Performing Department
(N/A)
Non Technical Summary
Clostridium perfringens-induced and coccidiosis-predisposed necrotic enteritis (NE) has reemerged as a prevalent poultry disease worldwide because of restricting prophylactic antimicrobial practice. It is estimated that NE costs $6 billion per year worldwide. Few affordable antimicrobial alternatives are available to prevent NE. Our long-term goal is to contribute toward the development of mechanism-based interventions to mitigating NE in chickens. The overall objective is to develop anti-C. perfringens vaccines and bile acid regiments to prevent NE. Our central hypothesis is that NE will be attenuated through inhibiting C. perfringens virulence by host immune response to vaccines or by specific microbe-metabolized bile acids. We formulate this hypothesis based on our findings and current state of knowledge that C. perfringens sporulation vaccine or dietary deoxycholic acid (DCA) effectively reduced chicken NE. Proteomics, immunoinformatics, and ELISA assays identified lead peptides for development of multiepitope fusion antigen (MEFA) vaccines against C. perfringens virulence. DCA, isoallolithocholic acid or microbe-metabolized chenodeoxycholic acid reduced C. perfringens growth and virulence. Based on these findings, we plan to attain the overall objective by completing the following specific objectives: Objective 1: Develop multiepitope fusion antigens vaccines of C. perfringens polyvalent factors to reduce chicken NE. Objective 2: Develop microbe-metabolized bile acid regiments to reduce C. perfringens virulence and chicken NE. At the completion of the proposed research, we will have developed MEFA vaccines and microbe-metabolized bile acid regiments to effectively reduce NE. Ultimately, the results of this continuum of research will have an important positive impact by contributing meaningfully toward the development of mechanism-based strategies to prevent
Animal Health Component
100%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Goals / Objectives
Our long-term goal is to contribute toward the development of mechanism-based interventions to mitigating poultry necrotic enteritis. We plan to fulfill the long term goal with the following specific objectives. 1) We will develop multiepitope fusion antigens vaccines of C. perfringens polyvalent factors to reduce chicken NE. 2) We will develop microbe-metabolized bile acid regiments to reduce C. perfringens virulence and chicken NE. At the completion of the proposed research, the results of this continuum of research will have an important positive impact by contributing meaningfully toward the development of mechanism-based strategies to prevent poultry NE.
Project Methods
1. Develop multiepitope fusion antigen vaccines of C. perfringens virulence factors to reduce chicken NE.1-1. Construct and evaluate the immunogenicity of CPE-backbone MEFA vaccines in vitro.Following the protocol by Li et al., 2022, MEFA vaccines will be constructed in silico by substituting the backbone of avirulent 107-319 CPE at once with 6 of the identified 10-mer epitopes. The resultant MEFA vaccine protein will be examined with ExPASy, Phyre2, PyMol, and CHARMM as described by Duan et al., 2017. The in silico synthesized CPE-MEFA vaccine will be reverse-translated into DNA sequence and synthesized into protein expression plasmids. The vaccine plasmids will be over-expressed in E. coli BL21 and purified by His-tag and Ni-NTA agarose. After dialyzed and concentrated, CPE-MEFA protein vaccines will be aliquoted and stored at -80 ºC and will be evaluated using Western Blot.1-2. Evaluate the immunogenicity of CPE-MEFA vaccines in chickens.In this section, the immunogenicity of CPE-MEFA protein vaccines will be evaluated in chickens. To estimate necessary chicken replication numbers, we used G*Power software (Faul et al., 2009). The chicken experiment will be performed similar to our report (Wang et al., 2019). Briefly, a cohort of one day-old broiler chicks will be transferred to John Kirkpatrick Skeeles Poultry Health Laboratory (up to Biosafety level 3) at University of Arkansas at Fayetteville. Birds at one day of age will be assigned to one control and 10 vaccine groups. The birds will be immunized by s.c. on days 1 (50 µg/bird) and 10 (booster, 250 µg/bird). The birds will be euthanized on days 23 and 25. Serum samples will be collected for ELISA assay of vaccine efficacy.1-3. Evaluate the CPE-MEFA vaccines against chicken NE.In this section, we will assess the MEFA vaccines against NE challenge. Briefly, birds at one day of age will be assigned to 6 vaccine groups. Each group will consist of 3 challenge groups of noninfected, Eimeria maxima, and NE (E. maxima and C. perfringens). The birds will be immunized as in section 1-2. At 18 days of age, chicks will be gavaged with 20,000 sporulated oocysts/bird E. maxima to induce coccidiosis and then infected with 109 CFU/chick C. perfringens at 23 and 24 days of age to induce NE. Feed intake, body weight, and mortality will be measured at days 0, 18, 23 and 25. Birds will be followed for clinical signs of watery to bloody diarrhea, severe depression, decreased appetite, closed eyes, ruffled feathers, and mortality. The broilers will be euthanized on day 23 for early coccidiosis and the