptomycin-resistant strain of Salmonella enteritidis. The ability of the vaccine to protect the mice against virulent challenge correlated with the ability of the strain to establish a persisting population in the liver and spleen. Enumeration of the liver and spleen populations in the challenged mice revealed that extensive growth of S. enteritidis occurred in animals which showed "protection," as assessed by progressive mortality data. No evidence was obtained for a major role of humoral factors in the cross-protection against intravenous S. enteritidis challenge.     

Heat-labile antigens of Salmonella enteritidis. II. Mouse-protection studies

Collins, F. M. (University of Adelaide, Adelaide, South Australia), and Margaret Milne. Heat-labile antigens of Salmonella enteritidis. II. Mouse-protection studies. J. Bacteriol. 92:549-557. 1966.-A number of extracts prepared from a virulent and an avirulent strain of Salmonella enteritidis were used to immunize mice. Living and alcohol-killed whole-cell vaccines were also used to compare the relative protective value of the various preparations. All mice were challenged intravenously with 100 to 1,000 ld(50) of S. enteritidis. Daily counts of the liver, spleen, and blood populations of vaccinated and control mice revealed that the challenge organism was rapidly eliminated only in those mice which had been immunized with a living vaccine. Immunization with extracts resulted in rapid clearance of S. enteritidis from the blood, but, after a delay of 24 to 48 hr, the bacterial populations increased until a maximal liver and spleen population of approximately 5 x 10(8) was reached. Between 55 and 100% of the immunized animals died, compared with 95 to 100% of the controls. With all four extracts, it was only the first antigenic fraction eluted from diethylaminoethyl cellulose which had any detectable effect on host resistance. The ineffectiveness of vaccines prepared with the various extracts or with whole killed bacteria relative to the protection observed after immunization with living organisms is discussed.     

Cross-protection aginst Salmonella enteritidis infection in mice. I. Immunization trials.

Mice were immunized subcutaneously with live and killed vaccines, with and without complete adjuvant incorporating Salmonella typhi-murium M206, Salmonella gallinarum 9R, Salmonella pullorum Sp223 as well as homologous Salmonella enteritidis Se795. The animals were challenged 21 days post-vaccination with 100 LD50 of virulent S. enteritidis 5694 SMR subcutaneously along with unvaccinated control mice. To assess the immunity against acute and chronic infections, the percentage of absolute survivors i.e. survivors without lesions and without the challenge organism, was taken as the criterion. Live vaccines proved better than killed vaccines. Live vaccines with complete adjuvant induced a good protection. Cross-protection could be induced with the live vaccine with complete adjuvant against S. enteritidis infection in mice.     

Egg contamination by Salmonella serovar enteritidis following vaccination with Delta-aroA Salmonella serovar typhimurium

The efficacy of an aroA Salmonella serovar typhimurium modified live vaccine to decrease internal egg contamination after oral challenge of hens with egg-contaminating Salmonella serovar enteritidis was assessed. Challenge was with a mixed phenotype of S. enteritidis that had virulence characteristics previously associated with enhanced oral invasiveness and egg contamination in chickens. Immunized birds had fewer positive ovary/oviduct pools and lower cfu g(-1) cecal contents than did non-immunized birds, but the differences were not significant. The number of positive intestinal (duodenum, jejunum, ileum) and organ (spleen, kidney, liver) pools following challenge from each treatment group were equivalent. Most importantly, immunization did not decrease egg contamination. These results suggest that the ability of modified live vaccines to reduce internal egg contamination by S. serovar enteritidis can be assessed using characterized strains for challenge.     

Cross-protection against Salmonella enteritidis infection in mice. III. Delayed hypersensitivity reaction and clearance of the challenge organism.

Mice were immunized with live vaccines and with live vaccines with complete adjuvant incorporating Salmonella enteritidis, Salmonella typhi-murium, Salmonella gallinarum or Salmonella pullorum. On the 21st day after vacination, the hypersensitivity reactions elicited by the mice to extracts of the challenge organism (S. enteritidis 5694 SMR) were assessed. The degree of delayed hypersensitivity reaction was compared with the level of protection induced by the vaccine. The role in protection of delayed hypersensitivity is discussed. Clearance of the challenge organism from the liver of previously vaccinated and unvaccinated mice was assessed quantitatively.     

