MICROBIOME AS ANTIMICROBIAL ALTERNATIVES TO REDUCE FOOD BORNE CAMPYLOBACTERIOSIS AND TO IMPROVE POULTRY PRODUCTIVITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012366
• Project Start Date: Apr 18, 2017
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Project Director
Sun, XI.

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Poultry Sciences

Non Technical Summary
The long term goal of this project is to identify microbiota, its metabolites, andsignaling moleculesas an effective and sustainable antimicrobial alternatives to improve chicken health and productivity and to reduce food borne campylobacteriosis. Campylobacter jejuni is a poultry commensal microorganism but a food borne bacterial pathogen with increasing antibiotic resistance incidences. Cases of campylobacteriosis at millions ranked second among all food borne pathogens with limited treatment options and severe post-infectious complications. Antimicrobial free feeding regiments has been encouraged from consumers, food industries, and regulatory agencies. However, chicken production loss and increased food borne disease incidences have been reported in countries practicing antimicrobial free regiments. Based on our previous findings and current status of knowledge, I hypothesize that signaling molecules and specific microbiota from C. jejuni colonization resistant animals protects against C. jejuni colonization and improve growth performance in chickens. ?Upon completion, this project will improve food safety and broiler growth performance and could provide valuableoptions to implement novel antimicrobial alternatives for poultry industry.

Animal Health Component

20%

Research Effort Categories

Basic

50%

Applied

20%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3220	1090	50%
308	3220	1100	50%

Knowledge Area
311 - Animal Diseases; 308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3220 - Meat-type chicken, live animal;

Field Of Science
1090 - Immunology; 1100 - Bacteriology;

Keywords

broiler

campylobacteriosis

coccidosis

necrotic enteritis

microbiota

metabolite

innate

Goals / Objectives
A) Determine the efficacy of microbiota from animal resisting C. jejuni colonization on reducing C. jejuni colonization and improve growth performance in broilers. B) Identify groups of protective microbes on mitigating C. jejuni colonization improve growth performance in broilers. C) Assess the impact of microbes and their metabolites on attenuating chicken-transmitted C. jejuni-induced colitis (campylobacteriosis) using Il10-/- mouse infection model. D) Identify selected microbiota and metabolites improve growth performance and protect against coccidiosis- and C. perfringens-induced NE.

