MINIstock: Model for INsect Inclusion in sustainable agriculture: USDA-ARS's research approach to advancing insect meal development and inclusion in animal diets.

Animal agriculture is under pressure to increase efficiency, sustainability, and innovation to meet the demands of a rising global population while decreasing adverse environmental effects. Feed cost and availability are 2 of the biggest hurdles to sustainable production. Current diets depend on sources of grain and animal byproduct protein for essential amino acids which have limited sustainability. Insects have arisen as an attractive, sustainable alternative protein source for animal diets due to their favorable nutrient composition, low space and water requirements, and natural role in animal diets. Additionally, insects are capable of bioremediating waste streams including agricultural and food waste, manure, and plastics helping to increase their sustainability. The insect rearing industry has grown rapidly in recent years and shows great economic potential. However, state-of-the-art research is urgently needed to overcome barriers to adoption in commercial animal diets such as regulatory restrictions, production scale issues, and food safety concerns. To address this need, the USDA Agricultural Research Service "MINIstoc: Model for INsect Inclusion" project was created to bring together diverse scientists from across the world to synergistically advance insect meal production and inclusion in animal diets. Here, we provide a short review of insects as feed while describing the MINIstock project which serves as the inspiration for the Journal of Economic Entomology Special Collection "Insects as feed: sustainable solutions for food waste and animal production practices."

Mosquitoes harvested from rice fields as alternative protein ingredient in broiler feed: insights from the first pilot study.

Global population continuous growth and increasing consumers' demands for protein-rich diets have posed sustainability challenges for traditional livestock feed sources. Consequently, exploring alternative and sustainable protein sources has become imperative to address the environmental burden and resource limitations associated with conventional ingredients. With respect to food security assurance, insects have emerged as a promising solution due to their exceptional nutritional profile, rapid reproduction rates, and low environmental impact. In the present pilot study, 10% of a soybean meal-based diet was replaced by adult mosquitoes harvested from rice fields. The objective was to assess the effect of this partial substitution on meat quality aspects and consumer acceptance. A total of 40 Cobb hybrid broiler chickens were randomly placed in a control and a mosquito-fed group. The study was conducted for 42 days and carcass physicochemical, nutritional, and microbiological characteristics, as well as sensory attributes were evaluated. Overall, results regarding quality attributes were comparable between the control and the treatment group. The organoleptic evaluation showed that the thighs from the mosquito-fed group had the highest overall consumer acceptance. These outcomes indicate that mosquitoes could be successfully used as a protein source for broiler feed without compromising the quality and acceptability of the meat.

Cold stress initiates catecholaminergic and serotonergic responses in the chicken gut that are associated with functional shifts in the microbiome.

Climate change is one of the most significant challenges facing the sustainability of global poultry production. Stress resulting from extreme temperature swings, including cold snaps, is a major concern for food production birds. Despite being well-documented in mammals, the effect of environmental stress on enteric neurophysiology and concomitant impact on host-microbiome interactions remains poorly understood in birds. As early life stressors may imprint long-term adaptive changes in the host, the present study sought to determine whether cold temperature stress, a prominent form of early life stress in chickens, elicits changes in enteric stress-related neurochemical concentrations that coincide with compositional and functional changes in the microbiome that persist into the later life of the bird. Chicks were, or were not, subjected to cold ambient temperature stress during the first week post-hatch and then remained at normal temperature for the remainder of the study. 16S rRNA gene and shallow shotgun metagenomic analyses demonstrated taxonomic and functional divergence between the cecal microbiomes of control and cold stressed chickens that persisted for weeks following cessation of the stressor. Enteric concentrations of serotonin, norepinephrine, and other monoamine neurochemicals were elevated (P < 0.05) in both cecal tissue and luminal content of cold stressed chickens. Significant (P < 0.05) associations were identified between cecal neurochemical concentrations and microbial taxa, suggesting host enteric neurochemical responses to environmental stress may shape the cecal microbiome. These findings demonstrate for the first time that early life exposure to environmental temperature stress can change the developmental trajectory of both the chicken cecal microbiome and host neuroendocrine enteric physiology. As many neurochemicals serve as interkingdom signaling molecules, the relationships identified here could be exploited to control the impact of climate change-driven stress on avian enteric host-microbe interactions.

