The Salmonella Pathogenicity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens

Using a deletion mutant in the regulator of SPI-2, ssrA, we investigated the role of SPI-2 in invasion, intestinal colonization and reproductive tract infection of chickens by Salmonella Enteritidis. The ssrA mutant was fully invasive in phagocytic and non-phagocytic cells but failed to persist within chicken macrophages. The ability of Salmonella Enteritidis to cause disease in orally infected 1-day-old chicks was not altered when ssrA was deleted. Furthermore, caecal colonization was not affected, while spleen and liver showed reduced colonization. Following intra-peritoneal and intravenous infection of 1-day-old chicks, internal organ colonization was strongly reduced. After intravenous inoculation in adult laying hens bacterial numbers of the ssrA mutant were significantly lower in oviducts and ovaries as compared to the wild type strain. The chickens showed less reproductive tract lesions and the recovery of egg production were faster compared to the wild type strain infected chickens. These findings indicate that the SPI-2 regulator ssrA promotes reproductive tract colonization, but is not essential for intestinal colonization of chickens with the host non-specific serotype Enteritidis.     

A limited role for SsrA/B in persistent Salmonella Typhimurium infections in pigs

Virulence genes regulated by the SsrA/B system are indispensable for systemic disease in BALB/c mice. The role of this regulating system in the pathogenesis of Salmonella Typhimurium infections in pigs is not documented. In the present study, the interactions of Salmonella Typhimurium and an ssrA/B mutant were compared in vitro and in vivo. The ssrA/B mutant strain displayed decreased Salmonella Pathogenicity Island 2 (SPI-2) expression levels, showed a replication defect in mouse macrophages and was attenuated in a mouse model after oral inoculation. Using real time qRT-PCR and a porcine ileal loop model, it was shown that the ssrA/B mutant strain was not significantly attenuated in overall virulence and SPI-1 expression in specific. Flowcytometric analysis demonstrated that the ssrA/B mutant strain was defective in intracellular replication in porcine macrophages. After oral inoculation of piglets with the wild type strain or the ssrA/B mutant strain, the animals of both groups excreted Salmonella and were colonized by Salmonella to the same extent. In an intravenous mixed infection model, the ssrA/B mutant strain was defective in the colonization of several internal organs. These results suggest that the ssrA/B gene of Salmonella Typhimurium plays a limited role in the persistent intestinal colonization of pigs.     

Role of SPI-1 in the interactions of Salmonella Typhimurium with porcine macrophages

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in mice after oral challenge. These genes mediate invasion in intestinal epithelial cells and induce cell death in murine macrophages. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections in food producing animals is not known. It was the aim of the present study to characterize the interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants in the SPI-1 genes hilA, sipA and sipB with porcine macrophages. SPI-1 was found to be important in the invasion of porcine pulmonary alveolar macrophages (PAM) and the induction of the formation of spacious phagosomes. Both early and delayed cytotoxicity were seen in PAM, but only the early cytotoxicity was SPI-1 dependent. Exposure of PAM to Salmonella Typhimurium induced the production of reactive oxygen species (ROS) and interleukin-8, but no differences were noticed between the induction mediated by the wild type strain and its SPI-1 mutant strains. In conclusion, invasion of porcine macrophages and the induction of early, but not delayed, cytotoxicity by Salmonella Typhimurium is SPI-1 dependent. SPI-1 dependent invasion, however, is not a prerequisite to induce a pro-inflammatory response.     

Salmonella enterica serovar Enteritidis colonization of the chicken caecum requires the HilA regulatory protein

