Polynucleotide phosphorylase (PNPase) is required for Salmonella enterica serovar Typhimurium colonization in swine.

The pnp gene encodes polynucleotide phosphorylase, an exoribonuclease involved in RNA processing and degradation. A mutation in the pnp gene was previously identified by our group in a signature-tagged mutagenesis screen designed to search for Salmonella enterica serovar Typhimurium genes required for survival in an ex vivo swine stomach content assay. In the current study, attenuation and colonization potential of a S. Typhimurium pnp mutant in the porcine host was evaluated. Following intranasal inoculation with 10(9) cfu of either the wild-type S. Typhimurium χ4232 strain or an isogenic derivative lacking the pnp gene (n = 5/group), a significant increase (p < 0.05) in rectal temperature (fever) was observed in the pigs inoculated with wild-type S. Typhimurium compared to the pigs inoculated with the pnp mutant. Fecal shedding of the pnp mutant was significantly reduced during the 7-day study compared to the wild-type strain (p < 0.001). Tissue colonization was also significantly reduced in the pigs inoculated with the pnp mutant compared to the parental strain, including the tonsils, ileocecal lymph nodes, Peyer's Patch region of the ileum, cecum and contents of the cecum (p < 0.05). The data indicate that the pnp gene is required for S. Typhimurium virulence and gastrointestinal colonization of the natural swine host.

Evaluation of the impact of quorum sensing transcriptional regulator SdiA on long-term persistence and fecal shedding of Escherichia coli O157:H7 in weaned calves.

Escherichia coli O157:H7 (O157) colonization of bovine intestine is mediated through the locus of enterocyte effacement (LEE)-encoded type III secretion system and secreted virulence proteins that promote colonization of the recto-anal junction (RAJ) of the large intestine of cattle. The quorum sensing transcriptional regulator SdiA, a homolog of LuxR, has been shown in vitro to repress LEE strongly when overexpressed from a multi-copy recombinant plasmid or when its activity is enhanced by the binding of N-acyl-L-homoserine lactones (AHLs), the quorum sensing signals that are detected by SdiA. Since LEE has been shown to be essential for colonization and persistence of O157 in bovine intestine, we examined whether a mutation in sdiA, which normally represses LEE in vitro, would also exert negative effect on colonization and long-term persistence of O157 in weaned calves. Ten-week old weaned calves (n = 4/group) were inoculated orally with 10(10) cfu of either the wild-type or sdiA mutant strain. Initial fecal shedding of the sdiA mutant and the wild-type strain were similar in magnitude and declined during the first 2 weeks post-inoculation. The sdiA mutant was detected in feces of only one of the four calves at low levels (≥10(2) cfu/g feces) from days 19 - 27 post-inoculation, whereas, the fecal shedding of the wild-type strain persisted at approximately 4-logs in all four calves from days 19 - 27. We also confirmed that SdiA represses ler, which encodes a positive transcriptional regulator of LEE, in response to AHLs, and reduces adherence of O157 to HEp-2 cells. In conclusion, this study demonstrates that although in vitro the sdiA gene represses LEE and LEE-mediated adherence to cultured cells, the presence of sdiA is necessary for colonization of bovine large intestine that in turn promotes persistent fecal shedding of O157 by these animals.

Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle.

The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1ß was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves.

Integrating comparative expression profiling data and association of SNPs with Salmonella shedding for improved food safety and porcine disease resistance.

Salmonella in swine is a major food safety problem, as the majority of US swine herds are Salmonella-positive. Salmonella can be shed from colonized swine and contaminate (i) neighbouring pigs; (ii) slaughter plants and pork products; (iii) edible crops when swine manure is used as a fertilizer; and (iv) water supplies if manure used as crop fertilizer runs off into streams and waterways. A potentially powerful method of addressing pre-harvest food safety at the farm level is through genetic improvement of disease resistance in animals. In this research, we describe a successful strategy for discovering genetic variation at candidate genes associated with disease resistance in pigs. This involves integrating our recent global gene expression analysis of the porcine response to Salmonella with information from the literature about important candidate genes. We identified single-nucleotide polymorphisms (SNPs) in these functional candidate genes and genotyped three independent pig populations that had data on Salmonella faecal shedding or internal burden (total n = 377) at these loci. Of 31 SNPs genotyped, 21 SNPs segregated in at least two populations with a minor allele frequency of 15% or greater. Statistical analysis revealed thirteen SNPs associated with Salmonella faecal shedding or tissue colonization, with an estimated proportion of false positives (PFP) ≤0.2. The genes with associated SNPs included GNG3, NCF2, TAP1, VCL, AMT, CCR1, CD163, CCT7, EMP1 and ACP2. These associations provide new information about the mechanisms of porcine host response to Salmonella and may be useful in improving genetic resistance to this bacterium.

