The importance of motility and chemotaxis for extra-animal survival of Salmonella enterica serovar Typhimurium and Dublin.

AIMS: This study investigated the importance of flagella and motility of Salmonella enterica serovar Typhimurium and Dublin in models of extra-animal survival. METHODS AND RESULTS: The study was performed using transposon mutants in flagella genes fliC and fljB and in chemotaxis genes cheA, cheB and cheR. Flagella and chemotaxis were found to be of minor importance for attachment to plant leaves, survival in liquid manure and interaction with the nematode C. elegans, while differences were observed between the fliC mutant and the wild-type strain of S. Dublin in interactions with amoebae. CONCLUSIONS: The study shows that flagella and chemotaxis play a minor role in extra-animal survival of these two serovars of Salmonella under the conditions tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Extra-animal survival is important in the full infection cycle for zoonotic salmonellae. Such serovars are motile. Even though the current study was only based on the characterization of two serovars, it strongly suggests that motility and chemotaxis are of minor importance during the spread of Salmonella from one animal to the next through the external environment.

Chitin hydrolysis by Listeria spp., including L. monocytogenes.

Listeria spp., including the food-borne pathogen Listeria monocytogenes, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear beta-1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that L. monocytogenes and other Listeria spp. are able to hydrolyze alpha-chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30 degrees C than at 37 degrees C. In L. monocytogenes EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (chiB) and lmo1883 (chiA), but not on a gene encoding a putative chitin binding protein (lmo2467). The chiB and chiA genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of Listeria are discussed.

Increased amount of Bifidobacterium thermacidophilum and Megasphaera elsdenii in the colonic microbiota of pigs fed a swine dysentery preventive diet containing chicory roots and sweet lupine.

AIMS: To investigate which specific bacterial species that were stimulated or inhibited in the proximal colon of pigs when a fructan-rich diet was compared with a diet that contained resistant carbohydrates. The study focussed especially on Bifidobacterial species by using a noncultureable approach. METHODS AND RESULTS: Terminal restriction fragment length polymorphism (T-RFLP) was used to describe differences in the total colonic microbiota as well as in the populations of Bifidobacterium spp. in pigs fed with a fructan-rich diet and a diet containing resistant carbohydrates. The fructan-rich diet has previously been shown to prevent swine dysentery caused by Brachyspira hyodysenteriae. The T-RFLP profiling, 16S rRNA gene cloning and in situ hybridization showed that the pigs fed with the fructan-rich diet had a higher proportion of Bifidobacterium thermacidophilum subsp. porcinum and Megasphaera elsdenii. CONCLUSIONS: These findings suggested that the bacterial fructan fermentation occurring in the porcine colon might be cross-feeding of lactate produced by B. thermacidophilum and used by M. elsdenii. SIGNIFICANCE AND IMPACT OF THE STUDY: B. thermacidophilum and M. elsdenii may be the course of the inhibition of the pathogenic bacteria Brach. hyodysenteriae in colon of pigs when they are fed fructan-rich diets.

The effect of inulin on new and on patent infections of Trichuris suis in growing pigs.

The objective of this experiment was to investigate the potential influence of inulin on the establishment of new and patent infections of Trichuris suis in growing pigs. Two experimental diets were formulated based on barley flour with either added insoluble fibre from oat husk (Diet 1) or a pure inulin (16%) supplementation (Diet 2). Twenty-eight 10-week-old pigs were divided randomly into 4 groups (Groups 1-4) each of 7 pigs. After 3 weeks adaptation to the experimental diets all pigs were infected with a single dose of 2000 infective T. suis eggs. Group 1 was fed Diet 1 until 7 weeks post-infection (p.i.) and Group 3 until 9 weeks p.i., Group 2 was fed Diet 2 until 7 weeks p.i., Group 4 was fed Diet 1 until week 7 p.i. and was switched-over from Diet 1 to Diet 2 until week 9 p.i. Seven weeks p.i. pigs in Groups 1 and 2 were slaughtered, and pigs in Groups 3 and 4 were slaughtered at 9 weeks p.i. Trichuris suis worm burdens were determined for all pigs. Inulin-fed pigs (Group 2) exhibited an 87% reduction in EPG, compared to the pigs on standard diet (Group 1) (P < 0.0001). The number of worms recovered at week 7 p.i. from pigs on the inulin diet (Group 2) was significantly reduced by 71%, compared to the pigs on standard diet (Group 1) (P < 0.01). At week 9, worm recovery in pigs on the inulin diet switch protocol (Group 4) was reduced by 47% compared to the control pigs in Group 3 (P < 0.01). Further, the inulin-fed pigs exhibited a significant reduction in female worm fecundity and worm large intestine location was more distal compared to those from pigs on standard diet. These results demonstrate that inclusion of the highly degradable fructose polymer inulin in the diet leads to significant reductions in T. suis establishment, egg excretion, and female worm fecundity and can be used as a treatment for patent infections.

