Source: UNIVERSITY OF ARKANSAS submitted to NRP
MODULATING MICROBIOME AND ITS DOWNSTREAM TARGETS TO ATTENUATE CHICKEN NECROTIC ENTERITIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1019059
Grant No.
2020-67016-31346
Cumulative Award Amt.
$350,000.00
Proposal No.
2018-06686
Multistate No.
(N/A)
Project Start Date
May 1, 2020
Project End Date
Apr 30, 2024
Grant Year
2020
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703
Performing Department
Poultry Science
Non Technical Summary
Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide because of restricting usage of prophylactic antibiotics under consumer preferences and regulatory pressures. It is estimated that NE costs poultry industry $6 billion every year worldwide. Few effective antimicrobial alternatives are available to prevent and treat NE. The long term goal of this proposal is to identify specific microbiome and its downstream targets as effective and sustainable antimicrobial alternatives to reduce necrotic enteritis. Our preliminary data showed that microbiota metabolic product deoxycholic acid (DCA) prevented Eimeria maxima and C. perfringens-induced NE, productivity loss, severe intestinal inflammation, and cell death. Following mechanism studies revealed that C. perfringens sporulation and inflammatory COX signaling drove NE development and DCA reduced them. Basing on these data and current state of knowledge, we hypothesize that microbiota metabolic product DCA attenuates NE through reducing inflammatory response and C. perfringens sporulation. In current proposal, we will investigate mechanism on how inflammatory signaling pathways and C. perfringens virulence modulate NE outcome at molecular levels and in chickens. More specifically, we aim to implement the following objectives.Objective 1: Evaluate the contribution of DCA-attenuated inflammatory COX-2 signaling on NE. Objective 2: Determine the role of DCA-reduced C. perfringens sporulation on NE.Upon completion, this project could significantly improve poultry productivity and provide effective and new approaches for antimicrobial free feeding regiments.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113220116025%
3113299109025%
3113299110025%
3113299111025%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3299 - Poultry, general/other;

Field Of Science
1090 - Immunology; 1160 - Pathology; 1100 - Bacteriology; 1110 - Parasitology;
Goals / Objectives
Necrotic enteritis (NE) caused by coccidia and Clostridium perfringens infections has reemerged as a prevalent poultry disease worldwide because of restricting usage of prophylactic antibiotics under consumer preferences and regulatory pressures. It is estimated that NE costs $6 billion /year worldwide. Few effective antimicrobial alternatives are available to prevent and treat necrotic enteritis. The long term goal of this proposal is to identify specific microbiome and its downstream targets as effective and sustainable antimicrobial alternatives to reduce necrotic enteritis. We plan to fulfill the long term goal with the following objectives. 1) We will evaluate the contribution of DCA-attenuated inflammatory COX-2 signaling on necrotic enteritis. 2) We will determine the role of DCA-reduced C. perfringens sporulation on necrotic enteritis. Upon completion, this project could significantly improve poultry productivity and provide effective approaches for antimicrobial free feeding regiments. The mechanism insight of necrotic enteritis pathogenesis obtained from this project will pave the way for targeted prevention and treatment of this disease.
Project Methods
1. Evaluate the contribution of DCA-attenuated inflammatory COX-2 signaling on NE. 1-1. Prevent coccidiosis and NE by blocking inflammatory COX-2 signaling using celecoxib To further define the role of inflammatory COX-2 signaling in coccidiosis and NE, a cohort of one day old broiler chicks (20 chicks/pen) will be transferred to our farm. 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. For preventing coccidiosis study, at 17 days of age, the birds will be assigned to 6 dietary groups of 0, 0.06, 0.12, or 0.24 g/kg celecoxib, 0.12 g/kg aspirin, or 0.05 g/kg bacitracin methylene disalicylate (BMD). For preventing NE study, at 22 days of age, the birds will be fed the 6 diets. Each dietary group consist of triplicate pens of noninfected (PBS), E. maxima, and NE (E. maxima and C. perfringens). Feed intake, body weight, and mortality will be measured at days 0, 18, 23 and 26. Birds will be followed for clinical signs and mortality. The broilers will be sacrificed at day 23 for early coccidiosis and the rest of birds will be sacrificed at day 26 for final NE or coccidiosis evaluation. Ileal tissue samples will be assayed for intestinal inflammation on mRNA accumulation of Ifnγ, Il1β, Litaf (Tnfα), Il8, Cxcl2, Il17a, and Mmp9. Ileal tissue will also be Swiss-rolled, fixed and processed for H&E staining and histopathological score. C. perfringens colonization and adherence/invasion in intestinal tissue or lumen will be determined by FISH technique enhanced for spores and real time PCR of 16S rDNA. CPE in intestinal digesta will be quantified using CPE ELISA kit.1-2. Treat NE using celecoxibIn this subobjective, we will examine the efficacy of celecoxib on treating coccidiosis and NE. The bird pen setup and bird infection are the same as section C-1-2. For treating NE study, at 24 days of age, the birds will be fed the 6 diets. Feed intake, body weight, mortality, inflammatory mRNA, C. perfringens colonization, sporulation, and invasion, CPE