Use of bioinformatic SNP predictions in differentially expressed genes to find SNPs associated with Salmonella colonization in swine.

Asymptomatic Salmonella-carrier pigs present a major problem in preharvest food safety, with a recent survey indicating >50% of swine herds in the United States have Salmonella-positive animals. Salmonella-carrier pigs serve as a reservoir for contamination of neighbouring pigs, abattoir pens and pork products. In addition, fresh produce as well as water can be contaminated with Salmonella from manure used as fertilizer. Control of Salmonella at the farm level could be through genetic improvement of porcine disease resistance, a potentially powerful method of addressing preharvest pork safety. In this research, we integrate gene expression profiling data and sequence alignment-based prediction of single nucleotide polymorphisms (SNPs) to successfully identify SNPs in functional candidate genes to test for the associations with swine response to Salmonella. A list of 2527 genes that were differentially regulated in porcine whole blood in response to infection with Salmonella enterica serovar Typhimurium were selected. In those genes, SNPs were predicted using ANEXdb alignments based on stringent clustering of all publically available porcine cDNA and expressed sequence tag (EST) sequences. A set of 30 mostly non-synonymous SNPs were selected for genotype analysis of four independent populations (n = 750) with Salmonella faecal shedding or tissue colonization phenotypes. Nine SNPs segregated with minor allele frequency ≥15% in at least two populations. Statistical analysis revealed SNPs associated with Salmonella shedding, such as haptoglobin (HP, p = 0.001, q = 0.01), neutrophil cytosolic factor 2 (NCF2 #2, p = 0.04, q = 0.21) and phosphogluconate dehydrogenase (p = 0.066, q = 0.21). These associations may be useful in identifying and selecting pigs with improved resistance to this bacterium.

Methods for transcriptomic analyses of the porcine host immune response: application to Salmonella infection using microarrays.

Technological developments in both the collection and analysis of molecular genetic data over the past few years have provided new opportunities for an improved understanding of the global response to pathogen exposure. Such developments are particularly dramatic for scientists studying the pig, where tools to measure the expression of tens of thousands of transcripts, as well as unprecedented data on the porcine genome sequence, have combined to expand our abilities to elucidate the porcine immune system. In this review, we describe these recent developments in the context of our work using primarily microarrays to explore gene expression changes during infection of pigs by Salmonella. Thus while the focus is not a comprehensive review of all possible approaches, we provide links and information on both the tools we use as well as alternatives commonly available for transcriptomic data collection and analysis of porcine immune responses. Through this review, we expect readers will gain an appreciation for the necessary steps to plan, conduct, analyze and interpret the data from transcriptomic analyses directly applicable to their research interests.

Correlating blood immune parameters and a CCT7 genetic variant with the shedding of Salmonella enterica serovar Typhimurium in swine.

The porcine response to Salmonella infection is critical for control of Salmonella fecal shedding and the establishment of Salmonella carrier status. In this study, 40 crossbred pigs were intranasally inoculated with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and monitored for Salmonella fecal shedding and blood immune parameters at 2, 7, 14 and 20 days post-inoculation (dpi). Using a multivariate permutation test, a positive correlation was observed between Salmonella Typhimurium shedding levels at 2 and 7dpi and serum interferon-gamma (IFNgamma) levels at 2dpi (p<0.05), with Salmonella being shed in greater numbers from animals with higher IFNgamma levels. A positive correlation was also observed between IFNgamma levels and the number of banded neutrophils (2dpi), circulating neutrophils (7 and 14dpi), monocytes (7dpi), and white blood cells (WBCs) (7, 14 and 20dpi). We have further performed association studies on these immune response parameters as well as shedding status of the Salmonella-infected pigs with a single nucleotide polymorphism (SNP) in the porcine gene CCT7, previously shown by our group to be transcriptionally up-regulated in swine experimentally inoculated with Salmonella Typhimurium. Our analyses with the 40 pigs suggest a positive association (p=0.0012) of SNP genotype A/G at position AK240296.c1153G>A of the CCT7 gene with Salmonella shedding at 7dpi compared to the G/G homozygote genotype. Linking specific genes and genetic polymorphisms with the porcine immune response to Salmonella infection and shedding may identify potential markers for carrier pigs as well as targets for disease diagnosis, intervention and prevention.

