Porcine in vitro and in vivo models to assess the virulence of Salmonella enterica serovar Typhimurium for pigs

Salmonella Typhimurium infections in pigs pose an important human health hazard. One promising control measure is the development of live attenuated vaccine strains using defined knockout mutants. Preferably, screening of candidate knockout vaccine strains for attenuation should first be done in models allowing testing of a large number of strains. Thereafter, a limited number of selected strains should be further characterized in an experimental infection model in pigs. The aim of the present study was to develop such models. The invasive and proliferative characteristics of S. Typhimurium were assessed in both a non-polarized and a polarized porcine intestinal epithelial cell line. Neutrophils obtained from porcine blood were used to study the capacity of Salmonella to withstand killing by these phagocytes. The ability to induce an intestinal inflammatory response was investigated in a terminal intestinal loop model. The systemic phase of infection was mimicked by studying the uptake and intracellular survival of S. Typhimurium in porcine pulmonary alveolar macrophages and peripheral blood monocytes. These models should allow screening for attenuated strains. For further characterization, an experimental infection model was established, providing extensive data on the course of an oral infection and the optimal time points for colonization (day 5 postinoculation [pi]) and persistency (days 21-28 pi) in pigs. In conclusion, screening for virulence of S. Typhimurium strains with subsequent confirmation for a subset of strains in a well-defined experimental infection model would significantly reduce the number of experimental pigs required.     

Survival and transmission of Salmonella enterica serovar typhimurium in an outdoor organic pig farming environment

It was investigated how organic rearing conditions influence the Salmonella enterica infection dynamics in pigs and whether Salmonella persists in the paddock environment. Pigs inoculated with S. enterica serovar Typhimurium were grouped with Salmonella-negative tracer pigs. Bacteriological and serological testing indicated that organic pigs were susceptible to Salmonella infections, as 26 of 46 (56%) tracer pigs turned culture positive. An intermittent and mainly low-level excretion of Salmonella (<100 cells g-1) partly explains why the bacteriological prevalence appeared lower than the seroprevalence. Salmonella persisted in the paddock environment, as Salmonella was isolated from 46% of soil and water samples (n=294). After removal of pigs, Salmonella was found in soil samples for up to 5 weeks and in shelter huts during the entire test period (7 weeks). Subsequent introduction of Salmonella-negative pigs into four naturally Salmonella-contaminated paddocks caused Salmonella infections of pigs in two paddocks. In one of these paddocks, all tracer pigs (n=10) became infected, coinciding with a previous high Salmonella infection rate and high Salmonella excretion level. Our results showed that pigs reared under organic conditions were susceptible to Salmonella infections (just like conventional pigs) and that Salmonella persisting in the paddock environment could pose an infection risk. A driving force for these infections seemed to be pigs with a high Salmonella excretion level, which caused substantial contamination of the environment. This suggests that isolation of animals as soon as a Salmonella infection is indicated by clinical symptoms of diarrhea could be a means of reducing and controlling the spread and persistence of Salmonella in outdoor organic pig production environments.     

Protagonists of innate immunity during in Salmonella infections

Salmonella are facultative intracellular Gram-negative bacteria that are found ubiquitously in nature and have the ability to infect a wide range of hosts including humans, domesticated, wild mammals, and birds. The principal clinical manifestations associated with Salmonella infection in humans are enteric fever (typhoid and paratyphoid) and a self-limiting gastroenteritis (salmonellosis). Additionally, silent carriage of this bacterium is frequent and contributes to disease dissemination. Typhoid fever still represents a major public health problem in many developing countries. On the other hand, industrialized countries experience an increased incidence of nontyphoidal Salmonella infections with most cases tracing back to food contamination. Studies using mouse model of infection with a highly virulent Salmonella typhimurium serotype have provided important insight into the complexity of the innate immune response to infection. The players are numerous but emphasis was placed on the genes that were discovered using genetic approaches and in vivo assay with live pathogen and include positional cloning of mouse mutations and manipulation of genes in the context of whole animal either by transgenesis or knockout technologies. Some of the critical genes include those known to play a role in the detection of the bacteria (Cd14, Lbp, Tlr4 and Tlr5) and in microbicidal activity (Slc11a1, Nos2, NADPH oxidase and cryptdins). These discoveries have already initiated the search for the contribution of particular genetic pathways in the innate immune response of humans to infection with Salmonella and other intracellular microorganisms.     

Glycerol supplementation enhances L. reuteri's protective effect against S. Typhimurium colonization in a 3-D model of colonic epithelium

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.     