Insect frass composition and potential use as an organic fertilizer in circular economies.

Insect manure or "frass" has emerged as an alternative nutrient source for alleviating the dependence on fossil fuel-based fertilizers, reducing food waste, and promoting food security. Yet, research on insect frass chemical composition is in its infancy. Here, we assessed the chemical properties of yellow mealworm (Tenebrio molitor L.) frass compared with poultry litter (PL). Insect frass was obtained from the National Biological Control Laboratory (NBCL; IF-L) and an insect-rearing company (IF-C). PL was collected from facilities in Arkansas (PL-AR) and North Carolina (PL-NC). Samples were analyzed for pH, electrical conductivity, macro- and micronutrients, heavy metals, pathogens, and indicator microorganisms. On average, insect frass had 43% and 47% higher C and N than PL, respectively (P < 0.05). Considering a 5 mg/ha application rate, IF-C can supply 159 kg N/ha, twice the N supply of PL-AR (78 kg/ha). IF-L had a 53% higher P supply than PL-NC. Mean K, Ca, S, and micronutrient contents were higher in PL than in frass (P < 0.05), whereas As, Cd, Cr, and Pb were nearly absent in frass. Chemical composition and pathogens in fertilizer sources were largely affected by insect-rearing substrate and supplements used in poultry and insect production. Insect frass utilized in this study had optimum C and N rates relative to PL, suggesting a promising soil amendment for improving soil health and C sequestration, thus contributing to sustainable agricultural intensification and reuse of food waste in circular economies.

Draft genome sequences of Campylobacter species isolated from the cecal contents of broiler chickens.

Here, we report the draft genomes of 10 Campylobacter strains isolated from the cecal contents of market-age broiler chickens naturally colonized with Campylobacter. Through a comprehensive analysis of these draft genomes, we have unveiled their core genetic elements and several antimicrobial resistance genes.

Inclusion of trans-cinnamaldehyde and caprylic acid in feed results in detectable concentrations in the chicken gut and reduces foodborne pathogen carriage.

Poultry act as a major reservoir host for Salmonella and Campylobacter spp., the 2 leading causes of foodborne illnesses globally and in the United States. Preharvest stage interventions to reduce foodborne pathogen carriage in poultry are increasingly informed by consumer preference for antibiotic-free poultry production. The in-feed inclusion of plant-derived antimicrobial compounds is a promising antibiotic alternative strategy to reduce foodborne pathogen load in the broiler chicken gut. Yet, the fate of these phytochemicals through the broiler chicken gastrointestinal tract is unknown. Likewise, while in-feed phytochemicals have been widely demonstrated in challenge models to reduce foodborne pathogen carriage, little is known regarding efficacy to curb natural routes of infection. As such, the aim of the present study was 2-fold. We sought to determine the concentrations of 2 phytochemicals, trans-cinnamaldehyde and caprylic acid, in each region of the chicken gastrointestinal tract following their in-feed inclusion over a 6-wk production period. In addition, we investigated how the in-feed provision of these phytochemicals may protect against environmental acquisition of Campylobacter jejuni and Salmonella spp. Trans-cinnamaldehyde and caprylic acid were detected in crop, gizzard, duodenal, jejunal, and ileal contents. Crop and gizzard concentrations were not significantly (P > 0.05) different. A significant (P < 0.05) decrease in phytochemical concentration was observed in intestinal regions compared to crop and gizzard. Trans-cinnamaldehyde was consistently identified in cecal and colon contents, while caprylic acid was not detectable in these regions. Trans-cinnamaldehyde and caprylic acid were found to reduce (P < 0.05) Salmonella load. Together, our data establish that the in-feed addition of trans-cinnamaldehyde and caprylic acid, 2 phytochemicals that have previously been shown to exert antimicrobial activity against poultry-associated foodborne pathogens, results in detectable concentrations in the broiler chicken gastrointestinal tract. By providing researchers with a gastrointestinal region-by-region map of phytochemical concentrations, the present study is expected to inform the choice of in-feed phytochemicals targeting foodborne pathogen carriage in the broiler chicken gastrointestinal tract.

