Identification of active deubiquitinases in the chicken tissues.

The existing protein annotation in chicken is mostly limited to computational predictions based on orthology to other proteins, which often leads to a significant underestimation of the function of these proteins. Genome-scale experimental annotation can provide insight into the actual enzymatic activities of chicken proteins. Amongst post-translational modifications, ubiquitination is of interest as anomalies in ubiquitination are implicated in such diseases as inflammatory disorders, infectious diseases, or malignancies. Ubiquitination is controlled by deubiquitinases (DUBs), which remove ubiquitin from protein substrates. However, the DUBs have not been systematically annotated and quantified in chicken tissues. Here we used a chemoproteomics approach, which is based on active-site probes specific to DUBs, and identified 26 active DUBs in the chicken spleen, cecum, and liver.

Effects of broiler feed medications on Salmonella.

This pilot analysis was conducted with data from 52 conventional grow-out broiler flocks in a prospective field observational study in the southeastern United States during 2003-2006. Each flock was sampled for Salmonella 1 wk before the end of grow-out, upon arrival at the processing plant, and during processing (prior to and immediately after carcass chilling). The broiler litter was sampled on the day of bird harvest. The grow-out feeding programs, including the medications delivered in feed, were surveyed with questionnaires completed by the broiler managers and feedmill managers. Each detail of the feeding program was tested for statistical association with the frequency of Salmonella in the flock at each sampling point, after accounting for variation in Salmonella frequency between the farms, broiler complexes, and companies. Significant associations were found between Salmonella frequency in the broiler flock pre- and postharvest and the inclusion of feeds containing individual coccidiostats and other antimicrobial growth promoters, days on feed, and total consumption of feeds containing these products, as well as with practices such as a mash feed and a nonmedicated withdrawal feed. The analysis provided testable hypotheses for how broiler feed medications impact the frequency of Salmonella in the flocks.

Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon.

BACKGROUND: Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum. RESULTS: We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies. CONCLUSIONS: This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.

An atlas of the catalytically active liver and spleen kinases in chicken identified by chemoproteomics.

Phosphorylation is a post-translational protein modification regulating most known cellular processes. While protein kinases constitute a large family of highly conserved enzymes, identification of active kinases is challenging due to a low abundance of some of these signaling molecules. Although chicken is the first agricultural animal to have a sequenced genome, annotation of the kinome, i.e., a complement of all protein kinases in the genome is limited. We used chemical probes consisting of ATP and ADP derivatives binding to specific lysine (Lys) residues within the ATP-binding pocket of kinases, combined with proteomics, to identify 267 peptides labeled with the ATP and ADP acyl derivatives and 188 corresponding chicken kinases in chicken spleen and liver. Our description of active chicken kinases and ATP binding sites will support future studies focused on identifying the role of this important class of enzymes in chicken health and disease. SIGNIFICANCE: Advances made in understanding chicken enzymes are critical for the improved knowledge of the regulatory pathways controlling physiological processes in chicken. Since protein phosphorylation controls multiple aspects of cell fate, it is often linked to pathological conditions, and understanding of the kinase expression in chicken is essential for future therapeutic approaches. We coupled proteomics and labeling with active-site probes binding to Lys residues within the ATP-binding pocket of kinases to identify 188 kinases and corresponding 267 peptides labeled with the ATP and ADP acyl derivatives in chicken spleen and liver. Results of the present study describing catalytically active kinases is a starting point for chemoproteomic-based interrogation of kinases in chicken exposed to different conditions. Kinases identified in this study are available through the Chickspress genome browser that has previously published mRNA, miRNA, and shotgun proteomics data.

NEGATIVE REGULATORY EFFECTS OF PHOSPHATIDYLINOSITOL3-KINASE PATHWAY ON PHAGOCYTOSIS AND MACROPINOCYTOSIS IN BOVINE MONOCYTES.

Recent studies have shown that monocytes and macrophages not only present antigens to effector T cells and stimulate and shape T cell-mediated immune responses, but they also prime naïve T cells, thus initiating adaptive immune responses. Phosphatidylinositol 3-kinase functions at an early phase of toll-like receptor signaling pathways, modulates the magnitude of the primary immune responses, and is involved in the reorganization of the actin cytoskeleton during macropinocytic and phagocytic antigen uptakes, important early steps in triggering adaptive immune responses. We assessed by flow cytometry the endocytic capacities of bovine monocytes by using endocytic tracers and Salmonella transformed with a green fluorescence plasmid GFP to evaluate macropinocytosis, mannose receptor-mediated endocytosis, and phagocytosis in bovine professional antigen presenting cells, respectively. Our data reveal that wortmannin, an inhibitor of phosphatidylinositol 3-kinase signaling pathway, significantly increased macropinocytosis and phagocytosis but did not affect the mannose receptor-mediated antigen uptake in bovine monocytes. Protein expression data support these findings by showing decreased levels of phosphoinositide 3-kinase in the presence of wortmannin during macropinocytosis. We expanded further the key role of phosphatidylinositol 3-kinase as an endogenous suppressor of primary immune responses, suggesting a novel mechanism of phosphatidylinositol 3-kinase antigen uptake modulation that may provide a unique therapeutic target for controlling excessive inflammation.

Lighting during grow-out and Salmonella in broiler flocks.

BACKGROUND: Lighting is used during conventional broiler grow-out to modify bird behaviour to reach the goals of production and improve bird welfare. The protocols for lighting intensity vary. In a field study, we evaluated if the lighting practices impact the burden of Salmonella in broiler flocks. METHODS: Conventional grow-out flocks reared in the states of Alabama, Mississippi and Texas, USA in 2003 to 2006 were sampled 1 week before harvest (n = 58) and upon arrival for processing (n = 56) by collecting feathered carcass rinsate, crop and one cecum from each of 30 birds, and during processing by collecting rinsate of 30 carcasses at pre-chilling (n = 56) and post-chilling points (n = 54). Litter samples and drag swabs of litter were collected from the grow-out houses after bird harvest (n = 56). Lighting practices for these flocks were obtained with a questionnaire completed by the growers. Associations between the lighting practices and the burden of Salmonella in the flocks were tested while accounting for variation between the grow-out farms, their production complexes and companies. RESULTS: Longer relative duration of reduced lights during the grow-out period was associated with reduced detection of Salmonella on the exterior of birds 1 week before harvest and on the broiler carcasses at the post-chilling point of processing. In addition, starting reduced lights for > or = 18 hours per day later in the grow-out period was associated with decreased detection of Salmonella on the exterior of broilers arriving for processing and in the post-harvest drag swabs of litter from the grow-out house. CONCLUSIONS: The results of this field study show that lighting practices implemented during broiler rearing can impact the burden of Salmonella in the flock. The underlying mechanisms are likely to be interactive.