toxin, tissue histopathology will be collected and measured as in section 1-1.1-3. Evaluate inflammatory COX-2 signaling pathways activated by C. perfringens infection using splenocytesTo examine COX-2 up- and down-stream signaling pathways, isolated chicken splenocytes will be challenged with chicken IFN-γ in the presence of celecoxib. After RNA is isolated, celecoxib-inhibited gene expression of Litaf, Il1β, and Mmp9, and Gapdh will measured by real time PCR. To examine COX-2 related signaling pathways of mTOR and necrosis, we have to use Western Blot systems. Because of the limited reliable chicken antibodies for our research, we will use mouse splenocytes and Raw 264.7 macrophage, and rat small intestinal epithelial cell line IEC-18. Mouse splenocytes, Raw macrophage, and IEC-18 cells will be challenged with C. perfringens, C. perfringens sporulation supernatant, mouse IFN-γ, TNFα, and IL1β. COX-2, mTOR, necrosis, or NF-κB signaling will be blocked with select inhibitors celecoxib, rapamycin, IM-54, or Bay 11-7082, respectively. Proteins of COX-2, mTOR downstream target of p-p70S6K, necrosis marker RIP3, total and p-IκBα, and β-ACTIN will be measured using Western Blot.1-4. Prevent lethal NE using DCA or celecoxib. To examine how effective DCA and celecoxib on preventing lethal NE, birds will be assigned to 7 dietary groups with diets at 0, 0.5, 1, or 1.5 g/kg microencapsulated (improving utilization) DCA, 1.5 g/kg DCA, 0.12 g/kg celecoxib or 0.05 g/kg BMD. At 18 days of age, chicks will be gavaged with 50,000 sporulated oocysts/bird E. maxima to induce severe coccidiosis and at 22 days of age, the birds will be fed the 6 diets and then infected with 1010 CFU/chick C. perfringens at 23 and 24 days of age to induce NE. Feed intake, body weight, mortality, inflammatory mRNA, and tissue histopathology will be collected and measured.2. Determine the role of DCA-reduced C. perfringens sporulation on NE.2-1. Induce NE using C. perfringens sporulation supernatant To assess the role of C. perfringens sporulation CPE on development of NE, C. perfringens will be cultured in DSSM medium (sporulation) or DSSM-P (DSSM without phosphate, no sporulation) or at 42°C and anaerobic condition for 24 hours. The media will be centrifuged and collected for supernatant (supe.) of DSSM and DSSM-P. The DSSM-P supe. and DSSM supe. will be inoculated to Raw 264.7 macrophages for induction of cell death using PI staining. For induction of NE, a cohort of one day old broiler chicks will be transferred to our farm. The birds will be assigned to 4 groups of PBS, DSSM, DSSM-P supe. and DSSM supe. At 9 days of age, half birds of each group will be infected with 5,000 oocysts/bird E. maxima and the rest of bird will be gavaged with PBS. From 11 to16 days of age, the rest 3 groups of birds will be gavaged four times a day with DSSM, DSSM-P supe. and DSSM supe (1ml/bird/time). Body weight, and mortality will be measured at days 0, 9, 11 and 16. Birds will be followed for clinical signs as in section 1-1 and will be sacrificed at day 16 for NE evaluation. Feed intake, body weight, mortality, inflammatory mRNA, CPE toxin, tissue histopathology will be collected and measured.2-2. Prevent C. perfringens sporulation supernatant-induced NE using DCA or celecoxib. To examine how effective DCA or COX-2 inhibitor celecoxib prevent C. perfringens supernatant-induced NE, birds will be assigned to 7 dietary groups with diets at 0, 0.1, 0.5, or 1 g/kg microencapsulated DCA, 1.5 g/kg DCA, 0.12 g/kg celecoxib or 0.05 g/kg BMD. Half birds in each group will be gavaged with E. maxima once at d 9 and C. perfringens DSSM supe. four times a day (1ml/bird/time) from 11 to 16 days of age. Feed intake, body weight, mortality, inflammatory mRNA, CPE toxin, tissue histopathology will be collected and measured.2-3. Determine DCA inhibiting C. perfringens sporulation and toxin production in vitro.To assess the role of DCA on inhibiting C. perfringens sporulation and CPE, C. perfringens will be sporulated for 24 hours in DSSM medium in the presence of 0, 0.2, 0.5, and 1.8 mM DCA. We will examine the bacterial sporulation under microscope and OD600 reading. To quantify the toxin production, we will measure it using ELISA kit from TechLab. To evaluate the toxin induced cell death, equal dose of each supernatant will be inoculated into Raw 264.7 cell medium to induce cell death overnight. The cells will be stained with PI to label dead cells and DAPI for total cell count. We will also collect cell protein for WB of necrosis marker RIP3. To further functionally assess the toxin toxicity to birds, a cohort of one day old broiler chicks will be transferred to our farm. Half birds in each group will be gavaged with E. maxima once at d 9 and C. perfringens DSSM supe. four times a day from 11 to 16 days of age. Feed intake, body weight, mortality, inflammatory mRNA, CPE toxin, tissue histopathology will be collected and measured.2-4. Evaluate the role of coccidiosis on C. perfringens sporulation and NE. To examine the role of coccidiosis on C. perfringens sporulation, we will induce various severity of coccidiosis and then induce NE with C. perfringens. Briefly, birds will be assigned to 6 groups. At 18 days of age, chicks will be gavaged with 0, 500, 2,000, 5,000, 20,000 and 50,000 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. The birds will be sacrificed at d 26. Feed intake, body weight, mortality, inflammatory mRNA, C. perfringens colonization, sporulation, and invasion, CPE toxin, tissue histopathology will be collected and measured.