Computational integration of structural and functional genomics data across species to develop information on the porcine inflammatory gene regulatory pathway.

We are investigating the porcine gut immune response to infection through gene expression profiling. Porcine Affymetrix GeneChip data was obtained from RNA prepared from mesenteric lymph node of swine infected with either Salmonella enterica serovar Typhimurium (ST) or S. Choleraesuis (SC) for 0, 8, 24, 48 or 504 hours post-inoculation (hpi). In total, 2365 genes with statistical evidence for differential expression (DE; p < 0.01, q < 0.26, fold-change > 2) between at least two time-points were identified. Comparative Gene Ontology analyses revealed that a high proportion of annotated DE genes in both infections are involved in immune and defence responses. Hierarchical clustering of expression patterns and annotations showed that 22 of the 83 genes upregulated from 8-24 hpi in the SC infection are known NF-kappaB targets. The promoter sequences of human genes orthologous to the DE genes were collected and TFM-Explorer was used to identify a set of 72 gene promoters with significant over-representation of NF-kappaB DNA-binding motifs. All 22 known NF-kappaB target genes are in this list; we hypothesize that the remaining 51 genes are un-recognized NF-kappaB targets. Integration of these results and verification of putative target genes will increase our understanding of the porcine response pathways responding to bacterial infection.

A self-propelled, constant-speed spray vaccinator for commercial layer chickens.

Vaccination of commercial layer chickens is labor intensive and often results in poor rates of seroconversion, which, in turn, generally correlate with decreased flock uniformity and performance. Attempts to improve the vaccination process include numerous variations of individual shop-built vaccinators in use by the layer sector of the poultry industry. Each of these vaccinators has limitations that contribute to poor vaccinations. Major problems include the nonuniform speed of the applicator system and pressure fluctuations at the spray nozzles, which contribute to sporadic dispersion of the vaccine as the vaccinator is pushed or carried past the cages. A battery-powered, self-propelled, constant-speed vaccinator was designed and constructed that operates with constant nozzle pressure. In field use, this vaccinator has resulted in both labor savings (reduction of manpower from five to one to vaccinate 75,000 chickens) and time savings (from 45 min to 7.5 min/poultry house) as well as improved vaccination results (higher positive seroconversion rates) against the poultry pathogen Mycoplasma gallisepticum (MG), a bacterium associated with losses of 15.7 eggs/hen over a 45-wk laying period in MG-infected layers as compared with layers maintained free from infection with MG.

Mycoplasma gallinarum infection in commercial layers and onset of fatty liver hemorrhagic syndrome.

Fatty liver hemorrhagic syndrome (FLHS) was observed in each of three trials in which commercial layers were utilized to determine the effect of Mycoplasma gallinarum (MGn) on egg and eggshell quality parameters and egg production. In each of three trials, FLHS occurred 31-54 days later in MGn-inoculated hens as compared with the Mycoplasma-clean (control) hens. In trials 1 and 2, no therapeutic intervention was initiated to ameliorate FLHS. In trial 3, therapeutic intervention was instituted and consisted of the addition of 1 pound of choline chloride/ton of feed. Total mortality recorded throughout the duration of each trial and attributable to FLHS was not significantly different between the control and the MGn-inoculated treatment. However, FLHS-associated mortality in each of the three trials was numerically greater for the control treatment.

The effects of 6/85 live Mycoplasma gallisepticum vaccine in commercial layer hens over a 43-week laying cycle on egg production, selected egg quality parameters, and egg size distribution when challenged before beginning of lay.

In each of two trials, 80 commercial leghorn-type pullets were separated into two treatments with four replicates of 10 chickens in each treatment. Forty pullets were designated as controls and received no inoculation, whereas the remaining 40 pullets received the 6/85 vaccine strain of Mycoplasma gallisepticum (MG) at 10 wk of age. Hen-day egg production, egg weight, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded weekly in each trial through an entire laying cycle of 43 wk. Further, eggs from all treatments were collected daily, Monday-Thursday, and individually weighed. No significant difference was observed between the treatments for 43-wk means for hen-day egg production, for any of the monitored egg or eggshell quality parameters, or for the number of extra large, large, medium, small, pee wee, or undergrade egg sizes. A significant (P < or = 0.05) difference was observed for the number of jumbo-sized eggs between the two treatments. Results of this study suggest that vaccination of commercial layer chickens at 10 wk of age with 6/85 strain MG does not detrimentally impact egg production, egg size distribution, or ovary/oviduct function as evidenced by selected egg parameters monitored in this study.

