Salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection

Abstract Susceptibility to Salmonella typhimurium infection was compared in H (high Ab responder) and L (low Ab responder) mice obtained by several selective breeding experiments (Selections I, II, III, IV and IV A) [10,19,22]. H mice were always much more susceptible to infection than their L mice counterparts within a continuous LD 50 variation range. In three of the selections (I, II and IV A) the low responsiveness character is known to result mainly from rapid Ag degradation in L mice macrophages. It was hypothesized that resistance to multiplication of intracellular pathogens could be related to an increased catabolic activity towards Ag. This was actually demonstrated, in F₂ segregant hybrids of selection IV A, by the significant inverse correlation between capacity for Ab production and resistance to infection.     

Early antibody response in mice to either infection or immunization with Salmonella typhimurium

After immunization with either live or heat-killed Salmonella typhimurium, mice responded with an extremely rapid production of bactericidal antibody which was correlated with the appearance of immunity to a heavy challenge dose (100 ld(50)) of the virulent bacteria. Inactivation of sera with mercaptoethanol along with Sephadex fractionation indicated that the observed bactericidal activity was associated with a macroglobulin which was completely mercaptoethanol-sensitive. The unexpected finding, that a heat-killed vaccine gave excellent protection from a challenge dose which killed all unimmunized control mice, seriously challenges the theory attributing immunity against typhoid infection entirely to a cellular host factor produced only in response to a live vaccine.     

Macrophage activity in rabies virus infection of genetically selected high and low antibody responder lines of mice

After infection with the Pasteur strain of fixed rabies virus, the onset of disease, mortality, interferon (IFN) synthesis and interaction of the virus with macrophages were investigated in high (H_I) and low (L_I) antibody responder lines of mice. The H_I mice were shown to be more resistant than the L_I mice, and resistance was age-dependent, since mice from both mouse lines were fully susceptible up to 2 weeks of age. IFN synthesis studies of the serum indicated that, after rabies infection, H_I mice produced a slightly higher amount of IFN, which was determined to be predominantly IFN-gamma. In the brains of L_I mice, only IFN-alpha/beta was found, in contrast to the mixture of IFN-alpha/beta and IFN-gamma observed in the brains of H_I mice. Although macrophages from the two mouse lines expressed the same degree of extrinsic activity, their intrinsic activities were quite different; the L_I mice showed a greater ability to uptake and process the virus or ingest C3 (IgM) sheep red blood cells. The present findings attribute the higher antibody response and IFN-gamma synthesis observed in H_I mice during rabies infection to slower processing of the rabies antigen in their macrophages, thus conferring upon them a greater ability to present it to the immune system, leading to a higher degree of resistance to rabies infection.     

Co-localization of quantitative trait loci regulating resistance to Salmonella typhimurium infection and specific antibody production phenotypes

Salmonella enterica serotype typhimurium is a facultative intracellular bacteria that induces systemic infection in mice. Resistance to this pathogen is under polygenic control in which Nramp1 is the major gene involved. Lines of mice obtained by selective breeding for high (HIII) or low (LIII) antibody response to flagellar antigens of salmonellae showed significant susceptibility differences, although both the lines display Nramp1(R) alleles. The HIII line was extremely susceptible to infection, while the LIII line was resistant. In order to examine the cellular and genetic mechanisms involved in this distinct pattern of resistance, HIII and LIII mice were analyzed for IFNgamma and IL4 production and screened for quantitative trait loci involved in S. typhimurium infection, using several polymorphic microsatellites. In the present work, HIII mice showed an IFNgamma downregulation in the early phase of infection when compared with LIII animals. No interline differences in IL4 production were verified. The loci screening was performed on immunized F2 intercrosses obtained from HIII and LIII mice. Three antibody-controlling chromosomal regions were coincident, and another was mapped near one of the four loci known to affect susceptibility to S. typhimurium. These results indicate a major role of IFNgamma in our model, and suggest the co-localization of quantitative trait loci modulating both infection and antibody production phenotypes.     

Inverse modification of antibody responsiveness to RGG in lines of mice selected for high or low responses to somatic antigen of Salmonella

High (H/s) and low (L/s) antibody responder lines of mice selected according to their response to the somatic (s) antigen of Salmonella (Selection IV) have unexpected inverse capacity for antibody production to rabbit gamma globulin (RGG): H/s mice are low or even nonresponders to this antigen, whereas L/s mice are high responders. It was shown that the phenotypic variability within each line is due to environmental factors. RGG was a selection antigen in Selection V; the high (H/p) and low (L/p) responder mice are therefore considered as homozygous for the RGG genes. Responsiveness to RGG was investigated in F₁ and F₂ hybrids obtained by crossing the phenotypically similar RGG responder or nonresponder mice of Selections IV and V. The results support the hypothesis that the same genes control the response to RGG in L/s and H/p lines as well as in H/s and L/p lines. This means that the genes specific for RGG responsiveness were independent from those regulating responses to the s antigen. Unaffected by the selective breeding in Selection IV, they have been fixed by chance in an inverse way in H/s and L/s lines.     