Effect of plant-derived antimicrobials, eugenol, carvacrol, and ß-resorcylic acid against Salmonella on organic chicken wings and carcasses.

Organic poultry constitutes a sizeable segment of the American organic commodities market. However, processors have limited strategies that are safe, effective, and approved for improving the microbiological safety of products. In this study, the efficacy of 3 plant-derived antimicrobials (PDAs), eugenol (EG), carvacrol (CR), and ß-resorcylic acid (BR) was evaluated against Salmonella on organic chicken wings and carcasses. Wings inoculated with Salmonella (6 log10 CFU/wing) were treated with or without the treatments (BR [0.5%, 1% w/v], EG [0.5%, 1% v/v], CR [0.5%, 1% v/v], chlorine [CL; 200 ppm v/v], or peracetic acid [PA; 200 ppm v/v]) applied for 2 min at 54°C (scalding study) or 30 min at 4°C (chilling study). Homogenates and treatment water were evaluated for surviving Salmonella. Six wings or carcasses per treatment were analyzed in each study. All treatments, except CL and 0.5% BR in the scalding study, yielded significant reductions of Salmonella on wings compared to the positive control (PC-Salmonella inoculated samples not treated with antimicrobials). To follow, carcasses inoculated with Salmonella (higher inoculum [106 CFU/carcass] or lower inoculum [104 CFU/carcass]) and immersed in antimicrobials (CR 1% [v/v] and industry controls [CL {200 ppm}, or PA [200 ppm]) for 30 min at 4°C were stored until analysis. For the higher inoculum study, 1% CR resulted in a 3.9 log10 CFU/g reduction of Salmonella on the carcass on d 0 compared to PC (P < 0.05); however, CL yielded no reduction. On d 3, CR and PA resulted in 0.9 and 1.2 log10 CFU/g reduction of Salmonella, respectively (P < 0.05). For the lower inoculum study, consistent Salmonella reductions were obtained with CR and PA (1.4-2.1 log10 CFU/g) on d 0 and 7. High reductions of Salmonella in processing water were obtained in all studies. CR effectively controls Salmonella on wings and carcasses and in processing water immediately after application. Follow-up studies on the organoleptic characteristics of PDA-treated chicken carcasses are necessary.

A Multiomic Analysis of Chicken Serum Revealed the Modulation of Host Factors Due to Campylobacter jejuni Colonization and In-Water Supplementation of Eugenol Nanoemulsion.

Campylobacter jejuni is a foodborne pathogen that causes campylobacteriosis globally, affecting ~95 million people worldwide. Most C. jejuni infections involve consuming and/or handling improperly cooked poultry meat. To better understand chicken host factors modulated by Campylobacter colonization, we explored a novel LCMS-based multiomic technology using three experimental groups: (1) negative control, (2) positive control, and (3) eugenol nanoemulsion (EGNE) treatment (supplemented with 0.125% EGNE in the water) of broiler chickens (n = 10 birds/group). Birds in groups two and three were challenged with C. jejuni on day 7, and serum samples were collected from all groups on day 14. Using this multiomic analysis, we identified 1216 analytes (275 compounds, seven inorganics, 407 lipids, and 527 proteins). The colonization of C. jejuni significantly upregulated CREG1, creatinine, and 3-[2-(3-Hydroxyphenyl) ethyl]-5-methoxyphenol and downregulated sphingosine, SP d18:1, high mobility group protein B3, phosphatidylcholines (PC) P-20:0_16:0, PC 11:0_26:1, and PC 13:0_26:2. We found that 5-hydroxyindole-3-acetic acid significantly increased with the EGNE treatment when compared to the positive and negative controls. Additionally, the treatment increased several metabolites when compared to the negative controls. In conclusion, this study revealed several potential targets to control Campylobacter in broiler chickens.