Project Methods
For subproject of Campylobacter jejuni microbiota: Microbiota will be prepared by collecting and preparing from stools of healthy donors of mice. Selective bacteria and microbial metabolites will also be isolated using specific agar plates or identified from 16S sequencing. Day of hatch chicks will be obtained from a local hatcher. Chicks will be orally gavaged with candidate microbiota. After standard colonization for 2 weeks, chicks in one of the two pens of pre-colonized microbiota or PBS will be gavaged with 109 CFU/chick C. jejuni strain 81-176. Fecal samples from individual chicks will be collected at 0, 7, and 21 days post C. jejuni-gavaging using sterile tubes. Feed intake and body weight will be measured at the same days. The broilers will be humanely sacrificed by CO2 inhalation at 21 days post gavaging. Segments of ceca, proximal and distal colon tissues will be snap-frozen for assessing mRNA expression of proinflammatory cytokines, mucin, and barrier integrity: mRNA of Il1β, Tnfα, Il8, Cxcl2, Il17a, Muc2, Tff3, and Klf4 as well as signaling proteins p-p70S6K (T389) and barrier proteins of E-cadherin and β-catenin. Cecal and colon tissue will be Swiss-rolled, fixed and processed for H&E staining. Intestinal inflammation will be assessed by histological score using mouse colitis score matrix as described before. C. jejuni colonization and adherence in intestinal tissue or lumen will be determined by culture, FISH techniques or real time PCR. C. jejuni virulence will be assessed in intestinal tissue and luminal content using real time PCR on genes of KatA, FlgQ, and AnsB. Cecal luminal content will be collected for 16S rRNA or RNA sequencing. This experiment will need to be repeated up to 4 times in order to confirm the results due to expected variability based on breeder flock source, breeder age, and hatcher variability.After isolating transmitted C. jejuni from chickens of above experiments, 8-12 weeks of age C57BL6 Il10-/- mice will be transferred from breeding room to BSL2 infection room. The mice will be treated with a broad spectrum antibiotics cocktail (streptomycin 2 g/L, bacitracin 1 g/L, gentamicin 0.5 g/L and ciprofloxacin 0.125 g/L) for 1 week and 24h after this regimen (with group 1 being the exception) orally gavaging (0.1mL) with the isolated 109 CFU/ml C. jejuni. Mice will be followed clinically for evidence of diarrhea, failure to thrive and mortality. Mice will be sacrificed at 21-day post infection. Segments of proximal and distal colon tissues will be snap-frozen for assessing mRNA expression of proinflammatory cytokines, mucin, and barrier integrity: mRNA of Il1β, Tnfα, Cxcl1, Cxcl2, Il17a, Muc2, Tff3, and Klf4 as well as signaling proteins p-p70S6K (T389) and barrier proteins of E-cadherin and β-catenin. Colon tissue will be Swiss-rolled, fixed and processed for H&E staining. Intestinal inflammation will be assessed by histological score. C. jejuni colonization and invasion into intra- and extra-intestinal tissue (colon, spleen and mesenteric lymph nodes (MLN) will be determined by culture, FISH techniques and real time PCR. Campylobacteriosis marker of neutrophil infiltration into colon will be assessed by immunohistochemistry (IHC) of myeloperoxidase (MPO) as showed previously. This experiment will need to be repeated up to 2 times in order to confirm the results due to expected variability based on age, breeders and antibiotic cocktail efficiency.For subproject of necrotic enteritis pathogenesis: Day of hatch chicks will be obtained from a local hatchery. On day 14, groups of chicks will be orally gavaged with oocysts of live coccidiosis vaccine consisted of Eimeria maxima, E. tenella, and E. acervulina. Chicks will be fed respective diets listed below. On day 18, chicks will be gavaged with the following colony forming unit (CFU) doses of C. perfringens.Fecal samples from individual chicks will be collected at 0, 4, and 14, 21 days post C. perfringens-gavaging using sterile tubes. Feed intake and body weight will be measured at the same days. The broilers will be humanely sacrificed by CO2 inhalation at 21 days post gavaging. Segments of small intestine, ceca, and colon tissues will be snap-frozen for assessing mRNA expression of proinflammatory cytokines, mucin, and barrier integrity: mRNA of Il1β, Tnfα, Il8, Cxcl2, Il17a, Muc2, Tff3, and Klf4 as well as barrier proteins of E-cadherin and β-catenin. Small intestine, cecal and colon tissue will be Swiss-rolled, fixed and processed for H&E staining. Intestinal inflammation will be assessed by histological score. C. perfringens colonization and adherence in intestinal tissue or lumen will be determined by culture, FISH techniques or real time polymerase chain reaction (PCR). C. perfringens virulence of germination, growth and sporulation will be assessed in intestinal tissue and luminal content using real time PCR. This experiment will need to be repeated up to 4 times in order to confirm the results due to expected variability based on breeder flock source, breeder age, and hatcher variability.For in vitro experimental evaluation, the broilers will be used to isolate peritoneal macrophages and neutrophils, splenocytes, intestinal epithelial cells and fibroblast. These primary cells will be used for in vitro experiments on germination, growth and sporulation, bacterial clearance, proinflammatory cytokine secretion, and migration assay. This experiment will evaluate C. perfringens in vitro germination, growth and sporulation using primary chicken cells.