Dietary Bifidobacterium lactis (HN019) enhances resistance to oral Salmonella typhimurium infection in mice

The ability of a newly identified probiotic lactic acid bacterial strain, Bifidobacterium lactis (HN019), to confer protection against Salmonella typhimurium was investigated in BALB/c mice. Feeding mice with B. lactis conferred a significant degree of protection against single or multiple oral challenge with virulent S. typhimurium, in comparison to control mice that did not receive B. lactis. Protection included a ten-fold increase in survival rate, significantly higher post-challenge food intake and weight gain, and reduced pathogen translocation to visceral tissues (spleen and liver). Furthermore, the degree of pathogen translocation showed a significant inverse correlation with splenic lymphocyte proliferative responses to mitogens, blood and peritoneal cell phagocytic activity and intestinal mucosal anti-S. typhimurium antibody titers in infected mice; all of these immune parameters were enhanced in mice fed B. lactis. Together, these results suggest that dietary B. lactis can provide a significant degree of protection against Salmonella infection by enhancing various parameters of immune function that are relevant to the immunological control of salmonellosis. Thus dietary supplementation with B. lactis provides a unique opportunity for developing immune-enhancing probiotic dairy food products with proven health benefits.     

Nifedipine administration impairs natural resistance of mice to Salmonella typhimurium

Administration of Ca²⁺ channel blockers in cardiac disorders and the central role of Ca ²⁺ in modulating neutrophil functions, prompted us to investigate whether administration of nifedipine to mice would alter their natural resistance to infectious agents like Salmonella typhimurium. Neutrophil chemiluminescence (CL) in response to S. typhimurium was significantly (p < 0.01) decreased in mice fed with nifedipine (0.015 mg/kg body weight) over a period of six months. Intracellular killing of S. typhimurium by isolated neutrophils also decreased significantly (p < 0.01) and exponentially with nifedipine administration, representing a 42% fall at six months. In addition the drug administration lowered the survival rate of animals following challenge by a lethal dose of S. typhimurium (LD₅₀ = 1 × 10⁴ bacteria/animal). Our data suggest that long term administration of nifedipine lowers the natural resistance of mice to S. typhimurium owing to impaired neutrophil functions.Accepted for presentation at XIII Congress of International Society for Heart Research, Ann Arbor, Michigan (USA). Accepted for publication in Molec. Cellular Biochem. (1989). Nifedipine impairs neutrophil respiratory burst by a mechanism other than calcium channel blockade.     

Soluble plasma antigen in experimental Salmonella typhimurium infection in mice

Abstract To detect and characterize Salmonella antigen in blood, outbred CF-1 female mice were inoculated intraperitoneally with S. typhimurium LT-2 and blood was assayed by ELISA for Salmonella common structural antigen. Plasma antigen was detectable early in the course of infection and increased in quantity later in the course of illness when animals showed high grade bacteremia and high counts of splenic bacteria. Antigen was associated with a cell-free plasma fraction of blood, passed through filters with cut-offs of 0.2 μ and molecular mass of 1000 kDa, and was enhanced in detectability after heating to 100°C for 15 min. Antigen was concentrated by diluting plasma 1:4 in 0.1 M EDTA, heating to 100°C, and concentrating the supernate with an ultrafiltration membrane with a molecular mass cut-off of 15 kDa. By gel filtration, antigen was associated with a peak at about molecular mass 300 kDa in heated plasma and a peak at about 380 kDa in unheated plasma. These results indicate that murine typhoid infection results in circulating soluble plasma antigen, which is heat-stable with a molecular mass of approximately 300 kDa.     