Ability of Garlic and Ginger Oil to Reduce Salmonella in Post-Harvest Poultry.

Approximately 1.35 million human salmonellosis cases are reported in the United States every year, resulting in over 26,000 hospitalizations and 400 deaths. Consumption of contaminated poultry products is one of the leading causes of human salmonellosis. Poultry meat becomes contaminated when feces from an infected bird comes into contact with the carcass during processing. Additional carcasses can then become cross-contaminated along the processing line. While chemicals such as peracetic acid are currently used to kill microbes such as Salmonella, consumers are increasingly calling for more natural alternatives. Our objective for this study was to determine the ability of the phytochemicals garlic and ginger oil to reduce Salmonella prevalence in the processing environment. In a simulated scalding tank environment, dipping contaminated chicken skin samples in a solution containing both garlic and ginger oil reduced Salmonella by up to 2 log CFU. Furthermore, the oils prevented Salmonella growth in the tank solution. The mechanism of action of garlic and ginger was evaluated using the sub-inhibitory concentration of each oil individually. While both were found to decrease autoinducer-2 (AI-2) levels, no effect was seen on expression of 10 genes involved in Salmonella virulence and survival. In total, this work demonstrates the potential of garlic and ginger to reduce Salmonella prevalence in the post-harvest environment. However, more work remains to be done to understand the mechanism of action.

Effect of Salmonella Typhimurium Colonization on Microbiota Maturation and Blood Leukocyte Populations in Broiler Chickens.

Reducing Salmonella in commercial chickens is vital to decreasing human salmonellosis infections resulting from contact with contaminated poultry and poultry products. As the intestinal microbiota plays an important role in preventing pathogen colonization, we sought to understand the relationship between Salmonella infection and the cecal microbiota and the host immune system. Day-of-hatch broiler chicks were assigned to three treatments: control, artificial (SA), and natural (SN) Salmonella infection. At seven days of age, control and SA birds were inoculated with PBS or Salmonella Typhimurium, respectively. Five SA birds were transferred to SN cages to facilitate natural infection. Cecal content and blood samples were collected at 0, 8, 14, and 21 days of age for microbiota and leukocyte analysis, respectively. A significant change in microbiota composition was observed in both groups as noted by a decrease in Lactobacillus and Escherichia and an increase in Bacteroides. Leukocyte analysis revealed a decrease in the percentage of circulating monocytes at 7 days post-infection while a decrease in thrombocyte and an increase in heterophil percentages were seen at 14 days post-infection. Taken together, these results demonstrate the ability of Salmonella to modulate the intestinal microbiota to facilitate colonization. Additionally, results indicated an early role of monocytes and thrombocytes during colonization, followed by heterophils.

A neurochemical biogeography of the broiler chicken intestinal tract.

The study of neurochemical-based interkingdom signaling and its impact on host-microbe interaction is called microbial endocrinology. Neurochemicals play a recognized role in determining bacterial colonization and interaction with the gut epithelium. While much attention has been devoted to the determination of neurochemical concentrations in the mammalian gut to better understand tissue and region-specific microbial endocrinology-based mechanisms of host-microbe interaction, little is known regarding the biogeography of neurochemicals in the avian gut. Greater resolution of avian gut neurochemical concentrations is needed especially as recent microbial endocrinology-based investigations into bacterial foodborne pathogen colonization of the chicken gut have demonstrated neurochemicals to affect Campylobacter jejuni and Salmonella spp. in vivo and in vitro. The aim of the present study was to determine the concentrations of stress-related neurochemicals in the tissue and luminal content of the duodenum, jejunum, ileum, cecum, and colon of the broiler intestinal tract, and to investigate if this biogeography changes with age of the bird. While all neurochemicals measured were detected in the intestinal tract, many displayed differences in regional concentrations. Whereas the catecholamine norepinephrine was detected in each region of the intestinal tract, epinephrine was present only in the cecum and colon. Likewise, dopamine, and its metabolite 3,4-dihydroxyphenylacetic acid were found in the greatest quantities in the cecum and colon. Serotonin and histamine were identified in each gut region. Region-specific age-related changes were observed (P < 0.05) for serotonin, its metabolite 5-hydroxyindole acetic acid as well as for histamine. Several neurochemicals, including norepinephrine, were found in the contents of each gut region. Epinephrine was not detected in the gut content of any region. Salsolinol, a microbial-produced neuroactive compound was detected in the gut content but not in tissue. Together, our data establish a neurochemical biogeography of the broiler chicken intestinal tract. By providing researchers with a region-by-region map of in vivo gut neurochemical concentrations of a modern broiler chicken breed, this neurochemical map is expected to inform future investigations that seek to utilize avian enteric neurochemistry.