Invasion of Salmonella into intestinal epithelial cells is believed to be essential for the pathogenesis of Salmonella infections. Invasion is mediated by genes located on the Salmonella pathogenicity Island I (SPI-1), which are needed for assembling a type three secretion system, that mediates injection of bacterial proteins into the cytosol of epithelial cells, resulting in cytoskeletal rearrangements and as a consequence invasion. HilA is the key regulator of the Salmonella Pathogenicity Island I. To assess the role of hilA in colonization of gut and internal organs in poultry, animals were infected with 10(8) CFU of a delta hilA mutant of S. Enteritidis and its parent strain at day of hatch. Very low numbers of delta hilA mutant strain were able to colonize the internal organs shortly after infection, but they were not eliminated from internal organs at 4 weeks post-infection. At that time, the colonization level of the wild type bacteria in internal organs was decreased to the same low level compared with delta hilA mutant strain bacteria. Shedding of the delta hilA mutant strain and colonization of the caeca was seriously decreased relative to the parent strain starting from Day 5 post-infection. At 4 weeks post-infection, the delta hilA mutant strain was more or less eliminated from the chicken gut, while the parent strain was still shed to a high level and colonized the caeca to a high extent (more than 10(7) CFU/g). It is concluded that hilA is involved in long-term shedding and colonization of S. Enteritidis in the chicken caeca.     

Only one of the two type VI secretion systems encoded in the Salmonella enterica serotype Dublin genome is involved in colonization of the avian and murine hosts

The type VI secretion system (T6SS) is a virulence factor for many Gram-negative bacteria. Salmonella genus harbors five phylogenetically distinct T6SS loci encoded in Salmonella Pathogenicity Islands (SPIs) SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22, which are differentially distributed among serotypes. The T6SSs encoded in SPI-6 and SPI-19 contribute to pathogenesis of serotypes Typhimurium and Gallinarum in mice and chickens, respectively. Salmonella Dublin is a pathogen restricted to cattle where it causes a systemic disease. Also, it can colonize other hosts such as chickens and mice, which can act as reservoirs of this serotype. Salmonella Dublin harbors the genes for both T6SS_SPI-6 and T6SS_SPI-19. This study has determined the contribution of T6SS_SPI-6 and T6SS_SPI-19 to host-colonization by Salmonella Dublin using avian and murine models of infection. Competitive index experiments showed that, a mutant strain lacking both T6SSs (∆T6SS_SPI-6/∆T6SS_SPI-19) presents a strong colonization defect in cecum of chickens, similar to the defect observed for the ∆T6SS_SPI-6 mutant, suggesting that this serotype requires a functional T6SS_SPI-6 for efficient colonization of the avian gastrointestinal tract. Colonization of mice was also defective, although to a lesser extent than in chickens. In contrast, the T6SS_SPI-19 was not necessary for colonization of either chickens or mice. Transfer of T6SS_SPI-6, but not T6SS_SPI-19, restored the ability of the double mutant to colonize both animal hosts. Our data indicate that Salmonella Dublin requires only the T6SS_SPI-6 for efficient colonization of mice and chickens, and that the T6SS_SPI-6 and T6SS_SPI-19 are not functionally redundant.     

SPI-1-encoded type III secretion system of Salmonella enterica is required for the suppression of porcine alveolar macrophage cytokine expression

ABSTRACT: Genes localized at Salmonella pathogenicity island-1 (SPI-1) are involved in Salmonella enterica invasion of host non-professional phagocytes. Interestingly, in macrophages, SPI-1-encoded proteins, in addition to invasion, induce cell death via activation of caspase-1 which also cleaves proIL-1β and proIL-18, precursors of 2 proinflammatory cytokines. In this study we were therefore interested in whether SPI-1-encoded type III secretion system (T3SS) may influence proinflammatory response of macrophages. To test this hypothesis, we infected primary porcine alveolar macrophages with wild-type S. Typhimurium and S. Enteritidis and their isogenic SPI-1 deletion mutants. ΔSPI1 mutants of both serovars invaded approx. 5 times less efficiently than the wild-type strains and despite this, macrophages responded to the infection with ΔSPI1 mutants by increased expression of proinflammatory cytokines IL-1β, IL-8, TNFα, IL-23α and GM-CSF. Identical macrophage responses to that induced by the ΔSPI1 mutants were also observed to the infection with sipB but not the sipA mutant. The hilA mutant exhibited an intermediate phenotype between the ΔSPI1 mutant and the wild-type S. Enteritidis. Our results showed that the SPI-1-encoded T3SS is required not only for cell invasion but in macrophages also for the suppression of early proinflammatory cytokine expression.     