Progress 05/01/20 to 05/01/23

Outputs
Target Audience:Poultry Science researchers and Poultry and allied Industries Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students have experienced onchicken experiment designing, running, and analyzing.Ying Fu has extracted and analyzed bile acid data. Ying Fu and Tahrir Alenezi have run molecular biological works on genecloning, vaccination, and monoclonal antibody produciton. How have the results been disseminated to communities of interest?The above findings have been reported on professional conference meetings including the Poultry Science Association (PSA) and Conference of Research Workers in Animal Diseases (CRWAD). The results also have been published on leading journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A. Impact statement: Chicken necrotic enteritis (NE) is estimated to be responsible for losses of around six billion dollars in the poultry industry worldwide every year. The main pathogens are Eimeria and a Gram-positive bacterium Clostridium perfringens, which is also a foodborne pathogen. Antimicrobial alternatives against NE are urgently needed because of increasing antimicrobial resistance and pressure from consumers and regulations. Scientists and experts of poultry gut health from the Center of Excellence for Poultry Science at the Division of Agriculture in the University of Arkansas System have developed antimicrobial alternatives to control NE. The research showed that microbiota metabolite deoxycholic acid effectively inhibited C. perfringens infection and reduced chicken NE. Furthermore, antiinflammation agent rapamycin enhanced deoxycholic acid efficacy on reducing NE. The results suggest that specific bile acids coupling with antiinflammation agents could be used as an effective antibiotics alternative to control NE and improve chicken productivity. B. Accomplishment: To investigate objective 1, we have conducted a number of chicken and laboratory experiments. Briefly, in chicken experiments, day-old broiler chicks were assigned to various diets supplemented with bile acids or COX2 inhibitor Celecoxib. The bile acids included deoxycholic acid (DCA), cholic acid (CA), ox bile (mainly conjugated cholic acid), chicken bile (mainly taurochenodeoxycholic acid), and lithocholic acid (LCA). We also investigated COX2 upstream signaling pathway of mammalian target of rapamycin (mTOR) on NE development using mTOR inhibitor rapamycin in feed or subcutaneous injection. The birds were challenged with Eimeria maxima at d 18 and C. perfringens at d 23 to 25 to induce NE. The birds were individually weighed at d 0, 7, 16, 23 and 26. The birds were euthanized at d 26 when ileal tissue and digesta were collected for analyzing histopathology, fluorescence in situ hybridization (FISH), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and other assays. The dates of challenge, weighing, and euthanization were sometimes slightly different among experiments. Notably, the double challenges often induced clinical and subclinical NE. At the cellular level, birds infected developed NE showed shortening villi, crypt hyperplasia and immune cell infiltration in ileum. Dietary DCA prevented NE and improved chicken body weight gain loss in NE birds, while the combination of DCA and rapamycin had the best improvement. NE birds fed with Celecoxib showed reduced (but not significant) body weight gain reduction compared to NE control birds, while Celecoxib exacerbated NE-induced bloody diarrhea and worsened NE-induced mortality. Dietary CA, ox bile, chicken bile, and LCA didn't improve chicken NE-induced body weight gain loss. At the cellular level, DCA, LCA and rapamycin reduced NE-induced intestinal inflammation. At the molecular level, DCA and rapamycin reduced NE-induced proinflammatory gene expression such as Infγ, Mmp9, and Il23. The dietary DCA reduced NE-induced intestinal cell apoptosis, revealed through TUNEL assays. Importantly, NE reduced the total bile acid level in ileal digesta compared to uninfected birds, which might contribute to NE development. Dietary bile supplementation increased total bile level but only DCA reduced NE-induced body weight gain loss and intestinal inflammation. At the cellular level, blocking mTOR signaling pathway by rapamycin strongly reduced immune cell infiltration into intestinal lamina propria. Upon mechanism study with cell culture system, we found that rapamycin reduced mouse immune cell migration in response to C. perfringens presence. Interestingly, rapamycin delayed the intestinal epithelial cell wound healing using mouse CMT-93 cells. These results suggest that mTOR signaling in immune cells exacerbates C. perfringens induced intestinal inflammation by promoting immune cell infiltration, while the mTOR signaling in epithelial cells is indispensable for wound healing. Together, these results suggest that coupling specific bile acids and antiinflammation agents could effectively reduce chicken NE as effective antibiotics alternatives. To investigate objective 2, a number of chicken and laboratory experiments were carried out. C. perfringens sporulation vaccines were developed from the bacterial sporulation supernatant. The presence of C. perfringens enterotoxin (CPE) in the supernatant was detected using Dot-Blot and Western Blot assays with anti-CPE antibody. Broiler chicks were immunized with the CPE vaccines at d 0 and boosted at d 10. The birds were challenged with E. maxima and C. perfringens similar to described above. The body weight and sample collection and analysis were similar to the description above. In addition, blood samples were collected for assaying antibody generation. Consistently, challenged birds developed