Pathology of Trichuris suis infection in pigs fed an inulin- and a non-inulin-containing diet.

Pathology of the colon in pigs infected with Trichuris suis and fed an inulin and a non-inulin containing diet was studied to investigate the inflammatory response induced and the influence of inulin on this. Twenty-nine pigs were allocated into four groups (N-7, I-7, N-9 and N/I-9). Groups N-7, N-9 and N/I-9 were given a diet with non-fermentable carbohydrates, and group I-7 was fed a diet including fermentable carbohydrates (inulin). After 2 weeks, all pigs were inoculated with 2000 T. suis eggs. Seven weeks post-infection (p.i.), groups N-7 and I-7 were killed and group N/I-9 changed to inulin diet to study the influence of inulin on already established T. suis. Nine weeks p.i., groups N-9 and N/I-9 were killed. Trichuris suis were collected from the colon for enumeration. Tissues from the colon of each pig were taken for histological and immunohistochemical quantitative and semi-quantitative evaluations of heterophils, eosinophils, mast cells, IgA(+), IgG(+), IgM(+) and CD3(+) cells. The findings were compared with tissues from seven uninfected pigs of the same age. Pigs fed inulin had significantly fewer worms compared with pigs on the non-fermentable carbohydrate diet. The number of inflammatory cells in the colonic mucosa was increased in all T. suis-infected pigs compared with uninfected controls. The numbers of eosinophils, mast cells, IgA(+), IgG(+) and CD3(+) cells were significantly lower in pigs fed inulin than pigs fed non-fermentable carbohydrate. Whether this is a direct effect of inulin or a secondary effect due to the lower parasitic burden in inulin fed pigs is not known.

The effect of dietary carbohydrates and Trichuris suis infection on pig large intestine tissue structure, epithelial cell proliferation and mucin characteristics.

Two experiments (Exps. 1 and 2) were performed to study the influence of Trichuris suis infection and type of dietary carbohydrates on large intestine morphology, epithelial cell proliferation and mucin characteristics. Two experimental diets based on barley flour were used; Diet 1 was supplemented with resistant carbohydrates from oat hull meal, while Diet 2 was supplemented with fermentable carbohydrates from sugar beet fibre and inulin. In Experiment 1, 32 pigs were allocated randomly into four groups. Two groups were fed Diet 1 and two groups Diet 2. Pigs from one of each diet group were inoculated with a single dose of 2000 infective T. suis eggs and the other two groups remained uninfected controls. In Experiment 2, 12 pigs were allocated randomly into two groups and fed Diet 1 or Diet 2, respectively, and inoculated with a single dose of 2000 infective T. suis eggs. All the pigs were slaughtered 8 weeks post inoculation (p.i.). The worm counts were lower in pigs fed Diet 2 in both experiments, but not significantly so. Both diet and infection status significantly influenced the tissue weight of the large intestine. In both experiments, pigs fed Diet 2 had heavier large intestines than pigs fed Diet 1 and in Experiment1 the infected pigs of both diets had heavier large intestines than their respective control groups. Diet and infection also significantly affected the morphological architecture and mucin production in both experiments. Pigs fed Diet 1 had larger crypts both in terms of area and height than pigs fed Diet 2 and T. suis infected pigs on both diets in Experiment 1 had larger crypts than their respective control groups. The area of the mucin granules in the crypts constituted 22-53% of the total crypt area and was greatest in the T. suis infected pigs fed Diet 1. Epithelial cell proliferation was affected neither by diet nor infection in any of the experiments. The study showed that both T. suis infection and dietary carbohydrates significantly influence the morphological architecture and the production and composition of mucins in the large intestine of pigs and suggests that both factors are important in large intestine function and that carbohydrates may play a role in the susceptibility to intestinal helminth infections.