T-2 toxin induced Salmonella Typhimurium intoxication results in decreased Salmonella numbers in the cecum contents of pigs, despite marked effects on Salmonella-host cell interactions

ABSTRACT: The mycotoxin T-2 toxin and Salmonella Typhimurium infections pose a significant threat to human and animal health. Interactions between both agents may result in a different outcome of the infection. Therefore, the aim of the presented study was to investigate the effects of low and relevant concentrations of T-2 toxin on the course of a Salmonella Typhimurium infection in pigs. We showed that the presence of 15 and 83 μg T-2 toxin per kg feed significantly decreased the amount of Salmonella Typhimurium bacteria present in the cecum contents, and a tendency to a reduced colonization of the jejunum, ileum, cecum, colon and colon contents was noticed. In vitro, proteomic analysis of porcine enterocytes revealed that a very low concentration of T-2 toxin (5 ng/mL) affects the protein expression of mitochondrial, endoplasmatic reticulum and cytoskeleton associated proteins, proteins involved in protein synthesis and folding, RNA synthesis, mitogen-activated protein kinase signaling and regulatory processes. Similarly low concentrations (1-100 ng/mL) promoted the susceptibility of porcine macrophages and intestinal epithelial cells to Salmonella Typhimurium invasion, in a SPI-1 independent manner. Furthermore, T-2 toxin (1-5 ng/mL) promoted the translocation of Salmonella Typhimurium over an intestinal porcine epithelial cell monolayer. Although these findings may seem in favour of Salmonella Typhimurium, microarray analysis showed that T-2 toxin (5 ng/mL) causes an intoxication of Salmonella Typhimurium, represented by a reduced motility and a downregulation of metabolic and Salmonella Pathogenicity Island 1 genes. This study demonstrates marked interactions of T-2 toxin with Salmonella Typhimurium pathogenesis, resulting in bacterial intoxication.     

Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages

ABSTRACT: Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Circumvention of the host's immune system by Salmonella might contribute to persistent infection of pigs. In the present study, we found that Salmonella Typhimurium strain 112910a specifically downregulated MHC II, but not MHC I, expression on porcine alveolar macrophages in a Salmonella pathogenicity island (SPI)-1 and SPI-2 dependent way. Salmonella induced downregulation of MHC II expression and intracellular proliferation of Salmonella in macrophages were significantly impaired after opsonization with Salmonella specific antibodies prior to inoculation. Furthermore, the capacity to downregulate MHC II expression on macrophages differed significantly among Salmonella strains, independently of strain specific differences in invasion capacity, Salmonella induced cytotoxicity and altered macrophage activation status. The fact that strain specific differences in MHC II downregulation did not correlate with the extent of in vitro SPI-1 or SPI-2 gene expression indicates that other factors are involved in MHC II downregulation as well. Since Salmonella strain dependent interference with the pig's immune response through downregulation of MHC II expression might indicate that certain Salmonella strains are more likely to escape serological detection, our findings are of major interest for Salmonella monitoring programs primarily based on serology.     

Immunization of chickens with Salmonella enterica subspecies enterica serovar Enteritidis pathogenicity island-2 proteins

Several structural components of the type III secretion systems (T3SS) encoded by Salmonella pathogenicity island (SPI)-1 and SPI-2 are exposed to the host's immune system prior to/during the infection/invasion process, making them potential vaccine candidates. In this study we evaluated whether chickens vaccinated with SPI-2 T3SS components could mount a significant humoral immune response (as measured by serum IgG titres) and whether these antibodies could be transferred to progeny (as measured by egg yolk IgG titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with SE. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgG that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered Salmonella enterica subspecies enterica serovar Enteritidis (SE) per bird in most situations.     