Pregnancy impairs the innate immune resistance to Salmonella typhimurium leading to rapid fatal infection

Typhoid fever and gastroenteritis caused by Salmonella enterica species are increasing globally. Pregnancy poses a high risk, but it is unclear how maternal immunity to infection is altered. In mice, susceptible strains die of S. enterica serovar typhimurium (ST) infection within 7 days whereas resistant mice (129 x 1/SvJ) develop a chronic infection. We found that virulent ST infection during pregnancy, in normally resistant 129 x 1/SvJ mice, evoked approximately 100% fetal loss and surprisingly >60% host fatality, with a median survival of 6 days. Splenic bacterial load was 1000-fold higher in pregnant mice. This correlated to a diminished splenic recruitment/expansion of innate immune cells: dendritic cells, neutrophils, and NK cells. In particular, the splenic expansion and activation of NK cells postinfection seen in nonpregnant mice was lacking in pregnancy. Most notably, pregnant-infected mice had decreased production of serum IL-12 and increased IL-6 levels. Moreover, uteroplacental tissue of pregnant-infected mice exhibited an approximately 40-fold increase in IL-6 mRNA expression relative to noninfected placenta, whereas IL-12p40 was not increased. In vivo blocking of IL-6 significantly reduced the splenic bacterial burden in pregnant mice yet failed to prevent fetal loss. Fetal demise correlated to the rapidity of infection; by 14 h, ST expanded to >10(5) in the placenta and had reached the fetus. Therefore, the preferential placental expansion of ST plausibly altered the inflammatory response toward IL-6 and away from IL-12, reducing the recruitment/activation of splenic innate immune cells. Thus, highly virulent pathogens may use placental invasion to alter systemic host resistance to infection.     

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection

Abstract Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE ) gene of Escherichia coli , a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB -like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium .     

Genetic control of the innate resistance of mice to Salmonella typhimurium: Ity gene is expressed in vivo by 24 hours after infection

The early response of inbred mice to infection with S. typhimurium is controlled by the mouse Chromosome 1 locus, Ity. To better understand the expression of this gene, the initial interactions between the reticuloendothelial system (RES) and i.v. injected salmonellae were compared in resistant (Ityr) and susceptible (Itys) mice. In both mouse strains 99% of the bacteria was cleared from the blood within 2 hr, and uptake of S. typhimurium by splenic and hepatic macrophages was similar regardless of Ity genotype. In vivo phagocytosis of bacteria was followed by a 30 to 60% decline in viable bacteria, which was attributed to the bactericidal activity of RES macrophages. Experiments with radiolabeled S. typhimurium strains TML and TML/TS27 (a temperature-sensitive mutant) confirmed that the efficiency of this early phase killing was not under Ity control. Despite the equivalent uptake and initial bactericidal activity by resident macrophages, bacterial numbers in the RES organs of Itys mice were significantly greater than in Ityr mice by approximately 24 hr after infection. These data suggest that Ity regulates the level of surviving intracellular bacteria that accumulate within resident macrophages of the liver and spleen.     

Nematospiroides dubius: direct and correlated responses to selection for high and low immune responsiveness in mice

High and low immune responder lines of mice were bred selectively from an allogeneic stock over 10 generations, based on their fecal parasite egg count assayed 3 weeks after reinfection with 100 Nematospiroides dubius larvae. By generation 10, (F10), the low immune response mice voided about 10 times as many fecal N. dubius eggs as the high immune response mice. Realized heritability for the selected trait, fecal egg count after secondary infection (= protective immunity), was 0.35 at F7. F7 was considered the selection limit. Selection for change in fecal egg count did not significantly influence the conformational nor reproductive characteristics of these mice. Significant phenotypic and genetic correlations were evident between the selected character and innate immunity to N. dubius, humoral antibody response to N. dubius infection, and establishment, growth, and reproduction of N. dubius in the selected mice.     

Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection

Two aspects of acquired resistance to Salmonella typhimurium infection in BALB/c mice, i.e., the ability to clear the primary inoculum from the spleen and resistance to a secondary challenge, were studied with the use of mAb against T cell subsets. The ability to clear a temperature-sensitive mutant of S. typhimurium from the spleen (assessed at day 21) was abrogated by in vivo treatment with anti-CD4 mAb. Accelerated bacterial clearance could be adoptively transferred into naive mice. In vitro depletion experiments also showed the role of CD4+ T cells in this phenomenon. Depletion of CD8+ T cells had only a marginal effect. Resistance to reinfection in the late phase of the primary infection (day 50) was markedly depressed by in vivo treatment with anti-CD4 mAb, whereas this was not the case during the early phase (day 14). Furthermore, during the early phase of infection athymic nude mice showed increased nonspecific resistance to reinfection. Taken together these results suggest that T-independent mechanisms play a major role in acquired resistance during the early phase of infection.     

Changes in the bactericidal activity of macrophages from mice with various degrees of resistance to Salmonella typhimurium infection as affected by interferon

The bactericidal activity of mouse macrophages with different sensitivity to Salmonella infection has been studied. The sensitivity of BALB/c mice to S. typhimurium infection is associated with the low bactericidal activity of their macrophages. The introduction of interferon stimulates the bactericidal activity of macrophages sensitive to Salmonella infection of mice, which sharply enhances the resistance of the animals to this infection.