Salmonella Enteritidis Colonization Research Articles

Bacteriophage Treatment Reduces Salmonella Colonization of Infected Chickens

Three different lyric bacteriophages (BPs) were isolated from the sewage system of commercial chicken flocks and used to reduce Salmonella Enteritidis (SE) colonization from experimental chickens. Ten-day-old chickens were challenged with 9.6 x 10(5) colony-forming units (CFU)/ml of a SE strain and treated by coarse spray or drinking water with a cocktail of the three phages at a multiplicity of infection (MO1) of 10(3) plaque-forming units (PFU) 24 hr prior to SE challenge. Chickens were euthanatized at day 20 of age for individual SE detection, quantitative bacteriology, and phage isolation from the intestine and from a pool of organs. SE detection was performed by both bacteriologic culture and genome detection by polymerase chain reaction (PCR). Qualitative bacteriology showed that aerosol-spray delivery of BPs significantly reduced the incidence of SE infection in the chicken group (P = 0.0084) to 72.7% as compared with the control group (100%). In addition, SE counts showed that phage delivery both by coarse spray and drinking water reduced the intestinal SE colonization (P < 0.01; P < 0.05, respectively). BPs were isolated at 10 days postinfection from the intestine and from pools of organs from BP-treated chickens. We conclude that the phage treatment, either by aerosol spray or drinking water, may be a plausible alternative to antibiotics for the reduction of Salmonella infection in poultry.

Global gene expression profile after Salmonella enterica Serovar enteritidis challenge in two F8 advanced intercross chicken lines

A chicken 13K cDNA microarray was used to profile global gene expression after Salmonella enteritidis (SE) challenge of young chickens. Two F8 advanced intercross lines (AIL), broiler by Leghorn, and broiler by Fayoumi, were studied. Day-old chicks were orally inoculated with SE, and spleens were harvested at day 7 or 8 post-inoculation. The SE bacteria burden in the spleen was quantified. The 20% high and 20% low SE burden birds within each AIL and harvest time were studied by microarray. The loop design was used for pair-comparison between high and low SE burden challenged birds and unchallenged birds, within each AIL and harvest time. The signal intensity of each gene was globally normalized and expressed on the natural log scale. A mixed model including line, treatment, time, array (random effect), dye, and all two-way interactions among treatment, time, and line was used to identify differentially expressed candidate genes at the 1% significance level. The results suggest that genetics, time, and interaction between genetics and time play important roles in gene regulation of SE infection and colonization in chickens. The differentially expressed genes identified in the current study are candidates for detailed hypothesis-driven investigation of genes determining resistance to SE in chickens.

Effect of a Defined Competitive Exclusion Culture for Prophylaxis and Reduction of Horizontal Transmission of Salmonella enteritidis in Broiler Chickens

Effective Competitive Exclusion (CE) cultures have been shown to accelerate development of normal microflora in chicks and poults, providing increased resistance to infection by some enteric bacterial pathogens. Our objective was to develop a CE culture for prophylaxis and reduced horizontal transmission of Salmonella enteritidis (SE) in broiler chickens. In the present study, seven members of the family Enterobacteriaceae and 2 lactic acid bacteria isolates, each capable of in vitro and in vivo inhibition of SE, were selected and combined to form the putative CE culture. In the first experiment, day-of-hatch chicks were randomly divided into four pens. All treated chicks were orally gavaged with the CE culture and 3 pens were treated with the CE culture in the drinking water for four consecutive days. Treated and control-non treated chicks were challenged with SE 48 hr later. All 3 groups of birds that were treated with the CE culture had a significant decrease (p<0.05) in cecal colonization compared with non-CE-treated SE-challenged chicks. Two additional experiments were designed to measure the efficacy of the CE culture in reducing SE horizontal transmission from infected to uninfected chicks when commingled. SE was recovered in the cecal tonsils with a significantly lower incidence at days 7 and 14 in Experiment 2 and day 7 in Experiment 3 from the groups that received the CE in the drinking water as compared to controls respectively. These results suggest that a relatively simple and defined CE culture can reduce SE colonization in neonatal chicks.