Effect of caprylic acid alone or in combination with peracetic acid against multidrug-resistant Salmonella Heidelberg on chicken drumsticks in a soft scalding temperature-time setup.

The antimicrobial efficacy of caprylic acid (CA), a medium-chain fatty acid, against multidrug-resistant Salmonella Heidelberg (MDR SH) on chicken drumsticks in a soft-scalding temperature-time setup was investigated. Based on the standardization experiments in nutrient media and on chicken breast fillet portions, intact chicken drumsticks were spot inoculated with MDR SH and immersed in water with or without antimicrobial treatments at 54°C for 2 min. The treatments included 0.5% CA, 1% CA, 0.05% peracetic acid (PAA), 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA. Additionally, the efficacy of the potential scald treatments against MDR SH survival on drumsticks for a storage period of 48 h at 4°C was determined. Furthermore, the effect of these treatments on the surface color of the drumsticks was also evaluated. Appropriate controls were included for statistical comparisons. The antimicrobial treatments resulted in a significant reduction of MDR SH on drumsticks. For the lower inoculum (∼2.5 log10 CFU/g) experiments, 0.5% CA, 1% CA, 0.05% PAA, 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA resulted in 0.7-, 1.0-, 2.5-, 1.4-, and 1.5- log10 CFU/g reduction of MDR SH on drumsticks, respectively (P < 0.05). The same treatments resulted in 0.9-, 1.3-, 2.5-, 2.2-, and 2.6- log10 CFU/g reduction of MDR SH when the drumsticks were contaminated with a higher inoculum (∼4.5 log10 CFU/g) level (P < 0.05). Moreover, the antimicrobial treatments inactivated MDR SH in the treatment water to undetectable levels, whereas 2.0- to 4.0- log10 CFU/mL MDR SH survived in the positive controls (P < 0.05). Also, the treatments were effective in inhibiting MDR SH on the drumsticks compared to the respective controls during a storage period of 48 h at 4°C; however, the magnitude of reduction remained the same as observed during the treatment (P < 0.05). Additionally, none of the treatments affected the color of the drumsticks (P > 0.05). Results indicate that CA could be an effective natural processing aid against MDR SH on chicken products.

Select Phytochemicals Reduce Campylobacter jejuni in Postharvest Poultry and Modulate the Virulence Attributes of C. jejuni.