rest of birds will be euthanized at day 25 for final NE or coccidiosis evaluation. Intestinal tissue and content samples will be collected. Targeted metabolomics will be used to quantify ileal bile acids. The small intestinal tissue samples will be assessed for inflammatory response on mRNA accumulation of Ifnγ, Il1β, Litaf (Tnfα), Il8, Cxcl2, Il17a, and Mmp9. The small intestinal tissue will also be Swiss-rolled, fixed and processed for H&E staining. C. perfringens colonization and adherence/invasion in intestinal tissue or lumen will be determined by FISH technique and real time PCR of 16S rDNA as described before (Bansal et al., 2021). Intestinal inflammation will be assessed by histopathological score as described before (Bansal et al., 2020). Serum samples will be collected at days 17, 23 and 25 for evaluating MEFA vaccine efficacy.1-4. Construct Bacillus subtilis-expressed MEFA vaccines.Because the CPE-MEFA vaccines have high affinity for intestinal absorption, it is possible to deliver the CPE-MEFA vaccine to birds orally, which is essential for poultry farm practice. To express the CPE-MEFA vaccines in Bacillus subtilis, we will subclone the MEFA vaccine sequences into pDR111-BSH with signal peptide (SP) regions of AmyE. The resultant plasmid pDR111-MEFA will be transformed into Bacillus subtilis 168, named B. subtilis-CPE-MEFA. Secretary CPE-MEFA vaccine proteins will be evaluated using Dot Blot and WB analysis with Histag antibody.1-5. Evaluate B. subtilis-CPE-MEFA vaccines against chicken NE.In this section, we will evaluate the secretory B. subtilis-CPE-MEFA vaccines against NE challenge in chickens. Briefly, at one day of age, birds will be assigned to 6 B. subtilis-CPE-MEFA vaccine groups. Each group will consist of 3 challenge groups of noninfected (PBS), E. maxima, and NE (E. maxima and C. perfringens). The birds will be immunized by feeding B. subtilis-CPE-MEFA at 108 CFU/g feed in feed at days of 1 and 10. The bird infection, vaccination efficacy, and intestinal sample collection and analysis will be similar to section 1-3.2: Develop microbe-metabolized bile acid regiments to reduce C. perfringens virulence and chicken NE.2-1. Assess the efficacy of isoalloLCA against chicken NE.Because CDCA microbe derivative, isoalloLCA, effectively inhibited C. perfringens in vitro growth (Alenezi et al., 2023), we reason that isoalloLCA will effectively reduce chicken NE. To exam this hypothesis, we will conduct chicken NE challenge experiments. Briefly, a cohort of one day old broiler chicks will be transferred to the farm. To prevent NE, at one day of age, the birds will be assigned to 4 dietary groups with diets at 0, 0.006, 0.03, and 0.06 g/kg isoalloLCA. Each dietary group will consist of 3 challenge groups of noninfected (PBS), E. maxima, and NE (E. maxima and C. perfringens). The bird infection and intestinal sample collection and analysis will be similar to section 1-3 in addition to isoalloLCA quantification.2-2. Assess the efficacy of bile acid metabolizing bacteria against chicken NE.CDCA is the main bile acid in chicken small intestine. We hypothesize that birds colonized with the specific bile acid metabolizing bacteria will metabolize endogenous CDCA and be protected against NE. Briefly, at one day of age, birds will be assigned to 4 bacterial colonization groups with 0 or 108 CFU/bacterium/kg P. merdae, E. lenta, or C. sardiniense. Each treatment group will consist of 3 challenge groups of noninfected (PBS), E. maxima, and NE (E. maxima and C. perfringens). The bird infection and intestinal sample collection and analysis will be similar to section 2-1.2-3. Identify CDCA metabolites by Pm and El using metabolomics.In this section, we use untargeted and targeted metabolomics to identify microbial metabolized bile acids. Briefly, bile acids and their metabolites will be extracted from the digesta by chloroform and methanol as described before (Bansal et al., 2020). For targeted HPLC/MS-MS metabolomics of bile acids, MRM methods have been developed for bile acids isotopically labeled and unlabeled standards (Bansal et al., 2020). For untargeted metabolomics, we will generate HPLC/MS-MS data of all metabolites in samples with intensity and retention time. We will then compare the metabolomics results between control and CDCA groups. Based on the mass spectra (MS, m/z) and retention time (min), the CDCA metabolites will be assessed. To determine what the exact bile acid is, we will use the standard bile acids and targeted metabolomics.2-4. Evaluate the efficacy of the newly identified CDCA metabolites against chicken NE.In this section, we will use the newly identified CDCA metabolites to prevent chicken NE. First, we will assess the CDCA metabolite dose response on reducing C. perfringens growth and virulence as described in the previous report (Alenezi et al., 2023). Based on the results, the CDCA metabolite doses in feed will be determined. To prevent NE, at one day of age, birds will be assigned to CDCA metabolite dietary groups with diets at 0 and various CDCA metabolite doses. Each dietary group will consist of 3 challenge groups of noninfected (PBS), E. maxima, and NE (E. maxima and C. perfringens). The bird infection and intestinal sample collection and analysis will be similar to section 2-1.