Foodborne Salmonella ecology in the avian gastrointestinal tract.

Foodborne Salmonella continues to be a major cause of salmonellosis with Salmonella Enteritidis and S. Typhimurium considered to be responsible for most of the infections. Investigation of outbreaks and sporadic cases has indicated that food vehicles such as poultry and poultry by-products including raw and uncooked eggs are among the most common sources of Salmonella infections. The dissemination and infection of the avian intestinal tract remain somewhat unclear. In vitro incubation of Salmonella with mammalian tissue culture cells has shown that invasion into epithelial cells is complex and involves several genetic loci and host factors. Several genes are required for the intestinal phase of Salmonella invasion and are located on Salmonella pathogenicity island 1 (SPI 1). Salmonella pathogenesis in the gastrointestinal (GI) tract and the effects of environmental stimuli on gene expression influence bacterial colonization and invasion. Furthermore, significant parameters of Salmonella including growth physiology, nutrient availability, pH, and energy status are considered contributing factors in the GI tract ecology. Approaches for limiting Salmonella colonization have been primarily based on the microbial ecology of the intestinal tract. In vitro studies have shown that the toxic effects of short chain fatty acids (SCFA) to some Enterobacteriaceae, including Salmonella, have resulted in a reduction in population. In addition, it has been established that native intestinal microorganisms such as Lactobacilli provide protective mechanisms against Salmonella in the ceca. A clear understanding of the key factors involved in Salmonella colonization in the avian GI tract has the potential to lead to better approach for more effective control of this foodborne pathogen.

Behavioral responses of laying hens to different alfalfa-layer ration combinations fed during molting.

Induced molting by feed withdrawal has been a common practice in the commercial layer industry and usually involves the removal of feed for a period of up to 14 d. However, this is a practice that is believed to adversely influence the welfare of the hens and there is a need to examine behavioral responses to alternative molt regimens. The behavioral patterns of hens on 90% alfalfa:10% layer ration, 80% alfalfa:20% layer ration, and 70% alfalfa:30% layer ration molt diets were compared with feed withdrawal (FW) hens, and fully fed (FF) hens. The White Leghorn laying hens were approximately 54 wk old and were placed in 3 identical climate-controlled rooms. The hens were individually housed in 2-tier wire battery cages and provided treatment rations and water ad libitum. Nonnutritive pecking, walking, drinking, feeder activity, preening, aggression, and head movement were quantified during two 10-min periods each day for 6 hens from each treatment. Over the 9-d treatment period, hens in the FW, 70% alfalfa:30% layer ration, and 80% alfalfa:20% layer ration groups spent significantly more time walking than hens in the 90% alfalfa:10% layer ration group. The FF and 70% alfalfa:30% layer ration hens spent half as much time preening, whereas the FW hens displayed nearly twice as much nonnutritive pecking when compared with other treatments. Most differences in head movements occurred at the beginning of the molt period, whereas during the last half of molt, alfalfa-fed hens exhibited feeder activity similar to FF hens, and all were significantly higher than that of FW hens. After some initial adjustment by the hens, consumption of alfalfa molt diets appeared to reduce nonnutritive pecking behavior, which is characteristically associated with FW hens.

Behavior of laying hens on alfalfa crumble molt diets.

Several dietary alternatives to feed withdrawal have been proposed to induce a molt in laying hens. This study compared the behavior of laying hens on an alfalfa crumble diet (ALC) to hens that were either on a conventional layer diet (FF) or hens that had feed withdrawn (FW) during a 9-d trial. Each treatment consisted of 24 hens (3 hens per battery cage), and treatment began after a 2-week acclimation period. Video cameras connected to a digital multiplexer recorded the behavior of the hens. The percentages of observations performing nonnutritive pecking, feeder activity, drinking, walking, preening, head movement, and aggression were quantified for two 10-min periods at daily intervals. The FF hens spent significantly more (P < or = 0.05) time drinking than the other treatments, whereas FW hens displayed the most head movements. From d 1 through 7, FW hens walked less than ALC hens except on d 8 when FW hens walked more than ALC and FF hens. On d 4 and 6, the FW hens spent an increased amount of time preening compared with FF hens until the last few days of the molt period. For the most part, FW hens generally displayed more nonnutritive pecking than ALC and FF hens throughout the molt period. However, FW hen visits to the feeders declined as the trial proceeded, whereas ALC and FF hens generally spent more time at the feeder. In summary, the ALC diet showed potential as an alternative to FW for inducing a molt in laying hens based on reduced nonnutritive pecking behavior, head movements, and greater feeding activity.

