M2 Polarization and Inhibition of Host Cell Glycolysis Contributes Intracellular Survival of Salmonella Strains in Chicken Macrophage HD-11 Cells.

Salmonella enterica is a group of facultative, gram-negative bacteria. Recently, new evidence indicated that Salmonella could reprogram the host metabolism to increase energy or metabolites available for intracellular replication. In this study, using a chicken-specific kinomic immunometabolism peptide array analysis, we found that infection by S. Enteritidis induced significant phosphorylation changes in many key proteins of the glycolytic pathway in chicken macrophage HD-11 cells, indicating a shift in glycolysis caused by Salmonella infection. Nitric oxide production and changes of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) represented by extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively, were measured in chicken macrophages infected with three Salmonella strains (S. Enteritidis, S. Heidelberg, and S. Senftenberg). The infection reduced glycolysis and enhanced OXPHOS in chicken macrophages as indicated by changes of ECAR and OCR. Salmonella strains differentially affected macrophage polarization and glycolysis. Among three strains tested, S. Enteritidis was most effective in downregulating glycolysis and promoting M2 polarization as measured by ECAR, ORC, and NO production; while S. Senftenberg did not alter glycolysis and may promote M1 polarization. Our results suggested that downregulation of host cell glycolysis and increase of M2 polarization of macrophages may contribute to increased intracellular survival of S. Enteritidis.

Leukocyte Response to Campylobacter Intra-Abdominal Infection in One Day Old Leghorn Chickens.

Using a previously characterized and described abdominal model to define the avian immune response to Salmonella intra-abdominal challenge in chickens, we have adapted this technique for the study of chickens' immune response to a Campylobacter intra-abdominal challenge. The intra-abdominal Campylobacter infection model facilitates the characterization of peripheral blood leukocyte dynamics and abdominal cell infiltrates. Day-of-hatch Leghorn chickens were injected intra-abdominally (IA) with Campylobacter jejuni [(CJ)1 × 108 colony-forming units (CFUs)]. Changes in peripheral blood leukocyte numbers and abdominal cell infiltrates were monitored at 0, 4, 8, and 24 h post-injection. Peripheral blood leukocyte numbers were also determined for 2 h post-injection. For mortality studies, birds were injected intra-abdominally with 1 × 108 CFUs CJ and mortalities were recorded for 72 h post-injection. In the peripheral blood of CJ-injected chicks, total white blood cell (WBC) numbers began increasing by 2 h post-injection, peaking at 4 h post-injection with the predominant cell type being polymorphonuclear leukocytes (heterophils). Total WBCs declined after 8 h and this decline continued at 24 h, with total WBC numbers approaching control values. The injection of CJ into the abdominal cavity caused a rapid rise in abdominal cell infiltrates with the predominant infiltrating leukocytes being heterophils. Peak abdominal heterophil infiltrates were observed at 8 h post-injection, declining only slightly by 24 h post-injection. Mortality in the CJ challenge groups reached 37%. Mortality in the Salmonella enteritidis positive control groups were greater than 50%. The data suggest that Campylobacter infection does stimulate the innate immune response in chickens when administered IA, however, the immune response and infection is not characterized with the high levels of mortality observed with a Salmonella infection. These data provide a basis for a more definitive characterization of chickens' immune response to Campylobacter and a model to evaluate intervention strategies to prevent the infection and colonization of poultry.

Addition of a protected complex of biofactors and antioxidants to breeder hen diets confers transgenerational protection against Salmonella enterica serovar Enteritidis in progeny chicks.