Progress 04/18/17 to 09/30/21

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 have experienced onmouse and chicken experiment designing, running, and analyzing. Recently, Ying Fuhasextracted 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? Nothing Reported

Impacts
What was accomplished under these goals? Throughout the four and half years, we have successfully accomplished the four goals in the project using chicken and mouse experiments, in vitro assay, molecular biology, HPLC/MS-MS. We have also discovered new leads for the ongoing and further research. The following are the specific achievements. To investigate goals A and B, we have conducted a number of chicken experiments. We found that C. jejuni 81-176 was readily colonized intestinal tract at d 16 and reached an almost plateau at d 21. Remarkably, dietary deoxycholic acid (DCA) excluded C. jejuni cecal colonization below the limit of detection at 16 and 28 days of age. Neither chicken ages of infection nor dietary lithocholic acid or ursodeoxycholic acid altered C. jejuni AR101 chicken colonization level, while DCA reduced 91% of the bacterium in chickens at d 28. Notably, DCA diet reduced phylum Firmicutes but increased Bacteroidetes compared to infected control birds. Importantly, DCA modulated anaerobes attenuated 93% of C. jejuni colonization at d 28 compared to control infected birds. In conclusion, DCA shapes microbiota composition against C. jejuni colonization in chickens, suggesting a bidirectional interaction between microbiota and microbial metabolites. These findings were published on PLoS One. 2019. 5;14(7):e0214705. doi: 10.1371/journal.pone.0214705. eCollection. We have also conducted chicken experiments using microbiota from specific pathogen free (SPF) mice, named SPF anaerobes (SPF-Anaerobe) and aerobes (SPF-Aerobe). We found that SPF-Aerobe and SPF-Anaerobe increased cecal phylumBacteroidetesand reduced phylumFirmicutescompared to those in the nontransplanted birds. Interestingly, microbiota from noninfected chickens, SPF-Aerobe, or SPF-Anaerobe inhibited AR101 in vitro growth, whereas microbiota from infected birds alone failed to reduce pathogen growth. The bacteriumEnterobacter102 isolated from infected birds transplanted with SPF-Aerobe inhibited AR101 in vitro growth and reduced pathogen gut colonization in chickens. Together, SPF mouse microbiota was able to colonize chicken gut and reduceC. jejunichicken colonization. The findings may help the development of effective strategies to reduceC. jejunichicken contamination and campylobacteriosis. These findings were published on Pathogens. 2021. 27;10(11):1387. doi: 10.3390/pathogens10111387. To investigate goal C, we isolated C. jejuni AR101 passage 1 (Cj-P1) from chickens infected with C. jejuniAR101 (Cj-P0C. jejuniCj-P1-DCA-Anaero was isolated from Cj-P0-infected birds transplanted with DCA-modulated anaerobic microbiota. Specific pathogen freeIl10−/−mice were gavaged with antibiotic clindamycin and then infected with Cj-P0, Cj-P1, or Cj-P1-DCA-Anaero, respectively. After 8days post infection,Il10−/−mice infected with Cj-P1 demonstrated severe morbidity and bloody diarrhea and the experiment had to be terminated. Cj-P1 induced more severe histopathology compared to Cj-P0, suggesting that chicken transmission increasedC. jejunivirulence. Importantly, mice infected with Cj-P1-DCA-Anaero showed attenuation of intestinal inflammation compared to Cj-P1. At the cellular level, Cj-P1 induced moreC. jejuniinvasion and neutrophil infiltration into theIl10−/−mouse colon tissue compared to Cj-P0, which was attenuated with Cj-P1-DCA-Anaero. At the molecular level, Cj-P1 induced elevated inflammatory mediator mRNA accumulation ofIl17a,Il1β, andCxcl1in the colon compared to Cj-P0, while Cj-P1-DCA-Anaero showed reduction of the inflammatory gene expression. In conclusion, our data suggest that DCA-modulated anaerobes