Growth and genetic responses of Salmonella Typhimurium to pH-shifts in an anaerobic continuous culture.

Salmonella infection of chickens that leads to potential human foodborne salmonellosis continues to be a concern. Changes in the pH of poultry gastrointestinal tract could influence Salmonella growth and virulence response. In the current study, growth responses of a chicken isolate Salmonella enterica serovar Typhimurium (ST) to three incremental pH-shifts (6.17-7.35) in continuous cultures (CC) were evaluated. The expression of rpoS and hilA was determined by real time-polymerase chain reaction (RT-PCR) as well. Increases in pH resulted in higher cell protein concentrations, glucose disappearance, and glucose and ATP yields. Although with some inconsistency between the two trials, the data indicated that the ammonia release into media was favored by low pH. The pH shifts did not significantly affect acetate biosynthesis. No consistent trends of pH influence on propionate and butyrate production could be detected. In all three pH shifts, relative expression of hilA was dominant at 0h which represented CC steady state. In pH shift 7.35-6.86 (Trial 1), the relative expression of rpoS at time 0 and 1h were over five-fold higher than after 3 and 6h of growth. Overall, the results suggest that ST physiology is altered by changes in pH, which could be determinant factors for ST survival in the poultry gastrointestinal ecosystems.

Molting in Salmonella enteritidis-challenged laying hens fed alfalfa crumbles. III. Blood plasma metabolite response.

The objective of this study was to examine an alfalfa crumble diet as an alternative molt diet and compare the physiological response of hens to the responses of feed-deprived molted hens. Hens >50 wk old were placed into 6 treatment groups (12 hens per group in trial 1 and 10 hens per group in trial 2): nonmolted Salmonella enterica serovar Enteritidis positive (FF+), nonmolted Salmonella Enteritidis negative (FF-), feed withdrawal Salmonella Enteritidis positive (FW+), FW Salmonella Enteritidis negative (FW), alfalfa Salmonella Enteritidis positive (ALC+), and ALC Salmonella Enteritidis negative (ALC-). Each hen in the Salmonella Enteritidis-positive groups was challenged on the fourth day of the study with 1 mL of 10(6)-cfu Salmonella Enteritidis. Blood was collected on d 2, 5, 9, and 12 of the trial. Blood plasma was collected and metabolite concentrations were analyzed for glucose, calcium, cholesterol, uric acid, total protein, and triglycerides. The feed intakes of the FF hens were 4- to 6-fold greater (P

Molting in Salmonella enteritidis-challenged laying hens fed alfalfa crumbles. IV. Immune and stress protein response.

Immunological responses of molting hens either infected or not infected with Salmonella enterica serovar Enteritidis were compared in 2 trials with Single Comb White Leghorn hens >50 wk old. The hens were placed into 6 treatment groups with 12 hens per group: nonmolted Salmonella Enteritidis positive (FF+), non-molted Salmonella Enteritidis negative (FF-), feed withdrawal Salmonella Enteritidis positive (FW+), FW Salmonella Enteritidis negative (FW-), alfalfa Salmonella Enteritidis positive (ALC+), and ALC Salmonella Enteritidis negative (ALC-). Each hen in the Salmonella Enteritidis-positive groups was challenged on d 4 of the study with 1 mL of 10(6)-cfu Salmonella Enteritidis, and diets were administered for 12 d. Blood samples were collected on d 2, 5, 9, and 12, and blood smears were enumerated for heterophil to lymphocyte (H:L) ratios. Serum samples were also analyzed for alpha(1)-acid glycoprotein (AGP) levels and antibody level. On d 12, hens were euthanized and bile samples from the gall bladder and sections of the ileum and the ceca were collected, and an ELISA was used to determine the intestinal, serum, and bile antibody responses. The FW+ hens produced more (P

Molting in Salmonella Enteritidis-challenged laying hens fed alfalfa crumbles. II. Fermentation and microbial ecology response.

