DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) AS A RAPID METHOD FOR ASSESSING GASTROINTESTINAL TRACT MICROFLORA RESPONSES IN LAYING HENS FED SIMILAR ZINC MOLT INDUCTION DIETS

Induced molting through feed withdrawal can change the microenvironment of crop and ceca sufficiently to allow them to be the sites of Salmonella colonization in the chicken intestine. This study compares the denaturing gradient gel electrophoresis (DGGE) profiles of microbial crop and cecal communities among molted hens fed similar zinc acetate or zinc propionate amended molt diets to hens either undergoing feed withdrawal or hens full fed and not molted. Dendrograms of DGGE amplicon patterns indicated over 85% similarity of cecal communities between zinc acetate fed hens and zinc propionate fed hens and over 60% similarity of crop communities between zinc acetate fed hens and zinc propionate fed hens. Rapid comparison of complex gastrointestinal microflora profiles in laying hens fed similar diets is possible using DGGE.     

Alfalfa as a single dietary source for molt induction in laying hens

Molting is a process by which a hen's reproductive tract is rejuvenated prior to the beginning of a laying cycle. This process is often artificially induced in commercial settings in order to extend the productive life of a flock of hens. The most common method for the induction of molt is feed withdrawal for a period of several days. It has been noted that feed withdrawal, while effective in inducing molt and allowing an adequate reproductive rest period for the hen, may cause deleterious effects on the animal. This has prompted the investigation of alternatives to feed deprivation for the induction of molt in commercial laying hens. This study involved feeding alfalfa to hens to assess its ability to induce molt. Results show that alfalfa meal and alfalfa pelleted diets were equally effective as feed withdrawal in causing ovary weight regression in birds. Molted hens induced by alfalfa diets exhibited postmolt levels of egg production over a twelve week period that were similar to that of hens molted by feed withdrawal. The postmolt eggs laid by hens molted by alfalfa were of comparable quality to eggs from feed deprived hens. Alfalfa, a fibrous feed with low metabolizable energy, may be provided to hens on an ad libitum basis for an effective molt induction that retains comparable egg quality and production.     

Immunological cell and serum metabolite response of 60-week-old commercial laying hens to an alfalfa meal molt diet

The practice of induced molting involves the restriction of light, feed removal and optionally water for 5-14 days. However, there is growing concern regarding feed removal and animal welfare issues. With this in mind, alternative diets have been developed to produce similar molting effects as that of feed deprivation. Alfalfa, which largely consists of insoluble fiber, can be used as a molting diet. In this study, heterophil and lymphocyte counts, serum chemistry, and organ weight parameters were evaluated in hens that were deprived of feed or fed alfalfa during a nine day induced molt. Full-fed hens were used as the control. Blood serum parameters assessed included calcium, magnesium, glucose, total protein, ketone bodies, uric acid, and cholesterol. White blood cells were counted and categorized by cell type. On the ninth day of the trial, the hens were euthanized and the liver, spleen, heart, intestine, pancreas, ovary, oviduct, and kidney were collected and weighed. On day 8 birds molted with alfalfa or by feed deprivation had significantly higher (P<0.05) levels of ketone bodies and cholesterol and lower levels of calcium, and magnesium compared to the full-fed hens while birds molted by feed deprivation exhibited significantly lower levels of uric acid. Birds molted by both methods exhibited significant reductions in ovary, oviduct, liver and pancreas weights and increased spleen weights when compared to the non-molted hens. On days 0, 2, and 6 there were no significant differences (P>0.05) in either heterophil or lymphocyte percentages. However, heterophil percentages were higher in feed withdrawal birds than full-fed birds on day 4 but lymphocyte percentages were higher in full-fed birds compared to feed withdrawal birds. On day 8 of the induced molt lymphocyte percentages were higher from full-fed birds when compared to feed withdrawal birds but no significant differences were detectable for heterophil percentages. Based on reproductive organ weight loss and changes in serum and immunological responses of birds during molt, it appears that alfalfa meal can be an effective molt induction alternative.     

Using a feed-grade zinc propionate to achieve molt induction in laying hens and retain postmolt egg production and quality

A commercial-feed-grade form of zinc propionate was examined as a potential feed amendment at a concentration of 1% zinc to induce molt in 90-wk-old hens. Dietary treatments consisted, of 4 treatment groups of 28 birds each randomly assigned to either (1) molted conventionally by feed withdrawal, (2) 1% zinc as Zn acetate, (3) 1% zinc as Zn propionate, or (4) nonmolted control for 9 d. Ovary weights of hens fed Zn acetate or Zn propionate were not significantly different from each other, but hens fed Zn acetate or Zn propionate were significantly (p<0.05) lighter than the ovary weight of nonmolted control hens. Zinc concentrations in the kidney and liver were significantly (p<0.05) increased in both Zn acetate- and Zn propionate-molted hens when compared to either nonmolted control-fed hens or feed-withdrawal molted hens. Over the entire 3-mo postmolt period, there were no significant differences in interior or exterior egg qualities among the four treatments Egg production of hens fed Zn acetate was significantly lower than feed-withdrawal hens, Zn propionate-fed hens, or nonmolted control hens (p<0.05). The data of the current study demonstrated that feeding a feed grade of Zn propionate (1% Zn)-supplemented diet can induce molt and retain postmolt egg quality and production comparable to hens molted by feed withdrawal.     

