Heat stress impairs egg production in commercial laying hens infected by fowl typhoid.

Salmonella Gallinarum (SG) is an avian-restricted pathogen that causes fowl typhoid in poultry. Although it has been reported frequently over many decades in poultry flocks worldwide, the microorganism is more commonly associated with poultry in developing countries, particularly those with high ambient temperatures, where the acute form of the disease results in considerable economic losses. A more detailed investigation of environmental factors that affect the course of disease may assist in identifying effective prevention and control measures. Heat stress is known to impair the immunological response to a variety of pathogens and clearly may be an important contributory factor in the prevalence of disease in countries with warm or hot climates. Thus, the objective of the present study was to evaluate the effects of heat stress on chickens infected with SG. For this, light and semi-heavy commercial laying hens were distributed randomly within four groups as follows: infected and non-infected groups in rooms held at ambient temperature, and infected and non-infected groups under heat stress. Clinical signs, egg production, and mortality were recorded daily. Bacteriological counts in liver and spleen samples were estimated at 2, 5, 7, and 14 days post-infection. The results showed that both SG infection and heat stress had similar effects on egg production and a synergistic effect of the two stressors was observed. The data show an interaction between disease and heat stress which could point towards environmental and biosecurity approaches to resolving the possible 30% fall in production observed in such countries.

Butyrate-mediated autophagy inhibition limits cytosolic Salmonella Infantis replication in the colon of pigs treated with a mixture of Lactobacillus and Bacillus.

Probiotics as an effective and safe strategy for controlling Salmonella infection are much sought after, while autophagy is a central issue in eliminating intracellular pathogens of intestinal epithelial cells. In this study, an animal model of colitis has been developed by infecting weaned pigs orally with a strain of Salmonella Infantis in order to illuminate the potential efficacy of a mixture of Lactobacillus and Bacillus (CBB-MIX) in the resistance to Salmonella infection by regulating butyrate-mediated autophagy. We found that CBB-MIX alleviated S. Infantis-induced colitis and tissue damage. Autophagy markers ATG5, Beclin-1, and the LC3-II/I ratio were significantly enhanced by S. Infantis infection, while treatment with CBB-MIX suppressed S. Infantis-induced autophagy. Additionally, S. Infantis-induced colonic microbial dysbiosis was restored by this treatment, which also preserved the abundance of the butyrate-producing bacteria and the butyrate concentration in the colon. A Caco-2 cell model of S. Infantis infection showed that butyrate had the same effect as the CBB-MIX in restraining S. Infantis-induced autophagy activation. Further, the intracellular S. Infantis load assay indicated that butyrate restricted the replication of cytosolic S. Infantis rather than that in Salmonella-containing vacuoles. Suppression of autophagy by knockdown of ATG5 also attenuated S. Infantis-induced cell injury. Moreover, hyper-replication of cytosolic S. Infantis in Caco-2 cells was significantly decreased when autophagy was inhibited. Our data demonstrated that Salmonella may benefit from autophagy for cytosolic replication and butyrate-mediated autophagy inhibition reduced the intracellular Salmonella load in pigs treated with a probiotic mixture of Lactobacillus and Bacillus.

Salmonella infection induced intestinal crypt hyperplasia through Wnt/ß-catenin pathway in chicken.

S. Pullorum is a causative agent of enteric disease of poultry with serious diarrhea. However, the detailed mechanism behind its injury to intestinal mucosa barrier, especially for intestinal stem cells, is unclear. In this study, S. Pullorum were orally administrated to 3 days old chicken to investigate the pathogenesis of S. Pullorum on intestinal mucosal barrier, especially on the proliferation of epithelial cells. We found that S. Pullorum could colonize in the cecum and invade into the liver through intestinal mucosa damage, which caused obvious pathological changes in liver and intestine and even leaded to death, as well as significant reduction of body weight. We also found that S. Pullorum infection enhanced the mRNA expression of IL-1ß and IL-6 through TLR4/MyD88 pathway, which was also further verified by the increased lipopolysaccharide (LPS) levels in serum. Furthermore, S. Pullorum increased the depth of crypt and density of PCNA+ cells significantly through the over-activation of Wnt/ß-catenin signaling pathway. The expression of intestinal stem cells markers Lgr5 and Bmi1 was also increased after S. Pullorum infection to support the crypt hyperplasia. In addition, we verified that S. Pullorum infection enhanced the mRNA expression of IL-1ß, TLR4, Lgr5 and Bmi1. Our study indicated that S. Pullorum infection damaged the intestinal mucosa barrier to induce diarrhea, affected the abnormal proliferation of intestinal stem cells by over-activation of Wnt/ß-catenin pathway in chicken.

Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle.

Salmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness.

Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

Comparison of the clinical signs, pathological and immuohistochemical findings in visceral organs of chickens experimentally infected with Salmonella Zega through three routes.

Salmonella Zega isolated from natural outbreaks that were characterized by high mortality in poultry farms in three Southwestern States of Nigeria was used to inoculate two week-old chicks through different routes in order to determine and compare the clinical signs, pathological and immunohistochemical changes in each route of infection. The birds were divided into 4 groups of 25 each as groups A (orally inoculated), B (intraperitoneally inoculated), C (inoculated per cloaca) and D (uninoculated control). All the birds were inoculated with 0.2 ml of 1 × 108 cfu of the bacteria. Clinical signs were observed and recorded according to the route of infection, and with the days post-infection from day 0 till day 10 post-infection. Two birds from each group were sacrificed every 24 h and examined for gross lesions, which were described and scored according to the route of infection and days post-infection. Samples of visceral organs were collected for bacteriology, histopathology and immunohistochemistry. Clinical signs in chicks infected orally and intraperitoneally were weakness, anoraexia lethargy, somnolescence, yellowish diarrhoea observed from 4 days till day 10 post infections. Mild sign of weakness was observed in chickes infected per cloaca, from day 3 to 7. The gross lesions were congestion, oedema and enlargement and necrosis in visceral organs from day 4 to 10 post infection in orally and intraperitoneally infected chicks, but mild vascular changes were observed in chicks infected per cloaca, except in the caecum were lesions of necrosis and infiltration of inflammatory cells were moderate to severe. Microscopic lesions were necrosis of host cells and infiltration by lymphocytes, heterophils and macrophages in multiple organs observed from day 4 to 10 post infection in orally and intraperitoneally infected chicks. Immunoreactions were observed in all the visceral organs examined. Clinical signs, pathological and immunohistochemical findings were mild in chicks infected per cloaca, except caecal lesions. Salmonella Zega isolated from an outbreak in poultry farms in Abeokuta, Nigeria was highly pathogenic in chicken and produced similar findings in oral and intraperitoneal infections; while per cloacal infection showed a localized infection of the caecum.

I-FABP, Pig-MAP and TNF-α as biomarkers for monitoring gut-wall integrity in front of Salmonella Typhimurium and ETEC K88 infection in a weaned piglet model.

Having sensitive serum biomarkers able to determine the structural changes of the small intestine suffering from bacterial digestive diseases could be a valuable tool particularly in piglets at weaning, when intestinal infections are highly prevalent. We evaluated the usefulness of three inflammatory and gut-wall-integrity biomarkers to assess the degree of intestinal histo-morphological damage in piglets. Piglets were orally challenged with Salmonella Typhimurium or enterotoxigenic Escherichia coli (ETEC) to get a variable range of response according to individual variability. Forty-eight piglets were challenged with Salmonella Typhimurium and seventy-two with enterotoxigenic Escherichia coli K88. Clinical signs and faecal score were recorded. At Days 4 and 8 post-inoculation, blood was sampled, animals euthanised and distal ileum dissected. Morphological measures were obtained from the gut tissue, and serum tumour necrosis factor-alpha (TNF-α), pig major acute-phase protein (Pig-MAP) and intestinal fatty acid-binding protein (I-FABP) were determined. Animals developed mild-to-severe diarrhoea after the challenge. When analysing the complete set of analytical results, a high correlation was found among the three serum biomarkers. The most representative morphological indicator was the villus:crypt ratio (V:C), which showed a strong negative correlation with all three biomarkers. Regression analyses between faecal score and the previous variable showed linear relations. When the range of V:C was analysed, based on the quartile distribution of each serum variable, a marked increase in their concentration was observed with greater villus damage. Summarising, the combination of I-FABP, Pig-MAP and TNF-α may be useful for determining the intestinal injury degree and barrier integrity in recently weaned pigs.

