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Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S . Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S . Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S . Enteritidis. HTC cells were infected with S . Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S . Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S . Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S . Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S . Enteritidis invasion.

Due to its tropical origin and adaptation, rice is significantly impacted by cold stress, and consequently sustains large losses in growth and productivity. Currently, rice is the second most consumed cereal in the world and production losses caused by extreme temperature events in the context of \"major climatic changes\" can have major impacts on the world economy. We report here an analysis of rice genotypes in response to low-temperature stress, studied through physiological gas-exchange parameters, biochemical changes in photosynthetic pigments and antioxidants, and at the level of gene and protein expression, towards an understanding and identification of multiple low-temperature tolerance mechanisms. The first effects of cold stress were observed on photosynthesis among all genotypes. However, the tropical japonica genotypes Secano do Brazil and Cypress had a greater reduction in gas exchange parameters like photosynthesis and water use efficiency in comparison to the temperate japonica Nipponbare and M202 genotypes. The analysis of biochemical profiles showed that despite the impacts of low temperature on tolerant plants, they quickly adjusted to maintain their cellular homeostasis by an accumulation of antioxidants and osmolytes like phenolic compounds and proline. The cold tolerant and sensitive genotypes showed a clear difference in gene expression at the transcript level for OsGH3-2, OsSRO1a, OsZFP245, and OsTPP1, as well as for expression at the protein level for LRR-RLKs, bHLH, GLYI, and LTP1 proteins. This study exemplifies the cold tolerant features of the temperate japonica Nipponbare and M202 genotypes, as observed through the analysis of physiological and biochemical responses and the associated changes in gene and protein expression patterns. The genes and proteins showing differential expression response are notable candidates towards understanding the biological pathways affected in rice and for engineering cold tolerance, to generate cultivars capable of maintaining growth, development, and reproduction under cold stress. We also propose that the mechanisms of action of the genes analyzed are associated with the tolerance response.

Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.

Clostridium perfringens is a prevalent gut bacterial pathogen in humans and animals. This study investigated the role of the mechanistic targets of rapamycin (mTOR) and deoxycholic acid (DCA) on C. perfringens intestinal infection. Chickens were sequentially infected with Eimeria maxima and received the mTOR inhibitor rapamycin and DCA. C. perfringens-induced necrotic enteritis (NE) was evaluated using body weight gain (BWG), histopathology, bile acids, pathogen colonization, cell infiltration and death, and gene expression. The significant difference of p < 0.05 was analyzed by one-way ANOVA and multiple comparisons. Notably, rapamycin strongly reduced the subclinical and clinical NE histopathologies. DCA and DCA combined with rapamycin alleviated clinical NE and BWG loss. Rapamycin, DCA, and DCA + rapamycin attenuated bile acid reduction in NE birds, and they also reduced immune cell infiltration into the intestinal lamina propria as well as immune cell migration in vitro. At molecular levels, DCA and DCA + rapamycin reduced proinflammatory IFNγ, MMP9, IL23, and IL17 gene expression. Rapamycin, DCA, and DCA + rapamycin reduced NE-induced intestinal cell apoptosis. Together, these results suggest that mTOR signaling mediates C. perfringens-induced ileitis, and combining mTOR inhibition and DCA improves the intervention efficacy against NE ileitis and BWG loss.

Bacterial Chondronecrosis with Osteomyelitis (BCO) is a specific cause of lameness in commercial fast-growing broiler (meat-type) chickens and represents significant economic, health, and wellbeing burdens. However, the molecular mechanisms underlying the pathogenesis remain poorly understood. This study represents the first comprehensive characterization of the proximal tibia proteome from healthy and BCO chickens. Among a total of 547 proteins identified, 222 were differentially expressed (DE) with 158 up- and 64 down-regulated proteins in tibia of BCO vs. normal chickens. Biological function analysis using Ingenuity Pathways showed that the DE proteins were associated with a variety of diseases including cell death, organismal injury, skeletal and muscular disorder, immunological and inflammatory diseases. Canonical pathway and protein–protein interaction network analysis indicated that these DE proteins were involved in stress response, unfolded protein response, ribosomal protein dysfunction, and actin cytoskeleton signaling. Further, we identified proteins involved in bone resorption (osteoclast-stimulating factor 1, OSFT1) and bone structural integrity (collagen alpha-2 (I) chain, COL2A1), as potential key proteins involved in bone attrition. These results provide new insights by identifying key protein candidates involved in BCO and will have significant impact in understanding BCO pathogenesis.