The objective of this study was to examine microbial population shifts and short-chain fatty acid (SCFA) responses in the gastrointestinal tract of Salmonella Enteritidis-challenged molted and nonmolted hens fed different dietary regimens. Fifteen Salmonella-free Single Comb Leghorn hens (>50 wk old) were assigned to 3 treatment groups of 5 birds each based on diet in 2 trials: 100% alfalfa crumbles (ALC), full-fed (FF, nonmolted) 100% commercial layer ration, and feed withdrawal (FW). A forced molt was induced by either a 12-d alfalfa diet or FW. In all treatment groups, each hen was challenged by crop gavage orally 4 d after molt induction with a 1-mL inoculum containing 10(6) cfu of Salmonella Enteritidis. Fecal and cecal samples (d 4, 6, 8, 11, and necropsy on d 12) were collected postchallenge. Microbial population shifts were evaluated by PCR-based 16S ribosomal RNA gene amplification and denaturing gradient gel electrophoresis, and SCFA concentrations were measured. Total SCFA in fecal and cecal contents for FW molted hens were generally lower (P < or = 0.05) in the later stages of the molt period when compared to ALC and FF treatment groups. The overall trend of SCFA in cecal and fecal samples exhibited similar patterns. In trials 1 and 2, hens molted with ALC diet generally yielded more similar amplicon band patterns with the FF hens in both fecal and cecal samples by the end of the molting period than with FW hens. The results of these studies suggest that ALC molted hens supported microflora and fermentation activities, which were more comparable to FF hens than FW hens by the end of the molting period.

Molting in Salmonella enteritidis-challenged laying hens fed alfalfa crumbles. I. Salmonella enteritidis colonization and virulence gene hilA response.

The objectives of this study were to enumerate Salmonella enterica serovar Enteritidis colonization in fecal, cecal, and internal organs, and to compare the level of virulence gene expression (hilA) of experimentally challenged laying hens fed different dietary molt-induction regimens. Twelve Salmonella-free Single Comb Leghorn hens (>50 wk old) hens were randomly assigned to each of 6 treatment groups designated based on diet in 2 trials: 1) feed withdrawal Salmonella Enteritidis-positive (FW+), 2) fully fed Salmonella Enteritidis-positive (FF+), 3) 100% alfalfa crumble Salmonella Enteritidis-positive (ALC+), 4) feed withdrawal Salmonella Enteritidis-negative, 5) fully fed Salmonella Enteritidis-negative, and 6) 100% alfalfa crumble Salmonella Enteritidis-negative. A forced molt was induced by a 12-d alfalfa diet and a feed-withdrawal regimen. On d 4 of the molt, all hens in groups 1, 2, and 3 were challenged by crop gavage with 1 mL of inocula containing approximately 10(6) cfu of nalidixic acid- and novobiocin-resistant Salmonella Enteritidis (phage type 13A). At the conclusion of both trials, all hens were euthanized and Salmonella Enteritidis colonization was enumerated in the cecal contents, liver, spleen, and ovaries. In addition, fecal (d 4 and 8) and cecal samples (necropsy at d 12) were collected postchallenge from treatment groups 1, 2, and 3 (Salmonella Enteritidis-positive) to quantify hilA expression by PCR. In both trials, all nonchallenged birds were Salmonella Enteritidis-negative; therefore, no further analysis was done. In trial 1, a 2-fold reduction in Salmonella Enteritidis colonization was observed in the ALC+ hens (log10 Salmonella Enteritidis of 1.99) compared with the FW+ hens (log(10) Salmonella Enteritidis of 3.89). In trial 2, a 4-fold reduction in Salmonella Enteritidis colonization was observed in the ALC+ hens (log(10) Salmonella Enteritidis of 1.27) compared with the FW+ hens (log(10) Salmonella Enteritidis of 5.12). In trial 2, Salmonella Enteritidis colonization in spleens was higher (P 0.05). In trial 2, hilA expression in FW+ hens was higher (P

Comparison of in vitro fermentation and molecular microbial profiles of high-fiber feed substrates incubated with chicken cecal inocula.

