Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Determine the molecular mechanisms of virulence within and across Eimeria strains affecting poultry and investigate the genetic and phenotypic responses by the bird. 1.1. Produce high-quality full genome sequences of Eimeria species of economic importance in commercial poultry. 1.2. Use the rapid, high throughput molecular screening assay to differentiate Eimeria species in a sample and determine if they are of vaccine or field origin. 1.3A. Elucidate the relationship between host redox (oxidative stress) status and development of E. maxima. 1.3B. Understand host resistance/tolerance mechanisms in the development of intestinal lesions during Eimeria maxima infection. 1.3C. Identification, characterization, and assessment of non-pathogenic bacterial species from reused litter used as ⿿proLitterbiotics⿿ during E. maxima infection. 2. Determine the molecular mechanisms of virulence to Clostridium perfringens-based enteritis and genotypic and phenotypic responses by the bird. 2.1. Sequencing and analysis of virulent field strains of C. perfringens. 2.2. Investigate host genotype and environmental interaction that predisposes young birds to C. perfringens-induced enteritis. 2.3A. Synthesize chitosan nanoparticle vaccines, loaded with antigens from field strains of C. perfringens and surface-tagged with E. maxima antigens. 2.3B. Identify the anti-C. perfringens IgA and IgG and T cell response curves in broilers inoculated orally with different doses of chitosan nanoparticle vaccine entrapped with C. perfringens and E. maxima proteins. 2.3C. Quantify the chitosan nanoparticle vaccine efficacy in decreasing the colonization of C. perfringens and disease score in broilers induced with necrotic enteritis. 3. Develop alternatives to antibiotics for preventing or treating Eimeria- and C. perfringens-based enteritis. 3.1A. Evaluate the impact of dietary antibiotic alternatives on intestinal physiology and microbial ecology in each segment of the gastrointestinal tract of genetically diverse broiler chickens in response to Eimeria- and C. perfringens-based necrotic enteritis. 3.1B. Characterization of starch digestibility along the digestive tract, digesta oligosaccharides, and SCVFAs in broiler chickens receiving different types of resistant starch (RS). 3.2. Investigate mechanisms by which probiotics influence intestinal physiology and microbial ecology of genetically diverse broilers in response to Eimeria- and C. perfringens-based necrotic enteritis. 3.3. Investigate the modes of action of dietary amino acids and nutrients on gut development/integrity, and host physiological response to Eimeria- and C. perfringens-induced enteritis. 3.3A. Potentiating the protective effects of RS with low protein and amino acid supplemented diets. 3.3B. Determine the response of broiler chickens challenged with Eimeria when fed diets with RS and low protein, AA-fortified diet. 3.3C. Investigating prebiotic-probiotic symbiosis using RS as functional fiber in broilers induced with NE. **See uploaded post plan for sub-objectives 3.1C, 3.1D, 3.3D, 3.3E and 3. 3F. Approach (from AD-416): The approach outlined in this integrated project is divide between three interrelated objectives. The project will employ several approaches to (1) produce high-quality full genome sequences of Eimeria species of economic importance in commercial poultry; (2) develop rapid, high throughput molecular screening assays to differentiate Eimeria species in a sample, as well as determine their origins [i.e. vaccine vs. field]; and (3) improve production efficiency by studying the influence of host genetics on resistance, susceptibility, and tolerance to Eimeria spp., and the impact of dietary redox potential (e.g. cysteine) and enteric and litter microbiota on the pathology of Eimeria spp. in chickens. To better understand the incidence of NE, which is often predisposed by coccidiosis, the project will continue collecting field isolates of C. perfringens from the southeast and mid-Atlantic regions and produce full genome sequencing and complete comparative analyses of those NE-causing strains. This approach will allow identification of predominant virulence factors in C. perfringens of commercial poultry that could serve as targets for designing and developing vaccines as alternative control measures to antibiotics. Using an established nanoparticle vaccine platform, the project will continue building this unique design and further develop and test anti-C. perfringens vaccines with the potential to be adopted by the poultry industry. In dealing with the urgent need to identify, test, and employ effective antibiotic alternatives for poultry, the project will conduct detailed activities to better understand the mechanistic actions of several candidate interventions on performance, physiological, microbial, immunological, and metabolic responses of the host. The application of well-defined probiotics, prebiotics, phytogenics, and specific nutrients during coccidiosis and NE will be used in vivo and in ovo to study the critical physiological changes that directly impact host health and performance. Parameters at the enteric and systemic levels will collectively provide strong host response correlates that can be utilized in refining the application of these potential alternatives in commercial settings. ARS researchers in Athens, Georgia, conducted an experiment to understand why some chickens are resistant and others susceptible to Eimeria maxima infection. Fourteen-day-old broiler chickens were inoculated via oral gavage with 2x105 E. maxima sporulated oocysts/bird, while the control groups were mock-infected with water. E. maxima infective doses were acquired from a single oocyst cloning. Chickens in the infected groups displayed clear signs of coccidiosis starting at 4 days post-infection (dpi), and growth reduction was observed by 6 dpi. Oocyst shedding confirmed the infection in these groups. All the chickens were housed in wired-floor cages and ad libitum supplied with water and a non-medicated standard grower diet. Five chickens were randomly selected from each treatment group at 6 (dpi) and euthanized by cervical dislocation to sample ~1 cm of ileum tissue from Meckel⿿s diverticulum. Ileum samples were snap-frozen in liquid nitrogen and then, stored at -80°C for later RNA extraction and sequencing using the NGS Illumina sequencing platform. A major finding was the positive regulation of calcium ion transport and regulation of calcium mediated signaling in the host. The amino acid tryptophan also showed up to be critical for the development of susceptibility of chickens to Eimeria spp. infection. Two studies were conducted to evaluate the efficacy of chitosan nanoparticles loaded with native (CN) or toxoids (CT) of C. perfringens proteins in protecting birds against necrotic enteritis (NE). In testing the stability of these nanoparticle vaccines, both CN and CT formulations demonstrated stability in a series of in vitro assays. For the evaluation in chickens, 180 broiler chicks were randomly assigned to treatment groups: sham-vaccinated (control), CN-vaccinated (CN), and CT-vaccinated (CT). Each chick received oral gavages of the respective control or vaccine on days 0 and 14. By 21 days of age, birds vaccinated with CN and CT nanoparticles exhibited 29% and 125% higher antigen specific T cells compared to the control group, respectively. Samples