Development of a Salmonella Typhimurium challenge model in weaned pigs to evaluate effects of water and feed interventions on fecal shedding and growth performance.

The aim of this study was to develop a Typhimurium (ST) challenge model in weaned pigs suitable to evaluate effects of water and feed interventions on fecal shedding and growth performance. Two studies were performed. In Exp. 1 weaned pigs were fed either a standard diet (CON) or a diet with a high buffer capacity (HB) and challenged for either 3 or 7 consecutive days in a Latin square design with 4 × 8 individually housed pigs. In Exp. 2, the CON 7-d challenge method was chosen for further model development and validation. Thirty-two individually housed weaned pigs were divided over 4 treatments: a nonchallenged control group (NCON), a challenged positive control group (PCON), a challenged intervention group with acidified water (WATER), and a challenged intervention group with acidified feed (FEED). Pigs were orally challenged once daily on d 7 to 9 or d 7 to 13 after weaning (d 0) with 1 ×10 cfu ST. From d 0 to 28, rectal temperature and occurrence of diarrhea were recorded daily, and BW and feed intake were measured weekly. Fecal samples were collected on d 0, 2, 7, 9, 13, 16, 20, 23, and 27 in Exp. 1 and d 0, 2, 7, 8, 9, 13, 15, and 27 in Exp. 2 for quantification. The results of both experiments showed quantifiable fecal shedding (average peak shedding of approximately 3.5 log and 5.5 log cfu/g, respectively), accompanied by a transient 0.5°C increase in rectal temperature and an increase in occurrence of diarrhea. In Exp. 2 during the week of challenge (i.e., d 7 to 14), a reduction in growth performance (ADG: -157 to 200 g/d and G:F: -0.22 to 0.25 g/d; < 0.01) in PCON and FEED was observed compared to NCON, with WATER showing an intermediate response. The WATER treatment also showed a numerically lower peak shedding (difference of -1.3 to 1.4 log cfu/g) compared to PCON and FEED. To conclude, we repeatedly infected weaned pigs successfully with 1 × 10 cfu of ST for 7 consecutive days, resulting in detectable and quantifiable fecal shedding. This ST challenge model may be suitable for evaluation of effects of water and feed interventions on peak fecal shedding and growth performance.

Probiotics in Salmonella-challenged Hy-Line brown layers.

A total of 192 Hy-Line Brown 40-week-old Salmonella-free layers were randomly assigned to 4 dietary treatments in a 5-wk experiment to test the efficacy of probiotics on egg production and quality, excreta and intestinal microbiota of laying birds challenged with Salmonella gallinarum. Dietary treatment comprised of (1) NC: ; basal diet, negative control, (2) PC: ; basal diet + oral S. gallinarum administration, positive control, (3) T1: ; basal diet + 0.1% Bacillus subtilis RX7 1 × 10(9) cfu/g + S. gallinarum administration and (4) T2: ; basal diet + 0.1% Bacillus methylotrophicus C14 1 × 10(9) cfu/g + S. gallinarum administration. All birds (n = 144) except NC were orally challenged with 1 ml suspension of 10(8) cfu/mL S. gallinarum KVCC BA 0700722 once at d 28 after the initiation of experiment. The egg production improved in post Salmonella-challenged birds whereas egg equality was improved during pre-challenge in probiotic supplemented birds compared to NC and PC. The Salmonella counts in the excreta were lower (P < 0.05) in T1 and T2 than PC at the end of the experiment whereas the Lactobacillus counts in the excreta were higher (P < 0.05) in T1 compared with NC. The Escherichia coli counts in excreta were numerically lower in T1 and T2 than PC. In the small and large intestine, there was slight increase in Lactobacillus counts in T2 compared with PC. The Salmonella counts in small and large intestine tended to be lower in T1 and T2 as compared with PC. However, Salmonella counts in challenged birds not supplemented with probiotics were significantly higher than non-challenged birds.