The influence of dietary carbohydrates on experimental infection with Trichuris suis in pigs.

Two experiments (Exps 1 and 2) were carried out to study the effect of dietary carbohydrates on the establishment of Trichuris suis in pigs. Two experimental diets based on barley flour were used; Diet 1 was supplemented with non-fermentable carbohydrates from oat hull meal, while Diet 2 was supplemented with fermentable carbohydrates from sugar beet fibre and inulin. In Exp. 1, thirty-two pigs were allocated randomly into 4 groups. Two groups were fed Diet 1 and 2 groups were fed Diet 2. Pigs from one of each diet group were inoculated with 2000 infective T. suis eggs each and the other two groups were uninfected controls. All pigs were slaughtered 8 weeks post-inoculation (p.i.). In Exp. 2, twenty-four pigs were allocated randomly into 2 groups and fed Diet 1 or Diet 2, respectively. All the pigs were inoculated with 2000 infective T. suis eggs. Six pigs from each group were slaughtered 8 weeks p.i. and the remaining 6 pigs from each group were slaughtered 12 weeks p.i. Infections were followed by faecal egg counts and worm burdens were assessed at necropsy. Pigs fed Diet 2 had lower egg counts in both experiments; in Exp. 2 the difference was significant (P<0.05). No differences were found in worm burdens 8 weeks p.i. in both experiments, however, worms from pigs on Diet 2 were significantly shorter (P<0.0001). Pigs fed Diet 2 and slaughtered 12 weeks p.i. had significantly lower worm counts (P<0.01) compared to pigs fed Diet 1. The results indicate that fermentable carbohydrates do not affect the establishment of T. suis in naïve pigs, but result in earlier expulsion and reduced growth of the established worms. Thus, diets with highly fermentable carbohydrates may be used in the control of T. suis.

ClpP is involved in the stress response and degradation of misfolded proteins in Salmonella enterica serovar Typhimurium.

Components of the ATP-dependent Clp protease complex are found in a wide range of prokaryotic cells and they are often expressed as part of the cellular stress response. To investigate the physiological role of the proteolytic subunit, ClpP, in Salmonella enterica serovar Typhimurium (S. typhimurium) an in-frame deletion of the clpP gene was constructed. Growth experiments revealed that clpP is important for the ability of S. typhimurium to grow under various stressful conditions, such as low pH, elevated temperature and high salt concentrations. Since the stationary-phase sigma factor, RpoS, is a target of the Clp proteolytic complex, the effect of the clpP deletion in the absence of RpoS was examined; it was observed that growth of the S. typhimurium clpP mutant is affected in both an RpoS-dependent and an RpoS-independent manner. Analysis of the degradation of abnormal puromycyl-containing polypeptides showed that ClpP participates in the proteolysis of such proteins in S. typhimurium. These findings prompted an investigation of the growth of an Escherichia coli clpP mutant under various stress conditions. The growth of this E. coli mutant was affected by heat, salt and low pH, although not to the same extent as observed for the S. typhimurium clpP mutant. The results of this study indicate that the S. typhimurium clpP mutant is generally more sensitive to environmental stress than the E. coli clpP mutant and it is proposed that this is due to a reduced ability to degrade misfolded proteins generated under these conditions.

The influence of stocking rate on transmission of helminth parasites in pigs on permanent pasture during two consecutive summers.