collected from an experiment conducted in FY2023 in which broilers were left unchallenged or challenged with Eimeria- and C. perfringens-induced subclinical necrotic enteritis in the absence or presence of subtherapeutic levels of the antibiotic bacitracin methylene disalicylate (BMD) or a commercially available sodium butyrate product were analyzed. Histological analysis was used to assess small intestinal tissue damage by determining villus height and crypt depth in two distal most sections. Microbial community composition, organic acid profiles, and amino acid levels were determined in the distal small intestinal sections and ceca. Expression of barrier function, cytokine, and nutrient transporter genes in small intestine and hormone receptor and nutrient transporter genes in liver and breast muscle was evaluated. Limiting the use of antibiotic growth promoters in the poultry industry has led to an increased incidence of necrotic enteritis (NE). A study was conducted to assess the impact of phytogenic blends on broiler performance and mortality during a co-infection with Eimeria maxima and Clostridium perfringens. 600 day-old Ross 708 male chicks were divided into four treatment groups (6 replicate pens, 25 birds/pen): negative control (NC) fed a corn-soybean meal diet; positive control (PC) fed NC + 15 ppm Avilamycin and 125 ppm Amprolium, and two phytogenic additive groups PHY1 and PHY2. On day 14, all birds were orally gavaged with 2,000 E. maxima sporulated oocysts, followed by one dose of approximately 1ÿ108 CFU of C. perfringens on day 19. Birds were weighed on days 14, 21, 28, and 42 on a per pen basis. Average daily gain and feed intake as well as feed conversion ratio were calculated during each feeding phase and adjusted for daily mortality. Additionally, on days 14, 21, and 42, jejunal samples were collected to assess mRNA abundance of key marker genes of the intestinal immune response. The data were analyzed using ANOVA and significance (P=0.05) was determined by the LSD test. In our efforts to mitigate enteric challenges with non-drug alternatives, the potential dietary application of algal polysaccharides on broiler responses to NE was examined. This study employed the same experimental NE model involving co-infection with E. maxima and C. perfringens per above to assess the effectiveness of a sulfate polysaccharide extracted from marine algae in mitigating the adverse effects of NE in broilers. A total of 600 day-old Ross 708 male broilers were randomly assigned to one of four treatment groups: NC (negative control, fed a corn-soybean meal diet); PC (positive control, fed NC + 15 ppm Avilamycin and 125 ppm Amprolium); AGS (Algoguard Standard, fed NC + Algoguard added at 0.1% of the diet); and AGH (Algoguard High, fed NC + Algoguard added at 0.2% of the diet). Performance parameters were measured weekly. On day 21, the jejunum and ileum of four birds per pen were examined for NE lesions. On days 14, 21, and 42, jejunum samples from one bird per pen were collected to measure mRNA abundance of gut integrity and immune response marker genes. Data were analyzed using JMP, and significance between treatments was identified by LSD (P = 0.05). Another study examined the effects of in ovo administration of a postbiotic with or without post-hatch water application on performance, intestinal lesion scores, and mRNA levels of mucosal integrity-related genes of broilers during subclinical NE. At embryonic day 18, Ross 708 eggs were injected into the amnion with 0.2 mL of either water or postbiotic. Male hatchlings (n=288; 12 birds/cage; 6 replicate cages per group) were assigned to one of the following four post-hatch treatment groups : 1) NC (in ovo water injection, no challenge); 2) PIW (postbiotic in ovo and in drinking water, no challenge); 3) NC+ (NC with NE challenge) ; and 4) PIW+ (PIW with NE challenge). On d 14, all birds were orally gavaged with 3,000 Eimeria maxima sporulated oocysts followed by two doses of ~1x108 CFU/mL/bird of C. perfringens on d 19 and d 20. Hatchability (day 1), weekly performance, and NE lesion scores (day 21) were measured weekly. Jejunum tissues were collected to assess mRNA abundance of immune response and mucosal integrity-related genes. This report presents results from one experiment with resistant starch (RS). Two RS sources and levels were identified as practical and optimum from previous objectives (objectives 3-1B and 3-1C) and used in this experiment. Raw potato starch (RPS, 2.5% or 5.0%) and high amylose corn starch (HCS, 5.0%) were incorporated into a corn-soybean meal-based control diet and fed to broiler chickens for 35 days. Half of the birds were challenged with mixed-Eimeria oocysts on day 9 of age for a total of 8 treatments. During the acute phase of infection, birds that received 5% HCS gained the most weight and had the lowest FCR. All the birds that received RS diets were heavier than the birds on the control diet on day 35. In addition, the birds that received diets with RS had greater ileal digestibility of amino acids, partly explaining the higher weight gain in the birds. The inclusion of the RS in diets did not impact the cecal short-chain fatty acids profile and had no effect on the morphology of the jejunal. Analysis of cecal content for microbiota profile is currently ongoing. An experiment was conducted where eggs were injected with saline (negative control) or probiotic and then hatched chicks from each group were given no additives (negative control) subtherapeutic levels of an antibiotic (positive control), or the probiotic. Birds in each group were then left unchallenged or challenged with Eimeria- and C. perfringens- induced subclinical necrotic enteritis. Growth performance, mortality, and lesion scores were used to determine severity of the challenge model and efficacy of the probiotic intervention. Blood and tissue samples were collected on 0, 24, and 48 hours post challenge. Sample analyses are underway and should be completed within the next two reporting periods. Histological analysis will be used to determine villus height and crypt depth to assess intestinal tissue damage. Microbial community composition will be determined by sequencing, and analysis of intestinal amino acid and organic acid levels will be used to determine impacts of host and microbial metabolism on intestinal ecology. Mechanisms driving effects on growth performance will be evaluated by measuring levels of circulating hormones and metabolites as well as expression of genes regulating uptake and utilization of dietary nutrients. ACCOMPLISHMENTS 01 ARS researchers in Athens, Georgia, were able to identify calcium as a major element in the progression of Eimeria spp. infection. The hypothesis was that dietary calcium level may significantly affect resistance or susceptibility to coccidiosis. Also, the upregulated genes contributing to the metabolism of the key amino acid tryptophan through secondary cellular pathways intricately affects the progression of coccidiosis. Taken together, reducing the cellular pool of nutrients and ions (tryptophan and calcium) that are scavenged by E. maxima for development and replication may explain the birds⿿ resistance or susceptibility. 