Colonization control of lactose-fermenting Salmonella typhimurium in young broiler chickens by use of dietary lactose

Inclusion of lactose in the diets of chickens has been determined to reduce cecal colonization with Salmonella typhimurium. We hypothesized, therefore, that dietary lactose may be a practical means for reducing the prevalence of Salmonella contamination of chicken products. Because some strains of Salmonella are atypical and ferment lactose, we investigated the effects of dietary lactose on cecal colonization with lactose-fermenting S typhimurium. Broiler chicks were inoculated intracloacally with Lac+ S typhimurium selected for resistance to novobiocin and rifampicin. The chicks also were inoculated orally with certain anaerobes that do not effectively inhibit colonization by S typhimurium, but do appear essential for lactose mediated inhibition of cecal colonization. Control chicks were not given dietary lactose, and chicks in the experimental group were fed a diet containing 7% lactose. Enumeration of Lac+ S typhimurium in cecal contents revealed dietary lactose to be effective at controlling this organism. Control was correlated with changes in cecal pH and increases in undissociated volatile fatty acids, especially propionic acid.     

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.     

Campylobacter and Salmonella control in chickens and the role of fermented food

Salmonella and Campylobacter are undesirable pathogens on poultry. Therefore the effect of fermented feed on the colonization in the gastro-intestinal tract of the chicken, the introduction of both bacteria in a chicken flocks, and the transmission between chickens was studied. Broilers that were fed with fermented feed were significantly less susceptible for Salmonella and Campylobacter than chickens on a standard chicken feed. The spread of Salmonella between broiler chickens was reduced. However, the results also showed, like for other known control measures, that this feed can not absolutely guarantee the absence of Salmonella and Campylobacter. Therefore fermented feed must be seen as one of the hurdles in a so called multiple hurdle strategy. The combination of different hurdles should prevent the introduction and transmission. The effect of fermented feed on Campylobacter and Salmonella is partially caused by the presence of high concentrations of organic acids. In chickens fed with liquid feed the acidic barrier in the first part of the GI-tract was clearly improved. Besides organic acids there are other changes in the GI-tract. Changes in colonization levels of indicator organisms, changes in levels of organic acids and an increased pH in ileum and ceacum. These changes indicate a stabilised GI-flora in fermented feed fed poultry. The research confirmed that by changes in the composition of the feed (carbohydrates, acids, or micro-organisms) the GI-health can be promoted and therewith can contribute to the control of food pathogens in farmed animals.     

Candidate genes for resistance to Salmonella enteritidis colonization in chickens as detected in a novel genetic cross

Salmonellosis is a zoonotic disease that is problematic for both animal production and food safety. A novel genetic cross, named the Iowa Salmonella response resource population (ISRRP), was established to elucidate the genetic control of resistance to Salmonella enteritidis (SE) colonization in young chicks, to characterize unique resistance alleles, and to estimate gene interaction effects. Outbred broiler sires were mated with dams of diverse, highly inbred, light-bodied lines to produce an F(1) generation that was informative for all heterozygous alleles of the sires. Mating F(1) sires back to dams of the corresponding inbred line produced a backcross generation. To mimic the natural route of exposure and thus afford the opportunity to investigate mucosal immunity, pathogenic SE were inoculated into the esophagus of day-old chicks. After 1 week, the SE colonizing the cecal lumen and the spleen were enumerated. Candidate genes were selected for analysis based upon one of the two criteria. Functional candidates were genes with reported activity related to the tested traits. Positional candidates were genes mapped near microsatellites that were linked, in other phases of this project, with antibody levels to SE vaccine. Broiler sire alleles of the MHC class I, NRAMP1, PSAP, and IAP1 genes showed association with SE colonization in the F(1) generation of this novel disease resistance resource population.     

鸡源性多重耐药沙门氏菌 类整合子与耐药基因研究

本试验旨在了解鸡源性沙门氏菌分离株多重耐药与Ⅰ类整合子及耐药基因的携带关系。采用K-B纸片法对29株鸡源性沙门氏菌分离菌株进行10种抗菌药物敏感试验;应用PCR技术对分离菌株进行Ⅰ类整合子及耐药基因检测。29株分离株中有13株对2种以上抗菌药物耐药,属于多重耐药株,氨苄西林-四环素-头孢唑啉-复合磺胺是主要多重耐药谱;13株多重耐药菌中有8株携带Ⅰ类整合子,blatem-1、tetA和tetB基因检出最高。结果表明沙门氏菌多重耐药性与整合子的携带之间关系密切,耐药表型测定结果与耐药基因检测结果基本一致。     