attenuate chicken-transmitted campylobacteriosis in mice and it is important to control the elevation ofC. jejunivirulence during chicken transmission process. To investigate goal D, we fed birds with microbial metabolic byproduct of secondary bile acid deoxycholic acid DCA diet and induced necrotic enteritis (NE) using co-infection of Eimeria maxima and Clostridium perfringens. We found that DCA, at as low as 50 µM, inhibited 82.8% ofC. perfringensgrowth in Tryptic Soy Broth (P < 0.05). SequentialE. maximaandC. perfringenschallenges significantly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished (P < 0.05) by 1.5 g/kg DCA diet. At the cellular level, DCA alleviated NE-associated ileal epithelial death and significantly reduced lamina propria cell apoptosis. Interestingly, DCA reducedC. perfringensinvasion into ileum (P < 0.05) without altering the bacterial ileal luminal colonization. Molecular analysis showed that DCA significantly reduced inflammatory mediators ofInfγ,Litaf,Il1β, andMmp9mRNA accumulation in ileal tissue. Mechanism studies revealed thatC. perfringensinduced (P < 0.05) elevated expression of inflammatory mediators ofInfγ,Litaf, andPtgs2(Cyclooxygenases-2 (COX-2) gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin significantly attenuated INFγ-induced inflammatory response in the splenocytes. Consistent with thein vitroassay, chickens fed 0.12 g/kg aspirin diet protected the birds against NE-induced BW loss, ileal inflammation, and intestinal cell apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through attenuating inflammatory response. These novel findings of microbiome protecting birds against NE provide new options on developing next generation antimicrobial alternatives against NE. These findings were published on Sci Rep. 2019. 10;9(1):14541. doi: 10.1038/s41598-019-51104-0. In summary, after conducted a variety of in vitro and in vivo experiments, we have reached our proposed goals and are performing continuous works toward finding new interventions controlling C. jejuni infection and NE.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Antibiotic alternative deoxycholic acid alleviates chicken necrotic enteritis. Mohit Bansal, Mussie Abraha, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 21.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Microbial metabolite deoxycholic acid in drinking water prevents chicken necrotic enteritis. Mussie Abraha, Mohit Bansal, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 22
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Blocking intestinal inflammation by aspirin alleviates chicken necrotic enteritis. Mohit Bansal, Mussie Abraha, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 159.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: The effect of different bile salts on Campylobacter jejuni colonization in chickens. Bilal Al-Rubaye, Mussie Abraha, Ayidh Almansour, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 211
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Deoxycholic acid modulates anaerobes to reduce Campylobacter jejuni chicken colonization. Bilal Al-Rubaye, Mussie Abraha, Ayidh Almansour, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. ABI annual meeting in Little Rock, Arkansas, September 25, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: SPF-Anaerobe microbiota mediates Campylobacter jejuni clones and campylobacterosis in broiler chickens. Ayidh Almansour, Bilal Al-Rubaye, Mussie Abraha, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. Symposium on Gut Health in Production of Food Animals in St. Louis, Missouri, November 5-7, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Deoxycholic acid modulating anaerobes reduces Campylobacter jejuni chicken colonization. Bilal Al-Rubaye, Mussie Abraha, Ayidh Almansour, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. 99th Conference of Research Workers in Animal Diseases (CRWAD) in Chicago, Illinoi, December 2-4, 2018.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Alrubaye, B., Abraha, M., Almansour, A., Bansal M., Wang, H., Kwon, YM., Huang, Y., Hargis, B., and Sun, X. 2019. Microbial metabolite deoxycholic acid shapes microbiota against Campylobacter jejuni chicken colonization. PLoS One. 14(7):e0214705. doi: 10.1371/journal.pone.0214705. eCollection 2019. (Corresponding author)