Gene Expression Profiles of Chicken Embryo Fibroblasts in Response to Salmonella Enteritidis Infection.

The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in ß-oxidation of fatty acids in mitochondria.

Monitoring of behavior using a video-recording system for recognition of Salmonella infection in experimentally infected growing pigs.

Behavior is one of the most commonly used indicators of illness; however, few studies have investigated how different common diseases affect animal behavior. This experiment was conducted to investigate behavioral and clinical alterations in growing pigs experimentally infected with Salmonella spp. during a 4-week post-infection period. A total of 48 growing pigs were divided into one of the three treatment groups (1) control, (2) infection with Salmonella Typhimurium or (3) infection with Salmonella Enteritidis. Individual pigs' behavior was recorded daily (0900 to 1100 and 1600 to 1800 h) using a video-recording system. Pigs in both infected groups had lower weight gain and feed intake during week 0 to 2 and 0 to 4 experimental period. Bacteriological data revealed that pigs in both infected groups persistently shed bacteria throughout the period of study. Oral infection of growing pigs with S. Typhimurium and S. Enteritidis significantly reduced the frequency of morning large (except week 1) and small movement throughout the study period. In the evening, significantly lowest frequency of movements were observed in the S. Enteritidis-infected group compared with the control. The standing and sitting frequency were significantly lower in both infected groups only at the morning of week 4. Infection with Salmonella spp. led to a significant reduction in the frequency and duration of morning eating and drinking throughout the experimental period, with the exception of 4th week drinking duration. The lowest frequency of evening eating during week 1 and 4 was recorded in both infected groups; whereas, the duration differed only at week 1. The evening drinking frequency only tended to decrease in response to S. Typhimurium infection at week 1. This study shows that, pigs infected with Salmonella spp. had poor performance, shedding high levels of Salmonella with their feces and reduced feeding and drinking activity, which are adaptive responses to infection and may help caretakers to detect ill health.

Effect of zinc on growth performance, gut morphometry, and cecal microbial community in broilers challenged with Salmonella enterica serovar typhimurium.

To evaluate the effects of supplemental zinc on growth performance, gut morphometry, and the cecal microbial community in broilers challenged with Salmonella typhimurium, 180, 1-day-old male Cobb 500 broiler chicks were randomly assigned to 3 treatments with ten replicates for a 42 day experiment. The 3 treatments were: unchallenged, S. typhimurium-challenged, and S. typhimurium-challenged with 120 mg/kg of zinc supplementation in the diet. Salmonella infection caused a reduction in body-weight gain and feed intake, disrupted the intestinal structure by decreasing the villus-height/crypt-depth ratio of the ileum and increasing the apoptotic index of ileal epithelial cells. Moreover, the cecal microbial community was altered by Salmonella infection, as demonstrated by a reduced number of Lactobacillus and total bacteria. Dietary zinc supplementation improved growth performance by increasing the body-weight gain and feed intake in the challenged broilers. In addition, zinc repaired intestinal injury by reducing the apoptotic index of ileal epithelial cells, enhancing villus height and the villus-height/crypt-depth ratio of the ileum, and the proliferation index of ileal epithelial cells. Finally, zinc regulated the cecal microbial community by increasing the number of total bacteria and beneficial Lactobacillus bacteria, and reducing the number of Salmonella. The results indicated that dietary zinc supplementation improved growth performance, intestinal morphology, and intestinal microbiota in S. typhimurium-challenged broilers.

Bone Mineralization of Broiler Chicks Challenged with Salmonella enteritidis Fed Diet Containing Probiotic (Bacillus subtilis).