02 In the necrotic enteritis (NE) vaccine study, NE was induced in the vaccinated birds, leading to increased lesion scores, mortality, and reduced body weight gain. Additionally, the NE challenge resulted in compromised gut integrity and increased colonization of pathogenic and foodborne bacteria. However, the synthesized vaccines were found to mitigate some of these adverse effects, demonstrating a reversal of the negative impacts observed upon NE challenge. This study by ARS researchers in Athens, Georgia, reinforces the potential of CN and CT nanoparticle vaccines as effective candidates for protecting poultry against necrotic enteritis, supporting the promising results obtained in the previous year's research. 03 Impacts of dietary antibiotic alternatives on broilers during a subclinical necrotic enteritis challenge. ARS researchers in Athens, Georgia, demonstrated that supplementation with low levels of the antibiotic bacitracin methylene disalicylate (BMD) and a commercial feed additive of sodium butyrate prevented upregulation of select barrier proteins as a response to enteric challenge. This indicates that both additives could have reduced intestinal tissue damage resulting from subclinical necrotic enteritis, findings that were supported by the histological analysis of intestinal sections. Enteric challenge downregulated gene expression of key nutrient transporters in intestinal tissue and breast muscle, and the reduction in some of these genes was prevented by BMD and sodium butyrate. There was also a reduction in certain circulating hormone (e.g., corticosterone and glucocorticoid) receptors when BMD was fed, though this was not similar in birds fed butyrate. These findings suggest that the partial mitigation of breast muscle yield observed because of enteric challenge could be a result of hormonal signaling impacting nutrient partitioning at multiple levels, indicating that strategies aimed at preventing performance losses due to necrotic enteritis should focus on mitigating reduced nutrient uptake and utilization in both the intestine and breast muscle. 04 Phytogenics supplementation resulted in positive effects on performance and responses of broilers during necrotic enteritis (NE). Compared to the control group (NC), ARS researchers in Athens, Geogia, found that PHY2 significantly improved performance parameters similar to the antibiotic growth promoters group, especially during the peak challenge as well as the cumulative grow-out periods. Also, NE-associated mortality was significantly reduced by the phytogenic supplementation compared to NC birds. Further, gene expression levels of key inflammatory markers was significantly lower in PHY2 compared to all treatments on d 21. Based on these results, the phytogenic blend (PHY2) as supplemented in the diets of broilers improved performance and was as effective as the antibiotic and coccidiostat combination applied in this challenge setting. Also, during peak infection, a decrease in various inflammatory cytokines could enhance tolerance against infection, while the release of anti-inflammatory mediators can resolve inflammation and restore homeostasis. Collectively, with the enhanced performance, reduced pathology, and positive impact on gut integrity, these findings demonstrated the usefulness of phytogenic blends in this NE model, and present potential to diminish the intrusion of pathogens and enhance broilers' ability to counteract the adverse effects of NE. 05 Assessed the effectiveness of algal polysaccharides in mitigating the negative impact of necrotic enteritis (NE) in both the test article and growth promoter groups.. ARS researchers in Athens, Georgia, observed significantly lower mortality and improved performance of broilers in the supplemented groups, Additionally, these supplements equally reduced NE lesions compared to the NC group. While no significant differences were observed in gene expression of gut integrity markers on day 21 among all treatments, the algal supplements resulted in greater levels of these markers on day 42 compared to both control groups, indicating a healthier return to homeostasis following the enteric challenge. Further, the supplemented birds exhibited reduced levels of inflammatory markers on day 21, another indication of a positive effect on gut health. This reduction in inflammation could potentially alleviate NE symptoms in broiler chickens. Collectively, the enhancement in performance, reduction in lesion scores, and positive regulation of key intestinal genes demonstrated the potential of this marine algae-derived dietary supplement to alleviate the negative impacts of the disease; yet, further investigations into its exact mode of action under various enteric challenges are warranted. 06 Additional studies on in ovo or dietary supplementation of antibiotic alternatives during necrotic enteritis demonstrated differential effectiveness of the various defined supplements. Overall, the postbiotic treatment applied by ARS researchers in Athens, Georgia, enhanced early post-hatch performance (weight gain and feed efficiency) of broilers similarly to the antibiotic growth promoter. Additionally, it reduced necrotic enteritis (NE) lesion scores in a comparable fashion to the drug control. The postbiotic positively influenced gene expression of several gut integrity markers compared to the negative control (non-supplemented) birds. Collectively, in ovo injection of postbiotic enhanced early performance, reduced pathology, and showed similar or better performance compared to antibiotic-supplemented birds suggesting that this postbiotic application may be an effective antibiotic alternative to mitigate NE. 07 University of Georgia (UGA) researchers were able to characterize the effect of the different levels of resistant starch (RS) in birds challenged with coccidiosis. The obtained data showed that moderate inclusion of RPS (raw potato starch) and HCS (high amylose corn starch) benefited the growth of broiler chickens both before, during, and after the coccidiosis challenge, although the effect of RS after the coccidiosis challenge is less apparent. The beneficial effect of RS is related more to its effect on amino acid digestibility than to its effect on modifying the profile of cecal short-chain fatty acids and branched-chain fatty acids.
Impacts
(N/A)
Publications
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Determine the molecular mechanisms of virulence within and across Eimeria strains affecting poultry and investigate the genetic and phenotypic responses by the bird. 1.1. Produce high-quality full genome sequences of Eimeria species of economic importance in commercial poultry. 1.2. Use the rapid, high throughput molecular screening assay to differentiate Eimeria species in a sample and determine if they are of vaccine or field origin. 1.3A. Elucidate the relationship between host redox (oxidative stress) status and development of E. maxima. 1.3B. Understand host resistance/tolerance mechanisms in the development of intestinal lesions during Eimeria maxima infection. 1.3C. Identification, characterization, and assessment of non-pathogenic bacterial species from reused litter used as ⿿proLitterbiotics⿿ during E. maxima infection. 2. Determine the molecular mechanisms of virulence to Clostridium perfringens-based enteritis and genotypic and phenotypic responses by the bird. 2.1. Sequencing and analysis of virulent field strains of C. perfringens. 2.2. Investigate host genotype and environmental interaction that predisposes young birds to C. perfringens-induced enteritis. 