Role of peritoneal macrophages in the development of an experimental Salmonella infection

The effect produced on the course of Salmonella infection in mice by the removal of peritoneal macrophages with agarose has been studied. Peritoneal macrophages have been shown to control the multiplication of faintly virulent and virulent S. typhimurium strains in the spleen of mice. In immune mice the elimination of the virulent strain of the causative agent of superinfection may occur without the control of peritoneal macrophages.     

Correlation of in vitro properties of Rhodococcus (Corynebacterium) equi with virulence for mice

To study the virulence of Rhodococcus (Corynebacterium) equi, seven ATCC strains of different serotypes were tested for their LD50 in mice, clearance of the organism from the lungs and spleen following intravenous or intratracheal inoculation, and in vitro interaction with murine peritoneal macrophages. Strains ATCC 33704 and 33705 were virulent for mice and multiplied in the lungs and spleen, resulting in death of the animal in 5 days. The other five strains were avirulent for mice. The number of bacteria in the lungs and spleen of mice given these five strains decreased immediately. Pulmonary clearance of strains ATCC 33703, 33706, and 33707 was significantly more rapid than that of the virulent strains ATCC 33704 and 33705 12 hr after inoculation. Complete clearance of the avirulent strain ATCC 33707 occurred by day 14, while that of virulent ATCC 33704 and 33705 strains occurred by day 30. The virulent strains ATCC 33704 and 33705 were resistant not only to phagocytosis but also to intracellular killing by macrophages. Strains ATCC 33702 and 33706 were rapidly killed by macrophages although they were rather resistant to phagocytosis. Strain ATCC 33703 was easily phagocytized though resistant to killing by macrophages. The most avirulent strains, ATCC 33707 and 6939, were easily phagocytized and rapidly killed by macrophages. These results indicate that virulence appeared to be related to the ability of the organisms to resist clearance from the lungs and spleen and to resist phagocytosis and intracellular killing by macrophages.     

Evaluation of the Lon-deficient Salmonella strain as an oral vaccine candidate

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.     

Increased sensitivity of immune macrophages to the cytotoxic action of virulent shigellae

The in vitro interaction of live bacteria belonging to virulent and avirulent Shigella and Salmonella strains with peritoneal macrophages obtained from mice immunized by the intragastric administration of these bacteria has been studied. In contrast to Salmonella-activated macrophages capable of resisting the intracellular proliferation and the cytopathic action of homologous bacteria, Shigella-activated macrophages become more sensitive to the cytopathic action of virulent shigellae. The ability of shigellae to render an aggravating cytopathic effect on the activated macrophages correlates with the virulence of dysentery bacilli and is practically absent in avirulent strains, including S. flexneri 2a No. 516 M vaccine strain.     

Differential expression of IL-1 and TNF receptors in murine macrophages infected with virulent vs. avirulent Legionella pneumophila

Infection of macrophages from genetically susceptible A/J mice with Legionella pneumophila induces high levels of various cytokines in serum as well as in cultures of spleen or peritoneal cells from the mice. However, modulation of receptor expression for these cytokines during infection has not been studied in detail, even though these receptors on macrophages have a critical role in inflammatory responses during the infection. In the present study, the differential expression of mRNA for TNF and IL-1 receptors as well as receptor antigens during infection of macrophages with virulent vs. avirulent L. pneumophila was investigated. Mouse thioglycollate-elicited peritoneal macrophages showed by RT-PCR constitutive steady-state levels of mRNA for TNF-type I and -type II receptors as well as IL-1 type I receptor. However, IL-1 type II receptor mRNA was not expressed in thioglycollate-elicited macrophages. Infection of macrophages with virulent bacteria caused an upregulation of IL-1 type I and TNF type I receptor mRNA, but had no effect on TNF type II receptor message. Avirulent L. pneumophila infection caused much less induction of these receptor mRNAs. The amount of receptor antigen of IL-1 type I on the surface of macrophages was also increased by infection with virulent L. pneumophila determined by flow cytometric analysis. These results indicate that L. pneumophila infection not only causes induction of various cytokines, but also modulation of certain cytokine receptors, which may regulate the susceptibility to infection.     