Background Three-dimensional models of cell culture such as organoids and mini organs accord better advantage over regular cell culture because of their ability to simulate organ functions hence, used for disease modeling, metabolic research, and the development of therapeutics strategies. However, most advances in this area are limited to mammalian species with little progress in others such as poultry where it can be deployed to study problems of agricultural importance. In the course of enterocyte culture in chicken, we observed that intestinal mucosal villus-crypts self-repair and form spheroid-like structures which appear to be useful as ex vivo models to study enteric physiology and diseases. Results The villus-crypts harvested from chicken intestinal mucosa were cultured to generate enteroids, purified by filtration then re cultured with different chemicals and growth factors to assess their response based on their morphological dispositions. Histochemical analyses using marker antibodies and probes showed the enteroids consisting different cell types such as epithelial, goblet, and enteroendocrine cells typical to villi and retain functional characteristics of intestinal mucosa. Conclusions We present a simple procedure to generate avian crypt-villous enteroids containing different cell types. Because the absorptive cells are functionally positioned outwards, similar to the luminal enterocytes, the cells have better advantages to interact with the factors present in the culture medium. Thus, the enteroids have the potential to study the physiology, metabolism, and pathology of the intestinal villi and can be useful for preliminary screenings of the factors that may affect gut health in a cost-effective manner and reduce the use of live animals.

The sheared avian intestinal villus-crypts exhibit high tendency to self-repair and develop enteroids in culture. Presuming that this transition process involves differential biomolecular changes, we employed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF–MS) to find whether there were differences in the spectral profiles of sheared villi versus the enteroids, assessed in the mass range of 2–18 kDa. The results showed substantial differences in the intensities of the spectral peaks, one particularly corresponding to the mass of 4963 Da, which was significantly low in the sheared villus-crypts compared with the enteroids. Based on our previous results with other avian tissues and further molecular characterization by LC-ESI-IT-TOF–MS, and multiple reaction monitoring (MRM), the peak was identified to be thymosin β4 (Tβ4), a ubiquitously occurring regulatory peptide implicated in wound healing process. The identity of the peptide was further confirmed by immunohistochemistry which showed it to be present in a very low levels in the sheared villi but replete in the enteroids. Since Tβ4 sequesters G-actin preventing its polymerization to F-actin, we compared the changes in F-actin by its immunohistochemical localization that showed no significant differences between the sheared villi and enteroids. We propose that depletion of Tβ4 likely precedes villous reparation process. The possible mechanism for the differences in Tβ4 profile in relation to the healing of the villus-crypts to developing enteroids is discussed.

Human zoonotic infection with Campylobacter is a major cause of gastroenteritis in the United States and worldwide. Listeria monocytogenes causes a potentially fatal infection in humans and is often attributed to contaminated food. Genetic typing has demonstrated that Campylobacter infection is often associated with the consumption of contaminated poultry products, with Campylobacter often colonizing the poultry gastrointestinal tract, while listeriosis is commonly associated with the consumption of contaminated ready-to-eat (RTE) foods. In this study, a strain of endospore-forming bacterium (strain NH) that is bactericidal towards the human food pathogens Campylobacter jejuni and Listeria monocytogenes was identified and characterized. Transwell experiments demonstrated that the bactericidal effect on both C. jejuni and L. monocytogenes is due to secretions from the spore former. These foodborne pathogens consistently exhibited 7 log reductions in growth when exposed to the NH cell-free culture filtrate. Sequencing of the 16s rRNA gene V4 variable region and analysis of the full-length 16s rRNA gene sequence from the WGS indicated that strain NH belongs to the species Aneurinibacillus aneurinilyticus. A microplate bioassay demonstrated that a bactericidal substance that is sensitive to protease could be collected from cell-free filtrates by salting out with ammonium sulfate. Gel filtration chromatography indicated a native molecular weight for the bactericidal protein of ca. 50 kDa, consistent with a class III bacteriocin. The active protein bound strongly to a cation-exchange resin and with an isoelectric point of ten, suggesting a positively charged protein. Both cation-exchange chromatography and isoelectric focusing indicated the enrichment of an 11 kDa protein on SDS-PAGE. This protein was identified through mass spectroscopy as the flgM protein, an anti-sigma factor. Analysis of whole genome sequencing (WGS) of the strain NH genome indicated the presence of a number of non-conservative amino acid substitutions in the flgM-gene-derived amino acid sequence of strain NH and A. aneurinilyticus compared to other members of the Aneurinibacillus genus. Further investigation is needed to determine whether these substitutions are correlated with the bactericidal activity. The identified strain may be useful as a feed additive for the pre-harvest control of Campylobacter jejuni in poultry.