2.3A. Synthesize chitosan nanoparticle vaccines, loaded with antigens from field strains of C. perfringens and surface-tagged with E. maxima antigens. 2.3B. Identify the anti-C. perfringens IgA and IgG and T cell response curves in broilers inoculated orally with different doses of chitosan nanoparticle vaccine entrapped with C. perfringens and E. maxima proteins. 2.3C. Quantify the chitosan nanoparticle vaccine efficacy in decreasing the colonization of C. perfringens and disease score in broilers induced with necrotic enteritis. 3. Develop alternatives to antibiotics for preventing or treating Eimeria- and C. perfringens-based enteritis. 3.1A. Evaluate the impact of dietary antibiotic alternatives on intestinal physiology and microbial ecology in each segment of the gastrointestinal tract of genetically diverse broiler chickens in response to Eimeria- and C. perfringens-based necrotic enteritis. 3.1B. Characterization of starch digestibility along the digestive tract, digesta oligosaccharides, and SCVFAs in broiler chickens receiving different types of resistant starch (RS). 3.2. Investigate mechanisms by which probiotics influence intestinal physiology and microbial ecology of genetically diverse broilers in response to Eimeria- and C. perfringens-based necrotic enteritis. 3.3. Investigate the modes of action of dietary amino acids and nutrients on gut development/integrity, and host physiological response to Eimeria- and C. perfringens-induced enteritis. 3.3A. Potentiating the protective effects of RS with low protein and amino acid supplemented diets. 3.3B. Determine the response of broiler chickens challenged with Eimeria when fed diets with RS and low protein, AA-fortified diet. 3.3C. Investigating prebiotic-probiotic symbiosis using RS as functional fiber in broilers induced with NE. **See uploaded post plan for sub-objectives 3.1C, 3.1D, 3.3D, 3.3E and 3. 3F. Approach (from AD-416): The approach outlined in this integrated project is divide between three interrelated objectives. The project will employ several approaches to (1) produce high-quality full genome sequences of Eimeria species of economic importance in commercial poultry; (2) develop rapid, high throughput molecular screening assays to differentiate Eimeria species in a sample, as well as determine their origins [i.e. vaccine vs. field]; and (3) improve production efficiency by studying the influence of host genetics on resistance, susceptibility, and tolerance to Eimeria spp., and the impact of dietary redox potential (e.g. cysteine) and enteric and litter microbiota on the pathology of Eimeria spp. in chickens. To better understand the incidence of NE, which is often predisposed by coccidiosis, the project will continue collecting field isolates of C. perfringens from the southeast and mid-Atlantic regions and produce full genome sequencing and complete comparative analyses of those NE-causing strains. This approach will allow identification of predominant virulence factors in C. perfringens of commercial poultry that could serve as targets for designing and developing vaccines as alternative control measures to antibiotics. Using an established nanoparticle vaccine platform, the project will continue building this unique design and further develop and test anti-C. perfringens vaccines with the potential to be adopted by the poultry industry. In dealing with the urgent need to identify, test, and employ effective antibiotic alternatives for poultry, the project will conduct detailed activities to better understand the mechanistic actions of several candidate interventions on performance, physiological, microbial, immunological, and metabolic responses of the host. The application of well-defined probiotics, prebiotics, phytogenics, and specific nutrients during coccidiosis and NE will be used in vivo and in ovo to study the critical physiological changes that directly impact host health and performance. Parameters at the enteric and systemic levels will collectively provide strong host response correlates that can be utilized in refining the application of these potential alternatives in commercial settings. High throughput sequencing of multiple vaccine and field origin Eimeria isolates has been completed and data have been presented at multiple meetings. Overall, all vaccine isolates were nearly identical to each other within a species across manufacturers. One difference seen was with E. maxima in one vaccine that contains two different strains of maxima; in only this vaccine was a short form of the internal transcribed spacer- 1 ( ITS1) gene region detected. For field samples, some variability was seen across the three gene regions but not enough to truly differentiate a ⿿pathogenic isolate from a ⿿vaccine origin isolate. There was a difference seen for E. tenella in the CO1 gene, which may indicate strain differences, but more research is needed. Interestingly, using the PCR and NGS sequencing protocol developed in our laboratory, we were able to detect E. praecox in nearly every field sample tested. This Eimeria species is often cryptic and does not cause disease. It is also not included in any vaccine, so it represents a true ⿿field isolate. Prevalence is often overlooked so it is not known if this species plays any role in disease states. Additionally, we detected E. dispersa in several poultry houses on 1 farm. This is intriguing as E. dispersa is not a poultry species, but does have less host specificity than other Eimeria species and is found in some wild and game birds. The fact that it was present in high enough quantities to A) be collected in a random sampling from multiple chicken houses, and B) be detected by a traditional PCR assay across all three genes tested raises several questions, the most significant being, ⿿Does this species now infect poultry? E. maxima is one of the most pathogenic Eimeria species persistently invading the middle jejunum and ileum, damaging the intestinal mucosa of chickens. Heat stress (HS) is a common stressor and contributes to inflammation and oxidative stress in broilers. We investigated the effect of E. maxima infection and HS on ileal digestibility, mRNA expression of nutrient and amino acid transporters, and ileal tissue morphology in broiler chickens. There were four treatment groups: thermoneutral control (TNc), thermoneutral infected (TNi), heat stress control (HSc), and heat stress infected (HSi), 6 replicates each of 10 birds per treatment. At day 6 post infection, ileal contents and tissues were collected to quantify ileal digestibility of crude protein and fat, mRNA levels of nutrient transporters as well as histopathology. Necrotic enteritis (NE) is an enteric disease caused by C. perfringens toxins and is estimated to cost the global poultry industry billions of dollars annually. Modern and heritage breeds tend to exhibit differential responses to disease challenges. We evaluated the responses of broiler chicks from a modern (Cobb) and a heritage (ACRB) breed during a NE challenge. The design was a 2ÿ2 factorial with breed (ACRB and Cobb) and challenge (no challenge and NE) as main factors. A total of 96 day-of- hatch male chicks (48 ACRB and 48 Cobb) were allocated to the four experimental treatments with 8 replicate cages and 3 birds/cage. On day 14, birds in the NE-challenged groups were orally gavaged with 3,000 E. maxima sporulated oocysts followed by two doses of ~1ÿ108 CFU of C. perfringens on days 19 and 20. On day 21, blood and tissue samples were