High fiber and nonstarch polysaccharide-based poultry diets have received more interest recently for retaining or promoting beneficial gastrointestinal microbial populations. The objective of this study was to investigate and compare the in vitro potential fermentability of high-fiber feed substrates (HFFS) by laying hen cecal microflora. Feed sources examined included soybean meal, soybean hull, beet pulp, wheat middlings, ground sorghum, cottonseed meal, 100% alfalfa meal, 90% alfalfa + 10% commercial layer ration, 80% alfalfa + 20% commercial layer ration, and 70% alfalfa + 30% commercial layer ration. Cecal contents and HFFS were incubated anaerobically in serum tubes at 39 degrees C for 24 h. Samples from 2 trials were analyzed at 0 and 24 h for short-chain fatty acids (SCFA). Short-chain fatty acids in samples at 0 h were subtracted from 24-h samples to determine the net production of SCFA. In both trials involving HFFS incubations with cecal inocula, acetate production was highest followed by propionate and butyrate whereas isobutyrate and isovalerate production were in trace amounts. In trial 2, detectable valerate production appeared to consistently occur with alfalfa-based HFFS. It was clear that SCFA production was largely dependent upon HFFS, because cecal inoculum alone yielded little or no detectable SCFA production. For HFFS incubations without cecal inocula, acetate production was highest; propionate and butyrate were similar, and isobutyrate, valerate, and isovalerate production were in trace amounts. Polymerase chain reaction-based denaturing gradient gel electrophoresis results from both trials indicated 69 and 71% similarity for comparison of all feed mixtures in trials 1 and 2, respectively. All alfalfa-based HFFS yielded a higher similarity coefficient in trial 2 than in trial 1 with a band pattern of 90% similarity; diets containing 90% alfalfa + 10% commercial layer ration and 80% alfalfa + 20% commercial layer ration in trial 2 formed a subgroup with a 94% microbial similarity coefficient. These data suggest that high fiber sources may contribute to the fermentation and microbial diversity that occurs in the ceca of laying hens.

Comparison of spontaneous antibiotic resistance frequency of Salmonella Typhimurium growth in glucose amended continuous culture at slow and fast dilution rates.

The objective of the study was to determine the frequency of spontaneous acquisition of resistance to select antibiotics by Salmonella Typhimurium (ST) when grown in glucose amended continuous flow culture at slow (D = 0.025 h(-1)) or fast (D = 0.27 h(-1)) dilution rates. The bacterium was grown in LB minimal medium (pH 6.25) containing no antibiotics. Upon achieving steady state, samples were plated to tryptic soy agar (TSA) alone or supplemented (per ml) with 2 and 16 microg oxytetracycline, 4 and 16 microg tetracycline, 2 and 64 microg kanamycin, and 0.25 and 2 microg enrofloxacin. Regardless of growth rate, CFU of resistant ST from the TSA containing antibiotics was less than 2 x 10(1) except for 2 microg kanamycin and 0.25 microg enrofloxacin treatments (higher than 1 x 10(9) and 4 x 10(7) CFU of resistant ST for trials 1 and 2, respectively). Frequency of recovering resistant ST from the TSA containing the higher antibiotic concentrations was less than 1 in 10(9) for all antibiotics, but was higher on the media containing 2 microg kanamycin and 0.25 microg enrofloxacin at both slow and fast growth rates. In general, minimal susceptibility differences were detected for isolates from slow and fast dilution rates.