Characteristics of the antigen-binding properties of lymphocytes in experimental Salmonella infection

The data on the count of antigen-binding lymphocytes in the blood and lymphoid organs of mice in the course of Salmonella infection are presented. The reaction used for their detection is specific. Antigen-binding lymphocytes detected in mice in Salmonella infection belong to T- and B-cell populations and carry surface receptors belonging to IgA and IgM. Study of antigen-binding lymphocytes 1-20 hours after infection may be helpful in the specific diagnosis of Salmonella infections.     

The Salmonella spvB virulence gene encodes an enzyme that ADP-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells

ADP-ribosylating enzymes, such as cholera and diphtheria toxins, are key virulence factors for a variety of extracellular bacterial pathogens but have not been implicated previously during intracellular pathogenesis. Salmonella strains are capable of invading epithelial cells and localizing in macrophages during infection. The spvB virulence gene of Salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. Here, we present evidence that spvB encodes an ADP-ribosylating enzyme that uses actin as a substrate and depolymerizes actin filaments when expressed in CHO cells. Furthermore, site-directed mutagenesis demonstrates that the ADP-ribosylating activity of SpvB is essential for Salmonella virulence in mice. As spvB is expressed by Salmonella strains after invasion of epithelial cells or phagocytosis by macrophages, these results suggest that SpvB functions as an intracellular ADP-ribosylating toxin critical for the pathogenesis of Salmonella infections.     

Immunity to systemic Salmonella infections

Salmonella infections are a serious public health problem in developing countries and represent a constant concern for the food industry. The severity and the outcome of a systemic Salmonella infection depends on the "virulence" of the bacteria, on the infectious dose as well as on the genetic makeup and immunological status of the host. The control of bacterial growth in the reticuloendothelial system (RES) in the early phases of a Salmonella infection relies on the NADPH oxidase-dependent anti-microbial functions of resident phagocytes and is controlled by the innate resistance gene Nramp1. This early phase is followed by the suppression of Salmonella growth in the RES due to the onset of an adaptive host response. This response relies on the concerted action of a number of cytokines (TNFalpha, IFNgamma, IL12, IL18, and IL15), on the recruitment of inflammatory phagocytes in the tissues and on the activation of the recruited cells. Phagocytes control bacterial growth in this phase of the infection by producing reactive nitrogen intermediates (RNI) generated via the inducible nitric oxide synthase (iNOS). Clearance of the bacteria from the RES at a later stage of the infection requires the CD28-dependent activation of CD4+ TCR-alphabeta T-cells and is controlled by MHC class II genes. Resistance to re-infection with virulent Salmonella micro-organisms requires the presence of Th1 type immunological memory and anti-Salmonella antibodies. Thus, the development of protective immunity to Salmonella infections relies on the cross-talk between the humoral and cellular branches of the immune system.     

Persistent bacterial infections and primary immune disorders

Mycobacteria, Salmonella and Helicobacter species have all evolved mechanisms to evade host defenses and cause persistent infection in humans. Host control of mycobacteria and Salmonella is largely achieved by the IFN-gamma/IL-12 pathway. Immune disorders affecting this pathway are characterized by disseminated infections with environmental or nontuberculous mycobacteria. Helicobacter is a predominantly extracellular bacterium that uses its remarkable genetic diversity (as well as other mechanisms) in order to evade host defenses. The importance of humoral immunity in containing Helicobacter infections to the mucosal surface is illustrated by the primary immune disorder, X-linked agammaglobulinemia in which patients are prone to chronic bacteremia and skin infections by Helicobacter and related species such as Flexispira and Campylobacter. Exploration of these particular infections in their specific immune defects sheds light on both host and bacterial mechanisms that have implications for pathogenesis and therapy.     

Pyroptosis and host cell death responses during Salmonella infection

Salmonella enterica are facultatively intracellular pathogens causing diseases with markedly visible signs of inflammation. During infection, Salmonella interacts with various host cell types, often resulting in death of those cells. Salmonella induces intestinal epithelial cell death via apoptosis, a cell death programme with a notably non-inflammatory outcome. In contrast, macrophage infection triggers caspase-1-dependent proinflammatory programmed cell death, a recently recognized process termed pyroptosis, which is distinguished from other forms of cellular demise by its unique mechanism, features and inflammatory outcome. Rapid macrophage pyroptosis depends on the Salmonella pathogenicity island-1 type III secretion system (T3SS) and flagella. Salmonella dynamically modulates induction of macrophage pyroptosis, and regulation of T3SS systems permits bacterial replication in specialized intracellular niches within macrophages. However, these infected macrophages later undergo a delayed form of caspase-1-dependent pyroptosis. Caspase-1-deficient mice are more susceptible to a number of bacterial infections, including salmonellosis, and pyroptosis is therefore considered a generalized protective host response to infection. Thus, Salmonella-induced pyroptosis serves as a model to understand a broadly important pathway of proinflammatory programmed host cell death: examining this system affords insight into mechanisms of both beneficial and pathological cell death and strategies employed by pathogens to modulate host responses.     