collected from 1 bird/pen to measure mRNA abundance of cytokines, chemokines, receptors, and other immune response-related genes. Additionally, 2 birds/pen were necropsied and scored for intestinal NE lesions. In studying the effects of necrotic enteritis on performance and gut status, researchers characterized the production performance, cecal microbiome and cecal tonsil transcriptome of birds vaccinated with the synthesized nanoparticle vaccine during a necrotic enteritis challenge. Chitosan nanoparticles were formulated with native (CN) or toxoids (CT) of extracellular proteins (ECP) of C. perfringens, both surface-tagged with Eimeria maxima proteins. The CN and CT nanoparticles were stable at gut pH. The CN and CT nanoparticles released approximately 8 % of their surface proteins at pH 7.4 after 24 hours. CN and CT nanoparticles were biosafe. The vaccine induced protective immune response in vivo in chickens. Necrotic enteritis was induced in birds vaccinated with the synthesized vaccine. Necrotic enteritis challenge increased lesion scores, decreased body weight gain and increased mortality. The challenge also decreased gut integrity and increased loads of pathogenic and foodborne bacteria. The synthesized vaccine alleviated those negative effects. Two experiments were conducted during this reporting period. In the first, birds were left unchallenged or challenged with Eimeria- and C. perfringens-induced subclinical necrotic enteritis (NE) in the absence or presence of subtherapeutic levels of the antibiotic bacitracin methylene disalicylate (BMD) or a commercially available phytogenic product. Growth performance, mortality, and lesion scores were used to determine severity of the challenge model. Tissues were collected 24 h after the NE challenge, and expression of genes associated with barrier function, inflammatory status, immune response, and antimicrobial action were measured in intestinal sections. All of this work has been completed. The second experiment had the same setup but replacing the phytogenic product with a sodium butyrate product. Performance, mortality, and lesion scores were used to determine severity of the challenge model. Blood and tissue samples were collected on 0, 24, and 48 hours post challenge, and intestinal barrier integrity and inflammatory status were assessed. All of the aforementioned work has been completed. Additional analyses are underway and should be completed within the next reporting period. Mechanisms driving effects on growth performance will be evaluated by measuring levels of circulating hormones and metabolites as well as expression of genes regulating uptake and utilization of dietary nutrients. Histological analysis will be used to determine villus height and crypt depth to assess intestinal tissue damage. Microbial community composition will be determined by sequencing, and analysis of intestinal amino acid and organic acid levels will be used to determine impacts of host and microbial metabolism on intestinal ecology. Milestones 12/13. Results from two experiments on resistant starches (RS) in broilers are presented in this report period. In Experiment 1, the impact of different RS types, and levels, on growth performance, intestinal tract characteristics, and microbial metabolites (short- and branched-chain fatty acids) in broiler chickens was investigated. Three RS were tested: banana starch, raw potato starch, and chemically modified maize starch; each RS was fed at three levels (2.5, 5, or 10% of the diet) . For each of the three RS tested, including the RS at higher levels (10% of diet) produced the largest reduction in starch digestibility along the digestive tract. The modified maize starch was the least resistant of the three RS types. Nevertheless, birds that received the resistant maize starch had the most short-chain fatty acids in the caeca. Results from the experiment show that it is impractical to use high levels of RS in diets due to difficulty in diet peletting, negative impact on birds⿿ growth, and the high cost of some of the resistant starches, mainly the banana starch. In the second experiment, the objective was to test whether the length of time the birds were fed RS diets influenced the growth of the birds, nutrient utilization, microbial fermentation, or microbial profile. Three RS diets were formulated: 2.5 or 5.0 % raw potato starch or 3.5% modified corn starch. The three RS diets were fed to broiler chickens for 21, 14, or 7 days. A corn-soybean meal control diet was fed to another treatment group for 21 days. Birds that received RS diets for 14 or 7 days had received the control diet for the previous 7 or 14 days, respectively. All the treatments were terminated at 21 days of the age of the birds. The best growth performance (FCR) was observed in birds fed 3.5% modified corn starch, irrespective of the length of time they were fed the RS diet. The effect on intestinal structures also depended on the inclusion level of RS, not the feeding duration. Neither the inclusion level nor duration of the feeding of RS influenced the cecal short-chain fatty acids contents, but birds receiving RS for 21 days had the lowest cecal pH. Further analysis is ongoing regarding the treatment effects on microbial profiles. Two studies were conducted to assess the effects of amino acid supplementation to low protein diets on performance and response of broilers during an Eimeria challenge. Graded doses of E. maxima were employed in conjunction with varying inclusion rates of key amino acids namely arginine (Arg), threonine (Thr), and glutamine (Gln) separately or in combination in Study 1. Growth performance was recorded on 6- and 9- days post-infection (DPI), and liver samples collected on 6 DPI for oxidative status analyses. Under Eimeria infection, birds fed a normal diet and low CP diet tended to have the same growth performance. The supplementation in the low CP diet may adversely affect the growth performance while Arg supplementation could have a beneficial effect on growth performance and oxidative status of birds during an acute infection stage.Methionine (Met) was used in Study 2 where performance parameters, body composition, gut health, and oocyst shedding on Eimeria spp. were assessed. Increased Met supplementation would linearly improve growth performance of Eimeria infected birds. However, it might also favor the Eimeria reproduction and decreased BMD and BMC especially in low CP conditions. ACCOMPLISHMENTS 01 Heat stress (HS) and parasitic challenge in broiler chickens. ARS researchers in Athens, Georgia, analyzed gene expression of samples from coccidia (important poultry parasite) infection and heat stress studies revealed additional knowledge of this model system. Molecular analysis of nutrient transporters in intestinal tissues from thermoneutral and HS birds revealed distinct profiles of differentially regulated genes within each group of birds indicating that heat stress can putatively alleviate the adverse effects of parasitic infection. These results showed that exposing broiler chickens to heat stress can mitigate the disruptive effect of coccidia on the intestinal digestibility and absorptive capacity by limiting the parasite-induced tissue injury and suppressing oxidative damage of intestinal cells. 