Consumption or handling of poultry and poultry products contaminated with Campylobacter species are a leading cause of foodborne illness in humans. Current strategies employed to reduce Campylobacter in live chickens provide inconsistent results indicating the need for an alternative approach. This study investigated the efficacy of phytochemicals, namely, turmeric, curcumin, allyl sulfide, garlic oil, and ginger oil, to reduce Campylobacter jejuni in postharvest poultry and sought to delineate the underlying mechanisms of action. Two experiments were conducted on the thigh skin of the chicken, and each experiment was repeated twice. Samples were inoculated with 50 µl (∼107 CFU/sample) of C. jejuni strain S-8 and allowed to adhere for 30 min. Skin samples were dipped into their respective prechilled treatment solutions (0.25 and 0.5% in experiments 1 and 2, respectively) at 4°C for an hour to simulate chilling tank treatment, followed by plating to enumerate C. jejuni (n = 3 samples/treatment/trial). The mechanisms of action(s) were investigated using subinhibitory concentration (SIC) in adhesion, quorum sensing, and gene expression analyses. Adhesion assay was conducted on the monolayers of ATCC CRL-1590 chicken embryo cells challenged with C. jejuni and incubated in the presence or absence of phytochemicals for 1.5 h, followed by plating to enumerate adhered C. jejuni. The effects of phytochemicals on quorum sensing and cell viability were investigated using Vibrio harveyi bioluminescence and LIVE/Dead BacLightTM bacterial viability assays, respectively. In addition, droplet digital PCR determined the gene expression analyses of C. jejuni exposed to phytochemicals. Data were analyzed by GraphPad Prism version 9. C. jejuni counts were reduced by 1.0-1.5 Log CFU/sample with garlic oil or ginger oil at 0.25 and 0.5% (p < 0.05). The selected phytochemicals (except curcumin) reduced the adhesion of C. jejuni to chicken embryo cells (p < 0.05). In addition, all the phytochemicals at SIC reduced quorum sensing of C. jejuni (p < 0.05). The cell viability test revealed that cells treated with 0.25% of phytochemicals had compromised cell membranes indicating this as a mechanism that phytochemicals use to damage/kill C. jejuni. This study supports that the application of phytochemicals in postharvest poultry would significantly reduce C. jejuni in poultry meat.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

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.

Serotonin modulates Campylobacter jejuni physiology and invitro interaction with the gut epithelium.

Microbial endocrinology, which is the study of neurochemical-based host-microbe interaction, has demonstrated that neurochemicals affect bacterial pathogenicity. A variety of neurochemicals, including norepinephrine, were shown to enhance intestinal epithelial colonization by Campylobacter jejuni. Yet, little is known whether serotonin, an abundant neurochemical produced in the gut, affects the physiology of C. jejuni and its interaction with the host gut epithelium. Considering the avian gut produces serotonin and serves as a major reservoir of C. jejuni, we sought to investigate whether serotonin can affect C. jejuni physiology and gut epithelial colonization in vitro. We first determined the biogeographical distribution of serotonin concentrations in the serosa, mucosa, as well as the luminal contents of the broiler chicken ileum, cecum, and colon. Serotonin concentrations were greater (P < 0.05) in the mucosa and serosa compared to the luminal content in each gut region examined. Among the ileum, colon, and cecum, the colon was found to contain the greatest concentrations of serotonin. We then investigated whether serotonin may effect changes in C. jejuni growth and motility in vitro. The C. jejuni used in this study was previously isolated from the broiler chicken ceca. Serotonin at concentrations of 1mM or below did not elicit changes in growth (P > 0.05) or motility (P > 0.05) of C. jejuni. Next, we utilized liquid chromatography tandem mass spectrometry to investigate whether serotonin affected the proteome of C. jejuni. Serotonin caused (P < 0.05) the downregulation of a protein (CJJ81176_1037) previously identified to be essential in C. jejuni colonization. Based on our findings, we evaluated whether serotonin would cause a functional change in C. jejuni adhesion and invasion of the HT29MTX-E12 colonic epithelial cell line. Serotonin was found to cause a reduction in adhesion (P < 0.05) but not invasion (P > 0.05). Together, we have identified a potential role for serotonin in modulating C. jejuni colonization in the gut in vitro. Further studies are required to understand the practical implications of these findings for the control of C. jejuni enteric colonization in vivo.

Japanese quail (Coturnix japonica) as a novel model to study the relationship between the avian microbiome and microbial endocrinology-based host-microbe interactions.

