A review on development of novel strategies for controlling Salmonella Enteritidis colonization in laying hens: fiber-based molt diets.

Limiting Salmonella Enteritidis from table eggs can involve intervention approaches at several levels of the production cycle, beginning at the hatchery and ending at the processing or table egg production facilities. Likewise, interventions that limit Salmonella Enteritidis dissemination can be implemented at various stages during the life cycle of infection of Salmonella in the laying hen. However, achieving complete elimination of Salmonella infestation in egg products has remained elusive. There is a multitude of reasons for this, including adaptability of the organism, virulence properties, and persistence. Likewise, environmental factors in the layer house such as transmission routes, reservoirs, and feed sources can influence the exposure of susceptible laying hens to Salmonella Enteritidis. Consequently, successful applications of control measures depend not only on the timing of when they are applied but also on effective surveillance to detect frequency and level of infection of Salmonella. Several studies demonstrated that molt induction by feed withdrawal altered the immune system and the gastrointestinal tract of hens, making them susceptible to Salmonella Enteritidis colonization of the gastrointestinal tract. To alleviate this, the development of alternative methods to induce a molt became necessary. The use of several fiber-containing diets was shown to effectively induce a molt with alfalfa-based diets being the most extensively studied. Further reduction of Salmonella Enteritidis levels in eggs will probably require application of multiple interventions at several steps during egg production and processing as well as a better understanding of the mechanisms used by Salmonella Enteritidis to persist in laying flocks.

Salmonella enteritidis hilA gene fusion response after incubation in spent media from either S. enteritidis or a poultry Lactobacillus strain.

The purpose of this study was to determine if growth of a poultry probiotic lactobacilli strain can influence S. enteritidis virulence expression by measuring the response of a hilA-lacZY transcriptional fusion. beta-galactosidase activity was not detected when S. enteritidis was incubated in Lactobacillus-spent medium (24 h growth, pH 4.1, 50.4 mM lactate) but was detectable in spent medium from 4 h growth cultures of Lactobacillus sp. (final OD of 0.213, pH 5.7, 12 mM lactate) when pH and lactate were adjusted to that of the 24 h-pH 4 spent media levels. Adjusting the pH of the 24 h spent medium from 4 to 6, resulted in a measurable beta-galactosidase activity that was significantly higher than expression in LB broth. When S. enteritidis was grown in Salmonella-spent media (24 h growth, pH 4.2, 78 mM acetate), hilA expression was increased 4-fold over expression in the LB broth.

Expression of the hilA Salmonella typhimurium gene in a poultry Salm. enteritidis isolate in response to lactate and nutrients.

Pathogens express virulence genes in response to the combination of environmental conditions present in the host environment. The crop is the first gastrointestinal environment encountered in birds. However, feed withdrawal alters the crop environment resulting in an increased pH, and decreased concentrations of lactate, glucose and amino acids compared with unmoulted birds. Salmonella enteritidis infections increase significantly in hens that have been force moulted by feed withdrawal. The present study examined the effects of pH, carbohydrate sources, amino acids and lactate on expression of Salm. enteritidis virulence by measuring expression of hilA. The hilA gene encodes a transcriptional activator that regulates expression of Salmonella virulence genes in response to environmental stimuli. HilA expression was determined using a poultry isolate of Salm. enteritidis carrying a hilA-lacZY transcriptional fusion from Salm. typhimurium. The media used were Luria Bertani (LB) broth and LB broth diluted 1:5 (DLB). The expression of hilA was 2.9-fold higher in DLB broth compared with LB broth which suggested that there is a nutritional component to the regulation of hilA. Addition of 0.2% glucose, fructose or mannose to LB and DLB reduced hilA expression 1.5 to twofold. Addition of 0.2% Casaminoacids, arabinose, fucose, or lactose had little effect on hilA expression. Lactate (25 and 50 mmmol 1-1) reduced hilA expression at pH 6, 5 and 4, with the lowest expression occurring at pH 4. Based on these results it appears that the composition of the crop lumen could potentially influence Salm. enteritidis virulence expression.

Response of selected poultry cecal probiotic bacteria and a primary poultry Salmonella typhimurium isolate grown with or without glucose in liquid batch culture.

The objective of this study was to determine whether fermentation by a cecal probiotic co-culture of an Enterococcus sp. and Veillonella sp. would inhibit the in vitro growth of a S. typhimurium poultry isolate. The growth rates of S. typhimurium and Enterococcus were significantly reduced at pH 5. At the two pH levels, there was a significant (p < 0.001) increase at 24 h in colony forming units for each of the bacteria enumerated from the mixed culture compared to the respective pure culture enumerations. S. typhimurium was not inhibited in mixed cultures. The mixed cultures produced more acetate than any of the pure cultures and lactate produced by Enterococcus appeared to be utilized by Veillonella.