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Sun, X., and Wang, Hong. 2019. A new, economical, and easy protocol to culture 3D mouse hepatoid and cholangoid. International Journal of Clinical Gastroenterology and Hepatology (IJCGH): https://www.raftpubs.com/International-Journal-of-Clinical-Gastroenterology-and-Hepatology/articles/IJCGH_raft1002 1: 07-11. (Corresponding autho
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Clostridium perfringens enterotoxin induces chicken necrotic enteritis. Mussie Abraha, Mohit Bansal, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Symposium on Gut Health in Production of Food Animals in St. Louis, Missouri, November 5-7, 2018.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Pope JL, Yang Y, Newsome RC, Sun W, Sun X, Ukhanova M, Neu J, Issa JP, Mai V, Jobin C. 2019. Microbial Colonization Coordinates the Pathogenesis of a Klebsiella pneumoniae Infant Isolate. Sci Rep. 9(1):3380. doi: 10.1038/s41598-019-39887-8.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang H, Cardenas J D, Bansal M, Abraha M, Al-Rubaye B, Telle G, Hargis B, and Sun X. 2019. Microbial metabolite deoxycholic acid controls Clostridium perfringens-induced chicken necrotic enteritis through attenuating inflammatory cyclooxygenase signaling. Sci Rep 9: 14541. DOI: 10.1038/s41598-019-51104-0. (Corresponding author)
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Modulating microbiota and metabolome to reduce Campylobacter jejuni colonization in chickens. A.M. Almansour, M. Abraha, B. Alrubaye, M. Bansal, H. Wang, B.M. Hargis, X. Sun. CRWAD Proceedings. 2019. P129.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Increased ileal deoxycholic acid levels were associated with reduced necrotic enteritis in broiler chickens. M. Bansal, R. Liyanage, M. Abraha, A. Almansour, H. Wang, A. Gupta, B. Hargis, and X. Sun. Poult. Sci. 98(E-Suppl. 1). 94
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Microbiota metabolite deoxycholic acid-modulated anaerobes attenuate chicken transmission-exacerbated campylobacteriosis in Il10?/? mice. X. Sun, H. Wang, B. Alrubaye, M. Abraha, A. Almansour, and M. Bansal. Poult. Sci. 98(E-Suppl. 1). 203
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dietary deoxycholic acid reduces necrotic enteritis and modifies bile acid composition. M. Bansal, A. Almansour, H. Wang, A. Gupta, B.M. Hargis, X. Sun, M. Abraha, R. Liyanage. CRWAD Proceedings. 2019. p72.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dietary deoxycholate modifies chicken intestinal bile acid and reduces Campylobacter jejuni colonization. M. Bansal, R. Liyanage, B. Alrubaye, A. Almansour, H. Wang, Y. Fu, A. Gupta, X. Sun, M. Abraha, T. Alenezi. CRWAD Proceedings. 2019. P224.
• 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: Book Chapters Status: Published Year Published: 2020 Citation: Sun X. 2020. Chapter 7: Role of Gut Microbiome in Colorectal Cancer. Book: Gut Microbiome and Its Impact on Health and Diseases. pp 153-165. https://www.springer.com/gp/book/9783030473839