The relative efficacy of different strain combinations of lactic acid bacteria in the reduction of populations of Salmonella enterica Typhimurium in the livers and spleens of mice

Multispecies probiotics have been reported to be more effective than monostrain probiotics in health promoting for the host. In this study, 12 lactic acid bacteria (LAB) strains were selected based on the level of induction of tumor necrosis factor (TNF)-α in RAW 264.7 macrophage cells. Their adherence to Caco-2 cells and inhibitory effects on Salmonella invasion of Caco-2 cells were compared. Strains with different probiotic properties were then combined and BALB/c mice were fed with LAB strains for 63 days; then the mice were challenged with Salmonella on day 64. For Salmonella-unchallenged mice that received a multistrain combination of LAB strains that have greater TNF-α production in macrophages, greater adherence and inhibit Salmonella invasion of Caco-2 cells to a greater extent, their peritoneal macrophages had greater phagocytic activity. For Salmonella-challenged mice, a significant reduction of Salmonella cells in the livers and spleens of the mice was observed 8 days post challenge. The addition of 12% skim milk powder together with LAB strain combinations significantly enhanced the reduction of Salmonella cells in the mice livers and spleens. In conclusion, we have shown that LAB strain combinations with particular probiotic properties when fed to mice can inhibit Salmonella invasion of the liver and spleen.     

Human antigen-specific IgA responses in blood and secondary lymphoid tissue: an analysis of help and suppression

Immunization with a virulent Salmonella typhimurium, strain SL3235, has been found to provide high levels of protection against challenge with virulent Salmonella in hypersusceptible mouse strains in the C3H lineage. These mouse strains include the lipopolysaccharide-hyporesponsive C3/HeJ mouse and the closely related but lipopolysaccharide-responsive C3HeB/FeJ mouse. To assess the role of cellular immunity in the protection elicited by this attentuated organism, delayed-type hypersensitivity (DTH) was measured in these mouse strains and in inherently resistant mice. Of the mouse strains tested, only the inherently resistant CD-1 and C3H/HeNCrlBR mice developed significant DTH responses, as assessed by footpad swelling tested at various times after immunization with SL3235. The hypersusceptible C3H/HeJ and C3HeB/FeJ mice failed to exhibit significant DTH responses despite their high levels of immunity.     

Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis.

The ability of macrophages to release cytokines is crucial to the host response to intracellular infection. In particular, macrophage-derived TNF plays an important role in the host response to infection with the intracellular pathogen Mycobacterium tuberculosis. In mice, TNF is indispensable for the formation of tuberculous granulomas, which serve to demarcate the virulent bacterium. TNF is also implicated in many of the immunopathological features of tuberculosis. To investigate the role of TNF in the local immune response, we infected human alveolar macrophages with virulent and attenuated mycobacteria. Infection with virulent strains induced the secretion of significantly higher levels of bioactive TNF than attenuated strains correlating with their ability to multiply intracellularly. Treatment of infected macrophages with neutralizing anti-TNF Abs reduced the growth rate of intracellular bacteria, whereas bacterial replication was augmented by addition of exogenous TNF. Infected and uninfected macrophages contributed to cytokine production as determined by double-staining of M. tuberculosis and intracellular TNF. The induction of TNF by human alveolar macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism of human pathogens.     

Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection

Salmonella enterica serovar Typhimurium (S. typhimurium) infects a wide variety of mammalian hosts and in rodents causes a typhoid-like systemic disease involving replication of bacteria inside macrophages within reticuloendothelial tissues. Previous studies demonstrated that the mig-14 and virK genes of Salmonella enterica are important in bacterial resistance to anti-microbial peptides and are necessary for continued replication of S. typhimurium in the liver and spleen of susceptible mice after orogastric inoculation. In this work we report that inflammatory signalling via interferon-gamma (IFN-gamma) is crucial to controlling replication of mig-14 mutant bacteria within the liver and spleen of mice after oral infection. Using a Salmonella persistence model recently developed in our laboratory, we further demonstrate that mig-14 contributes to long-term persistence of Salmonella in the spleen and mesenteric lymph nodes of chronically infected mice. Both mig-14 and virK contribute to the survival of Salmonella in macrophages treated with IFN-gamma and are necessary for resistance to cathelin-related anti-microbial peptide (CRAMP), an anti-microbial peptide expressed at high levels in activated mouse macrophages. We also show that both Mig-14 and VirK inhibit the binding of CRAMP to Salmonella, and demonstrate that Mig-14 is an inner membrane-associated protein. We further demonstrate by transmission electron microscopy that the primary locus of CRAMP activity appears to be intracytoplasmic, rather than at the outer membrane, suggesting that Mig-14 may prevent the penetration of the inner membrane by CRAMP. Together, these data indicate an important role for mig-14 in anti-microbial peptide resistance in vivo, and show that this resistance is important to the survival of Salmonella in systemic sites during both acute and persistent infection.     