Addition of vitamins and antioxidants has been long associated with increased immunity and are commonly used in the poultry industry; however, less is known regarding their use in broiler breeder hens. The objective of this study was to determine if feeding a complex of protected biofactors and antioxidants composed of vitamins and fermentation extracts to broiler breeder hens conferred resistance against Salmonella enterica serovar Enteritidis (S. Enteritidis) in the progeny chicks. Three-day-old chicks from control- and supplement-fed hens were challenged with S. Enteritidis and necropsied 4- and 11-days postchallenge (dpc) to determine if there were differences in invasion and colonization. Serum and jejunum were evaluated for various cytokine and chemokine production. Fewer (P = 0.002) chicks from supplement-fed hens had detectable S. Enteritidis in the ceca (32.6%) compared to chicks from control-fed hens (64%). By 11 dpc, significantly (P < 0.001) fewer chicks from supplement-fed hens were positive for S. Enteritidis (liver [36%]; ceca [16%]) compared to chicks from the control hens (liver [76%]; ceca [76%]). The recoverable S. Enteritidis in the cecal content was also lower (P = 0.01) at 11 dpc. In additional to the differences in invasion and colonization, cytokine and chemokine production were distinct between the 2 groups of chicks. Chicks from supplement-fed hens had increased production of IL-16, IL-6, MIP-3α, and RANTES in the jejunum while IL-16 and MIP-1ß were higher in the serum of chicks from the control-fed hens. By 11 dpc, production of IFN-γ was decreased in the jejunum of chicks from supplement-fed hens. Collectively, these data demonstrate adding a protected complex of biofactors and antioxidants to the diet of broiler breeder hens offers a measure of transgenerational protection to the progeny against S. Enteritidis infection and reduces colonization that is mediated, in part, by a robust and distinct cytokine and chemokine response locally at the intestine and systemically in the blood.

Chicken-Specific Kinome Analysis of Early Host Immune Signaling Pathways in the Cecum of Newly Hatched Chickens Infected With Salmonella enterica Serovar Enteritidis.

Poultry is a major source of human foodborne illness caused by broad host range Salmonella serovars (paratyphoid), and developing cost-effective, pre-harvest interventions to reduce these pathogens would be valuable to the industry and consumer. Host responses to infectious agents are often regulated through phosphorylation. However, proteomic mechanisms of Salmonella acute infection biology and host responses to the bacteria have been limited concentrating predominately on the genomic responses of the host to infection. Our recent development of chicken-specific peptide arrays for kinome analysis of host phosphorylation-based cellular signaling responses provided us with the opportunity to develop a more detailed understanding of the early (4-24 h post-infection) host-pathogen interactions during the initial colonization of the cecum by Salmonella. Using the chicken-specific kinomic immune peptide array, biological pathway analysis showed infection with S. Enteritidis increased signaling related to the innate immune response, relative to the non-infected control ceca. Notably, the acute innate immune signaling pathways were characterized by increased peptide phosphorylation (activation) of the Toll-like receptor and NOD-like receptor signaling pathways, the activation of the chemokine signaling pathway, and the activation of the apoptosis signaling pathways. In addition, Salmonella infection induced a dramatic alteration in the phosphorylation events of the JAK-STAT signaling pathway. Lastly, there is also significant activation of the T cell receptor signaling pathway demonstrating the initiation of the acquired immune response to Salmonella infection. Based on the individual phosphorylation events altered by the early Salmonella infection of the cecum, certain conclusions can be drawn: (1) Salmonella was recognized by both TLR and NOD receptors that initiated the innate immune response; (2) activation of the PPRs induced the production of chemokines CXCLi2 (IL-8) and cytokines IL-2, IL-6, IFN-α, and IFN-γ; (3) Salmonella infection targeted the JAK-STAT pathway as a means of evading the host response by targeting the dephosphorylation of JAK1 and TYK2 and STAT1,2,3,4, and 6; (4) apoptosis appears to be a host defense mechanism where the infection with Salmonella induced both the intrinsic and extrinsic apoptotic pathways; and (5) the T cell receptor signaling pathway activates the AP-1 and NF-κB transcription factor cascades, but not NFAT.

A blend of microencapsulated organic acids and botanicals reduces necrotic enteritis via specific signaling pathways in broilers.