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mohit Bansal, Ying Fu, Ayidh Almansour, Mussie T. Abraha, Tahrir Aleneji, Danielle Graham, Anamika Gupta, Hong Wang, Rohana Liyanage, Billy M. Hargis, Xiaolun Sun. Evaluation of bile acids against chicken necrotic enteritis. PSA Annual meeting. Online, July 20-22, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ying Fu, Mohit Bansal, Ayidh Almansour, Tahrir Alenezi, Mussie T. Abraha, Hong Wang, Danielle Graham, Billy M. Hargis, Xiaolun Sun. Clostridium perfringens vaccine prevents chicken necrotic enteritis. PSA Annual meeting. Online, July 20-22, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mohit Bansal, Ying Fu, Ayidh Almansour, Tahrir Aleneji, Mussie T. Abraha, Danielle Graham, Anamika Gupta, Hong Wang, Billy M. Hargis, Xiaolun Sun. Mammalian target of rapamycin signaling modulates chicken necrotic enteritis. PSA Annual meeting. Online, July 20-22, 2020.
• 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: Journal Articles Status: Published Year Published: 2021 Citation: Nallasamy, P., Kang, Z. Y., Sun, X., Anandh Babu, P. V., Liu, D., Jia, Z. (2021). Natural Compound Resveratrol Attenuates TNF-Alpha-Induced Vascular Dysfunction in Mice and Human Endothelial Cells: The Involvement of the NF-$\kappa$B Signaling Pathway. International Journal of Molecular Sciences, 22(22), 12486.
• 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: 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: 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: Conference Papers and Presentations 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: 2017 Citation: Microbiome improves growth performance and attenuates Clostridium perfringens challenge. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. PSA in Orlando, Florida, July 17-20, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Relationship between the microbiome in different sections of the gastrointestinal tract of broiler chickens fed a corn versus a rye based diets. M. Baxter, JD Latorre, Si Hong, S. Ricke, X. Sun, B. Hargis and G. Tellez. PSA in Orlando, Florida, July 17-20, 2017.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Locoregional effects of microbiota in a preclinical model of colon carcinogenesis. 2017. Sarah Tomkovich, Ye Yang, Kathryn Winglee, Josee Gauthier, Marcus M�hlbauer, Xiaolun Sun, Mansour Mohamadzadeh, Xiuli Liu, Patricia Martin, Gary P. Wang, Eric Oswald, Anthony A. Fodor and Christian Jobin. Cancer Research (IF 9.6). DOI: 10.1158/0008-5472.CAN-16-3472
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Clostridium perfringens overgrowth, virulence and host response in chicken necrotic enteritis. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. ASM Microbe in New Orleans, LA June 1-5, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Intestinal diseases aggravate Campylobacter jejuni infection potential in chickens. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. IAFP in Tampa, Florida, July 912, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Microbial metabolite deoxycholic acid attenuates necrotic enteritis. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. Symposium on Gut Health in Production of Food Animals in St. Louis, Missouri, November 13-15.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Xiaolun Sun, K. W., Raad Z. Gharaibeh, Josee Gauthier, Zhen He, Prabhanshu Tripathi, Dorina Avram, Steven Bruner, Anthony A. Fodor, Christian Jobin. 2018. Microbiota-derived Metabolic Factors Reduce Campylobacteriosis in Mice. Gastroenterology (IF 20.8). 154 (6):1751-1763.e2. DOI: https://doi.org/10.1053/j.gastro.2018.01.042. (Co-Corresponding author)
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Xiaolun Sun and Zhenquan Jia. 2018. Microbiome modulates intestinal homeostasis against inflammatory diseases. Review. Veterinary Immunology and Immunopathology (IF 1.7). 205: 97-105. https://doi.org/10.1016/j.vetimm.2018.10.014 (Corresponding author)
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Al-Rubaye, B., Abraha, M., Almansour, A., Bansal, M., Wang, H., Kwon, Y.M., Huang, Y., Hargis, B., and Sun, Xiaolun. Microbiota metabolic product deoxycholic acid controls chicken necrotic enteritis. Submitted to Scientific Reports (IF 4.1) in Nov. 2018. (Corresponding author)