Salmonella infections in a marsupial, the quokka (Setonix brachyurus), in relation to seasonal changes in condition and environmental stress.

An unusual abundance of Salmonella infections was studied in an island population of a wild marsupial, the quokka (Setonix brachyurus), which experiences starvation in summer associated with significant mortality. The frequency of infections was found to vary seasonally over most parts of the island, with high infection rates (70 to 100%) in summer and low infection rates (0 to 30%) in winter. In some samples, there was an average of as many as two isolations per animal, and up to five isolations were made from a single animal. By the end of summer, virtually all animals excreted Salmonella spp., with a median rate of excretion of approximately 3,000 Salmonella organisms per g of feces. The seasonal changes occurred over intervals of only weeks. The infections are believed to be associated with disruption of the digestive physiology of the animals caused by the poor quality of feed available in summer. This conclusion was supported by a quantitative study of the infections and by a field manipulation experiment which delayed the initiation of the infections as long as a food supplement was available. The proliferation of Salmonella spp. is discussed in terms of the ecology of the quokka and of the use of Salmonella spp. as indicators of environmental stress acting on the animals.     

Salmonella infections in a marsupial, the quokka (Setonix brachyurus), in relation to seasonal changes in condition and environmental stress

An unusual abundance of Salmonella infections was studied in an island population of a wild marsupial, the quokka (Setonix brachyurus), which experiences starvation in summer associated with significant mortality. The frequency of infections was found to vary seasonally over most parts of the island, with high infection rates (70 to 100%) in summer and low infection rates (0 to 30%) in winter. In some samples, there was an average of as many as two isolations per animal, and up to five isolations were made from a single animal. By the end of summer, virtually all animals excreted Salmonella spp., with a median rate of excretion of approximately 3,000 Salmonella organisms per g of feces. The seasonal changes occurred over intervals of only weeks. The infections are believed to be associated with disruption of the digestive physiology of the animals caused by the poor quality of feed available in summer. This conclusion was supported by a quantitative study of the infections and by a field manipulation experiment which delayed the initiation of the infections as long as a food supplement was available. The proliferation of Salmonella spp. is discussed in terms of the ecology of the quokka and of the use of Salmonella spp. as indicators of environmental stress acting on the animals.     

Foodborne Salmonella ecology in the avian gastrointestinal tract

Foodborne Salmonella continues to be a major cause of salmonellosis with Salmonella Enteritidis and S. Typhimurium considered to be responsible for most of the infections. Investigation of outbreaks and sporadic cases has indicated that food vehicles such as poultry and poultry by-products including raw and uncooked eggs are among the most common sources of Salmonella infections. The dissemination and infection of the avian intestinal tract remain somewhat unclear. In vitro incubation of Salmonella with mammalian tissue culture cells has shown that invasion into epithelial cells is complex and involves several genetic loci and host factors. Several genes are required for the intestinal phase of Salmonella invasion and are located on Salmonella pathogenicity island 1 (SPI 1). Salmonella pathogenesis in the gastrointestinal (GI) tract and the effects of environmental stimuli on gene expression influence bacterial colonization and invasion. Furthermore, significant parameters of Salmonella including growth physiology, nutrient availability, pH, and energy status are considered contributing factors in the GI tract ecology. Approaches for limiting Salmonella colonization have been primarily based on the microbial ecology of the intestinal tract. In vitro studies have shown that the toxic effects of short chain fatty acids (SCFA) to some Enterobacteriaceae, including Salmonella, have resulted in a reduction in population. In addition, it has been established that native intestinal microorganisms such as Lactobacilli provide protective mechanisms against Salmonella in the ceca. A clear understanding of the key factors involved in Salmonella colonization in the avian GI tract has the potential to lead to better approach for more effective control of this foodborne pathogen.     