Short-chain volatile fatty acids modulate the expression of the hilA and invF genes of Salmonella typhimurium.

The ability of Salmonella typhimurium to invade the intestinal mucosal cells is an important step in pathogenesis. This invasion process requires genes encoded on the Salmonella pathogenicity island 1 (SPI1). Two transcriptional activators, HilA and InvF, encoded in SPII regulate the expression of invasion genes in response to environmental stimuli such as osmolarity, oxygen tension, and pH. During its pathogenic life cycle, Salmonella typhimurium is also exposed to short-chain fatty acids (SCFA), especially acetate, propionate, and butyrate, in the intestinal lumen, as well as the SCFA used as food preservatives. The effects of SCFA on the expression of hilA and invF-lacZY transcriptional fusions were examined to determine the potential role of SCFA in the pathogenesis of Salmonella typhimurium. Growth rates were reduced by increasing SCFA concentrations at pH 6 but not at pH 7. At pH 7, hilA and invF expression was induced by acetate but not by propionate or butyrate, while at pH 6, all SCFA induced hilA and invF expression at 1 h. In general, hilA and invF expression levels when compared to respective control responses were higher at 1 h than at 4 and 8 h in the presence of most SCFA concentrations at pH 6. However, expression levels at 4 and 8 h were either similar or higher than the 1-h responses for the hilA-lacZY fusion strain in the presence of acetate while exposure to 20 mM propionate yielded similar levels of expression at 1, 4, and 8 h. The pH-dependent manner of induction suggests that entry of SCFA into the cell was necessary for induction. We speculate that SCFA may serve as an environmental signal that triggers the expression of invasion genes in the gastrointestinal tract.

Short-chain fatty acids affect cell-association and invasion of HEp-2 cells by Salmonella typhimurium.

This study demonstrates that the growth of S. typhimurium in Luria Bertani broth supplemented with acetate, propionate, butyrate, or a mixture of the three SCFA, affected cell-association and the ability to invade cultured HEp-2 cells. Cell-association and invasion was determined after growth for 4 h of growth in the presence of the SCFA at pH 6 and 7. The results suggest that the growth rate of the culture may have affected cell-association and invasion since accompanying the significant decrease in growth rate in the presence of SCFA at pH 6 was a decrease in cell-association and invasion. However, the results also suggest that the individual SCFA may play a role in modulating cell-association and the invasion phenotype and the regulation of cell-association and invasion by the SCFA was dependent on the concentration and the pH of the medium. Although the growth rates were similar for S. typhimurium in the SCFA mixture, butyrate (100 mM) and propionate (50 mM) at pH 6, differences in cell-association and invasion were observed among these cultures. Also, at pH 7, differences were observed among the SCFA treatments even though the growth rates were similar.

Feed deprivation affects crop environment and modulates Salmonella enteritidis colonization and invasion of leghorn hens.

Leghorn hens over 50 weeks of age were assigned to two treatment groups designated as either unmolted controls or molted. A forced molt was induced by a 9-day feed withdrawal, and each hen was challenged orally with 10(5) Salmonella enteritidis organisms on day 4 of feed withdrawal. On days 4 and 9 of molt, the numbers of lactobacilli and the concentrations of lactate, acetate, propionate, and butyrate, and total volatile fatty acids in the crops decreased while crop pH increased significantly (P < 0.05) in the molted hens compared to the controls. S. enteritidis crop and cecal colonization, in addition to spleen and liver invasion, increased significantly (P < 0.05) in the molted hens compared to the controls. The invasive phenotype of Salmonella spp. is complex and requires several virulence genes which are regulated by the transcriptional activator HilA. Samples of the crop contents from the molted and unmolted birds were pooled separately, centrifuged, and filter sterilized. The sterile crop contents were then used to measure the expression of hilA. By using a lacZY transcriptional fusion to the hilA gene in S. enteritidis, we found that hilA expression was 1.6- to 2.1-fold higher in the crop contents from molted birds than in those from control birds in vitro. The results of the study suggest that the changes in the microenvironment of the crop caused by feed deprivation are important regulators of S. enteritidis survival and influence the susceptibility of molted hens to S. enteritidis infections. Furthermore, our in vitro results on the expression of hilA suggest that the change in crop environment during feed withdrawal has the potential to significantly affect virulence by increasing the expression of genes necessary for intestinal invasion.

Detection and quantification of poultry probiotic bacteria in mixed culture using monoclonal antibodies in an enzyme-linked immunosorbent assay.