Necrotic enteritis (NE) is a devastating disease that has seen a resurgence of cases following the removal of antibiotics from feed resulting in financial loss and significant animal health concerns across the poultry industry. The objective was to evaluate the efficacy of a microencapsulated blend of organic (25% citric and 16.7% sorbic) acids and botanicals (1.7% thymol and 1% vanillin [AviPlusP]) to reduce clinical NE and determine the signaling pathways associated with any changes. Day-of-hatch by-product broiler breeder chicks were randomly assigned to a control (0) or supplemented (500 g/MT) diet (n = 23-26) and evaluated in a NE challenge model (n = 3). Birds were administered 2X cocci vaccine on d 14 and challenged with a cocktail of Clostridium perfringens strains (107) on d 17 to 19. On d 20 to 21 birds were weighed, euthanized, and scored for NE lesions. Jejunal tissue was collected for kinome analysis using an immuno-metabolism peptide array (n = 5; 15/treatment) to compare tissue from supplement-fed birds to controls. Mortality and weight were analyzed using Student's t test and lesion scores analyzed using F-test two-sample for variances (P < 0.05). The kinome data was analyzed using PIIKA2 peptide array analysis software and fold-change between control and treated groups determined. Mortality in the supplemented group was 47.4% and 70.7% in controls (P = 0.004). Lesions scores were lower (P = 0.006) in supplemented birds (2.47) compared to controls (3.3). Supplement-fed birds tended (P = 0.19) to be heavier (848.6 g) than controls (796.2 g). Kinome analysis showed T cell receptor, TNF and NF-kB signaling pathways contributed to the improvements seen in the supplement-fed birds. The following peptides were significant (P < 0.05) in all 3 pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN- γ mRNA expression in tissue between control- and supplement-fed chickens. In conclusion, the addition of a microencapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways.

From crypts to enteroids: establishment and characterization of avian intestinal organoids.

Intestinal organoids (IO), known as "mini-guts", derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3ß inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.

Antagonistic Effects of Lipids Against the Anti-Escherichia coli and Anti-Salmonella Activity of Thymol and Thymol-ß-d-Glucopyranoside in Porcine Gut and Fecal Cultures In Vitro.

Strategies are sought to reduce the carriage and dissemination of zoonotic pathogens and antimicrobial-resistant microbes within food-producing animals and their production environment. Thymol (an essential oil) is a potent bactericide in vitro but in vivo efficacy has been inconsistent, largely due to its lipophilicity and absorption, which limits its passage and subsequent availability in the distal gastrointestinal tract. Conjugation of thymol to glucose to form thymol-ß-d-glucopyranoside can decrease its absorption, but in vivo passage of effective concentrations to the lower gut remains suboptimal. Considering that contemporary swine diets often contain 5% or more added fat (to increase caloric density and reduce dustiness), we hypothesized that there may be sufficient residual fat in the distal intestinal tract to sequester free or conjugated thymol, thereby limiting the availability and subsequent effectiveness of this biocide. In support of this hypothesis, the anti-Salmonella Typhimurium effects of 6 mM free or conjugated thymol, expressed as log10-fold reductions of colony-forming units (CFU) ml-1, were diminished 90 and 58%, respectively, following 24-h in vitro anaerobic fecal incubation (at 39°C) with 3% added vegetable oil compared to reductions achieved during culture without added oil (6.1 log10 CFU ml-1). The antagonistic effect of vegetable oil and the bactericidal effect of free and conjugated thymol against Escherichia coli K88 tested similarly were diminished 86 and 84%, respectively, compared to reductions achieved in cultures incubated without added vegetable oil (5.7 log10 CFU ml-1). Inclusion of taurine (8 mg/ml), bile acids (0.6 mg/ml), or emulsifiers such as polyoxyethylene-40 stearate (0.2%), Tween 20, or Tween 80 (each at 1%) in the in vitro incubations had little effect on vegetable oil-caused inhibition of free or conjugated thymol. Based on these results, it seems reasonable to suspect that undigested lipid in the distal gut may limit the effectiveness of free or conjugated thymol. Accordingly, additional research is warranted to learn how to overcome obstacles diminishing bactericidal activity of free and conjugated thymol in the lower gastrointestinal tract of food-producing animals.

Controlling the Colonization of Clostridium perfringens in Broiler Chickens by an Electron-Beam-Killed Vaccine.

Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p < 0.0001) in late challenge (JGS4064: 4 out of 10; JGS1473: 0 out of 10; JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.

Evaluation of Thymol-ß-d-Glucopyranoside as a Potential Prebiotic Intervention to Reduce Carriage of Zoonotic Pathogens in Weaned and Feeder Pigs.

The gut of food-producing animals is a reservoir for foodborne pathogens. Thymol is bactericidal against foodborne pathogens but rapid absorption of thymol from the proximal gut precludes the delivery of effective concentrations to the lower gut where pathogens mainly colonize. Thymol-ß-d-glucopyranoside is reported to be more resistant to absorption than thymol in everted jejunal segments and could potentially function as a prebiotic by resisting degradation and absorption in the proximal gut but being hydrolysable by microbial ß-glycosidase in the distal gut. Previous in vitro studies showed bactericidal effects of thymol-ß-d-glucopyranoside against Campylobacter, Escherichia coli, and Salmonella enterica serovar Typhimurium in the presence but not absence of intestinal microbes expressing ß-glycosidase activity, indicating that hydrolysis was required to obtain antimicrobial activity. Presently, the oral administration of thymol-ß-d-glucopyranoside was studied to examine the effects on intestinal carriage of Campylobacter, E. coli, and S. Typhimurium in swine. The effects of thymol-ß-d-glucopyranoside or thymol on antimicrobial sensitivity of representative E. coli isolates and characterized Salmonella strains were also explored. Results from two in vivo studies revealed little antimicrobial effects of thymol-ß-d-glucopyranoside on Campylobacter, E. coli, or S. Typhimurium in swine gut. These findings add credence to current thinking that hydrolysis and absorption of thymol-ß-d-glucopyranoside and thymol may be sufficiently rapid within the proximal gut to preclude delivery to the distal gut. Antibiotic susceptibilities of selected bacterial isolates and strains were mainly unaffected by thymol. Further research is warranted to overcome obstacles, preventing the delivery of efficacious amounts of thymol-ß-d-glucopyranoside to the lower gut.

Methylsulfonylmethane Exhibits Bacteriostatic Inhibition of Vancomycin-Resistant Enterococcus faecium, In Vitro.

The aim of this study was to evaluate the antibacterial properties of methylsulfonylmethane (MSM) on vancomycin-resistant Enterococcus faecium (VRE). Bacterial proliferation was measured spectrophotometrically during growth in brain heart infusion broth with 0%, 3%, 5%, 7%, 10%, 12%, and 16% MSM. To assess the mechanism of inhibition, VRE was grown overnight with 0-16% MSM and enumerated on unmedicated and medicated (3-16% MSM) brain heart infusion agar (BHIA). Viability studies were performed to evaluate the impact of 10-16% MSM on VRE over 7 days. Absorbance data indicated a dose-dependent inhibition from 0% to 7% MSM and no increase in optical density in 10-16% MSM. VRE enumerated on unmedicated BHIA from overnight cultures with 10-16% MSM partially recovered. No growth was observed when BHIA contained 10-16% MSM. There was little effect on VRE growth in 10% MSM over 7 days. VRE displayed a population rebound on day 6 when exposed to 12% MSM, and elimination by day 6 in 16% MSM. Regrowth after MSM removal may be indicative of a bacteriostatic mechanism of inhibition. Cell elimination in 16% MSM suggests inhibition of an essential metabolic function from which the bacterium could not recover.

Can the use of older-generation beta-lactam antibiotics in livestock production over-select for beta-lactamases of greatest consequence for human medicine? An in vitro experimental model.