Poor survival but high immunogenicity of IL-2-expressing Salmonella typhimurium in inherently resistant mice

An IL-2-expressing, attenuated strain of Salmonella typhimurium (strain GIDIL2) was previously shown to survive poorly and to have lower immunogenicity in susceptible mice than its parental, non-cytokine-expressing strain (designated BRD509). In the present study, we compared the immune responses induced by both bacterial strains in inherently Salmonella-resistant C3H/HeN mice. Analysis of the bacterial loads in the peritoneum and spleen revealed that colony-forming units (CFUs) of GIDIL2 were consistently lower than the corresponding BRD509 CFUs. As early as 48 h after inoculation, there were 60-fold lower CFUs of GIDIL2 than BRD509 organisms in the peritoneal cavity. Similarly, the differences in splenic CFUs of GIDIL2 were 20- to 50-fold lower than those of BRD509 over a period of 3-21 days post-injection. This rapid rate of clearance of the GIDIL2 organisms correlated with significantly decreased infection-induced splenomegaly and nitric oxide production by spleen cells. However, despite the poor survival of GIDIL2 organisms in vivo, they could activate peritoneal NK cells efficiently. As early as 48 h after immunization, equivalent levels of NK-mediated cellular cytotoxicity were induced by BRD509 and GIDIL2 strains. Direct evidence for priming of the immune response was shown by demonstrating increased production of IFN-gamma in a recall response by spleen memory T cells obtained 3 weeks after immunization. Finally, mice inoculated with a single dose of either BRD509 or GIDIL2 organisms were fully protected against a challenge of >100-fold the LD50 dose of virulent Salmonella. Taken together, our data demonstrate that despite their rapid clearance from the reticuloendothelial system, IL-2-expressing Salmonella are immunogenic and fully capable of affording excellent protection against virulent challenge in Salmonella-resistant C3H/HeN mice.     

Role of Macrophages in Acute Murine Cytomegalovirus Infection

It has been recognized that macrophages play an important role in controlling virus infection in experimental animal models. To evaluate the role of macrophages in acute murine cytomegalovirus infection, macrophages in the spleen and the liver were eliminated by an intravenous injection of liposomes containing a cytolytic agent, dichloromethylene diphosphonate. The depletion of macrophages led to a significant increase of virus titer in the spleen and lungs in both susceptible BALB/c and resistant C57BL/6 mice during the first three days after intravenous infection. In the spleen, the increase of virus titer in macrophage-depleted BALB/c mice was much greater than that in NK cell-depleted mice. These results suggest that macrophages contribute to protection mainly by the mechanisms which are independent of NK cells during the first three days after infection. The increase of virus titer in macrophage-depleted C57BL/6 mice was as great as that in NK cell-depleted mice because of the high contribution of NK cells to protection in C57BL/6 mice. In the liver in both strains of mice, the effects of macrophage depletion on virus titer were not as much as those in the spleen and lungs. Furthermore, the local depletion of peritoneal macrophages resulted in a great increase of virus titer in the spleen at three days after intraperitoneal infection. We conclude that macrophages greatly contribute to decreasing the virus load in some organs possibly through either or both intrinsic and extrinsic mechanisms in the early phase of primary infection with murine cytomegalovirus.     

Cytophilic immunoglobulins on the surface of neutrophilic granulocytes in mice perorally immunized with a live vaccine made from the supressor revertant S. typhimurium Rev 8]

The reaction of the spleen cell migration inhibition in the presence of monospecific antisera against mouse G, A and M immunoglobulins was used to detect cytophilic antibodies on the surface of mouse granulocytes. The oral administration of ACR live vaccine from suppressor revertant Salmonella typhimurium Rev. 8 protected the mice against infection induced by virulent species of mouse. Salmonella typhimurium. The immunized mice showed an increase in cytophilic IgG on the surface of neutrophile granulocytes.     

A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors

Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.     

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.     