BACKGROUND: Microbial endocrinology, which is the study of neuroendocrine-based interkingdom signaling, provides a causal mechanistic framework for understanding the bi-directional crosstalk between the host and microbiome, especially as regards the effect of stress on health and disease. The importance of the cecal microbiome in avian health is well-recognized, yet little is understood regarding the mechanisms underpinning the avian host-microbiome relationship. Neuroendocrine plasticity of avian tissues that are focal points of host-microbiome interaction, such as the gut and lung, has likewise received limited attention. Avian in vivo models that enable the study of the neuroendocrine dynamic between host and microbiome are needed. As such, we utilized Japanese quail (Coturnix japonica) that diverge in corticosterone response to stress to examine the relationship between stress-related neurochemical concentrations at sites of host-microbe interaction, such as the gut, and the cecal microbiome. RESULTS: Our results demonstrate that birds which contrast in corticosterone response to stress show profound separation in cecal microbial community structure as well as exhibit differences in tissue neurochemical concentrations and structural morphologies of the gut. Changes in neurochemicals known to be affected by the microbiome were also identified in tissues outside of the gut, suggesting a potential relationship in birds between the cecal microbiome and overall avian physiology. CONCLUSIONS: The present study provides the first evidence that the structure of the avian cecal microbial community is shaped by selection pressure on the bird for neuroendocrine response to stress. Identification of unique region-dependent neurochemical changes in the intestinal tract following stress highlights environmental stressors as potential drivers of microbial endocrinology-based mechanisms of avian host-microbiome dialogue. Together, these results demonstrate that tissue neurochemical concentrations in the avian gut may be related to the cecal microbiome and reveal the Japanese quail as a novel avian model in which to further examine the mechanisms underpinning these relationships. Video abstract.

Sodium Butyrate Reduces Salmonella Enteritidis Infection of Chicken Enterocytes and Expression of Inflammatory Host Genes in vitro.

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1ß, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

Production and characterization of avian crypt-villus enteroids and the effect of chemicals.

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.

Draft Genome Sequences of Campylobacter jejuni Strains Isolated from Poultry.

Four wild-type Campylobacter jejuni strains isolated from the cecal contents of broiler chickens were sequenced. The average genome size was 1,622,170 bp, with 1,667 to 1,761 coding sequences and 47 to 51 RNAs. Multiple genes encoding motility, intestinal colonization, toxin production, stress tolerance, and multidrug resistance were present in all the strains.

Trans-Cinnamaldehyde, Eugenol and Carvacrol Reduce Campylobacter jejuni Biofilms and Modulate Expression of Select Genes and Proteins.

Campylobacter jejuni is the leading cause of human foodborne illness globally, and is strongly linked with the consumption of contaminated poultry products. Several studies have shown that C. jejuni can form sanitizer tolerant biofilm leading to product contamination, however, limited research has been conducted to develop effective control strategies against C. jejuni biofilms. This study investigated the efficacy of three generally recognized as safe status phytochemicals namely, trans-cinnamaldehyde (TC), eugenol (EG), or carvacrol (CR) in inhibiting C. jejuni biofilm formation and inactivating mature biofilm on common food contact surfaces at 20 and 37°C. In addition, the effect of phytochemicals on biofilm architecture and expression of genes and proteins essential for biofilm formation was evaluated. For the inhibition study, C. jejuni was allowed to form biofilms either in the presence or absence of sub-inhibitory concentrations of TC (0.75 mM), EG (0.61 mM), or CR (0.13 mM) for 48 h and the biofilm formation was quantified at 24-h interval. For the inactivation study, C. jejuni biofilms developed at 20 or 37°C for 48 h were exposed to the phytochemicals for 1, 5, or 10 min and surviving C. jejuni in the biofilm were enumerated. All phytochemicals reduced C. jejuni biofilm formation as well as inactivated mature biofilm on polystyrene and steel surface at both temperatures (P < 0.05). The highest dose of TC (75.64 mM), EG (60.9 mM) and CR (66.56 mM) inactivated (>7 log reduction) biofilm developed on steel (20°C) within 5 min. The genes encoding for motility systems (flaA, flaB, and flgA) were downregulated by all phytochemicals (P < 0.05). The expression of stress response (cosR, ahpC) and cell surface modifying genes (waaF) was reduced by EG. LC-MS/MS based proteomic analysis revealed that TC, EG, and CR significantly downregulated the expression of NapA protein required for oxidative stress response. The expression of chaperone protein DnaK and bacterioferritin required for biofilm formation was reduced by TC and CR. Scanning electron microscopy revealed disruption of biofilm architecture and loss of extracellular polymeric substances after treatment. Results suggest that TC, EG, and CR could be used as a natural disinfectant for controlling C. jejuni biofilms in processing areas.