Progress 10/01/19 to 09/30/20

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 haveexperiencedon broiler and mouse experimentdesigning, running, and analyzing. Mohit Bansal andAyidh Almansour have extracted and analyzed bile acid data. Ying Fu and Tahrir Alenezi have runmolecularbiological 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 a leading journal. What do you plan to do during the next reporting period to accomplish the goals?We willidentify the virulence factors ofClostridium perfringenson induction of intestinal diseases in chickens and humans. We will developC. perfringensvaccine and monoclonal antibodies. We will continue to work on identifying specific microbes and microbial metabolites on preventingCampylobacterjejunicolonization.

Impacts
What was accomplished under these goals? For goal A, we have found that mouse or DCA-fed chicken microbiota prevented chickens againstC. jejunicolonization and the main protective microbiota was from anaerobes. We are working on identifying the specific individual or groups of protective bacteria using co-culturing and 16S DNA sequencing methods.We also have found that microbiota-metabolized primary bile acids inhibitedC. jejunigrowthwhile somemicrobiota-metabolized secondarybile acids facilitate its growth. For goal C, we have found thatDCA-fed chicken anaerobic microbiotaattenuatedchicken-transmittedC. jejuni-induced colitis (campylobacteriosis) usingIl10-/-mouse infection model. The reduction of campylobacteriosis was derived from reduced proinflammatory response and neutrophil infiltration. For goal D, we have found that secondary bile acid deoxycholic acid and lithocholic acid prevented Clostridium perfringens-induced necrotic enteritis. We aslo found that mTOR signaling mediatedClostridium perfringens-induced necrotic enteritis and blocking mTOR by rapamycin reduced intestinal inflammation. In addition, we found that aClostridium perfringensvaccine effectively reduced clinical severeClostridium perfringens-induced necrotic enteritis.

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: Book Chapters Status: Published Year Published: 2020 Citation: Sun X. 2020. Chapter 7: Role of Gut Microbiome in Colorectal Cancer. Book: Gut Microbiome and Its Impact on Health and Diseases. pp 153-165. https://www.springer.com/gp/book/9783030473839
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mohit Bansal, Ying Fu, Ayidh Almansour, Tahrir Aleneji, Mussie T. Abraha, Danielle Graham, Anamika Gupta, Hong Wang, Billy M. Hargis, Xiaolun Sun. Mammalian target of rapamycin signaling modulates chicken necrotic enteritis. PSA Annual meeting. Online, July 20-22, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mohit Bansal, Ying Fu, Ayidh Almansour, Mussie T. Abraha, Tahrir Aleneji, Danielle Graham, Anamika Gupta, Hong Wang, Rohana Liyanage, Billy M. Hargis, Xiaolun Sun. Evaluation of bile acids against chicken necrotic enteritis. PSA Annual meeting. Online, July 20-22, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ying Fu, Mohit Bansal, Ayidh Almansour, Tahrir Alenezi, Mussie T. Abraha, Hong Wang, Danielle Graham, Billy M. Hargis, Xiaolun Sun. Clostridium perfringens vaccine prevents chicken necrotic enteritis. PSA Annual meeting. Online, July 20-22, 2020.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The research results benefit poultry industry, acedemic field, consumers, and health field. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have trained five graduate students Mohit Bansal, Ayidh Almansour, Mussie Abraha, Ying Fu and Tahrir Alenezi on designing, running, and analyzing broiler and mouse experiments. They are also trained to run molecular and biological analysis. 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 of Scientific Reports and Plos One. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on identifying specific microbes and microbial metabolites on preventing C. jejuni colonization and necrotic enteritis in chickens.