02 Differential responses of broiler breeds to necrotic enteritis (NE). The Eimeria/Clostridium combination challenge model performed by ARS researchers in Athens, Georgia, induced NE lesions in birds from heritage (ACRB) and modern (Cobb) breeds, but without statistical differences. mRNA abundance of key immune response proteins showed greater levels in the peripheral blood of commercial birds compared to heritage breed bird. Additionally, mRNA abundance of other inflammatory markers and cellular receptors was greater in ACRB spleens compared to Cobb. In the bursa (key organ for antibody generation), a subset of immune response markers was more abundant in Cobb birds compared to ACRB. As the Cobb and ACRB birds exhibited differential responses to NE, it can be concluded that genetic selection programs may play an important future role in not only evaluating birds⿿ susceptibility to NE, but also as a means to help mitigate predisposition to the disease. 03 Nanoparticle vaccine research. A study was conducted by ARS researchers in Athens, Georgia, to identify the effects of chitosan nanoparticles synthesized with native (CN) or toxoids (CT) of C. perfringens proteins on protecting birds against necrotic enteritis (NE). In testing stability of these vaccines, scientists showed that both CN and CT nanoparticles were stable in a series of in vitro assays. To test their potential in chickens, 90 broiler chicks were randomly assigned to treatment groups including sham-vaccinated (control), CN-vaccinated (CN) , and CT-vaccinated (CT). Each bird was orally gavaged with the respective control or vaccine candidate on days 0 and 14. At 21 d of age, the CN and CT groups had 47% and 334% more antibodies than control birds, respectively. NE was induced in birds vaccinated with the synthesized vaccines. The NE challenge increased lesion scores and mortality and decreased body weight gain. The challenge also reduced gut integrity and increased loads of pathogenic and foodborne bacteria. The synthesized vaccine reversed some of the above negative effects. 04 Impacts of dietary antibiotic alternatives on broilers during a subclinical NE challenge. ARS researchers in Athens, Georgia, demonstrated that supplementation with low levels of the antibiotic bacitracin methylene disalicylate (BMD) and, to a lesser extent commercial phytogenic [Biostrong Protect] and protected sodium butyrate [GUSTOR N⿿RGY] products, improved growth performance in the face of a subclinical NE challenge. Breast muscle yields (% of body weight) were reduced by the enteric challenge, and this reduction was partially reversed by the addition of sodium butyrate. All three additives reduced clinical lesions, with the greatest improvements coming from sodium butyrate and BMD. Dietary BMD and the phytogenic alternative both led to upregulation of select barrier protein genes in the small intestine of challenged birds as compared to challenged birds fed the basal diet. Under NE challenge conditions, both antibiotic alternatives and BMD upregulated select cytokines (immune response proteins) mediating intestinal inflammation, though the upregulation in the presence of BMD was less than with the alternative products. The phytogenic alternative and BMD both also increased levels for a receptor that stimulates the innate immune response as well as expression of three different antimicrobial peptides in the gut. Together, these results indicate that antibiotic alternatives can have effects both similar to and distinct from dietary BMD when broilers are faced with an enteric challenge, and both phytogenic and organic acid antibiotic alternatives are effective at partially mitigating negative consequences of the disease on growth and body composition. 05 Impact of resistant starches on chickens. ARS researchers in Athens, Georgia, were able to characterize the influence of the different resistant starches used. High inclusion levels of resistant starches are impractical in the diets of broiler chickens, both from animal productivity and diet preparation points of view. Current results show that the resistant starches were not equally resistant to hydrolysis in the digestive tract of chickens and hence have variable effects on the growth performance, microbial fermentation products, and gut health of broiler chickens. In addition, when lower levels of two commercially available resistant starches were fed to broiler chickens for different lengths of time, the effect of the starches largely depended on the type of starch provided rather than on how long the birds were fed the resistant starch-containing diets.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Determine the molecular mechanisms of virulence within and across Eimeria strains affecting poultry and investigate the genetic and phenotypic responses by the bird. 1.1. Produce high-quality full genome sequences of Eimeria species of economic importance in commercial poultry. 1.2. Use the rapid, high throughput molecular screening assay to differentiate Eimeria species in a sample and determine if they are of vaccine or field origin. 1.3A. Elucidate the relationship between host redox (oxidative stress) status and development of E. maxima. 1.3B. Understand host resistance/tolerance mechanisms in the development of intestinal lesions during Eimeria maxima infection. 1.3C. Identification, characterization, and assessment of non-pathogenic bacterial species from reused litter used as ⿿proLitterbiotics⿿ during E. maxima infection. 2. Determine the molecular mechanisms of virulence to Clostridium perfringens-based enteritis and genotypic and phenotypic responses by the bird. 2.1. Sequencing and analysis of virulent field strains of C. perfringens. 2.2. Investigate host genotype and environmental interaction that predisposes young birds to C. perfringens-induced enteritis. 2.3A. Synthesize chitosan nanoparticle vaccines, loaded with antigens from field strains of C. perfringens and surface-tagged with E. maxima antigens. 2.3B. Identify the anti-C. perfringens IgA and IgG and T cell response curves in broilers inoculated orally with different doses of chitosan nanoparticle vaccine entrapped with C. perfringens and E. maxima proteins. 2.3C. Quantify the chitosan nanoparticle vaccine efficacy in decreasing the colonization of C. perfringens and disease score in broilers induced with necrotic enteritis. 3. Develop alternatives to antibiotics for preventing or treating Eimeria- and C. perfringens-based enteritis. 3.1A. Evaluate the impact of dietary antibiotic alternatives on intestinal physiology and microbial ecology in each segment of the gastrointestinal tract of genetically diverse broiler chickens in response to Eimeria- and C. perfringens-based necrotic enteritis. 3.1B. Characterization of starch digestibility along the digestive tract, digesta oligosaccharides, and SCVFAs in broiler chickens receiving different types of resistant starch (RS). 3.2. Investigate mechanisms by which probiotics influence intestinal physiology and microbial ecology of genetically diverse broilers in response to Eimeria- and C. perfringens-based necrotic enteritis. 3.3. Investigate the modes of action of dietary amino acids and nutrients on gut development/integrity, and host physiological response to Eimeria- and C. perfringens-induced enteritis. 3.3A. Potentiating the protective effects of RS with low protein and amino acid supplemented diets. 3.3B. Determine the response of broiler chickens challenged with Eimeria when fed diets with RS and low protein, AA-fortified diet. 3.3C. Investigating prebiotic-probiotic symbiosis using RS as functional fiber in broilers induced with NE. **See uploaded post plan for sub-objectives 3.1C, 3.1D, 3.3D, 3.3E and 3. 