Impact of a direct-fed microbial supplementation on intestinal permeability and immune response in broiler chickens during a coccidia challenge.

Maintaining intestinal health supports optimal gut function and influences overall performance of broilers. Microlife® Prime (MLP) contains a unique combination of four strains of Bacillus spp. selected to support a healthy gut which may improve performance. The aim of this study was to determine the effects of MLP supplementation on intestinal health and immunity of broilers challenged with a mixed coccidia infection during peak [0 to 6-day post-infection (dpi)] and recovery phases (6 to 13 dpi). A total of 120 male, 4 days-old Ross 708, broiler chicks were allotted to 3 treatment groups (8 replicate cages; 5 birds/cage) in a randomized complete block design. Treatments included a non-challenge (NEG), a coccidia challenge (POS), and coccidia challenge fed MLP (5 × 105 CFU/g of diet). Diets were corn-soybean meal-based. At 11 days of age, all birds, except for NEG, were orally gavaged with 15 doses (3 × the recommended commercial dose). On 6, 9, and 13 dpi, birds were orally gavaged with fluorescein isothiocyanate conjugate dextran (FITC-d). Plasma and mid-jejunum tissues were collected 2 h later. On 6 dpi, duodenal lesions from 2 birds/cage were scored and droppings were collected for oocyst enumeration. Body weight gain (BWG) and feed conversion ratio (FCR) were calculated over the experimental period. Data were analyzed with GLIMMIX procedure of SAS. During the peak phase, POS birds had reduced BWG (23%) and FCR (15%) compared to NEG birds (P < 0.05), while birds fed MLP had similar BWG (209 and 208 g) and FCR (1.17 and 1.21) compared to NEG (P > 0.05). On 6 dpi, POS birds had higher lesion scores and oocyst shedding, 2 × increase in serum FITC-d, and higher jejunum IL-10, and IFN-γ mRNA compared to NEG (P < 0.05). Birds fed MLP had reduced plasma FITC-d compared to POS birds (P < 0.05) and similar IL-10 and IFN-γ mRNA. On 13 dpi, birds fed MLP had lower plasma FITC-d, jejunum IL-10 and IFN-γ mRNA compared to POS birds (P < 0.05), but similar IL-10 to NEG birds (P > 0.05). This study confirms MLP improves intestinal health and positively modulates mucosal immune response post-coccidia challenge.

Informal nutrition symposium: leveraging the microbiome (and the metabolome) for poultry production.

Knowledge of gut microbiology of poultry has advanced from a limited ability to culture relatively few microbial species, to attempting to understand the complex interactions between the bird and its microbiome. The Informal Nutrition Symposium 2021 was intended to help poultry scientists to make sense of the implications of the vast amounts of information being generated by researchers. This paper represents a compilation of the talks given at the symposium by leading international researchers in this field. The symposium began with an overview of the historical developments in the field of intestinal microbiology and microbiome research in poultry. Next, the systemic effects of the microbiome on health in the context of the interplay between the intestinal microbiota and the immune system were presented. Because the microbiome and the host communicate and influence each other, the novel field of kinomics (the study of protein phosphorylation) as used in the study of the poultry microbiome was discussed. Protein phosphorylation is a rapid response to the complex of signals among the microbiome, intestinal lumen metabolites, and the host. Then, a description of why an understanding of the role of microbial endocrinology in poultry production can lead to new understanding of the mechanisms by which the gut microbiota and the host can interact in defined mechanisms that ultimately determine health, pathogenesis of infectious disease, and behavior was given. Finally, a view forward was presented underscoring the importance of understanding mechanisms in microbiomes in other organ systems and other species. Additionally, the importance of the development of new -omics platforms and data management tools to more completely understand host microbiomes was stressed.

Functional characterization of the chicken cationic amino acid transporter-2 isoforms.

Utilization of lysine and arginine by mammalian skeletal muscle is due in large part to the system y(+) cationic amino acid transporters (CAT), particularly CAT-2A. The chicken CAT-2 (cCAT-2) gene is alternatively spliced to produce three isoforms (cCAT-2A/B/C). Chicken (Gallus gallus) CAT-2 isoforms were transiently and stably expressed in mammalian cell lines to determine cCAT-2 isoform cellular localization and transport properties. The cCAT-2A protein localized to the plasma membrane and the cCAT-2B protein localized to the cytoplasm juxtaposed to the plasma membrane. The cCAT-2C protein localized non-specifically throughout the cytoplasm. The cCAT-2A protein exhibited saturable transport. The K(t) of cCAT-2A for lysine using transient transfection was 2.6mM and the V(max) was 11.9pmol/mg protein/min. The K(t) of cCAT-2A for lysine using stable transfection was 7.9mM and the V(max) was 12.8pmol/mg protein/min. Transient and stable transfections of cCAT-2B or cCAT-2C resulted in no lysine or arginine transport. These data indicate that cCAT-2A is a low affinity, high velocity transporter for lysine and arginine and is the cCAT-2 isoform responsible for lysine and arginine transport in avian skeletal muscle.