Activation of mouse peritoneal macrophages during infection with Salmonella typhimurium does not result in enhanced intracellular killing

Our study was performed to investigate whether macrophages become activated during an infection with Salmonella typhimurium and, if so, whether these activated macrophages kill S. typhimurium faster than resident macrophages. Mice received i.v. injections with a sublethal number of S. typhimurium; on about day 12 of the infection the numbers of bacteria in the liver and the spleen were maximal. During the infection, activation of peritoneal macrophages could be demonstrated on the basis of three criteria, i.e., the ability to inhibit the proliferation of Toxoplasma gondii, an enhanced production of H2O2 and an increased expression of Ia Ag. The rate of in vitro intracellular killing of S. typhimurium by these activated macrophages was not increased compared to that for resident macrophages. To determine the growth of S. typhimurium in activated mice a nalidixic acid-resistant mutant strain, called S. typhimurium 510R, was used. The net growth rates of the mutant S. typhimurium 510R in the spleen of S. typhimurium 510-activated and normal mice were similar. However, in the liver of S. typhimurium 510-activated mice the number of S. typhimurium 510R did not change during 3 to 48 h after injection. The role of specific antibodies during the initial phase of the infection was negligible, because only low levels of antibodies were detected during the first 15 days of infection and the growth rates of S. typhimurium 510 in the spleen and liver of mice with high titers of antibodies were not significantly different from the rates in normal mice. The results of this study demonstrate that although macrophages become activated during an infection with S. typhimurium, these cells do not display an enhanced bactericidal activity in vitro and in vivo no significant effect on the growth rate of S. typhimurium in the spleen and a bacteriostatic effect in the liver is found. Hence macrophage activation is probably not very important in the host defense against S. typhimurium.     

Specificity of Salmonella porin as an eliciting antigen for cell-mediated immunity (CMI) reaction in murine salmonellosis

The specificities of Salmonella porin on elicitations of delayed-type hypersensitivity (DTH) reaction and interleukin-2 (IL-2) production in BALB/c mice immunized with Salmonella typhimurium were examined. Only porin from S. typhimurium was capable of eliciting significant levels of DTH and IL-2 production in S. typhimurium-immunized mice, whereas no significant DTH and IL-2 production were induced by porin from Salmonella enteritidis or Escherichia coli. Our observations suggested that Salmonella porin was a serovar-specific antigen for the elicitation of cell-mediated immunity (CMI) in salmonellosis.     

Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypes

The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella.     

Protective effect of recombinant tumor necrosis factor-alpha in murine salmonellosis

The enhancement of resistance by i.p. administration of murine rTNF-alpha into mice against i.p. challenge with virulent Salmonella typhimurium was studied. Administration of TNF-alpha (5 x 10(4) U/mouse) into mice at 6 or 12 h before the challenge with S. typhimurium organisms enhanced the bactericidal capacity in the peritoneal cavities of the mice. The diminution of the infecting organism in the peritoneal cavities of the TNF-alpha-treated mice was not due to the systemic spread of the organism inasmuch as few organism were recovered from other areas such as the spleen and liver. The TNF-alpha treatment effected a slight increase of neutrophils in the peritoneal cavity, but did not effect an increase of macrophages, including Ia-bearing macrophages. The survival rate of mice infected with Salmonella was improved by the i.p. injection of TNF-alpha before infection. Co-administration of smaller doses of TNF-alpha (5 x 10(3) U) and murine rIFN-gamma (10(2) U) at 6 h before the challenge also effectively enhanced bactericidal activity and protectivity. The cooperative effect of TNF-alpha and IFN-gamma was only seen when these recombinant cytokines were injected together at the proper time before the challenge. Injection of rabbit anti-TNF-alpha serum abolished the effects of TNF-alpha and the cooperative effect of TNF-alpha and IFN-gamma. Furthermore, the serum could abolish the cooperative effect of IFN-gamma and LPS on bactericidal activity, suggesting participation of LPS-induced TNF-alpha in the cooperation.     

Separate transfer of mouse protection and delayed-type hypersensitivity with Salmonella typhimurium transfer factor

Delayed-type hypersensitivity (DTH) induced with Salmonella typhimurium transfer factor (TF) contributed to an increase in mean survival days of mice challenged with homologous organisms and afforded only a low level of host protection as determined by survival rate, compared with that obtained by active immunization. TF of other enteric bacteria could transfer DTH which is cross-reactive to salmonella antigen but did not afford host protection. Although TF of Listeria monocytogenes did not transfer the cross-reactive DTH, it could confer the significant increase in mean survival days against the lethal challenge with S. typhimurium. Listerial ribosomal vaccine conferred the high level of mouse protection without inducing DTH to salmonella antigen. The resistance generated upon active immunization with listerial ribosomal vaccine could be enhanced by the injection of S. typhimurium TF to the same level as that obtained after immunization with homologous ribosomal vaccine. Among salmonella TF, there could be no cross-reactive immunity between S. typhimurium and S. choleraesuis, although the cross-reactive DTH was observed. The DTH transfer ability of TF was sensitive to Pronase which could not affect the ability to transfer host immunity, but RNase could abolish the ability to transfer host immunity without impairing DTH transfer activity. These results suggest that in mouse typhoid infection, DTH is not associated with host protection as determined by survival rate.     

Type 1 fimbriae of Salmonella enteritidis.

Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21.     

Inhibition of the growth of Lewis lung carcinoma in syngeneic mice by Salmonella antigens.