Novel virulence properties of the Salmonella typhimurium virulence-associated plasmid: immune suppression and stimulation of splenomegaly

Mice infected subcutaneously with wild-type Salmonella typhimurium, SR11, developed a significant splenomegaly when compared with mice infected with an equal number of a plasmid-cured strain. Further, the bacterial load in the spleen at 14 days after infection, measured as colony-forming units per gram tissue, was significantly higher in mice infected with the parent strain than in mice infected with the plasmid-cured strain. These data confirm the previously reported plasmid-associated ability of Salmonella to multiply within the spleen. In addition, lymph node cells (LNC) from mice infected with the parent strain had a significantly reduced ability to proliferate in response to concanavalin A, a T-cell mitogen, and to heat-killed S. typhimurium cells when compared with LNC isolated from mice infected with the plasmid-cured strain. Finally, reintroduction of a functional Tn5-tagged 90-kb plasmid into a plasmid-free strain restored its capacity to cause a marked splenomegaly and to suppress lymph node cell proliferation in BALB/c mice. These data demonstrate that the 90-kb plasmid of highly virulent S. typhimurium strains mediates several novel pathogenic properties in infected mice: (1) enhancement of the ability of Salmonella to multiply within the spleen; (2) stimulation of a splenic inflammatory response as displayed by marked splenomegaly; and (3) a general suppression of lymphocyte responsiveness to both T-cell mitogens and specific Salmonella antigens.     

Characterization of immune responses during infection with Mycobacterium avium strains 100, 101 and the recently sequenced 104

Mycobacterium avium strain 104 was chosen as the M. avium isolate to sequence, as it is virulent to humans, stable and readily transfectable. As this strain has not been widely studied we sought to investigate the pattern of 104 infection in mice. Bacterial growth and the immune response generated were compared with infection with the low virulence M. avium strain 100, and the high virulence common laboratory strain, 101. Mycobacterium avium strains 104 and 101 grew progressively within mice, while strain 100 was gradually cleared. Strains 104 and 101 induced strong T cell activation and spleen cell cultures produced similar levels of IFN-gamma. In mice infected with strain 100 no significant T cell activation or IFN-gamma production was measured. Further, mice infected with strain 104 or 101 also displayed comparable inflammatory responses and similar granuloma formation, while only minimal inflammation was seen in mice infected with strain 100. Strains 101 and 104 also grew in a similar fashion in bone-marrow-derived macrophages and induced significant levels of TNF and nitric oxide. Thus infection with M. avium strain 104 induced an immunological response comparable to M. avium strain 101 and, with the availability of its sequence, should be a useful tool for designing new vaccines or drugs therapies to treat the increasing incidence of M. avium infection in humans.     

Mutation of flgM attenuates virulence of Salmonella typhimurium, and mutation of fliA represses the attenuated phenotype.

Salmonella typhimurium ST39 exhibits reduced virulence in mice and decreased survival in mouse macrophages compared with the parent strain SL3201. Strain ST39 is nonmotile, carries an indeterminate deletion in and near the flgB operon, and is defective in the mviS (mouse virulence Salmonella) locus. In flagellum-defective strains, the flgM gene product of S. typhimurium negatively regulates flagellar genes by inhibiting the activity of FliA, the flagellin-specific sigma factor. In this study, flgM of wild-type S. typhimurium LT2 was found to complement the mviS defect in ST39 for virulence in mice and for enhanced survival in macrophages. Transduction of flgM::Tn10dCm into the parent strain SL3201 resulted in attenuation of mouse virulence and decreased survival in macrophages. However, a flgM-fliA double mutant was fully virulent in mice and survived in macrophages at wild-type levels. Thus, the absolute level of FliA activity appears to affect the virulence of S. typhimurium SL3201 in mice. DNA hybridization studies showed that flgM-related sequences were present in species other than Salmonella typhimurium and that sequences related to that of fliA were common among members of the family Enterobacteriaceae. Our results demonstrate that flgM and fliA, two genes previously shown to regulate flagellar operons, are also involved in the regulation of expression of virulence of S. typhimurium and that this system may not be unique to the genus Salmonella.     

Lipopolysaccharide expression and virulence in mice of Australian isolates of Salmonella enteritidis

The lipopolysaccharide (LPS) of 54 Australian isolates, nine isolates acquired or isolated overseas, and two reference strains of Salmonella enteritidis was studied to assess its relation to pathogenicity. LPS was extracted by proteinase K digestion of whole cells, and analysed by polyacrylamide gel electrophoresis. All isolates possessed an LPS structure identical to that of a reference strain of Salm. enteritidis phage type 4. Representative strains of the clinically prevalent phage types 4, 14 and 26, which express long chain LPS, were assessed for their pathogenicity in mice. Salmonella enteritidis phage type 4 produced a lethal infection in BALB/c mice, but not in C3H/HeJ or Quackenbush (outbred) strains. Phage types 14 and 26 did not produce an obvious infection in any mice, suggesting Australian strains of phage type 4 are more virulent than phage types 14 and 26.