AMP-activated protein kinase and carbohydrate response element binding protein: a study of two potential regulatory factors in the hepatic lipogenic program of broiler chickens.

This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken (Gallus gallus) liver. Fasting for 24 or 48 h produced significant declines in plasma glucose (at 24 h), insulin and thyroid hormone (T3) levels that were accompanied by changes in mRNA expression levels of hepatic lipogenic genes. The mRNA levels of malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1) and thyroid hormone responsive Spot 14 (Spot 14) declined in response to fasting. Refeeding for 24 h increased mRNA levels for each of these genes, characterized by a significant increase ('overshoot') above fed control values. No change in mRNA expression of the two AMPK alpha subunit genes was observed in response to fasting or refeeding. In contrast, ChREBP and sterol regulatory element binding protein-1 (SREBP-1) mRNA levels decreased during fasting and increased with refeeding. Phosphorylation of AMPK alpha subunits increased modestly after a 48 h fast. However, there was no corresponding change in the phosphorylation of ACC, a major downstream target of AMPK. Protein level and DNA-binding activity of ChREBP increased during fasting and declined upon refeeding as measured in whole liver tissue extracts. In general, evidence was found for coordinate transcriptional regulation of lipogenic program genes in broiler chicken liver, but specific regulatory roles for AMPK and ChREBP in that process remain to be further characterized.

Increased glucose availability activates chicken thymocyte metabolism and survival.

Glucose metabolism in mammalian thymocytes is coupled to their development and selection in the thymus. In chickens, thymocytes develop in a low glucose concentration in ovo and a high glucose concentration posthatch. To determine the effect of glucose concentration on thymocyte glucose metabolism, embryonic thymic lobes were cultured in media containing varying glucose concentrations and thymocytes were isolated for analysis. Glucose transporter-1 (Glut-1) and Glut-3 mRNA abundance was at least 2-fold higher in thymocytes incubated with 10 and 20 mmol/L glucose than in those incubated with 0 mmol/L glucose (P < 0.05) and glucose uptake was greatest in thymocytes incubated with 20 mmol/L glucose (P < 0.05). Thymocytes incubated with 0 and 20 mmol/L glucose had 185% greater hexokinase activity than in those incubated with 10 mmol/L glucose (P < 0.05). Consequently, thymocyte glucose utilization was dependent upon glucose availability. Increased glucose utilization resulted in a higher mitochondrial membrane potential in thymocytes incubated with 15 mmol/L glucose than in those incubated with 5 mmol/L glucose (P < 0.05), indicating enhanced thymocyte energy status in response to high glucose concentrations. Additionally, thymocyte viability was lower in thymocytes incubated with 5 mmol/L glucose than in those incubated with 10 and 15 mmol/L glucose (P < 0.05) and rates of thymocyte apoptosis were higher in thymocytes incubated with 5 mmol/L glucose than in those incubated with 15 mmol/L glucose (P < 0.05). Glucose availability induced metabolic changes in thymocytes that altered their energy status and survival. Consequently, these data indicate that glucose availability may influence the development of naïve T cells in the chicken thymus.

Molecular cloning and characterization of the chicken cationic amino acid transporter-2 gene.

The system y(+) cationic amino acid transporters (CATs) play an important role in the regulation of lysine and arginine utilization. We have characterized the genomic organization and tissue transcription of the chicken CAT-2 (cCAT-2) isoforms from pectoralis muscle. The primary cCAT-2 transcript is alternatively spliced within the same position of the mRNA to produce cCAT-2A and cCAT-2B isoforms that share high nucleotide homology with their mammalian counterparts. We also identified a novel third CAT-2 isoform, cCAT-2C. This isoform contains a premature termination codon and studies in the chicken LMH cell line show that cCAT-2C mRNA is degraded by the nonsense-mediated mRNA decay pathway. All three cCAT-2 isoforms are transcribed in liver, pectoralis, gastrocnemius and heart. Chicken cCAT-2A was the predominant cCAT-2 isoform in liver, pectoralis and gastrocnemius. Analysis of the 5'-untranslated region of cCAT-2 identified multiple cCAT-2 promoters. Chicken CAT-2 promoter usage was tissue dependent and was not responsible for cCAT-2 isoform production. Analysis of genomic sequence upstream cCAT-2 promoter regions revealed binding sites for transcription factors involved in amino acid sensing, hormone signaling and immune function. These results provide insight into the role of the cCAT-2 gene and its regulation of lysine and arginine utilization in aves.

Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues.

Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not been identified in birds, the objectives of this work were to clone, sequence, and characterize the genomic organization of ChREBP and Mlx genes and to determine the expression of ChREBP, Mlx, and several related genes including liver X receptor (LXR), SREBP-1 and thyroid hormone responsive Spot 14 (Spot 14) in chickens. Alternative splicing resulted in two ChREBP mRNA transcript variants that code for predicted proteins of 895 and 869 amino acids. The chicken Mlx gene produced a single mRNA transcript that codes for a predicted protein of 245 amino acids. Chicken ChREBP and Mlx predicted proteins shared high amino acid homology with select portions of corresponding mammalian proteins. In chickens, Mlx, SREBP-1, and LXR were expressed at comparable levels in all tissues examined. However, ChREBP demonstrated significant tissue-specific expression with the highest mRNA levels found in liver and duodenum and Spot 14 was expressed predominantly in liver and abdominal fat. Using Western blotting, the presence of ChREBP protein was detected in chicken liver tissue. Our findings add new insight into a potential role for specific transcription factors such as ChREBP and Mlx in the glucose-dependent regulation of lipogenesis in birds.

Energy metabolism in developing chicken lymphocytes is altered during the embryonic to posthatch transition.

Adequate energy status in lymphocytes is vital for their development. The ability of developing chicken lymphocytes to acquire and metabolize energy substrates was determined during embryonic days (e) and neonatal days (d) of life when primary-energy substrate metabolism is altered at the whole-animal level. In 3 experiments, bursacytes and thymocytes were isolated on e17, e20, d1, d3, d7, or d14 to analyze markers associated with glucose, glutamine, and lipid metabolism. Bursacyte glucose transporter-3 (Glut-3) mRNA abundance increased from d1 to d14 and hexokinase-1 (HK-1) mRNA abundance was maximum on e20 (P<0.05). Thymocyte Glut-1, Glut-3, and HK-1 mRNA abundance increased from e17 to d14 (P<0.05). HK enzyme activity increased from e20 to d3 in bursacytes and d3 to d7 in thymocytes (P<0.05). Glucose uptake by bursacytes and thymocytes was greater on d14 compared to d1 and d7 (P<0.05). Bursacyte and thymocyte sodium coupled neutral amino acid transporter-2 and glutaminase (GA) mRNA abundance increased from e20 to d7 (P<0.05). GA enzyme activity increased from e20 to d7 in bursacytes (P<0.05) and did not change in thymocytes. Carnitine palmitoyl transferase enzyme activity did not change over time in either cell type. These studies suggest that developing B and T lymphocytes adapt their metabolism during the first 2 wk after hatch. Developing lymphocytes increase glucose metabolism with no change in fatty acid metabolism and bursacytes, but not thymocytes, increase glutamine metabolism. Understanding the factors that regulate lymphocyte development in neonatal chicks may help promote their adaptive immune responses to pathogens in early life.

Lysine deficiency and feed restriction independently alter cationic amino acid transporter expression in chickens (Gallus gallus domesticus).

The effect of a lysine-deficient diet on cationic amino acid transporter (CAT1-3) mRNA expression was determined in broiler chickens. Chicks consumed a lysine-adequate (LA; 1.3% lysine) or lysine-deficient (LD; 0.7% lysine) diet. Pair-fed chicks consumed the LA diet in an amount equal to that consumed by LD chicks during the previous day (PLA). CAT 1-3 mRNA expression in the liver, pectoralis and bursa of LD chicks were lower than that of LA and PLA chicks (P<0.05), and levels were not detectable in LD chick thymus. High affinity CAT mRNA expression in isolated bursacytes was 16-fold higher in LD chicks than that of LA chicks (P<0.001). Thymocyte high affinity CAT mRNA expression was 5-fold lower than that of LA chicks (P<0.05). The summed amount of high affinity CAT-1 and CAT-3 mRNA expression in chicks fed a lysine adequate diet was highly correlated (r2=0.51; P<0.001) to a tissue's growth during a lysine deficiency or feed restriction. In the thymus and bursa of LD chicks, CAT mRNA levels differed between resident lymphocytes and their surrounding tissues. By expressing high affinity CAT isoforms, developing lymphocytes may have a greater ability to obtain lysine than their surrounding tissue during a lysine deficiency.

The acute phase response alters cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus).