The present study was carried out to determine the effect of probiotic, Bacillus subtilis, on ash and calcium contents of tibia bone in unchallenged and challenged broiler chicks with Salmonella enteritidis. In a completely randomized design, 160 chicks were divided into four groups. Each group had four replicates with 10 birds each. Treatments were control group, probiotic-treated group, challenged group and challenged probiotic-treated group. Ash and calcium contents of tibia at 21 and 42 days of age were determined. At 21 days of age, the highest contents of ash and calcium were related to probiotic-treated group and the lowest means to challenged chicks (P < 0.05). At this period, inclusion of probiotic to diet of challenged chick increased (P < 0.05) ash and calcium contents of tibia. With increases in age, the negative effects of challenging and beneficial effects of probiotic on bone mineralization diminished; since at 42 days of age, challenging or probiotic treatment had no effect on ash and calcium contents of tibia.

Características definidoras da Resposta Disfuncional ao Desmame Ventilatório como indicadores de acurácia do desmame ventilatório / Defining characteristics of the Dysfunctional Ventilatory Weaning Response as indicators of accuracy of ventilatory weaning / Características definitorias de la Respuesta Disfuncional al Destete Ventilatorio como indicadores de precisión del destete ventilatorio

Objetivou-se avaliar as características definidoras do diagnóstico Resposta Disfuncional do Desmame Ventilatório, como indicadores de acurácia das tentativas de desmame. Estudo observacional de 38 eventos de tentativa de desmame ventilatório em pacientes adultos internados em terapia intensiva. Para as características definidoras foram calculadas: sensibilidade, especificidade, valores preditivos positivos e negativos, acurácia ou sensibilidade, razão de verossimilhança e razão de chances diagnóstica. Também foram consideradas as medianas do número de características definidoras nos eventos de sucesso e insucesso. Foram consideradas acuradas: agitação, deterioração nos gases sanguíneos arteriais em relação aos parâmetros basais, uso moderado da musculatura acessória da respiração, aumento da frequência respiratória em relação aos parâmetros basais e frequência respiratória aumentada de forma significativa em relação aos parâmetros basais. Houve diferença estatística nas medianas do número de características definidoras observadas. Conclui-se que a característica definidora e o número delas influenciariam o sucesso da decisão sobre o desmame.

The study aimed to analyze the defining characteristics of the Dysfunctional Ventilatory Weaning Response as an indicator of the accuracy of ventilatory weaning. Observational study of 38 events of ventilatory weaning in adult patients admitted to intensive care. For the defining characteristics, it was calculated: sensitivity, specificity, positive and negative predictive values, accuracy or efficiency, likelihood ratio positive and negative, and diagnostic odds ratio. It was also considered the median number of defining characteristics in the event of success and failure. It was considered accurate: agitation, deterioration in arterial blood gases from baseline parameters, moderate use of accessory muscles of respiration, increased respiratory rate from baseline parameters and respiratory rate increases significantly with respect to baseline parameters. There was statistical difference in the median number of defining characteristics observed. It was concluded that the defining characteristic and the number of them would influence the success of the weaning decision.

El estudio tuvo como objetivo evaluar las características que definen el diagnóstico de Respuesta Disfuncional al Destete Ventilatorio como indicador de la exactitud del destete ventilatorio. Estudio observacional de 38 eventos de destete ventilatorio en pacientes adultos ingresados en cuidados intensivos. Para las características definitorias se calcularon: sensibilidad, especificidad, valores predictivos positivos y negativos, precisión o sensibilidad, cocientes de probabilidad y odds ratio diagnóstica. Fueran consideradas las medianas del número de características definitorias en casos de éxito o de fracaso. Se consideraron precisas: agitación, deterioro de los parámetros de gases en sangre arterial desde la línea de base, uso moderado de los músculos accesorios de la respiración, aumento de la frecuencia respiratoria a partir de parámetros de línea de base y frecuencia respiratoria aumentada significativamente en comparación con los parámetros de línea de base. Hubo diferencia estadísticamente significativa en la mediana del número de características definitorias observadas. Se concluye que la característica definitoria y el número de ellas influyen en el éxito de la decisión sobre el destete ventilatorio.

Effects of dietary clays on performance and intestinal mucus barrier of broiler chicks challenged with Salmonella enterica serovar Typhimurium and on goblet cell function in vitro.