Administration of a protein antigen preparation from Salmonella enteritidis 11RX at the site of challenge with Lewis lung carcinoma inhibited tumour development in mice previously immunised with live bacteria, but not control mice. The kinetics of tumour growth indicated that the inhibition of tumour development was due to a reduction of the initial tumour inoculum rather than the development of specific anti-tumour immunity.     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.     

The involvement of tumor necrosis factor in immunity to Salmonella infection

The role of TNF in immunity to Salmonella in mice was studied. Antiserum specific for murine TNF was raised and used to neutralize TNF activity in vivo. Injection of this serum into mice infected with the moderately mouse virulent Salmonella typhimurium strain M525 caused exacerbation of disease. Such treatment had no effect on the course of an infection with an attenuated S. typhimurium aroA (strain SL3261) mutant. However, the protection afforded by immunisation with live SL3261 against challenge with the virulent parent strain (SL1344) was abolished by anti-TNF antiserum. Interestingly both early (3 wk) immunity and late (10 wk) immunity was neutralized by such treatment. Inasmuch as early immunity is considered to be nonspecific and macrophage-mediated while late immunity is considered to be serotype-specific and T cell mediated, this suggests that TNF plays a role in protection from Salmonellosis in both cases.     

Salmonella typhimurium and Salmonella enteritidis induce gut growth and increase the polyamine content of the rat small intestine in vivo

Abstract The effects of infection by Salmonella enteritidis and S. typhimurium on the small and large intestines, liver, spleen and mesenteric nodules of rats were studied in vivo. Both Salmonella serotypes persisted and proliferated in the gastrointestinal tract and invaded sub-epithelial tissues, mainly the ileum, leading to the systemic distribution of these pathogens. Coincidental with the infection, the rate of crypt cell proliferation increased resulting in substantial growth of the small intestine. The extent of this and the accompanying accumulation of polyamines was particularly dramatic in the ileum where there was also some disruption of the villus epithelium. It is possible that these effects of the infection on the metabolism and morphology of the small bowel, which strongly resembled the changes induced by some plant lectins, may facilitate the colonisation and invasion of the gut by Salmonellae .     

Increased resistance to Salmonella infection of hypoferremic mice fed a low-protein diet

BALB/c and CBA/CA mice fed a protein-deficient diet developed a plasma hypoferremia corresponding to a 30 percent lowering of serum iron concentration. This hypoferremia persisted as long as the diet was maintained. Hypoferremic CBA/CA mice had increased resistance to Salmonella typhimurium C5 infection, as shown by the reduced lethal activity and the decreased growth of the bacteria in the spleen and in the peritoneal exudate of the deficient animals. This induced resistance was abolished after injection of iron or Desferal into the restricted animals. Such resistance was not observed with BALB/c mice fed a protein-deficient diet, in spite of the plasma hypoferremia. The growth of S. typhimurium C5 in the spleen and in the peritoneal exudate of these animals did not differ from the growth observed in control animals fed a protein-sufficient diet. This study suggests that hypoferremia induced by a protein-deficient diet is probably involved in the enhancement of resistance of CBA/CA mice to Salmonella infection, and that the phenomenon is host-strain dependent.     

Survival of Salmonella enteritidis PT4 and Salm. typhimurium Swindon in aerosols

A.S. McDERMID AND M.S. LEVER. 1996. Small particle aerosols of plate-grown Salmonella enteritidis and Salm. typhimurium were generated and maintained within a rotating drum at 75% relative humidity and 24°C for 2 h. Plate-grown organisms were found to be more aerosol-stable than broth-grown organisms. Differences were observed between the two species; plate-grown Salm. typhimurium retained 100% viability after 2 h compared to approximately 70% for plate-grown Salm. enteritidis . A large proportion of cells of both serotypes remained viable in aerosols after 2 h, confirming the potential for airborne transmission for these organisms, e.g. within henhouses and during food     

Enhancing effects of mini-cells prepared from Salmonella typhimurium on anti-tumor immunity in sarcoma 180-bearing mice

Summary The enhancing effect of mini-cells of Salmonella typhimurium which do not contain chromosomal DNA on anti-tumor immunity in mice was studied. The growth of sarcoma 180 cells which were subcutaneously transplanted into ICR mice was significantly retarded in mice treated with Salmonella mini-cells at the same time or 7 days after S180 transplantation, while no or only a little growth inhibition was observed in mice treated 7 days prior to S180 transplantation. Treatment with mini-cells inoculation alone did not increase the survival time of mice that had received intraperitoneal transplants of S180 cells. However, a statistically significant increase of survival time was observed in mice treated with a combination of mini-cells and surgical resection of subcutaneous tumors when S180 cells were injected 7 days after the surgical resection. The injection of mini-cells restored macrophage chemotaxis in S180-bearing mice in which macrophage chemotaxis was greatly retarded but lymphocyte activity was not.