贵州省猪源沙门氏菌对β-内酰胺类药耐药性及耐药基因分析

为了解贵州省猪源沙门氏菌对β-内酰胺类抗菌药物耐药性及其耐药基因的流行情况,本试验从贵州省9个地区规模养猪场中分离鉴定130株沙门氏菌,采用微量肉汤稀释法测定其对常用的8种β-内酰胺类抗菌药物的敏感性,并用PCR法对β-内酰胺酶耐药基因进行检测。结果显示,沙门氏菌对常用的β-内酰胺类抗菌药物耐药性十分严重,其中对头孢他啶的耐药率为100%,其次是氨苄西林和阿莫西林,耐药率分别为80.77%和76.15%,耐药率最低的是头孢噻呋和头孢氨苄,均为46.15%。所有菌株均为多重耐药,其中最少为二重,占总数的2.31%,最多为八重,占总数的4.62%,多重耐药主要集中在四至七重,占总数的88.46%。PCR结果显示,SHV耐药基因未检出,TEM、OXA、CTX-M 3种基因检出率分别是85%、75%和46%,细菌的耐药性与相关耐药基因的检出率基本呈正相关。结果表明,猪源沙门氏菌对β-内酰胺类药物具有普遍耐药性,其中头孢他啶尤为严重。TEM、OXA、CTX-M基因是贵州省猪源沙门氏菌主要耐药基因,临床日益严重的耐药现象与耐药基因的普遍存在有很大的关系。     

Host specific differences alter the requirement for certain Salmonella genes during swine colonization

The pathogenic potential of Salmonella is determined during the complex interaction between pathogen and host, requiring optimal regulation of multiple bacterial genetic systems within variable in vivo environments. The mouse model of systemic disease has been an extremely productive model to investigate the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium). Although the mouse model is a widely used paradigm for studying the pathogenesis of systemic disease caused by Salmonella, investigations concerning food safety interventions should employ natural hosts to examine gastrointestinal colonization by Salmonella. Recent research has demonstrated specific differences in the attenuation of certain S. Typhimurium mutants in mice compared to swine. This variation in pathogenesis between the mouse model and pigs for the S. Typhimurium mutants is presumably dependent upon either the requirements for specific gene products during systemic disease (mouse) versus gastrointestinal colonization (pig) or host specific differences. In addition, host specific diversity in Salmonella colonization of swine has also been described in comparison to other food-producing animals, including cattle and chickens. Differences in Salmonella colonization and pathogenesis across diverse animal species highlight the importance of species-specific studies of gastrointestinal colonization for the development of Salmonella interventions to enhance pork safety.     

Chicken gallinacin gene cluster associated with Salmonella response in advanced intercross line

Salmonella enterica serovar Enteritidis is a gram-negative bacterium that negatively affects human and animal health. Many eukaryotes use antimicrobial peptides (alpha-defensins, beta-defensins, gamma-defensins, and cathelicidins) in innate immune responses to fight bacterial infections. Poultry gallinacins are the functional equivalents of mammalian beta-defensins. Two related advanced intercross lines of chickens were analyzed for association of gallinacin genotypic variation with Salmonella Enteritidis burden levels in the cecum and spleen after infection. Thirteen genes of the chicken beta-defensin cluster (GAL1-13) were sequenced from individuals of each advanced intercross line, plus the founder broiler sire and representatives of the highly inbred Leghorn and Fayoumi founder dam lines. The mean was 17 single-nucleotide polymorphisms (SNPs) per kilobase. One single-nucleotide polymorphism per gene was genotyped with SNaPshot to test for statistical associations with Salmonella Enteritidis colonization after challenge. Among the 13 gallinacin genes evaluated, the single-nucleotide polymorphisms in all genes in a cluster of three adjacent genes (GAL11, GAL12, and GAL13) were associated with bacterial load in the cecal content in the broiler x Leghorn advanced intercross line (three-gene SNP genotype effect, P < 0.008). The results strongly suggest a role of the gallinacins in defense of poultry against enteric pathogens. The use of gallinacin single-nucleotide polymorphisms as molecular markers for genetic selection for Salmonella Enteritidis resistance might result in reduced bacterial burden via development of an enhanced innate immune response.