The effects of ts-11 strain Mycoplasma gallisepticum vaccination in commercial layers on egg production and selected egg quality parameters.

Live Mycoplasma gallisepticum (MG) vaccines have been USDA approved and licensed for use in commercial layer chickens since 1988; however, egg production and egg quality data exist only for the F strain of MG. Information pertinent to the effects of ts-11 MG on egg and eggshell quality parameters, as well as egg size distribution, is lacking. In this study, pullets were inoculated at 10 wk of age with ts-11 strain MG and placed in biological isolation units at 10 birds/unit. Hen-day egg production, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded in each trial through a 45-wk production cycle. Further, eggs from all treatments were collected daily, Monday-Thursday, and individually weighed. Results of this study indicate that no significant difference was observed between the treatments for the parameters measured or for egg size distribution. Therefore, these data should lessen producers' concerns pertaining to the impact of ts-11 strain MG on egg production, egg and eggshell quality parameters, and egg size distribution.

Regulation of sigma S degradation in Salmonella enterica var typhimurium: in vivo interactions between sigma S, the response regulator MviA(RssB) and ClpX.

The alternate sigma factor sigmaS plays an important role in the survival of Salmonella typhimurium following sudden encounters with a variety of stress conditions. The level of sigmaS is very low in rapidly growing cells but dramatically increases as those cells encounter environmental stress or enter into stationary phase. This increase is due in large measure to the stabilization of sigmaS protein against degradation by the ClpXP protease. The MviA protein, also known as RssB or SprE in Escherichia coli, is a putative member of a two component signal transduction system that plays a central role in facilitating sigmaS degradation by ClpXP. In contrast to most two-component systems, MviA does not appear to regulate gene expression but is believed to interact directly with sigmaS and somehow facilitate degradation. We now provide evidence that MviA(RssB) directly interacts both with sigmaS and ClpX in vivo, presumably enabling presentation of sigmaS to the ClpP protease. Interactions were demonstrated using a bacterial two-hybrid system in which sigmaS, MviA, and ClpX were fused to separate moieties of Bordetella pertussis CyaA (adenylate cyclase). Paired hybrid plasmids containing Cya'-MviA/RpoS-'Cya or Cya'-MviA/ClpX-'Cya successfully reconstituted adenylate cyclase activity in both S. typhimurium and E. coli. However, no direct interactions were detected between ClpX and RpoS. A second series of experiments has indicated that the interaction between MviA and sigmaS requires the N-terminus but not the C-terminus of MviA. Cellular levels of MviA appear to be very low in the cell based on lacZ fusion, Western blot and Northern blot analyses suggesting a catalytic role for MviA in sigmaS degradation. Mutagenesis of MviA residue D58, a canonical residue subject to phosphorylation in many two-component systems, decreased the ability of MviA to facilitate sigmaS turnover in vivo confirming that phosphorylation of MviA increases MviA activity.

Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases.

Periplasmic Cu, Zn-cofactored superoxide dismutase (SodC) protects Gram-negative bacteria from exogenous oxidative damage. The virulent Salmonella typhimurium strain ATCC 14028s has been found to contain two discrete periplasmic Cu, Zn-SOD enzymes that are only 57% identical at the amino acid level. SodCI is carried by a cryptic bacteriophage, and SodCII is closely related to the Cu, Zn-superoxide dismutase of Escherichia coli. All Salmonella serotypes appear to carry the sodCII locus, but the phage-associated sodCI gene is found only in certain strains belonging to the most highly pathogenic serotypes. Expression of either sodC locus appears to be enhanced during stationary phase, but only sodCII is regulated by the alternative sigma factor sigmas (RpoS). Mutants lacking both sodC genes are less lethal for mice than mutants possessing either sodC locus alone, indicating that both Cu, Zn-SOD enzymes contribute to Salmonella pathogenicity. The evolutionary acquisition of an additional sodC gene has contributed to the enhanced virulence of selected Salmonella strains.

Cyclic AMP receptor protein and TyrR are required for acid pH and anaerobic induction of hyaB and aniC in Salmonella typhimurium.

Two acid-inducible genes, aniC and aciK, that require anaerobiosis and tyrosine for expression were identified as orf326a encoding a potential amino acid/polyamine antiporter and hyaB encoding hydrogenase I, respectively. Cyclic AMP (cAMP) receptor protein, cAMP, and TyrR, regulator of aromatic amino acid metabolism, were strong positive regulators of both genes.