Prevention of Salmonella Infection in Poultry by Specific Egg-Derived Antibody

Salmonella enteritidis (SE) colonizes the intestinal tract of poultry and causes food born illness in humans. Reduction of (SE) colonization in the intestinal tract of poultry reduces potential carcass contamination during slaughter. The purpose of this study was to investigate the effect of SE-specific yolk immunoglobulin (IgY) on prevention of SE colonization in orally infected broiler chickens. Commercial Single Comb White Leghorn (SCWL) hens were hyperimmunized with SE whole cell antigens. The presence of anti-Salmonella antibody, IgY and IgG in egg yolk and serum respectively, was monitored by Enzyme Linked Sorbent Assay (ELISA). Two hundred forty male `Ross 308` day old chicks were randomly assigned to 8 groups and 3 replications of 10 birds were grown for 42 days of experiment. Eight experimental groups identified with, S, P, A, SP, SA, AP, SPA, C. Four birds from four challenged groups (S), were orally inoculated with 1 mL of bacterial suspension that contained 1×106 CFU mL-1 S. enteritidis at 3 day of age. The groups that supplemented with antibody (A) received 15 mL of yolk contained antibody mixed per 3.84 mL of drinking water on day 1 and continuing for duration of the experiment. The probiotic treated groups (P) were received probiotic, 0.1% of feed and 0.5% of feed, until day 21 and 56 respectively. One group as control (C) did not received any treatment of probiotic and antibody. A-treated and A-P treated groups had significantly lower fecal shedding (p<0.01) and lower concentration of SE cecal colonization (p<0.01). These groups also had a lower isolation of SE from the liver, spleen and ileum. The use of Salmonella enteritidis-specific IgY combined with probiotic had a beneficial effect in reducing the colonization of Salmonella in market-aged broiler under the condition of this study.

Effect of various combinations of alfalfa and standard layer diet on susceptibility of laying hens to Salmonella enteritidis during forced molt

Feed deprivation is commonly used by the poultry industry to induce molting and stimulate multiple egg-laying cycles. However, feed deprivation has been observed experimentally to increase susceptibility of poultry to Salmonella infections. Previous studies indicated that alfalfa was efficacious in reducing Salmonella; the present investigation was designed to evaluate the efficacy of combined alfalfa and layer diets on Salmonella colonization. Leghorn hens over 50 wk of age were divided into 12 groups of hens and placed in individual laying cages. One week prior to dietary changes, hens were put on an 8L:16D photoperiod that continued for the 9-d experiment. Hens were challenged orally with 104 cfu of Salmonella Enteritidis (SE) on d 4 of treatment and cultured for SE at the termination of the 9-d study. Two independent experiments were conducted consisting of the following treatment groups: nonfed hens, full-fed standard commercial layer diet, 100% alfalfa meal diet, a 90% alfalfa meal/10% standard commercial layer diet, and a 70% alfalfa meal/30% standard commercial layer diet. When evaluating SE colonization in the ceca (Exp. 1), a reduction (P < 0.05) was seen in the 100% alfalfa meal diet and the 70% alfalfa meal/30% standard commercial layer diet treatment groups when compared with the controls with Log10 values of 0.54, 0.44, and 2.82, respectively. Evaluation of physiological parameters showed the alfalfa treatment groups had reductions (P < 0.05) in weight loss, ovary weight, and feed consumption when compared with the full-fed standard commercial layer diet hens, and these results were comparable with the nonfed hens. In Exp. 2, all of the treatment groups had a reduction (P < 0.05) in SE colonization of the ceca when compared with the controls. There were also similar physiological reductions in weight loss, ovary weight, and feed consumption when birds were fed the alfalfa diets in Exp. 2. These data suggest that alfalfa can potentially be combined with layer ration to limit SE infection and still induce a molt comparable with feed withdrawal.

Effect of food protein supplements on Salmonella enteritidis infection and prevention in laying hens

Reduction of intestinal colonization of Salmonella enteritidis (SE) during the grow-out period is crucial to provide safer eggs, minimize economic losses, and reduce the spread of human salmonellosis. In the search for novel elimination and prevention methods based on feed supplementation, the effects of feed supplemented with nonimmunized egg yolk powder (did not contain anti-S. enteritidis antibodies), immunized egg yolk powder (with anti-S. enteritidis antibodies), egg yolk proteins, egg white, and skim milk powder were examined on laying hens. In the elimination study, the chickens were orally infected with SE then given a supplemented feed of 5, 10, or 15% (wt/wt) of each of the test samples. Fecal samples tested weekly showed an absence of SE after the first week of feeding nonimmunized egg yolk powder and a gradual decrease with the other samples. In the prevention study, Salmonella-free chickens were fed the supplemented feed for 4 wk and then infected orally. Fecal samples tested for 4 wk showed that SE was prevented from colonizing the intestinal tract throughout the test period by nonimmunized egg yolk powder, whereas the other samples only delayed the colonization. None of the fed supplements disrupted the balance of the intestinal microflora, and the counts in the feces remained constant. These results show that the administration of only 5.0% (wt/wt) of nonimmunized egg yolk powder can eliminate and prevent SE colonization in laying hens with no adverse effects. Furthermore, the present results indicate that hen egg yolk contains novel anti-adhesive or immunomodulatory components that may act to prevent SE infection.