This study was made to elucidate the transmission of nematode infections in outdoor pigs at different stocking rates during two consecutive seasons. Five pigs (Group 1A) inoculated with low doses of Oesophagostomum dentatum, Ascaris suum, and Trichuris suis and five helminth-naïve pigs (Group 1B) were turned out together in June 1996 on each of four pastures at stocking rates of 100, 240 (two pastures) and 576m(2) per pig, respectively. The pigs were slaughtered in early October, and pasture infectivity was subsequently measured using helminth-naïve tracer pigs (Tracer). In 1997, 10 helminth-naïve pigs were turned out on each pasture in May (Group 2) and again in August (Group 3), and allowed to graze for 12 weeks. The percentage of grass cover was reduced considerably at the high stocking rate in comparison to the other stocking rates. Transmission of all three helminths was observed on all pastures. In 1996, the O. dentatum faecal egg counts and worm burdens were significantly higher in pigs at the high stocking rate compared to pigs at the other stocking rates. O. dentatum did not survive the winter and pigs of Group 2 were inoculated with 3000 larvae each to reintroduce this parasite. Ascaris suum ELISA values and worm counts were highest at the high stocking rate in 1997 (Group 3). Transmission of T. suis was not significantly influenced by stocking rate. The results indicate that transmission of O. dentatum, and to some extent A. suum is influenced by stocking rate. However, both A. suum and T. suis eggs are still expected to constitute a high risk of infection on intensively used pastures where eggs may accumulate for years. The relationship between host density and helminth transmission seems more complex for grazing/rooting pigs than for grazing ruminants. This may be due to the differences in behaviour of the animals and the resulting differences in microclimate of the developing eggs/larvae.

Nose-rings and transmission of helminth parasites in outdoor pigs.

Five growing pigs experimentally infected with low doses of Oesophagostomum dentatum, Ascaris suum, and Trichuris suis were turned out with 5 helminth-naïve pigs on each of 3 pastures in June 1996 (Group 1). On one pasture all pigs received nose-rings. After slaughter of Group 1 in October, pasture infectivity was monitored using helminth-naïve, unringed tracer pigs. In 1997, helminth-naïve young pigs were turned out on the contaminated pastures in May (Group 2) and again in August (Group 3). Again all pigs on one pasture received nose-rings. All pigs and pastures were followed parasitologically and reduction in grass cover was monitored. Based on the acquisition of infection by the naïve pigs in Group 1, the estimated minimal embryonation times for eggs deposited on pasture were 23-25 days for O. dentatum, 5-6 weeks for A. suum and 9-10 weeks for T. suis. Results from tracer pigs and grass/soil samples indicated that pasture infectivity was light both years. Free-living stages of O. dentatum did not survive the winter. The nose-rings reduced rooting considerably, resulting in three-fold more grass cover on the nose-ring pasture compared to the control pastures by the end of the experiment. Nevertheless, the nose-rings did not significantly influence parasite transmission.

Protein-ligand interaction: grafting of the uridine-specific determinants from the CytR regulator of Salmonella typhimurium to Escherichia coli CytR.

Members of the LacI family of transcriptional repressors respond to the presence of small effector molecules. The binding of the ligands affect the proteins ability to repress transcription by stabilizing a conformation that, in most cases, is unfavorable for high-affinity DNA binding. The CytR anti-activator diverges from the other family members by relying on the cooperative DNA binding with the global regulator CRP. The inducers of CytR do not affect CytR-DNA binding per se, but alleviate repression by interrupting protein-protein interactions between the two regulators. Here, we have studied of the CytR-inducer interaction by exploring a discrepancy in the inducer response observed for the homologous CytR regulators of Escherichia coli and Salmonella typhimurium. CytR of S. typhimurium (CytRSt) appears to respond to the presence of both uridine and cytidine nucleosides, whereas E. coli CytR (CytREc) responds to cytidine only. We have used a combination of genetic and structural modeling studies to provide detailed information regarding the nature of this discrepancy. By analysis of hybrid CytR proteins followed by site-directed mutagenesis, we have successfully transferred the specificity determinants for uridine from CytRSt to CytREc, revealing that serine substitutions of only two residues (G131 and A152) in CytREc is required to make CytREc sensitive to uridine. In addition, by employing a genetic screen for induction of defective mutants, we have identified four amino acid residues in CytRSt that appear to be important for the response to uridine. The implications of these findings for the understanding of the ligand binding and induction of CytR are discussed in the context of the structural knowledge of CytR and homologous protein-ligand complexes.