Location: Endemic Poultry Viral Diseases Research
2024 Annual Report
Objectives
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
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.
Progress Report
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 1×108 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
1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