Effect of acidified drinking water on the recovery of Salmonella enteritidis from broiler crops

Crop is a known source of Salmonella contamination during broiler carcass processing. The effect of drinking water acidification by lactic acid or citric acid or a combination of those with cupric sulfate and d-limonene in the reduction of Salmonella Enteritidis (SE) recovered from the crop of broilers was evaluated. Treatments were administered during 8 hours of preslaughter fasting period (Experiments I and II) and during the last 32 hours of preslaughter (Experiment III). It was observed that acidification reduced water intake when treatments began at preslaughter feed withdrawal, and affected the possible reducing effect of these acids on SE recovering (Experiments I and II). Water intake during preslaughter feed withdrawal was not affected when treatment began 32 hours before slaughter (Experiment III). Treatments reduced SE recovering from crop (p<0.05). In Experiment III, 0.470% of lactic acid reduced the number of recovered SE in 99%. This study suggested that the addition of organic acids in the drinking water 24 hours before beginning the preslaughter feed withdrawal might reduce crop SE colonization and might be an important strategy to reduce SE contamination of broiler products during processing.

Association of INOS, TRAIL, TGF-β2, TGF-β3, and IgL genes with response to Salmonella enteritidis in poultry

Several candidate genes were selected, based on their critical roles in the host's response to intracellular bacteria, to study the genetic control of the chicken response to Salmonella enteritidis (SE). The candidate genes were: inducible nitric oxide synthase (INOS), tumor necrosis factor related apoptosis inducing ligand (TRAIL), transforming growth factor β2 (TGF-β2), transforming growth factor β3 (TGF-β3), and immunoglobulin G light chain (IgL). Responses to pathogenic SE colonization or to SE vaccination were measured in the Iowa Salmonella response resource population (ISRRP). Outbred broiler sires and three diverse, highly inbred dam lines produced 508 F1 progeny, which were evaluated as young chicks for either bacterial load isolated from spleen or cecum contents after pathogenic SE inoculation, or the circulating antibody level after SE vaccination. Fragments of each gene were sequenced from the founder lines of the resource population to identify genomic sequence variation. Single nucleotide polymorphisms (SNP) were identified, then PCR-RFLP techniques were developed to genotype the F1 resource population. Linear mixed models were used for statistical analyses. Because the inbred dam lines always contributed one copy of the same allele, the heterozygous sire allele effects could be assessed in the F1 generation. Association analyses revealed significant effects of the sire allele of TRAIL-Sty I on the spleen (P < 0.07) and cecum (P < 0.0002) SE bacterial load. Significant effects (P < 0.04) were found on the cecum bacterial load for TGF-β3-Bsr I. Varied and moderate association was found for SE vaccine antibody response for all genes. This is the first reported study on the association of SNP in INOS, TRAIL, TGF-β2, TGF-β3, and IgL with the chicken response to SE. Identification of candidate genes to improve the immune response may be useful for marker-assisted selection to enhance disease resistance.

Efeito protetor da microbiota cecal congelada e liofilizada sobre a infecção experimental de frangos de corte por Salmonella enterica sorovar Enteritidis

Estudaram-se os efeitos do tratamento de frangos de corte com microbiota cecal anaeróbia liofilizada (MCL) e congelada (MCC) sobre a infecção do trato digestivo das aves por Salmonella enterica sorovar Enteritidis. Foi usada microbiota intestinal sem prévia identificação bacteriana. A infecção foi persistente, em ordem, no ceco, inglúvio e duodeno. A infecção também foi autolimitante nos grupos tratados e no controle. Não ocorreu diferença entre o grupo-controle positivo e os tratados com MCL ou MCC. Houve redução da colonização do ceco no período de 12 dias após o desafio nos grupos tratados com MCL e MCC, o que não ocorreu no grupo-controle positivo. Não houve variação entre os tratamentos com MCL e MCC quanto às características pesquisadas. A S. Enteritidis reduziu o ganho de peso médio nas aves inoculadas. Os tratamentos com MCL e MCC minimizaram a redução de peso nos grupos infectados.

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.