Strains of genus Salmonella isolated from extraintestinal infections

Every year in Poland from tens to more than hundred bacteriologically verified extraintestinal infections caused by Salmonella species have been registered. These unusually located infections have substantially heavy course and in many cases hospitalisation and antibiotic therapy have to be involved. Cases of extraintestinal infections with these Gram-negative rods, which were described in the literature, concerned: pneumonias, lung abscesses and thoracic empyemas, and infections of: blood, bones and joints, wounds, fistulas and urinary tract. The aim of this study was to set extraintestinal Salmonella infections and to analyze a susceptibility of isolated strains to antimicrobial agents. Between 01.07.2002 and 31.12.2004 (30 months) 13 strains of Salmonella genus have been isolated, including 11 S. enteritidis and 2 S. Hadar. In general, with one exception, isolated strains were susceptible to tested antibiotics/chemotherapeutics. ESBL - positive strains were not detected. The tendency of Salmonella strains to cause extraintestinal infections has been noticed. The problem is still escalating, especially in group of patients chronically treated, with immunodeficiency and immunosuppression, after complicated medical procedures, also in the group of small children and aged persons. Therefore, it is necessary to evaluate a susceptibility to antibiotics/chemotherapeutics of every strain from confirmed case of Salmonella extraintestinal infection and it is important to apply a guided therapy.     

Salmonella persistence and transmission strategies

Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time and pose significant public-health problems. Multidrug-resistant Salmonella enteric serovar Typhi (S. Typhi) and non-Typhoidal Salmonella (NTS) are on the increase, and are often associated with HIV infection. Chronically infected hosts are often asymptomatic and transmit disease to na?ve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. Salmonella utilizes multiple strategies to evade and modulate host innate and adaptive immune responses in order to persist in the presence of a robust immune response. In addition, the intestinal microbiota plays a critical role in controlling Salmonella infection, disease, and transmissibility.     

Trends in the development of the epidemic process in salmonellosis caused by Salmonella typhimurium and Salmonella enteritidis

The dynamics and structure of the epidemic process of Salmonella infections among the population of Perm in 1983-1988 was studied and the results of evaluation of antibiotic resistance of the dominating Salmonella species analyzed. The study revealed that a decrease in salmonellosis morbidity caused by S. typhimurium was associated with a limited circulation of anthroponotic (antibiotic-resistant) variants of Salmonellae and a relative increase in the proportion of zoonotic (antibiotic-sensitive) strains. At the period of elevated morbidity this Salmonella infection affected mainly young children in cold months, whereas in recent years seasonal morbidity rises shifted to spring-summer and summer-autumn months, affecting older age groups of the population. The study also revealed that a rise in salmonellosis morbidity caused by S. enteritidis was due to increased circulation of zoonotic variants of Salmonellae. Changes in the epidemiological situation necessitate correction of the system of epidemiological surveillance on Salmonella infections with the emphasis on sanitation measures in stock-breeding farms with unfavorable epidemiological situation.     

Inside or outside: detecting the cellular location of bacterial pathogens

Salmonella are intracellular pathogens that infect and multiply inside macrophages. Although Salmonella are some of the best-studied pathogens, it is difficult to determine quickly and reliably whether the bacteria are intracellular or extracellular. We have developed a novel method using differential fluorescence of two fluorescent proteins to determine the cellular location of pathogenic bacteria in macrophage infection assays. Using the differential expression of two unique fluorescent proteins that are expressed under specific conditions, we have developed a real-time assay for macrophage infections. The critical advantages of this system are that it does not alter the bacterial surface, it is not toxic to either the bacteria or the host cell, and it may be used in real-time quantitative assays. This assay can be readily applied to any other model pathogenic systems such as Listeria, Mycobacteria, and Legionella in which intracellular gene expression has been characterized.     

The dark side of the salad: Salmonella typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle

Salmonella enterica serovar typhimurium contaminated vegetables and fruits are considerable sources of human infections. Bacteria present in raw plant-derived nutrients cause salmonellosis, the world wide most spread food poisoning. This facultative endopathogen enters and replicates in host cells and actively suppresses host immune responses. Although Salmonella survives on plants, the underlying bacterial infection mechanisms are only poorly understood. In this report we investigated the possibility to use Arabidopsis thaliana as a genetically tractable host system to study Salmonella-plant interactions. Using green fluorescent protein (GFP) marked bacteria, we show here that Salmonella can infect various Arabidopsis tissues and proliferate in intracellular cellular compartments. Salmonella infection of Arabidopsis cells can occur via intact shoot or root tissues resulting in wilting, chlorosis and eventually death of the infected organs. Arabidopsis reacts to Salmonella by inducing the activation of mitogen-activated protein kinase (MAPK) cascades and enhanced expression of pathogenesis related (PR) genes. The induction of defense responses fails in plants that are compromised in ethylene or jasmonic acid signaling or in the MKK3-MPK6 MAPK pathway. These findings demonstrate that Arabidopsis represents a true host system for Salmonella, offering unique possibilities to study the interaction of this human pathogen with plants at the molecular level for developing novel drug targets and addressing current safety issues in human nutrition.     

Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.