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ARS Home Southeast Area Athens, Georgia U.S. National Poultry Research Center Endemic Poultry Viral Diseases Research Research





- Dunn, John
- 2023
- 2022
- Animal Health (103)

Research Project: Genomic and Epidemiological Parameters to Inform Interventions Strategies for Enteric Diseases of Poultry **Location:
                            Endemic Poultry Viral Diseases Research** 2022 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**
                                

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 2×5log10 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**
                                

1. 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. 2. 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. 3. 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. 4. 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. 5. 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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