Acid stress responses in enterobacteria.

The enteric microogranisms Salmonella, Escherichia coli and Shigella flexneri prefer to grow in neutral pH environments. They nevertheless experience dramatic pH fluctuations in nature and during pathogenesis. In response to environmental encounters with acid, these organisms have evolved complex, inducible acid survival strategies. Regulatory features include an alternative factor (sigma S), 2- component signal transduction systems (PhoP/Q; MviA/?) and the major iron regulatory protein Fur. Specific survival mechanisms include emergency pH homeostasis by inducible amino acid decarboxylases and probable roles for DNA repair, chaparonins, membrane biogenesis as well as others that remain poorly defined. Continued study of acid survival in these organisms will provide insights regarding stress management and will have a direct impact on our understanding of pathogenesis.

The acid tolerance response of Salmonella typhimurium provides protection against organic acids.

Salmonella typhimurium encounters a variety of acid stress situations during pathogenesis and in the natural environment. These include the extreme low pH encountered in the stomach and a less acidic intestinal environment containing large amounts of organic weak acids (volatile fatty acids). The acid tolerance response (ATR) is a complex defence system that can minimize the lethal effects of extreme low pH (pH3). The data presented illustrate that the ATR can also defend against weak acids such as butyric, acetic or propionic acids. Although an acid shock of pH 4.4 induced the ATR, growth in subinhibitory concentrations of weak acids did not. Various mutations shown to affect tolerance to extreme acid conditions (pH 3) were tested for their effects on tolerance to weak acids. An rpoS mutant lacking the alternative sigma factor sigma s failed to protect cells against weak acids as well as extreme acid pH. The fur (ferric uptake regulator) and atp (Mg(2+)-dependent ATPase) mutants defective in extreme acid tolerance showed no defects in their tolerance to weak acids. Curiously, the atbR mutant that exhibits increased tolerance to extreme acid pH proved sensitive to weak acids. Several insertions that rendered cells sensitive to organic acids were isolated, all of which proved to be linked to the rpoS locus.

Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium.

Salmonella typhimurium encounters a variety of acid stress situations during growth in host and nonhost environments. The organism can survive potentially lethal acid conditions (pH <4) if it is first able to adapt to mild or more moderate acid levels. The molecular events that occur during this adaptive process are collectively referred to as the acid tolerance response and vary depending on whether the cells are in log- or stationary-phase growth. The acid tolerance response of logarithmically growing cells includes the participation of an alternate sigma factor, sigmaS (RpoS), commonly associated with stationary-phase physiology. Of 51 acid shock proteins (ASPs) induced during shifts to pH 4.4, 8 are clearly dependent on sigmaS for production (I. S. Lee, J. Lin, H. K. Hall, B. Bearson, and J. W. Foster, Mol. Microbiol. 17:155-167, 1995). The acid shock induction of these proteins appears to be the result of an acid shock-induced increase in the level of sigmaS itself. We have discovered that one component of a potential signal transduction system responsible for inducing rpoS expression is the product of the mouse virulence gene mviA+. MviA exhibits extensive homology to the regulatory components of certain two-component signal transduction systems (W. H. Benjamin, Jr., and P. D. Hall, abstr. B-67, p. 38, in Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993, 1993). Mutations in mviA (mviA::Km) caused the overproduction of sigmaS and sigmaS-dependent ASPs in logarithmically growing cells, as well as increases in tolerances to acid, heat, osmolarity and oxidative stresses and significant decreases in growth rate and colony size. Mutations in rpoS suppressed the mviA::Km-associated defects in growth rate, colony size, ASP production, and stress tolerance, suggesting that the effects of MviA on cell physiology occur via its control of sigmaS levels. Western blot (immunoblot) analyses of sigmaS produced from natural or arabinose-regulated promoters revealed that acid shock and MviA posttranscriptionally regulate sigmaS levels. Turnover experiments suggest that MviA regulates the stability of sigmaS protein rather than the translation of rpoS message. We propose a model in which MviA or its unknown signal transduction partner senses some consequence of acid shock, and probably other stresses, and signals the release of sigmaS from proteolysis. The increased concentration of sigmaS drives the elevated expression of the sigmaS-dependent ASPs, resulting in an increase in stress tolerance. The avirulent nature of mviA insertion mutants, therefore, appears to result from inappropriate sigmaS-dependent gene expression during pathogenesis.