The effect of an acute phase response (APR) on cationic amino acid transporter (CAT1-3) mRNA expression in liver, muscle, bursa and thymus was determined in broiler strain chickens. The APR was initiated by injecting Salmonella typhimurium lipopolysaccharide subcutaneously (LPS; 1 mg/kg bw). In Experiment 1, CAT1-3 mRNA expression was determined at multiple time points following LPS administration. LPS increased bursa and liver total and high affinity CAT mRNA expression (P<0.05) and transiently increased pectoralis total CAT mRNA expression (P<0.05). Total CAT mRNA expression in the thymus decreased 7.7-fold from 0 to 8 h after LPS injection (P<0.05). In Experiment 2, fasted chicks were uninjected or LPS-injected. LPS increased total and high affinity CAT mRNA 2-fold in both the bursa and liver (P<0.05) and did not change thymus total and high affinity CAT mRNA expression (P>0.05). LPS increased liver weight only (P<0.05) and did not alter the plasma lysine and arginine concentration (P>0.05). In Experiments 3 and 4, thymocyte proliferation and total protein content were dependent upon the media lysine concentration (P<0.001). The inability of the thymus to compete for lysine and arginine during the APR may limit the ability of thymocytes to develop during infections.

Glucose and cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus).

Tissue glucose transporter (GLUT1-3) and cationic amino acid transporter (CAT1-3) mRNA expression was determined in growing broiler chicks posthatch. In two experiments, tissues were either collected on days 1, 3 and 7 or days 1 and 14 posthatch. Heart and liver were the only tissues expressing a GLUT isoform on day 1. All tissues expressed a GLUT isoform on day 7 except for the thymus. Most tissues expressing a CAT isoform on day 1 decreased mRNA levels through day 7 (P<0.05), except for bursa CAT-1 which tended to increase (P=0.05). The thymus and spleen did not express any CAT isoform mRNA until day 7. The liver was the only tissue expressing GLUT-2 mRNA through day 14. On day 14, GLUT-1, CAT-1 and CAT-2 mRNA were differentially expressed across tissues (P<0.05). High-affinity GLUT and CAT mRNA expression was highest in the heart and bursa, respectively (P<0.05). Total CAT mRNA expression was greatest in the bursa (P<0.05). The thymus had the lowest high affinity GLUT and total CAT mRNA expression on day 14 posthatch. Therefore, T lymphocytes within the thymus may be most susceptible to glucose and cationic amino acid supply.

The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos).

Ducks produce a full length IgY and a truncated isoform (IgYDeltaFc). IgY and IgY(DeltaFc) were isolated from ducks vaccinated against Escherichia coli and used to make E.coli-Ig immune complexes (IC). Phagocytosis of IC by duck monocytes decreased directly with the proportion of IgYDeltaFc (p<0.001). IC containing IgY:IgY(DeltaFc) at ratios of 100:0, 50:50, 0:100, and 0:0 (E. coli alone) were injected intravenously into naïve mallard ducks. At 24 h after injection, plasma hemopexin levels were higher in ducks given either the 0:100 ratio or the 100:0 ratio than those given 50:50 or E. coli alone (p<0.005) Liver IL-1beta mRNA levels followed a similar pattern. Splenic IL-1beta mRNA decreased markedly as the proportion of IgY(DeltaFc) increased (p<0.01) Thus, IgY(DeltaFc) may shift the response to IC from the spleen to the liver as infections progress from acute to chronic.

Rice expressing lactoferrin and lysozyme has antibiotic-like properties when fed to chicks.

Two experiments were conducted to determine whether rice that has been genetically produced to express human lactoferrin (LF) or lysozyme (LZ) protects the intestinal tract similarly to subtherapeutic antibiotics (bacitracin + roxarsone; Antibiotics). Experiment 1 compared 10 corn-soy diets containing 20% of various proportions of LF, LZ or conventional rice (CONV). Chicks fed 5% LF + 10% LZ + 5% CONV had significantly better feed efficiency and thinner lamina propria in the duodenum than those fed 20% CONV. Experiment 2 compared five corn-soy diets containing experimental rice combinations totaling 15% rice. Chicks fed 10% LZ + 5% CONV or 5% LF + 10% LZ had significantly lower feed intake and significantly better feed efficiency than those fed 15% CONV. Chicks fed 10% LZ + 5% CONV, 5% LF + 10% LZ or Antibiotics had significantly greater villous height in the duodenum compared with chicks fed 15% CONV. The lamina propria of the ileum was thinner and contained fewer leukocytes in chicks fed 10% LZ + 5% CONV or Antibiotics compared with those fed 15% CONV. The results from these experiments demonstrate a potential of genetically produced LF and LZ rice to be used as a substitute for antibiotics in broiler diets.