3F. Approach (from AD-416): The approach outlined in this integrated project is divide between three interrelated objectives. The project will employ several approaches to (1) produce high-quality full genome sequences of Eimeria species of economic importance in commercial poultry; (2) develop rapid, high throughput molecular screening assays to differentiate Eimeria species in a sample, as well as determine their origins [i.e. vaccine vs. field]; and (3) improve production efficiency by studying the influence of host genetics on resistance, susceptibility, and tolerance to Eimeria spp., and the impact of dietary redox potential (e.g. cysteine) and enteric and litter microbiota on the pathology of Eimeria spp. in chickens. To better understand the incidence of NE, which is often predisposed by coccidiosis, the project will continue collecting field isolates of C. perfringens from the southeast and mid-Atlantic regions and produce full genome sequencing and complete comparative analyses of those NE-causing strains. This approach will allow identification of predominant virulence factors in C. perfringens of commercial poultry that could serve as targets for designing and developing vaccines as alternative control measures to antibiotics. Using an established nanoparticle vaccine platform, the project will continue building this unique design and further develop and test anti-C. perfringens vaccines with the potential to be adopted by the poultry industry. In dealing with the urgent need to identify, test, and employ effective antibiotic alternatives for poultry, the project will conduct detailed activities to better understand the mechanistic actions of several candidate interventions on performance, physiological, microbial, immunological, and metabolic responses of the host. The application of well-defined probiotics, prebiotics, phytogenics, and specific nutrients during coccidiosis and NE will be used in vivo and in ovo to study the critical physiological changes that directly impact host health and performance. Parameters at the enteric and systemic levels will collectively provide strong host response correlates that can be utilized in refining the application of these potential alternatives in commercial settings. Significant progress has been accomplished across several areas to better our understanding of enteric health, specifically during coccidiosis (Eimeria sp.) and necrotic enteritis (Clostridium perfringens) challenges. Such research encompasses characterizing field isolates and virulence factors of these enteric pathogens, host (chicken) responses to varying environmental/stress, dietary intervention strategies (enzymes, resistant starches, amino acids), and enteric challenge conditions, and efficacy of newly developed vaccines. The collective aim is to aid and overcome such challenges and lower severity and symptoms associated with reduced enteric health. This will allow further development of integrated dietary, environmental, and vaccine strategies to improve intestinal health and function, and bird growth performance, in the absence of antibiotics. Studies to assess the redox status and gut health of the host during Eimeria maxima infection. Ileum tissues from chickens infected with E. maxima and reared under either thermoneutral (TN) or heat stress (HS) conditions were used to study the molecular mechanisms that underlie responses to these stressors. Six hundred 14-d old commercial broiler chickens were divided into 2 groups and reared at 20°C (thermoneutral, TN) or 35°C (HS). Each temperature group comprised of 30 groups of 10 chicks. Within each temperature group, half of the chicks were infected via gavage with 2ÿ5log10 E. maxima sporulated oocysts suspended in water and the other half were mock infected with water. All chickens were raised in batteries with wired floor, with ad libitum access to feed and water. In all, there were 4 experimental groups: TN control [TNc], TN infected [TNi], HS control [HSc] and [HSi]. At 6-day-post infection, ileal content and tissue were collected to quantify ileal digestibility of crude protein, fat, amino acid mRNA levels of nutrient transporters and histopathology. Also at 6 dpi, 5 chickens per group were randomly sampled, and ileum tissues were collected for RNA extraction and then sequenced using Next Generation Sequencing and contained proper controls and analyses. Objective 2: Effects of necrotic enteritis on performance and gut status. We characterized the production performances, cecal microbiome and cecal tonsil transcriptome of birds vaccinated with the synthesized nanoparticle vaccine during necrotic enteritis infection. Necrotic enteritis was induced in birds vaccinated with the synthesized vaccine. Necrotic enteritis challenge increased lesion scores and decreased body weight gain and increased mortality. The challenge also decreased gut integrity and increased loads of pathogenic and foodborne bacteria. Necrotic enteritis challenge induced inflammatory genes and decreased ⿿good⿿ bacterial load and increased the relative abundance of foodborne bacteria. The synthesized vaccine partially reversed the above effects. Outcomes: Have a manuscript in revision. Published 5 articles related to NE or vaccine design. The filed patent was granted (US 17/167,893; Nanoparticle vaccine for Clostridium perfringens in poultry. Selvaraj, Akerele, Gourapura, and Renu. 2021). Objective 3 Impacts of dietary antibiotic supplementation on broilers during a subclinical enteric challenge. An experiment was conducted where birds were left unchallenged or challenged with Eimeria- and C. perfringens-induced necrotic enteritis in the absence or presence of subtherapeutic levels of the antibiotic bacitracin methylene disalicylate (BMD). Growth performance, mortality, and lesion scores were used to determine severity of the challenge model. Intestinal barrier integrity was measured through permeability assays with a fluorescent marker and mRNA expression of intestinal barrier proteins. Intestinal inflammatory status was assessed by measuring cytokine mRNA expression. Mechanisms driving effects on growth performance were evaluated by measuring levels of circulating hormones and metabolites as well as expression of genes regulating uptake and utilization of dietary nutrients. All of this work has been completed. Analysis related to intestinal microbial ecology is underway; this includes 16S sequencing to determine microbial community composition and analysis of intestinal amino acid and organic acid levels to determine impacts of host and microbial metabolism on intestinal ecology. Community-level physiological profiling was unable to be completed due to continued telework at ARS (see below). Outcomes: Presentations at scientific meetings (Ellestad and Bhattrai, 2022; Bhattrai and Ellestad, 2022a & 2022b); manuscripts in preparation. Effects of exogenous enzymes on enteric health and performance of broilers during enteric challenges. A series of studies delineated the effect of different dietary factors on the generation of prebiotic substances in-situ in the digestive tract of broilers. The factors studied were various levels of fiber, protein, xylanase and protease. Studies show that fiber levels had the most singular effect of increasing production of the prebiotic