clinical NE with severe diarrhea and body weight gain loss. Using FISH assay, we found that C. perfringens invaded and sporulated as ball-shaped, deep inside intestinal villi and lamina propria of the NE birds, while DCA reduced C. perfringens invasion and sporulation, showing a few red rod-shaped vegetative cells. Notably, the CPE vaccines reduced severe NE-induced body weight gain loss. The vaccines attenuated NE-induced intestinal inflammation, histopathology score, and C. perfringens colonization and invasion in the small intestine. The vaccine significantly reduced inflammatory gene Ifnγ expression in the intestinal tissue compared to that in NE birds. The success of the vaccination was also validated with ELISA assay using the immunized chicken sera. To evaluate the sporulation vaccine bioactivity, mouse intestinal epithelial cell CMT-93 and murine macrophage Raw 264.7 cells were challenged with the vaccines. After 24 h incubation, more cell death was observed in the CMT-93 and Raw cells challenged with the vaccines compared to vegetative cell supernatant. Consistently, the CPE vaccine induced stronger proinflammatory mRNA expression of Il1β, Tnfα, and Cxcl2 compared to vegetative cell supernatant. Using Western blot, we found that the vaccines induced necrosis but not apoptosis in the Raw cells showing increased necroptosis marker proteins RIP3 and p-MLKL but not cleaved Caspase 3 of apoptosis marker. Following these findings, we aimed to identify and develop recombinant C. perfringens vaccines. C. perfringens DNA was segmented and ligated to plasmid pComb3xSS and were transformed into TG1 cells to generate bacterial shotgun library (TG1-SGL-cp). The TG1-SGL-cp was then used to generate phage library (M13-SGL-cp) using M13O7 helper phage. Bio-panning of phages with the immunized chicken or mouse sera in a plate was performed to select M13- SGL-cp expressing proteins binding to the anti-C. perfringens antibodies in the sera. We found that dietary DCA reduced caged chicken NE but the accumulation of DCA in gut was less effective conjugated tauro-DCA (TDCA). We aimed to deconjugate TDCA using bile salt hydrolase (Bsh). The bsh genes were PCR-amplified from Bifidobacterium longum, cloned into plasmids pET-28a (pET-Bsh) and pDR111 (pDR-Bsh), and Sanger-sequenced. pDR-Bsh transformed into Bacillus subtilis was assessed by C. perfringens growth inhibition assay. After cloned, transformed, and expressed pET-Bsh in BL21, Bsh protein was His-tag purified, subjected to SDS PAGE, and showed an expected band of 32 kDa. Bsh expression was also verified by Dot Blot assay using anti-His-tag antibody. C. perfringens growth inhibition assay showed that TDCA reduced the bacterium growth in the presence of supernatant of B. subtilis transformed with pDR-Bsh. Following these findings, we are cloning and expressing additional bile acid metabolizing enzymes such as bile acid inducible (Bai) enzymes and Hydroxysteroid dehydrogenase (Hsdh).

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Alenezi, T., Fu, Y., Bansal, M., Almansour, A., Wang, H., Sun, X. (2022). Bile salt hydrolase improves taurodeoxycholic acid against Clostridium perfringens growth. Poult. Sci. 102 (E Supple 1). 81.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Fu, Y., Wang, H., Alenezi, T., Almansour, Sun, X. (2022). Constructing shotgun library for Clostridium perfringens vaccine. Poult. Sci. 102 (E Supple 1). 119.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ahmed, H., Amin, U., Sun., X, Pitts, DR., Li, Y., Zhu, H., Jia, Z. 2022. Triterpenoid CDDO-IM protects against lipopolysaccharide-induced inflammatory response and cytotoxicity in macrophages: The involvement of the NF-?B signaling pathway. Exp Biol Med (Maywood). 247(8):683-690. doi: 10.1177/15353702211066912.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Alenezi, T., Fu, Y., Almansour, A., Wang, H., Sun, X. (2022). Developing a simple method to make Bacillus subtilis competent cells with highly efficient electroporation transformation. Poult. Sci. 102 (E Supple 1). 179.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Fu, Y., Wang, H., Alenezi, T., Almansour, Sun, X. (2022). Microbiota modulated bile acid metabolites influence Campylobacter jejuni in vitro growth. Poult. Sci. 102 (E Supple 1). 239.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sun, X., Bansal, M., Alenezi, T., Fu, Y., Almansour, A., Wang, H. (2023). The role of bile metabolism disorder and inflammation on chicken necrotic enteritis. CRWAD proceedings. 103. P139.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Alenezi, T., Fu, Y., Bansal, M., Almansour, A., Wang, H., Sun, X. Cloning and expressing microbial bile acid metabolizing enzymes to reduce Clostridium perfringens infection. CRWAD proceedings. 103. P141.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Fu, Y., Almansour, A., Alenezi, T., Wang, H., Sun, X. Specific bacteria biotransform bile acids to influence Campylobacter jejuni in vitro growth. CRWAD proceedings. 103. P229.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Fu, Y., Alenezi, T., Sun, X. 2022. Clostridium perfringens-induced necrotic diseases: An Overview. Immuno 2 (2), 387-407.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Fu, Y., Bansal, M., Alenezi, T., Almansour, A., Wang, H., Sun, X. (2022). Vaccines using Clostridium perfringens sporulation proteins prevent chicken necrotic enteritis. Poult. Sci. 102 (E Supple 1). 59.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Fu, Y., Almansour, A., Bansal, M., Alenezi, T., Alrubaye, B., Wang, H., Sun, X. 2022. Vaccines using Clostridium perfringens sporulation proteins reduce necrotic enteritis in chickens. Microorganisms, 10(6), 1110.