Though carbapenems are not licensed for use in food animals in the U.S., carbapenem resistance among Enterobacteriaceae has been identified in farm animals and their environments. The objective of our study was to determine the extent to which older-generation ß-lactam antibiotics approved for use in food animals in the U.S. might differentially select for resistance to antibiotics of critical importance to human health, such as carbapenems. Escherichia coli (E. coli) strains from humans, food animals, or the environment bearing a single ß-lactamase gene (n = 20 each) for blaTEM-1, blaCMY-2, and blaCTX-M-* or else blaKPC/IMP/NDM (due to limited availability, often in combination with other bla genes), were identified, along with 20 E. coli strains lacking any known beta-lactamase genes. Baseline estimates of intrinsic bacterial fitness were derived from the population growth curves. Effects of ampicillin (32 µg/mL), ceftriaxone (4 µg/mL) and meropenem (4 µg/mL) on each strain and resistance-group also were assessed. Further, in vitro batch cultures were prepared by mixing equal concentrations of 10 representative E. coli strains (two from each resistance gene group), and each mixture was incubated at 37°C for 24 hours in non-antibiotic cation-adjusted Mueller-Hinton II (CAMH-2) broth, ampicillin + CAMH-2 broth (at 2, 4, 8, 16, and 32 µg/mL) and ceftiofur + CAMH-2 broth (at 0.5, 1, 2, 4, and 8µg/mL). Relative and absolute abundance of resistance-groups were estimated phenotypically. Line plots of the raw data were generated, and non-linear Gompertz models and multilevel mixed-effect linear regression models were fitted to the data. The observed strain growth rate distributions were significantly different across the groups. AmpC strains (i.e., blaCMY-2) had distinctly less robust (p < 0.05) growth in ceftriaxone (4 µg/mL) compared to extended-spectrum beta-lactamase (ESBL) producers harboring blaCTX-M-*variants. With increasing beta-lactam antibiotic concentrations, relative proportions of ESBLs and CREs were over-represented in the mixed bacterial communities; importantly, this was more pronounced with ceftiofur than with ampicillin. These results indicate that aminopenicillins and extended-spectrum cephalosporins would be expected to propagate carbapenemase-producing Enterobacteriaceae in food animals if and when Enterobacteriaceae from human health care settings enter the food animal environment.

Evaluation of two commercially-available Salmonella vaccines on Salmonella in the peripheral lymph nodes of experimentally-infected cattle.

BACKGROUND: Salmonella is a common inhabitant of the ruminant gastrointestinal tract, where it often resides asymptomatically and may be shed into the feces. More recently it was discovered that Salmonella may be contained within the peripheral, non-mesenteric lymph nodes, where it is impervious to in-plant pathogen control interventions and may serve as a source of Salmonella-contamination of ground beef. Over the past 10 years considerable research effort has been expended at understanding how this pathogen gets to these lymph nodes, the duration of infection, and, most importantly, screening and developing potential intervention strategies that may be employed on farm prior to the animal being presented for slaughter. METHODS: Utilizing an experimental model of Salmonella inoculation of bovine peripheral lymph nodes (PLNs), two pilot vaccine experiments were conducted to evaluate two Salmonella vaccines: Salmonella Newport Bacterial Extract (Experiment I) and Endovac-Bovi® (Experiment II) on preventing Salmonella acquisition by these nodes. In Experiment I, 4 months following the booster vaccination, 30 steers were inoculated with three Salmonella serotypes intradermally: Newport, Montevideo, and Anatum administered to the right legs, left legs, and to the caudal thorax and abdomen, respectively. Cattle were inoculated every other day over the course of five days (three total inoculation events) and 6 and 12 days following the final Salmonella inoculation, 16 and 14 head in each treatment were euthanized, respectively. In Experiment II, 12 head of Holstein steers were utilized. Seven days following the booster and weekly thereafter for 3 weeks (four total inoculation events), cattle were inoculated as above and euthanized 7 days following final inoculation. Right and left sub-iliac, popliteal and pre-scapular lymph nodes were collected in each experiment, weighed and cultured for Salmonella. RESULTS: In Experiment I, no treatment differences were observed in Salmonella prevalence 6 days post-inoculation (necropsy 1). However, in vaccinated cattle at the second necropsy, a reduction (p = 0.05) in Salmonella prevalence was observed in the sub-iliac and pre-scapular lymph nodes as well as when all nodes were evaluated collectively (p = 0.04). In Experiment II, the vaccine reduced (p = 0.03) Salmonella prevalence in the right popliteal and tended (p = 0.09) to decrease prevalence in both popliteal lymph nodes. CONCLUSION: Under these experimental conditions, the data generated provide evidence of a partial vaccine effect on Salmonella within PLNs and indicate that further research may be warranted.

