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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** 2023 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**
                                

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 2×2 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 ~1×108 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**
                                

1. 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. 2. 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. 3. 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. 4. 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. 5. 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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