Progress 05/01/21 to 04/30/22

Outputs
Target Audience:The research results benefit poultry industry, acedemic field, consumers, and healthcare field. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate studentsAyidh Almansour,Ying Fu and Tahrir Alenezi have experienced on broiler and mouse experiment designing, running, and analyzing. Ying Fu and Tahrir Alenezihave extracted and analyzed bile acid data. Ying Fu and Tahrir Alenezi have run molecular biological works on gene cloning and expression, vaccination, and monoclonal antibody produciton. How have the results been disseminated to communities of interest?The above findings have been reported on professional conference meetings including the Poultry Science Association (PSA) and Conference of Research Workers in Animal Diseases (CRWAD). The results also have been published on leading journals. What do you plan to do during the next reporting period to accomplish the goals?We will continue to intestigate which bile acids and their microbiota derivatives effectivelyC. perfringens in vitro growth and reduce BWG loss in NE birds. We will evaluate the bile acid derivativeson NE-inducedintestinal inflammation. We will also clone various bile acid metabolism enzymes to bio-transform the bile acids in vitro for reducing C. perfringensin vitro growth. We will continue to develop C. perfringenssporulaiton vaccines using Shotgun library and Phage Display.

Impacts
What was accomplished under these goals? To assess objective 1: Day old broiler chicks were assigned to dietary treatments of various rapamycin alone or coupled with 1.5 g/kg deoxycholic acid (DCA). The birds were induced NE by subsequential infection of Eimeria maxima and Clostridium perfringens. NE chickens experienced mild to severe intestinal histopathology compared to noninfected birds. Notably, blocking mTOR signaling with pharmacological inhibitor rapamycin strongly reduced C. perfringens-induced intestinal histopathology independent of NE severity. Interestingly, rapamycin failed to improve chicken body weight gain loss in birds infected with clinical NE. Dietary supplementation of both Rapamycin and DCA dramatically reduced clinial NE-induced body weight gain loss and histopathology. Molecular analysis of ileal tissue showed that inhibiting mTOR by rapamycin reduced proinflammatory mediators Infγ, Mmp9, and Il23 mRNA accumulation compared to NE birds. Cellular analysis of the ileal tissue revealed that blocking mTOR by rapamycin strongly reduced immune cell infiltration into intestinal lamina propria. Upon mechanism study with cell culture system, we demonstrated that mTOR inhibition by rapamycin reduced mouse immune cell migration in response to C. perfringens presence. Interestingly, rapamycin delayed the intestinal epithelial cell wound healing using mouse CMT-93 cells. Together, these results suggest that mTOR signaling in immune cells exacerbates C. perfringens-induced intestinal inflammation by promoting immune cell infiltration, while the signaling in epithelial cells is indispensable for wound healing. Rapamycin and DCA together effectively reduce chicken NE. In addition, we investigated the role of specific bile acids on NE development. Day-old broiler chicks were assigned to six groups: noninfected, NE, and NE with four bile diets of 0.32% chicken bile, 0.15% commercial ox bile, 0.15% lithocholic acid (LCA), or 0.15% deoxycholic acid (DCA). The birds were infected with Eimeria maxima at day 18 and C. perfringens at day 23 and 24. The infected birds developed clinical NE signs. The NE birds suffered severe ileitis with villus blunting, crypt hyperplasia, epithelial line disintegration, and massive immune cell infiltration, while DCA and LCA prevented the ileitis histopathology. NE induced severe body weight gain (BWG) loss, while only DCA prevented NE-induced BWG loss. Notably, DCA reduced the NE-induced inflammatory response and the colonization and invasion of C. perfringens compared to NE birds. Consistently, NE reduced the total bile acids in the ileal digesta, while dietary DCA and commercial bile restored it. Together, this study showed that DCA and LCA reduced NE histopathology, suggesting that secondary bile acids, but not total bile acid levels, play an essential role in controlling the enteritis. To assess objective 2: C. perfringens soluble proteins of vegetative cells (CP-super1 and CP-super2) and spores (CP-spor-super1 and CP-spor-super2) were prepared, and cell and chicken experiments were conducted. We found thatdeoxycholic acid reduced C. perfringens invasion and sporulation using E. maxima and C. perfringens co-infection necrotic enteritis (NE) model.C. perfringens enterotoxin (CPE) was detected in the CP-spor-super1&2. CP-spor-super1 or 2 induced cell death in mouse epithelial CMT-93 and macrophage Raw 264.7 cells. CP-spor-super1 or 2 also induced inflammatory gene expression and necrosis in the Raw cells. Birds immunized with CP-spor-super1 or 2 were resistant to C. perfringens induced severe clinical NE on histopathology and body weight gain loss. CP-spor-super1 vaccine reduced NE-induced proinflammatory Ifnγ gene expression as well as C. perfringens luminal colonization and tissue invasion in the the small intestine. Together, this study showed that CP-spor-super vaccines reduced NE histopathology and productivity loss. In previous study, we found that secondary bile acid DCA in feed reduces chicken NE but accumulation of DCA in gut is conjugated tauro-DCA (TDCA). We hypothesized that deconjugating TDCA by Bsh would improve dietary DCA efficacy against NE. C. perfringens growth inhibition assay was conducted with bile acids tauro-cholic acid (TCA), CA, TDCA or DCA. The tryptic soy agar (TSA) plates supplemented with 0.2% TDCA and 0.03% CaCl2 were used to examine the Bsh activity. The bsh genes were PCR-amplified, cloned