Inhibition of Salmonella Binding to Porcine Intestinal Cells by a Wood-Derived Prebiotic.

Numerous Salmonella enterica serovars can cause disease and contamination of animal-produced foods. Oligosaccharide-rich products capable of blocking pathogen adherence to intestinal mucosa are attractive alternatives to antibiotics as these have potential to prevent enteric infections. Presently, a wood-derived prebiotic composed mainly of glucose-galactose-mannose-xylose oligomers was found to inhibit mannose-sensitive binding of select Salmonella Typhimurium and Escherichia coli strains when reacted with Saccharomyces boulardii. Tests for the ability of the prebiotic to prevent binding of a green fluorescent protein (GFP)-labeled S. Typhimurium to intestinal porcine epithelial cells (IPEC-J2) cultured in vitro revealed that prebiotic-exposed GFP-labeled S. Typhimurium bound > 30% fewer individual IPEC-J2 cells than did GFP-labeled S. Typhimurium having no prebiotic exposure. Quantitatively, 90% fewer prebiotic-exposed GFP-labeled S. Typhimurium cells were bound per individual IPEC-J2 cell compared to non-prebiotic exposed GFP-labeled S. Typhimurium. Comparison of invasiveness of S. Typhimurium DT104 against IPEC-J2 cells revealed greater than a 90% decrease in intracellular recovery of prebiotic-exposed S. Typhimurium DT104 compared to non-exposed controls (averaging 4.4 ± 0.2 log10 CFU/well). These results suggest compounds within the wood-derived prebiotic bound to E. coli and S. Typhimurium-produced adhesions and in the case of S. Typhimurium, this adhesion-binding activity inhibited the binding and invasion of IPEC-J2 cells.

A microencapsulated feed additive containing organic acids, thymol, and vanillin increases in vitro functional activity of peripheral blood leukocytes from broiler chicks.

During the first week after hatch, young chicks are vulnerable to pathogens as the immune system is not fully developed. The objectives of this study were to determine if supplementing the starter diet with a microencapsulated feed additive containing citric and sorbic acids, thymol, and vanillin affects in vitro functional activity of peripheral blood leukocytes (PBLs). Day-old chicks (n = 800) were assigned to either a control diet (0 g/metric ton [MT]) or a diet supplemented with 500 g/MT of the microencapsulated additive. At 4 D of age, peripheral blood was collected (100 birds per treatment), and heterophils and monocytes isolated (n = 4). Heterophils were assayed for the ability to undergo degranulation and production of an oxidative burst response while nitric oxide production was measured in monocytes. Select cytokine and chemokine mRNA expression levels were also determined. Statistical analysis was performed using Student t test comparing the supplemented diet to the control (P ≤ 0.05). Heterophils isolated from chicks fed the microencapsulated citric and sorbic acids, thymol, and vanillin had higher (P ≤ 0.05) levels of degranulation and oxidative burst responses than those isolated from chicks on the control diet. Heterophils from the supplemented chicks also had greater (P ≤ 0.05) expression of IL10, IL1ß, and CXCL8 mRNA than those from control-fed chicks. Similarly, nitric oxide production was significantly (P ≤ 0.05) higher in monocytes isolated from birds fed the supplement. The cytokine and chemokine profile in monocytes from the supplement-fed chicks showed a significant (P ≤ 0.05) drop in IL10 mRNA expression while IL1ß, IL4, and CXCL8 were unchanged. In conclusion, 4 D of supplementation with a microencapsulated blend made up of citric and sorbic acids, thymol, and vanillin enhanced the in vitro PBL functions of degranulation, oxidative burst, and nitric oxide production compared with the control diet. Collectively, the data suggest feeding broiler chicks a diet supplemented with a microencapsulated blend of citric and sorbic acids, thymol, and vanillin may prime key immune cells making them more functionally efficient and acts as an immune-modulator to boost the inefficient and undeveloped immune system of young chicks.