Murine monoclonal antibodies were used in an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of selected probiotic bacteria present in a continuous-flow competitive exclusion culture known to be effective at reducing chicken cecal and crop colonization by Salmonella typhimurium. Veillonella, Enterococcus avium and S. typhimurium were grown anaerobically in batch culture of Viande Levure broth in pure culture and mixed culture. The mixed cultures produced significantly more acetate and propionate than any of the pure cultures with acetate and propionate being the predominant volatile fatty acids. The association in mixed culture resulted in a significant increase in cell numbers compared to the respective pure cultures. The ELISA was capable of detecting 10(4) cells per ml of the bacteria. The plots of cell numbers determined by the ELISA versus direct plating increased in accordance with increases in cell numbers with r2 values of 0.950, 0.922 and 0.940 for the pure culture incubations and 0.901, 0.924 and 0.905 in the mixed culture incubation for E. avium, S. typhimurium and Veillonella, respectively. The results indicate that the monoclonal antibodies can be used to quantitatively assay individual probiotic bacterial species grown in a mixed culture incubation.

Comparison of batch culture growth and fermentation of a poultry Veillonella isolate and selected Veillonella species grown in a defined medium.

The objective of this study was to develop a defined medium for quantitating nutritional requirements and fermentation products of a poultry cecal isolate of Veillonella and to compare these parameters with representative Veillonella species. The poultry isolate is one of 29 organisms from a continuous-flow culture that has been shown to be effective against Salmonella colonization in broilers. When the Veillonella species were grown in anaerobic batch culture, propionate and acetate were the only volatile fatty acids detected. Lactate was needed to provide energy for the growth of the Veillonella in the defined medium. The poultry isolate had significantly (p< 0.05) higher Y(lactate)(g of dry cell weight per mole of lactate utilized) and dry cell weight than the other Veillonella species when grown on amino acid supplemented defined media. Cultures of the Veillonella species in the defined medium grown with supplemented amino acids aspartate, threonine, arginine, and serine indicated that these amino acids were metabolized to acetate and propionate. Amino acid analysis on media inoculated with either V. atypica or the poultry isolate also indicated that these organisms may have different amino acid preferences. For nearly all of the amino acid supplemented media combinations the poultry isolate utilized significantly (p< 0.05) more threonine and serine whereas V. atypica utilized significantly (p< 0.05) more aspartate. The defined medium supported growth of all of the Veillonella species tested and should enable further in-depth physiological studies to be conducted on the poultry Veillonella studies.

Non-specific factors that may influence significance of urinary steroid excretion in breast cancer.

The urinary excretion of corticosteroids (17-oxogenic steroids) and adrenal androgens (11-deoxy-17-oxosteroids) was studied in women below the age of 50 in a variety of clinical situations for comparison with a normal group. The conditions studied were: chronic debility from non-malignant disease, weight reduction, admission to hospital and surgery for varicose veins, hepatic non-malignant disease, and non-mammary cancer.The objective of the study was to determine whether the changes found in early and advanced breast cancer and used to judge the prognosis of the disease are specific to the disease or are merely incidental to the degree of illness caused by the disease.Similar changes to those found in breast cancer-principally a reduction in the excretion of the androgens-were found in the women with severe hepatic disease and in advanced non-mammary cancer. These were also found to follow the effects of severe surgical stress.It is concluded that the changes found in breast cancer are a measure of the general systemic disturbance caused by the disease and are not due specifically to it. Nevertheless, the value of their prognostic significance remains unchallenged.

Corpus luteum deficiency as a cause of early recurrent abortion: a case history.

PIP: This paper presents a case history that suggests recurrent abortions in early pregnancy may be due to an inability to maintain an efficient corpus luteum. Pregnancy tests and the estimation of urinary human chorionic gonadotropin (HCG) titers were carried out for 62 days following the onset of the menstrual period preceding the study period. The patient was judged to have ovulated on the 19th or 20th day of the cycle and to have become pregnant at that point. The corpus luteum began to secrete progesterone in response to fertilization. Pregnanediol excretion rose from an average of 0.3 mg/day before ovulation to a high of 8.7 mg on day 31, but then fell sharply to only 1.0-3.0 mg/day, jeopardizing the chance of successful maintenance of the fetoplacental unit. HCG excretion was undetectable before day 43, then fluctuated between 1500-3500 IU/L, and became undetectable again on day 62. A heavy "menstrual" loss occurred on day 63. Progesterone replacement therapy in a subsequent pregnancy produced a successful outcome. This treatment should be administered as soon as the 1st period is missed. It is unclear whether corpus luteum dificiency is a direct cause of early recurrent abortion, or whether it is due to some others cause such as failure of the anterior pituitary to secrete sufficient luteinizing or luteotropic hormone.^ieng