into plasmids pET-28a (pET-Bsh) and pDR111 (pDR-Bsh), and Sanger-sequenced. After transformed pET-Bsh plasmid into E. coli DH5a and BL21 for amplification and protein expression, respectively, the His-tag purified Bsh was evaluated by SDS-PAGE, Dot-Blot, DCA plate precipitation, and C. perfringens inhibition assay. pDR-Bsh transformed into Bacillus subtilis was assessed by C. perfringens inhibition assay. Notably, the inhibition of C. perfringens growth by bile acids was in the order of DCA>TDCA>CA>TCA. Bifidobacterium longum in anaerobic air secreted Bsh showed as colonies surrounded with opaque precipitation zones on the TSA plates with TDCA and CaCl2. After cloned, transformed, and expressed pET-Bsh in BL21, Bsh protein was His-tag purified, subjected to SDS-PAGE, and showed an expected band of 32 kDa. Bsh expression was also verified by Dot Blot assay using anti-His-tag antibody. Bsh activity was then examined on the TSA plates, showing opaque crystallized DCA in the path of Bsh solution compared to transparent path from the elution buffer only. C. perfringens growth inhibition assay showed that TDCA reduced the bacterium growth in the presence of purified Bsh or supernatant of B. subtilis transformed with pDR-Bsh. In conclusion, DCA was more potent than other bile acids against C. perfringens growth. Bsh cloned from B. longum to E. coli or B. subtilis was able to inhibit C. perfringens growth in the presence of TDCA, suggesting potential new intervention against chicken NE.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Almansour, A., Fu, Y., Alenezi, T., Bansal, M., Alrubaye, B., Wang, H., Sun, X. (2021). Microbiota from Specific Pathogen-Free Mice Reduces Campylobacter jejuni Chicken Colonization. Pathogens, 10(11), 1387.
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Fu, Y., Alenezi, T., Almansour, A., Wang, H., Jia, Z., Sun, X. (2021). The Role of Immune Response and Microbiota on Campylobacteriosis. Campylobacter. IntechOpen.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Gupta, A., Bansal, M., Liyanage, R., Upadhyay, A., Rath, N., Donoghue, A., Sun, X. (2021). Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion. PLOS One, 16(4), e0250296.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Bansal, M., Alenezi, T., Fu, Y., Almansour, A., Wang, H., Gupta, A., Liyanage, R., Graham, D. B., Hargis, B. M., Sun, X. (2021). Specific Secondary Bile Acids Control Chicken Necrotic Enteritis. Pathogens, 10(8), 1041.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Fu, Y., Almansour, A., Alenezi, T., Wang, H., Sun, X. (2021). Chenodeoxycholic acid promotes susceptibility to campylobacteriosis in Il10-/- mice Conference of Research Workers in Animal Diseases (vol. 102, pp. 413).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Alenezi, T., Fu, Y., Bansal, M., Almansour, A., Wang, H., Sun, X. (2021). Cloning bile salt hydrolase to increase deconjugate bile acids for reducing Clostridium perfringens Conference of Research Workers in Animal Diseases (vol. 102, pp. 421).
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Fu, Y., Bansal, M., Almansour, A., Alenezi, T., Wang, H., Sun, X. (2021). Clostridium perfringens sporulation vaccines prevent chicken necrotic enteritis Conference of Research Workers in Animal Diseases (vol. 102, pp. 431).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Fu, Y., Wang, H., Alenezi, T., Almansour, A., Sun, X. (2021). Inflammation and bile acid composition determine C. perfringens-induced enteritis in mice Conference of Research Workers in Animal Diseases (vol. 102, pp. 473).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Almansour, A., Fu, Y., Alenezi, T., Bansal, M., Wang, H., Sun, X. (2021). Mouse specific pathogen free microbiota to reduce Campylobacter jejuni colonization in chickens Conference of Research Workers in Animal Diseases (vol. 102, pp. 384).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Sun, X., Bansal, M., Alenezi, T., Fu, Y., Almansour, A., Wang, H. (2021). Specific secondary bile acids control chicken necrotic enteritis Conference of Research Workers in Animal Diseases (vol. 102, pp. 417).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Fu, Y., Alenezi, T., Almansour, A., Wang, H., Sun, X. (2021). Using Phage Display to produce anti-Clostridium perfringens monoclonal antibodies Conference of Research Workers in Animal Diseases (vol. 102, pp. 424).


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The research results benefit poultry industry, acedemic field, consumers, and healthcare field. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five graduate students Mohit Bansal, Ayidh Almansour, Mussie Abraha, Ying Fu and Tahrir Alenezi have experienced on broiler and mouse experiment designing, running, and analyzing. Mohit Bansal and Ayidh Almansour have extracted and analyzed bile acid data. Ying Fu and Tahrir Alenezi have run molecular biological works on gene cloning, vaccination, and monoclonal antibody produciton. How have the results been disseminated to communities of interest?The above findings have been reported on professional conference meetings including the Poultry Science Association (PSA) and Conference of Research Workers in Animal Diseases (CRWAD). The results also have been published on leading journals. What do you plan to do during the next reporting period to accomplish the goals?We will continue to intestigate how to reduceBWG loss in NE birds when blocking inflammatory signaling pathways, such as dose titration of rapamycin and combining DCA with rapamycin and Celecoxib. We will evaluate different bile acid supplementation on NE-induced BWG loss and intestinal inflammation. We will continue to assess the role of C. perfringens sporulation on NE-induced BWG loss and intestinal inflammation, such as identifying specific protein in the Cp-spor-super.