Altered expression of lactate dehydrogenase and monocarboxylate transporter involved in lactate metabolism in broiler wooden breast.

Wooden breast (WB) results in significant losses to the broiler industry due to reductions in meat quality. While the etiology of WB is unknown, it is believed to be associated with localized hypoxia and decreased lactate levels in skeletal muscles, indicating the presence of altered lactate metabolism in WB. We hypothesized that the expression levels of the major signaling molecules that control lactate metabolism, including lactate dehydrogenases (LDHA and LDHB) and monocarboxylate transporters (MCT1 and MCT4), were altered in WB. Therefore, the objectives of this study were to evaluate whether there were changes in mRNA and protein levels of LDHA, LDHB, MCT1, and MCT4 in WB compared to normal breast (NB) muscles. Biochemical analysis for LDH enzyme activity in NB and WB muscles was studied. MicroRNA375 (miR-375) expression, known to be inversely associated with LDHB protein expression in human cells, was also investigated. The level of LDHA mRNA was 1.7-fold lower in WB tissues than in NB tissues (P < 0.0001). However, the LDHA protein levels were similar in WB and NB tissues. In contrast, the levels of LDHB mRNA and protein were 8.4-fold higher (P < 0.002) and 13.6-fold higher (P < 0.02) in WB than in NB tissues, respectively. The level of miR-375 was not different between WB and NB muscles. The specific LDH isoenzyme activity that converted lactate to pyruvate was 1.8-fold lower in WB compared to NB tissues (P < 0.01). The level of MCT1 mRNA was 2.3-fold higher in WB than those in NB muscles (P < 0.02). However, this upregulation was not observed with MCT1 protein expression levels. The expression levels of MCT4 mRNA and protein were elevated 2.8-fold (P < 0.02) and 3.5-fold (P < 0.004) in WB compared to NB tissues, respectively. Our current findings suggest the potential roles of LDHB and MCT4 on lactate metabolism and provide a unique molecular elucidation for altered lactate homeostasis in WB muscles of broilers.

Inhibition of calmodulin increases intracellular survival of Salmonella in chicken macrophage cells.

Calcium (Ca2+) is a pivotal intracellular second messenger and calmodulin (CaM) acts as a multifunctional Ca2+-binding protein that regulates downstream Ca2+ dependent signaling. Together they play an important role in regulating various cellular functions, including gene expression, maturation of phagolysosome, apoptosis, and immune response. Intracellular Ca2+ has been shown to play a critical role in Toll-like receptor-mediated immune response to microbial agonists in the HD11 chicken macrophage cell line. The role of that the Ca2+/CaM pathway plays in the intracellular survival of Salmonella in chicken macrophages has not been reported. In this study, kinome peptide array analysis indicated that the Ca2+/CaM pathway was significantly activated when chicken macrophage HD11 cells were infected with S. Enteritidis or S. Heidelberg. Further study demonstrated that treating cells with a pharmaceutical CaM inhibitor W-7, which disrupts the formation of Ca2+/CaM, significantly inhibited macrophages to produce nitric oxide and weaken the control of intracellular Salmonella replication. These results strongly indicate that CaM plays an important role in the innate immune response of chicken macrophages and that the Ca2+/CaM mediated signaling pathway is critically involved in the host cell response to Salmonella infection.

Inflammatory phenotypes in the intestine of poultry: not all inflammation is created equal.