oligosaccharides in the intestine and increasing the concentration of beneficial short chain fatty acids in the chickens⿿ ceca. Further studies showed that xylanase and xylo- oligosaccharides had similar beneficial effects in broilers challenged with coccidiosis. In addition, the supplementation of xylanase in diets containing high fiber helped reverse the negative effect of coccidiosis challenge by promoting increased concentration of beneficial short chain fatty acids and reducing the concentration of harmful branched chain fatty acids in the ceca of broiler chickens. Outcomes: Presentations at national and international scientific meetings, peer-review articles (Lin and Olukosi, 2021a,b). Effects of dietary amino acids on gut development/health during Eimeria challenge. A study was conducted to evaluate the effects of low crude protein diets supplemented with arginine, glutamine, methionine, and/or threonine on apparent ileal digestibility of amino acids, intestinal morphology, intestinal permeability, gene expression of nutrient transporters and tight junction proteins of broiler chickens challenged with mixed Eimeria spp. The results of amino acid and Eimeria study showed that the coccidia challenge reduced overall growth performance, but the low-crude protein had no adverse impacts on intestinal health and growth of Eimeria- infected birds compared to the Eimeria-challenged control. In summary, adding 0.75% of arginine, glutamine, or threonine in a low crude protein diet can improve the intestinal health of birds challenged with a mild coccidia infection. Outcomes: Presentations at national and international scientific meetings, peer-review papers (Kim et al., 2022; Teng et al., 2021). ACCOMPLISHMENTS 01 Assessing the redox status and gut health of the host during Eimeria maxima infection. There were 4 experimental groups performed by ARS researchers in Athens, Georgia: Temperature neutral control [TNc] at 20C, Temperature neutral infected [TNi] at 20C, Heat Stress control [HSc] at 35C and Heat Stress infected at 35C [HSi]. Multiple genes involved in energy and amino acid metabolism and immune functions were up or down regulated. Immune functions tended to be upregulated with coccidia infection with increase oxidative stress and reduction in the antioxidant capacity of the chicken. Also, chickens infected with E. maxima modify their metabolism to reduce the breakdown of several amino acids to limit available amino acids. 02 Effects of necrotic enteritis on performance and gut status. A study was conductedby ARS researchers in Athens, Georgia, to identify the effects of experimental necrotic enteritis (NE) infection on the performance, gut microbiome, and cecal tonsil transcriptome in broiler birds. NE was induced by inoculating Eimeria maxima at 14 d of age and Clostridium perfringens at 19, 20, and 21 d of age. NE challenge increased NE lesion score at 7 days post-E. maxima infection (dpi), decreased average weight gain and increased mortality at 7 and 14 dpi. NE challenge increased gut permeability at 5, 6, and 7 dpi and increased ileal C. perfringens load at 5dpi. NE challenge increased Eimeria oocyst shedding at 5, 6, 7, 8 and 14 dpi. NE challenge decreased the relative abundance of Lactobacillaceae and increased the relative abundance of Campylobacteriaceae, Comamonadaceae, and Ruminococcaceae at 6 dpi. NE challenge upregulated genes related to immune response and down-regulated genes related to lipid metabolism at 6 dpi. It can be concluded that NE infection decreased beneficial bacteria and increased gut permeability. The synthesized vaccine partially reversed the loss in gut permeability and loss in Lactobacillaceae. 03 Impacts of dietary antibiotic supplementation on broilers during a subclinical enteric challenge. ARS researchers in Athens, Georgia, demonostrated that supplementation with Bacitracin Methylene Disalicylate (BMD) improved growth performance in the face of a subclinical necrotic enteritis challenge but did not impact mortality or lesion score severity. Intestinal permeability, as assessed by passage of a fluorescent molecule into the blood, was not increased due to subclinical necrotic enteritis. This suggests that BMD does not influence clinical signs of necrotic enteritis but might impact physiological mechanisms driving reduced growth performance under subclinical conditions. However, mRNA levels of select barrier proteins were reduced in the face of enteric challenge, and effects on several of these were fully or partially mitigated with dietary BMD supplementations. Similarly, downregulation of several cytokines as a result of enteric challenge was at least partially reversed by the presence of BMD in the diet. Circulating hormone levels were not influenced by necrotic enteritis, but expression of several glucose transporters and an enzyme influencing utilization of dietary protein was reduced in challenged birds. Effects of necrotic enteritis that were fully or partially reversed by BMD represent targets for alternative strategies aimed at reducing impacts of enteric disease on broiler production efficiency. 04 Optimizing enteric health and performance of broilers with enzymes and resistant starches. In a series of studies conducted by ARS researchers in Athens, Georgia, to understand the role of exogenous enzymes and resistant starches (RS) on gut health of broiler chickens, it has become clear that exogenous enzymes can have roles similar to exogenous prebiotics in promoting gut health of broiler chickens. Among the dietary factors studied, dietary fiber played the most obvious role in enhancing generation of prebiotic oligosaccharides. More than that, the studies showed that the prebiotics generated are of sufficient quantity to elicit positive effects on microbial activities possibly via increasing the content of metabolites available for fermentation in the digestive tract. The metabolites, in turn, beneficially alter the relative concentrations of short- and branched-chain fatty acids especially in birds challenged with coccidiosis, a common poultry industry problem. Further, we were able to characterize the influence of the different resistant starches used. Resistant starches promoted lengthening of villi in jejunum but had minimal impacts on the weight and length of small intestine and other digestive organs. The current results showed that resistant starches do not elicit the same effect on animal growth performance or digestive organ growth and development. Work is ongoing to determine the site of digestion of the different RS and how the different RS used influenced efficiency of utilization of the short chain fatty acids in ceca and production of oligosaccharides in the digestive tract. This will help inform which RS to be studied further in the next planned experiments. 05 Dietary amino acids and their impact on gut development/health during Eimeria challenge. The results of amino acid and Eimeria study by ARS researchers in Athens, Georiga, showed that the coccidia challenge reduced overall growth performance, but the low-crude protein had no adverse impacts on intestinal health and growth of Eimeria-infected birds compared to the Eimeria-challenged control. In summary, adding 0. 75% of arginine, glutamine, or threonine in a low crude protein diet can improve the intestinal health of birds challenged with a mild coccidia infection.
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