Impacts
What was accomplished under these goals? To evaluate objective 1, day-old broiler chicks were assigned to 4 groups and fed diets supplemented with 0 (basal diet) and 4 mg/kg (on top of basal diet) COX2 inhibitor Celecoxib from d 17. Two groups of birds were challenged withEimeria maxima(20,000 oocysts/bird) at d 18 andC. perfringens(109 CFU/birdperday) at d 23, 24, and 25 to induce NE. The birds were sacrificed at d 26 when ileal tissue and digesta were collected. We found that COX2 inhibitor Celecoxib didn't significantly change chicken daily body weight gain (BWG) during d 0-26 compared to uninfected birds. Birds infected with E. maxima and C. perfringens developed clinical NE and suffered severe BWG loss. NE birds fed with Celecoxib showed reduced (but not significant) BWG compared to NE control birds. Celecoxib exacerbated NE-induced bloody diarrhea and worsened NE-induced mortality. The results suggest that blocking COX2 alone doesn't attenuate NE-induced intestinal inflammation, possibly due to interfering wound healing and bleeding from coccidiosis injury. We also investigated COX2 upstream signaling pathway of mammalian target of rapamycin (mTOR) on NE development. Day old broiler chicks were randomly allotted to five dietary treatments of 0, 0.075, 0.15, 0.3 0.45 mg/kg rapamycin. Feed supplementation was started from d 17. Additional two groups of chickens were subcutaneously injected with rapamycin of 8 and 64 µg/kg BW starting from d 17. At d 18, birds were orally infected withE. maxima(15,000 sporulated oocysts/ bird) to induce coccidiosis. The birds were subsequently infected with C. perfringensat d 23 and 24. Notably, birds infected withE. maximaandC. perfringensdeveloped acute NE and suffered severe growth performance reduction. Interestingly, rapamycin treatments alone did not significantly inhibit the BWG loss during NE phase of d 23-25. In vitro assay showed rapamycin inhibited immune cell migration. Notably, inhibiting mTOR signaling by rapamycin attenuated NE-induced intestinal inflammation. Together, these results suggest that inhibiting inflammatory signaling pathways alone reduce NE-induced intestinal inflammation but with limited impact on BWG loss, possibly from interfering coccidiosis-induced wound healing process. To assess objective 2, day-old broiler chicks were assigned to five groups and fed diets supplemented with 0 (basal diet), 0.8, 1.0 and 1.5 g/kg (on top of basal diet) DCA. Four groups of the birds were challenged withE. maxima(20,000 oocysts/bird) at d 18 andC. perfringens at d 23, 24, and 25 to induce NE. The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology, fluorescencein situhybridization (FISH), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and other assays. At the cellular level, birds infected withE. maximaandC. perfringensdeveloped subclinical NE and showed shortening villi, crypt hyperplasia and immune cell infiltration in ileum. Dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds. Subclinical NE birds suffered body weight gain reduction compared to the uninfected birds, an effect attenuated with increased doses of dietary DCA. At the molecular level, the highest dose of DCA at 1.5 g/kg reducedC. perfringensluminal colonization and sporulation compared to NE birds using PCR and FISH. Furthermore, the dietary DCA reduced subclinical NE-induced intestinal inflammatory gene expression and cell apoptosis using PCR and TUNEL assays. Subclinical NE reduced the total bile acid level in ileal digesta compared to uninfected birds. Notably, dietary DCA increased total bile acid and DCA levels in a dose-dependent manner compared to NE birds. These results indicate that DCA attenuates NE-induced intestinal inflammation and bile acid reduction and could be an effective antimicrobial alternative against the intestinal disease. To evaluate the role of C. perfringens sporulation on NE, two C. perfringens isolates of Cp1 and Cp2 were induced sporulation in Duncan Strong sporulation medium (DSSM). The C. perfringens sporulation supernatant was purified and saved as Cp1-spor-super and Cp2-spor-super. The presence of CPE in the Cp-spor-super was detected using Dot-Blot and Western Blot assays with anti-CPE antibody. Notably, CPE protein was expressed in the Cp1-spor-super and Cp2-spor-super. To examine whether and which cell type in the small intestine was influenced by Cp1-spor-super, mouse intestinal epithelial cell CMT-93 and murine macrophage Raw 264.7 cells were used. After challenged with the Cp1-spor-super for 48 hours, cell death was observed in CMT-93 cells, and more cell death was clearly observed in mouse macrophage Raw cells. We then examine whether the Cp1-spor-super induced inflammatory response in immune cells. Macrophage was challenged with the Cp1-spor-super for 4 hr and proinflammatory gene expression was assessed. Notably, the Cp1-spor-super induced more than 1000- and 400-fold increase of proinflammatory mRNA Il1β and Cxcl2, respectively. To assess which cell death pathway the Cp1-spor-super was induced, the Raw cells were challenged with the Cp1-spor-super for 5 hr. Unlike inflammatory cytokine TNFα, the Cp1-spor-super induced expression of p-MLKL but not cleaved Caspase 