The intestinal tract harbors a diverse community of microbes that have co-evolved with the host immune system. Although many of these microbes execute functions that are critical for host physiology, the host immune system must control the microbial community so that the dynamics of this interdependent relationship is maintained. To facilitate host homeostasis, the immune system ensures that the microbial load is tolerated, but anatomically contained, while remaining reactive to microbial invasion. Inflammation is the most prevalent manifestation of host defense in reaction to alterations in tissue homeostasis and is elicited by innate immune receptors that recognize and detect infection, host damage, and danger signaling molecules that activate a highly regulated network of immunological and physiological events for the purpose of maintaining homeostasis and restoring functionality. The efficacy, duration, and consequences of an inflammatory response is dependent upon the type of trigger that is recognized by the innate immune receptors. Further, because of different triggers, there are multiple phenotypes of inflammation. Physiological inflammation is the homeostatic balance between tolerance of the microbiota and the reactivity to pathogen invasion. Pathologic inflammation is usually an acute response that involves the host response to toxins and infection often resulting in collateral damage to surrounding tissue and increased metabolic energy use. Metabolic inflammation is a chronic low-grade inflammation generated by excessive nutrient intake and the metabolic surplus fosters metabolic dysfunction by integrating signals from both the immune and metabolic systems. Sterile inflammation is a low-grade chronic inflammation, in the absence of an infection, in response to chemical, physical, and metabolic stimuli. With a sterile inflammatory response, the stimulus persists without being eliminated suggesting that collateral damage is the cause of the disease. The common denominator with all intestinal inflammatory phenotypes is the central role of the gut microbiota whether it be microbial balance and diversity of microbial metabolic production or microbial turnover.

Nigella sativa L. as an alternative antibiotic feed supplement and effect on growth performance in weanling pigs.

BACKGROUND: Nigella sativa L. (NS) is a plant containing bioactive constituents such as thymoquinone. Extracts of NS improve performance and reduce enteropathogen colonization in poultry and small ruminants, but studies with swine are lacking. In two different studies oral administration of NS extracts at doses equivalent to 0, 1.5 and 4.5 g kg-1 diet was assessed on piglet performance and intestinal carriage of wildtype Escherichia coli and Campylobacter, and Salmonella Typhimurium. RESULTS: Wildtype E. coli populations in the jejunal and rectal content collected 9 days after treatment began were decreased (P ≤ 0.05). Populations recovered from pigs treated with extract at 1.5 and 4.5 g kg-1 diet were 0.72-1.31 log10 units lower than the controls (ranging from 6.05 to 6.61 log10 CFU g-1 ). Wildtype Campylobacter and Salmonella Typhimurium were unaffected by NS treatment. Feed efficiency over the 9 days improved linearly (P < 0.05) from 3.88 with 0 NS-treated pigs to 1.47 and 1.41 with pigs treated with NS at 1.5 and 4.5 g kg-1 diet, respectively, possibly due to high glutamine/glutamic acid content of the NS extract. CONCLUSION: NS supplementation of weanling pigs improved feed efficiency and helped control intestinal E. coli during this vulnerable production phase. © 2017 Society of Chemical Industry.

Chicken macrophages infected with Salmonella (S.) Enteritidis or S. Heidelberg produce differential responses in immune and metabolic signaling pathways.

Protein kinases act in coordination with phosphatases to control protein phosphorylation and regulate signaling pathways and cellular processes involved in nearly every functions of cell life. Salmonella are known to manipulate the host kinase network to gain entrance and survive inside host cells. The effect of Salmonella infection on the host kinase network has been studied in mammalian cells, but information is largely lacking in chicken immune cells. Our previous study indicated that chicken macrophage cells respond differentially to different Salmonella strains. In order to better understand the interaction between chicken macrophages and Salmonella, we used a peptide array-based kinome analysis to identify cellular process and signaling pathways that may play a critical role in the outcome of Salmonella infection. The kinome assay was performed on chicken HD11 macrophages collected at 1.5, 3, and 7h post-infection (hpi) with either S. Heidelberg or S. Enteritidis. A large number of peptides show significantly changed phosphorylation (p≤0.05) during the infection: 390, 449, and 575 peptides for S. Enteritidis and 185, 470, and 442 for S. Heidelberg at 1.5, 3, and 7 hpi, respectively. Many pathways involved in immunity, signal transduction, cellular process, and metabolism were significantly altered, in some case differentially, during the infection by the two Salmonella strains. Particularly, effects on lysosome process, iNOS, CARD9, NLRP3, and MAPK pathway provide significant insight to the inter play between pathogens and chicken macrophage cells during the infection.