In vivo and in vitro experiments were conducted to test for beneficial effects of dietary clays on broiler chicks challenged with Salmonella enterica serovar Typhimurium and to explore potential mechanisms. First, two hundred forty 1-d-old male broilers (initial BW: 41.6 ± 0.4 g) were allotted in a 2 × 4 factorial arrangement in a randomized complete block design. There were 2 infection treatments (with or without Salmonella) and 4 diets: basal (BAS), 0.3% smectite A (SMA), 0.3% smectite B, and 0.3% zeolite. The Salmonella reduced (P < 0.05) the growth rate of chicks fed the BAS, and feeding clay largely restored it (challenge × diet interaction, P < 0.05). Goblet cell number and size were increased (P < 0.05) by Salmonella in chicks fed the BAS and were reduced (P < 0.05) in Salmonella-challenged chicks by feeding SMA. Villus height was reduced by the Salmonella challenge in the chicks fed dietary clays (P < 0.01) but not in chicks fed the BAS (interaction P < 0.05). A human adenocarcinoma cell line (LS174T) was cultured in vitro in 3 separate experiments in the absence or presence of 3 concentrations (0.05, 0.10, and 0.50%) of SMA. Expression of mucin 2 (MUC2), resistin-like molecule ß (RELMß), and trefoil factor 3 (TFF3) were determined by real-time reverse-transcription PCR. The expression of RELMß was increased and expression of MUC2 was reduced (P < 0.05) by 0.10% SMA. Also, LS174T cells were cultured without or with SMA (0.05 and 0.10%) and the medium and cell lysate were analyzed for RELMß using an immunoblot assay. Protein expression of RELMß in the cell lysate was reduced (P < 0.05) by SMA addition but increased in the medium, indicating that SMA increased secretion of RELMß, thus depleting the cell and concentrating this protein in the medium. In conclusion, the dietary clays restored the growth depression caused by Salmonella, and changes in goblet cell function may contribute to the benefits of one of the clays, specifically SMA.

Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection.

BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

The embryonic expression patterns of zebrafish genes encoding LysM-domains.

The function and structure of LysM-domain containing proteins are very diverse. Although some LysM domains are able to bind peptidoglycan or chitin type carbohydrates in bacteria, in fungi and in plants, the function(s) of vertebrate LysM domains and proteins remains largely unknown. In this study we have identified and annotated the six zebrafish genes of this family, which encode at least ten conceptual LysM-domain containing proteins. Two distinct sub-families called LysMD and OXR were identified and shown to be highly conserved across vertebrates. The detailed characterization of LysMD and OXR gene expression in zebrafish embryos showed that all the members of these sub-families are strongly expressed maternally and zygotically from the earliest stages of a vertebrate embryonic development. Moreover, the analysis of the spatio-temporal expression patterns, by whole mount and fluorescent in situ hybridizations, demonstrates pronounced LysMD and OXR gene expression in the zebrafish brain and nervous system during stages of larval development. None of the zebrafish LysMD or OXR genes was responsive to challenge with bacterial pathogens in embryo models of Salmonella and Mycobacterium infections. In addition, the expression patterns of the OXR genes were mapped in a zebrafish brain atlas.

Constitutive expression of MMP9 in intestinal epithelium worsens murine acute colitis and is associated with increased levels of proinflammatory cytokine Kc.

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease, is a chronic inflammatory disease associated with an increased risk for colon cancer. Matrix metalloproteinases (MMPs) are the predominant proteinases expressed in the gut mucosa during active IBD. Our laboratory has previously demonstrated that epithelial-derived MMP9 is absent in normal colonic tissue but is upregulated during IBD. In this study MMP9 transgenic mice (Tg-villin-MMP9) are generated specifically to overexpress MMP9 in intestinal epithelium to examine the role and underlying mechanism by which it modulates the pathogenesis of acute colitis. Dextran sodium sulfate (3% DSS)- and Salmonella typhimurium (S.T.)-induced colitis models were used to study gut inflammation in Tg-villin-MMP9 and wild-type littermates (WT). Colonic tissue was analyzed via Western blot, histology, myeloperoxidase (MPO) assay, and quantitative PCR. Tg-villin-MMP9 mice expressed significantly increased MMP9 mRNA and protein expression at basal level. There was a significant decrease in the goblet cells, but a significant increase in proliferation and apoptosis were observed among Tg-villin-MMP9 mice compared with WT mice. There was also a significant increase in the proinflammatory chemokine Kc among Tg-villin-MMP9 compared with WT mice. Tg-villin-MMP9 exhibited a severe inflammatory response than WT mice in both DSS- and S.T.-induced colitis models as evident by greater weight loss and higher clinical score, histological score, and MPO activity, which correlated with relative levels of Kc mRNA. MMP9 expressed by intestinal epithelial cells mediates inflammation in colitis with simultaneous increase in proinflammatory cytokine Kc.