3, suggesting the Cp1-spor-super inducing necrosis but not apoptosis in Raw cells. These results suggest that the Cp1-spor-super induces inflammation and cell necrosis in immune cells. To further investigate the role of the Cp-spor-super on NE development, day-old broiler chicks were assigned to eight groups. Birds at d 1 were immunized with subcutaneous injection of Cp-spor-super without any adjuvant. The doses of vaccines were 40, 4, and 0.4 μg/bird for Cp1-spor-super 1-3 and 400, 40, and 4 μg/bird for Cp2-spor-super 1-3. The seven groups of the birds were challenged withEimeria maxima(20,000 oocysts/bird) at d 18 andC. perfringens at d 23, 24, and 25 to induce NE. The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology. A booster doses with ten times higher were administrated at d 10. Birds grew comparably between different groups during uninfected phase of d 0 to18. Notably, BWG in noninfected negative control (NC) birds was heavier compared to E. maxima-infected only and Cp2-spor-super 3 vaccinated birds during d 18 to 23. At NE phase of d 23-26, NE birds were losing body weight compared to NC birds. Remarkably, birds vaccinated with Cp1-spor-super 3 were able to continue to gain body weight compared to NE birds, while birds vaccinated with other vaccine regiments didn't gain significant body weight. E. maxima and C. perfringens induced acute NE is associated with ileal inflammation. Notably, NE birds displayed severe ileitis as seen by necrosis and fusion of villi and crypt, massive immune cell infiltration, and severe villus shortening. Remarkably, Cp1-spor-super 3 and Cp2-spor-super 3 vaccines dramatically attenuated NE-induced ileitis and histopathology score, while other vaccination doses didn't significantly lower ileitis and histopathology score. In summary, inhibiting inflammatory signaling pathways alone reduced NE-induced intestinal inflammation but with limited impact on BWG loss, possibly because of coccidiosis-induced wound healing process. DCA reduced subclinical NE on BWG loss, ileitis, intestinal inflammation, and C. perfringens colonization and sporulation at dose dependent manner. Vaccinating birds with Cp-spor-super prevented C. perfringens-induced severe clinical NE on BWG loss and histopathology.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Bansal M, Fu Y, Alrubaye B, Abraha M, Almansour A, Gupta A, Liyanage R, Wang H, Hargis B, Sun X. 2020. Dietary deoxycholic acid attenuates ileitis and bile acid deficiency in chicken subclinical necrotic enteritis. J Anim Sci Biotechnol. 2020; 11: 37. doi: 10.1186/s40104-020-00441-6. PMCID: PMC7069026. PMID: 32190299.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Shukla H, Lee HY, Koucheki A, Bibi HA, Gaje G, Sun X, Zhu H, Y. Li R & Jia Z. 2020. Targeting glutathione with the triterpenoid CDDO-Im protects against benzo-a-pyrene-1,6-quinone-induced cytotoxicity in endothelial cells. Molecular and Cellular Biochemistry. Published: 27 July 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gupta A, Bansal M, Wagle BR., Sun X, Rath N, Donoghue A and Upadhyay A. 2020. Sodium butyrate reduces Salmonella Enteritidis infection of chicken enterocytes and expression of inflammatory host genes in-vitro. Front. Microbiol. doi: 10.3389/fmicb.2020.553670.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fu Y, Almansour A, Bansal M, Alenezi T, Alrubaye B, Wang H, Hargis B, Sun X. 2020. Microbiota attenuates chicken transmission-exacerbated campylobacteriosis in Il10?/? mice. Sci Rep 10: 20841. DOI: 10.1038/s41598-020-77789-2.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: M. Bansal, Y. Fu, A. Almansour, M. Abraha, T. Alenezi, D. Graham, A. Gupta, H. Wang, R. Liyanage, B. Hargis, and X. Sun. 2020. Evaluation of bile acids against chicken necrotic enteritis. Poult. Sci. 99 (E Supple 1). 36.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: M. Bansal, Y. Fu, A. Almansour, T. Alenezi, M. Abraha, D. Graham, A. Gupta, H. Wang, B. Hargis, and X. Sun. 2020. Mammalian target of rapamycin signaling modulates chicken necrotic enteritis. Poult. Sci. 99 (E Supple1). 35.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Y. Fu, M. Bansal, A. Almansour, T. Alenezi, M. Abraha, D. Graham, A. Gupta, H. Wang, B. Hargis, and X. Sun. 2020. Clostridium perfringens vaccine prevents chicken necrotic enteritis. Poult. Sci. 99 (E Supple 1). 37.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: M. Bansal, Y. Fu, A. Almansour, T. Alenezi, A. Gupta, H. Wang, R. Liyanage, B. Hargis, and X. Sun. 2020. Assessment of primary and secondary bile acids against chicken necrotic enteritis. CRWAD proceedings. 101. p368.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: M. Bansal, Y. Fu, A. Almansour, T. Alenezi, A. Gupta, H. Wang, R. Liyanage, B. Hargis, and X. Sun. 2020. Inhibition of mTOR Signaling attenuates chicken necrotic enteritis induced intestinal inflammation. CRWAD proceedings. 101. p369.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Y. Fu, M. Bansal, A. Almansour, T. Alenezi, M. Abraha, D. Graham, A. Gupta, H. Wang, B. Hargis, and X. Sun. 2020. Vaccinating chickens with crude Clostridium perfringens sporulation product reduces necrotic enteritis. CRWAD Proceedings. 101, p419.