Background Clostridium perfringens -induced chicken necrotic enteritis (NE) is responsible for substantial economic losses worldwide annually. Recently, as a result of antibiotic growth promoter prohibition, the prevalence of NE in chickens has reemerged. This study was aimed to reduce NE through titrating dietary deoxycholic acid (DCA) as an effective antimicrobial alternative. Materials and methods Day-old broiler chicks were assigned to six groups and fed diets supplemented with 0 (basal diet), 0.8, 1.0 and 1.5 g/kg (on top of basal diet) DCA. The birds were challenged with Eimeria maxima (20,000 oocysts/bird) at d 18 and C. perfringens (10 9  CFU/bird per day) at d 23, 24, and 25 to induce NE. The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology, mRNA accumulation and C. perfringens colonization by real-time PCR, targeted metabolomics of bile acids, fluorescence in situ hybridization (FISH), or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results At the cellular level, birds infected with E. maxima and C. perfringens developed subclinical NE and showed shortening villi, crypt hyperplasia and immune cell infiltration in ileum. Dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds. Consistent with the increased histopathological scores, subclinical NE birds suffered body weight gain reduction compared to the uninfected birds, an effect attenuated with increased doses of dietary DCA. At the molecular level, the highest dose of DCA at 1.5 g/kg reduced C. perfringens luminal colonization compared to NE birds using PCR and FISH. Furthermore, the dietary DCA reduced subclinical NE-induced intestinal inflammatory gene expression and cell apoptosis using PCR and TUNEL assays. Upon further examining ileal bile acid pool through targeted metabolomics, subclinical NE reduced the total bile acid level in ileal digesta compared to uninfected birds. Notably, dietary DCA increased total bile acid and DCA levels in a dose-dependent manner compared to NE birds. Conclusion These results indicate that DCA attenuates NE-induced intestinal inflammation and bile acid reduction and could be an effective antimicrobial alternative against the intestinal disease.

Clostridium perfringens is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen C. perfringens. Assays were conducted to evaluate the inhibition of C. perfringens growth, hydrogen sulfide (H2S) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the bsh gene for cloning. The bsh gene from Bifidobacterium longum was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in E. coli BL21 and Bacillus subtilis 168 (B-sub-BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from B. subtilis was analyzed by a Dot-Blot. B-sub-BSH was evaluated for the inhibition of C. perfringens growth. C. perfringens growth reached 7.8 log10 CFU/mL after 24 h culture. C. perfringens growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced C. perfringens H2S production and the virulence gene expression of asrA1, netB, colA, and virT. BSH activity was observed in Lactobacillus johnsonii and B. longum under anaerobe but not L. johnsonii under 10% CO2 air. After the sequence alignment of bsh from ten bacteria, bsh from B. longum was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned bsh and amylase signal peptide sequence was inserted into pDR-BSH, B. subtilis was transformed and named B-sub-BSH. The transformation was evaluated using PCR with B. subtilis around 3 kb and B-sub-BSH around 5 kb. Secretory BSH expressed from B-sub-BSH was determined for His-tag using Dot-Blot. Importantly, C. perfringens growth was reduced greater than 59% log10 CFU/mL in the B-sub-BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against C. perfringens virulence, and recombinant secretory BSH from B-sub-BSH reduced C. perfringens growth, suggesting a new potential intervention against the pathogen-induced chicken NE.