Virulence and stress-related periplasmic protein (VisP) in bacterial/host associations.

Gram-negative bacteria have an outer membrane containing LPS. LPS is constituted of an oligosaccharide portion and a lipid-A moiety that embeds this molecule within the outer membrane. LPS is a pathogen-associated molecular pattern, and several pathogens modify their lipid-A as a stealth strategy to avoid recognition by the innate immune system and gain resistance to host factors that disrupt the bacterial cell envelope. An essential feature of Salmonella enterica Typhimurium pathogenesis is its ability to replicate within vacuoles in professional macrophages. S. Typhimurium modifies its lipid-A by hydroxylation by the Fe2+/α-ketoglutarate-dependent dioxygenase enzyme (LpxO). Here, we show that a periplasmic protein of the bacterial oligonucleotide/oligosaccharide-binding fold family, herein named virulence and stress-related periplasmic protein (VisP), on binding to the sugar moiety of peptidoglycan interacts with LpxO. This interaction inhibits LpxO function, leading to decreased LpxO-dependent lipid-A modifications and increasing resistance to stressors within the vacuole environment during intramacrophage replication promoting systemic disease. Consequently, ΔvisP is avirulent in systemic murine infections, where VisP acts through LpxO. Several Gram-negative pathogens harbor both VisP and LpxO, suggesting that this VisP-LpxO mechanism of lipid-A modifications has broader implications in bacterial pathogenesis. Bacterial species devoid of LpxO (e.g., Escherichia coli) have no lipid-A phenotypes associated with the lack of VisP; however, VisP also controls LpxO-independent phenotypes. VisP and LpxO act independently in the S. Typhimurium murine colitis model, with both mutants being attenuated for diverging reasons; ΔvisP is less resistant to cationic antimicrobial peptides, whereas ΔlpxO is deficient for epithelial cell invasion. VisP converges bacterial cell wall homeostasis, stress responses, and pathogenicity.

Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages.

More human illnesses caused by Salmonella enterica subspecies enterica serovar Enteritidis throughout the world have been linked to the consumption of contaminated eggs than to any other food vehicle. Deposition of this pathogen in the edible contents of eggs occurs when systemic infections of laying hens involve colonization of reproductive organs. In recent years, the consequences of different housing systems for laying flocks have become the focus of international attention from both animal welfare and public health perspectives. Nevertheless, many questions remain unresolved regarding the food safety implications of various laying hen production systems. The present study assessed the effects of 2 different housing types (conventional cages and colony cages enriched with perching, nesting, and scratching areas) on the invasion of internal organs by Salmonella Enteritidis in experimentally infected laying hens. In 2 trials, groups of laying hens housed in each cage system were orally inoculated with doses of 1.0 × 10(7) cfu of Salmonella Enteritidis. At 5 to 6 d postinoculation, hens were euthanized and samples of internal organs were removed for bacteriologic culturing. For both trials combined, Salmonella Enteritidis was recovered from 95.3% of cecal samples, with no significant differences observed between housing systems. However, Salmonella Enteritidis was detected at significantly (P < 0.05) higher frequencies from hens in conventional cages than from hens in enriched cages for samples of livers (96.9 vs. 75.0%), spleens (93.8 vs. 53.1%), ovaries (25.0 vs. 10.4%), and oviducts (19.8 vs. 2.1%). These results demonstrate that differences in housing systems for egg-laying flocks can affect the susceptibility of hens to colonization of internal organs by Salmonella Enteritidis.