Effect of melengestrol acetate as an alternative to induce molting in hens on the expression of yolk proteins and turnover of oviductal epithelium

Inducing hens to molt increases egg quality, egg production and extends the productive life of hens. It has been previously demonstrated that melengestrol acetate (MGA), an orally active progestin, decreased gonadotropic support for the ovary, which decreased the steroidogenic support for the oviduct and resulted in the cessation of lay. Estradiol produced by the theca cells of small follicles stimulates the production of the yolk proteins vitellogenin II and apolipoprotein II by the liver and supports the oviductal epithelial cells. The objective of the present experiment was to determine gene expression for yolk proteins and oviductal epithelial cell turn-over in response to a MGA-induced molt. Hy-Line W-36 laying hens were fed either 0 or 8mg MGA per day for 28 days in a balanced diet and then returned to a standard layer ration until day 36. Four birds per treatment on days 1, 8, 16, 28 and 36 were euthanized and the liver was removed and snap frozen in liquid nitrogen until RNA was extracted. Expression of vitellogenin II and apolipoprotein II genes was determined using real-time RT-PCR. A portion of the magnum was removed to determine proliferation and programmed cell death for secretory and ciliated luminal epithelium. Vitellogenin II and apolipoprotein II gene expression was reduced in hens fed 8mg MGA compared to those fed 0mg MGA. There was no effect of day on gene expression of either yolk protein. Cell proliferation was increased in the ciliated epithelial cells of the oviduct in hens receiving 8mg MGA compared to those receiving 0mg. However, programmed cell death of the ciliated epithelial cells was not different between controls and MGA treatment. Programmed cell death and proliferation increased in the secretory epithelial cells in hens receiving 8mg MGA compared to controls. Therefore, utilizing MGA as an alternative method to induce molt results in some, but not all, of the physiological changes previously described for hens molted by feed withdrawal. These findings lead us to suggest that some of the observed physiological changes resulting from feed withdrawal are required to increase egg quality and egg production following molt and other changes are not necessary, but are just a result of nutrient deprivation.     

Intestinal and eggshell calbindin, and bone ash of laying hens as influenced by age and molting

A series of trials was conducted in order to study the effects of age and molt on intestinal and eggshell gland (ESG) calbindin, and on bone ash. For this purpose an ELISA for chicken calbindin was developed. Age did not significantly affect duodenal or ESG calbindin. Bone ash increased (but not significantly in this study) from 8 to 16 months of age. During molt induction, egg laying was arrested, duodenal and ESG calbindin almost completely disappeared and ovary mass, plasma estradiol and total calcium (Ca) decreased markedly, whereas bone ash and body mass (BW) decreased moderately. During the non-laying period that followed the feed withdrawal period, duodenal and ESG calbindin remained low, whereas plasma estradiol and other estrogen-dependent variables, such as plasma total Ca and bone ash, increased slightly. At the onset of egg production following molting, duodenal and ESG calbindin levels were similar to pre molt level. Bone ash was higher than at the pre molt period. Body mass, small yellow follicles, ovary and oviduct mass and plasma estradiol were lower than their values prior to molt induction. Bone ash contents in the molted hens at the ages of 583 and 820 days were similar to or even slightly higher than those in the non-molted hens, whereas duodenal and ESG calbindin were not significantly different. These results suggest that the improvement of shell quality in the molted birds does not involve mechanisms associated with calbindin synthesis.     

Egg contamination by Salmonella serovar enteritidis following vaccination with Delta-aroA Salmonella serovar typhimurium

The efficacy of an aroA Salmonella serovar typhimurium modified live vaccine to decrease internal egg contamination after oral challenge of hens with egg-contaminating Salmonella serovar enteritidis was assessed. Challenge was with a mixed phenotype of S. enteritidis that had virulence characteristics previously associated with enhanced oral invasiveness and egg contamination in chickens. Immunized birds had fewer positive ovary/oviduct pools and lower cfu g(-1) cecal contents than did non-immunized birds, but the differences were not significant. The number of positive intestinal (duodenum, jejunum, ileum) and organ (spleen, kidney, liver) pools following challenge from each treatment group were equivalent. Most importantly, immunization did not decrease egg contamination. These results suggest that the ability of modified live vaccines to reduce internal egg contamination by S. serovar enteritidis can be assessed using characterized strains for challenge.     

Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co-ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI-1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI-1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI-1 gene regulation, we have discovered that the nucleoid-binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI-1 genes. A S. typhimurium fis mutant demonstrates a two- to threefold reduction in hilA:Tn5lacZY and a 10-fold reduction in invF:Tn5lacZY expression, as well as a 50-fold decreased ability to invade HEp-2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100-fold when administered orally, but has wild-type virulence when administered intraperitoneally. Expression of hilA:Tn5lacZY and invF:Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC-like regulator of Salmonella invasion genes.     

Potential of alfalfa as an alternative molt induction diet for laying hens: egg quality and consumer acceptability

Dietary molt induction to initiate additional egg laying cycles in commercial laying hen flocks is a wide spread practice in the United States. Feed deprivation is the most commonly used method but this practice has generated several concerns which has lead to research for viable alternative approaches. From a management standpoint a single ingredient molting diet consisting of high fiber-low energy represents an easily adaptable diet for large laying hen production units. Alfalfa meal is readily available in most commercial locations and possesses many of the desirable properties of an ideal laying hen molt diet. In the current study hens at a commercial laying facility were molted by both alfalfa and feed deprivation. After the hens had reentered post-molt commercial egg production, eggs were examined for egg quality performance. Egg shell strength, albumen height, yolk height, weight, length, and yolk color were all tested using various mechanical techniques. The eggs were also sampled for testing by consumer sensory panels that assessed the desirability of the eggs' color and flavor/texture. Eggs laid by hens molted by alfalfa had a significantly lower (p<0.05) "a*" level of colorimetry. Eggs laid by hens molted with alfalfa also exhibited significantly higher (p<0.05) egg weights and length. In the consumer sensory test, there was no significant difference (p>0.05) in color or flavor/texture scores in eggs from either feed deprived or alfalfa molted hens. The consumer sensory and mechanical quality attributes indicates that alfalfa shows promise as an alternative molt induction diet by providing a single diet option for extending egg production into a second egg laying cycle.     

Dietary thyroxine induces molt in chickens (Gallus gallus domesticus)

Thyroxine increases during a molt in wild and captive birds, and thyroidectomy prevents induction of molt. This trial examined the effect of dietary thyroxine on molt induction molt in chickens (laying hens, 59 weeks of age). In a completely randomized design (n=15 hens/replication; 6 replications/treatment), hens were randomly assigned to either a traditional molting program consisting of feed withdrawal (FWD), or to diets containing 40 mg thyroxine/kg diet (HT), 20 mg thyroxine/kg diet (LT), or 40 mg thyroxine from thyroactive iodinated casein/kg diet (TIC). The molting treatment lasted 7-13 d, until egg production reached 0%. After molt induction, birds had ad libitum access to the same diet, until egg production was re-initiated and maximized ( approximately 56 d). All treatments induced molt, based upon cessation of egg laying and regression of ovary and oviduct. Birds on FWD treatment lost more body weight during the molting period, but gained more after molt compared to thyroxine treatments (P<0.01 for each), although all body weights were similar when egg production was maximized. Data demonstrate that oral thyroxine, in purified or non-purified form, induces a molt and may enhance animal well-being by reducing the need for FWD.     

Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium

Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.     

RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium encounters numerous host environments and defense mechanisms during the infection process. The bacterium responds by tightly regulating the expression of virulence genes. We identified two regulatory proteins, termed RtsA and RtsB, which are encoded in an operon located on an island integrated at tRNA(PheU) in S. enterica serovar Typhimurium. RtsA belongs to the AraC/XylS family of regulators, and RtsB is a helix-turn-helix DNA binding protein. In a random screen, we identified five RtsA-regulated fusions, all belonging to the Salmonella pathogenicity island 1 (SPI1) regulon, which encodes a type III secretion system (TTSS) required for invasion of epithelial cells. We show that RtsA increases expression of the invasion genes by inducing hilA expression. RtsA also induces expression of hilD, hilC, and the invF operon. However, induction of hilA is independent of HilC and HilD and is mediated by direct binding of RtsA to the hilA promoter. The phenotype of an rtsA null mutation is similar to the phenotype of a hilC mutation, both of which decrease expression of SPI1 genes approximately twofold. We also show that RtsA can induce expression of a SPI1 TTSS effector, slrP, independent of any SPI1 regulatory protein. RtsB represses expression of the flagellar genes by binding to the flhDC promoter region. Repression of the positive activators flhDC decreases expression of the entire flagellar regulon. We propose that RtsA and RtsB coordinate induction of invasion and repression of motility in the small intestine.