Lactobacillus flora in the cloaca and vagina of hens and its inhibitory activity against Salmonella enteritidis in vitro

Lactobacilli in the cloaca and vagina of 40 normal laying hens were investigated quantitatively and qualitatively, and their ability to inhibit growth of Salmonella enteritidis (SE) was examined using a spot-the-lawn technique. All samples of cloacal contents and half the samples of vaginal mucus were positive for lactobacilli. The means ± SD of total Lactobacillus counts in the cloaca and those in the vagina were log10 5.5 +/- 1.1 and 2.5 +/- 2.6 cfu/g, respectively. In the cloaca, Lactobacillus acidophilus was isolated from 92.5% of hens, and Lactobacillus salivarius was isolated from 85.0% of hens, whereas Lactobacillus fermentum was isolated from only one hen. In the vagina, L. acidophilus and L. salivarius were isolated from 42.5% of hens. In the inhibition assay in vitro, all strains of Lactobacillus from cloacal contents and vaginal mucus inhibited growth of SE. There was a wide range of the inhibitory activity even in the same species. No difference of the growth inhibition zone was observed between lactobacilli from cloaca and those from vagina. The present study suggested that lactobacilli in the cloaca and vagina of hens might have a protective effect against SE colonization.

Variability in the Resistance of Four Chicken Lines to Experimental Intravenous Infection with Salmonella enteritidis Phage Type 4

The capacity of four chicken lines (Y11, L2, B13, PA12) to control Salmonella enteritidis (SE) phage type 4 (PT4) systemic colonization was investigated. Thirteen-week-old chickens were intravenously inoculated with 10(6) SE colony-forming units, and the levels of SE colonization were determined at various time intervals after inoculation in liver, spleen, genital organs, and ceca. The course of SE infection showed a rapid contamination of liver, spleen, and genital organs, whereas the ceca were infected later. A significant (P < 0.001) effect of the chicken line on levels of SE was detected on day 3 postinoculation (PI) in liver and ceca, on day 10 PI in ceca, and on day 15 PI in spleen. Because an early control of systemic Salmonella infection by the Ity/Nramp1 gene has been demonstrated in mice, we aimed to study the early resistance of chickens to SE. As a consequence, we then focused our study on the between- and within-line variabilities of SE levels on day 3 PI. According to the SE levels in liver on day 3 PI, the chicken lines could be classified as susceptible (Y11 and L2) or resistant (PA12 and B13). This early variability was explored in resistant B13 and susceptible L2 lines. Differences between these two lines were confirmed in liver but not in ceca. A large within-line variability was observed in all organs of these two lines. The genetic origin of this variability will have to be determined as a prerequisite to an eventual selection.

Estimated heritability of the resistance to cecal carrier state of Salmonella enteritidis in chickens

Previously, we have shown differences in susceptibility to the cecal carrier state in chicks orally infected with Salmonella enteritidis (SE) at 1 wk of age for four outbred lines: L2, B13, PA12, and Y11. The egg-type line L2 was one of the most susceptible lines and presented a large variability in cecal SE colonization. The heritability (h2) of the resistance to SE colonization in ceca was estimated in L2 chickens to determine whether genetic factors might be involved in its control. In three independent trials, a total of 819 L2 chicks produced from 88 sires and 232 dams were challenged orally with SE at 1 wk of age. Each week after inoculation, the frequency of cecal colonization was estimated. When this value had fallen to 50%, all the remaining animals were killed. The extent of cecal colonization by SE was estimated directly by counting the viable organisms in organs and determining the numbers of positive ceca. Enrichment culture was used in Trials 2 and 3. The effects of trial, of room within trial, and of cage within room on the frequency of SE contaminated ceca were often significant. No significant effect of sex was observed. Estimation of h2 using the frequency of SE positive ceca was low, 0.06 +/- 0.07, when results of direct culture were considered. In contrast, when considering the frequency obtained after enrichment, the h2 was estimated at 0.20 +/- 0.12. This result suggests a genetic basis for the expression of the resistance to colonization. An experiment of selection for resistance to SE carrier state in the chicken ceca should definitively confirm the genetic origin of the resistance.

Differences in Frequency, Level, and Duration of Cecal Carriage between Four Outbred Chicken Lines Infected Orally with Salmonella enteritidis

Four chicken lines, L2, B13, PA12 (egg-type), and Y11 (meat-type), were tested for experimental carrier state of Salmonella enteritidis (SE) in two identical trials. After oral inoculation of SE at 1 wk of age with 5 x 10(4) SE colony-forming units (CFU), 10 chickens per line were necropsied weekly for 6 wk and then every 8 or 15 days until the 12th week postinoculation (PI). Liver, spleen, ovary, and ceca were examined for level of SE colonization. Numbers of positive livers and spleens and levels of the challenge strain in these organs differed little between the four chicken lines. Only three positive ovaries were detected. According to the chicken line, ceca exhibited generally significant (P < 0.05) differences in the number of positive organs during weeks 5-11 PI, in the SE CFU levels (P < 0.05) in the first 5 wk PI and during weeks 8 and 10 PI, and in the duration of colonization. L2 and B13 chickens generally carried SE in their ceca at higher levels, in more animals, and for a longer time than PA12 and Y11 chickens. Y11 chickens were the most resistant to SE cecal colonization.