Impacts
What was accomplished under these goals? After a number of the experiments in 2019, many new results were found. Briefly, on objectives C, we found that chicken transmission exacerbates C. jejuni-induced enteritis, while deoxycholic acid (DCA)-modulated anaerobes attenuated the surge of C. jejuni infection ability. Briefly, after only 6 days post infection, Il10-/- mice infected with chicken-transmitted Cj-P1 showed clinical sign of enteritis as diarrhea, fur ruffling, and hunching, while mice infected with Cj AR101 didn't. At cellular level, AR101 induced mild intestinal inflammation and increased histopathology score in Il10-/- mice, showing as crypt hyperplasia and immune cell infiltration. Notably, Cj-P1 induced more severe campylobacteriosis compared to AR101, suggesting increased C. jejuni infection capacity after its chicken colonization and transmission. Importantly, mice infected with Cj-P1-DCA-Anaero (isolated from birds colonized with DCA anaerobes) showed attenuation of intestinal inflammation compared to mice infected with Cj-P1. At molecular level, Cj-P1 induced elevated inflammatory mediator mRNA accumulation of Il17a, Il1β, and Cxcl1 in the Il10-/- mouse colon compared to AR101, while Cj-P1-DCA-Anaero reduced the inflammatory gene expression. On objective A and B , we found that specific pathogen free anaerobes (SPF-Anaero) prevented against C. jejuni chicken colonization and improved body weight gain. Briefly, birds colonized with SPF-Anaero and infected with C. jejuni grew faster compared to infected bird at d28 (56.4 vs. 49.2 g/bird). SPF-Anaero reduced 99.6% and 98.6% of C. jejuni cecal colonization at d21 (1x 104 vs. 3 x 106) and d28 (4x104 vs. 3x106 CFU/bird), respectively, compared to infected birds. Notably, transplanting SPF-Anaero increased Bacteroidetes (47.60% vs. 12.33%) compared to infected control birds. Untargeted metabolomics analysis revealed that primary bile acid cholic acid and its isomers were enriched in cecal digesta of SPF-Anaero birds by 1000 folds compared to infected birds. Importantly, cholic acid inhibited C. jejuni in vitro growth. On objectives D, we found that DCA attenuates NE-induced intestinal inflammation and bile acid deficiency and could be an effective antimicrobial alternative against the intestinal disease. Briefly, at cellular level, birds infected with E. maxima and C. perfringens developed subclinical NE and showed shortening villi, crypt hyperplasia, and immune cell infiltration in ileum. Notably, dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds. Consistent with the increased histopathological scores, subclinical NE birds suffered body weight gain reduction compared to uninfected birds, an effect attenuated with increased doses of dietary DCA. At molecular level, the highest dose of DCA at 1.5 g/kg reduced C. perfringens luminal colonization 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 assay. Upon further examining ileal bile acid pool using targeted metabolomics, subclinical NE reduced 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. In a separate study, we used DCA in drinking water to prevent NE. The birds in the study experienced three levels of NE from no, subclinical, and to acute ileitis, in association with increased severity of coccidiosis respectively. Importantly, DCA at 0.075% drinking water prevented both subclinical and acute NE, showing attenuated body weight gain loss and intestinal inflammation. DCA with lower doses or as treatment was failed to reduce NE and body weight gain loss. As a summary, we found that SPF microbiota and its metabolite DCA prevented C. jejuni chicken colonization and improved bird growth performance. Notably, microbiota reduced chicken-transmission-exacerbated campylobacteriosis. DCA in feed and drinking water prevented acute and subclinical NE in chickens. The application of these findings will promote antimicrobial alternatives to combating infectious pathogens and their induction of intestinal diseases.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: 18. Alrubaye, B., Abraha, M., Almansour, A., Bansal M., Wang, H., Kwon, YM., Huang, Y., Hargis, B., and Sun, X. 2019. Microbial metabolite deoxycholic acid shapes microbiota against Campylobacter jejuni chicken colonization. PLoS One. 14(7):e0214705. doi: 10.1371/journal.pone.0214705. eCollection 2019. (Corresponding author)
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 15. Sun, X., and Wang, Hong. 2019. A new, economical, and easy protocol to culture 3D mouse hepatoid and cholangoid. International Journal of Clinical Gastroenterology and Hepatology (IJCGH): https://www.raftpubs.com/International-Journal-of-Clinical-Gastroenterology-and-Hepatology/articles/IJCGH_raft1002 1: 07-11. (Corresponding author)
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 16. Pope JL, Yang Y, Newsome RC, Sun W, Sun X, Ukhanova M, Neu J, Issa JP, Mai V, Jobin C. 2019. Microbial Colonization Coordinates the Pathogenesis of a Klebsiella pneumoniae Infant Isolate. Sci Rep. 9(1):3380. doi: 10.1038/s41598-019-39887-8.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 19. Wang H, Cardenas J D, Bansal M, Abraha M, Al-Rubaye B, Telle G, Hargis B, and Sun X. 2019. Microbial metabolite deoxycholic acid controls Clostridium perfringens-induced chicken necrotic enteritis through attenuating inflammatory cyclooxygenase signaling. Sci Rep 9: 14541. DOI: 10.1038/s41598-019-51104-0. (Corresponding author)
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: 17. Tomkovich S, Dejea CM, Winglee K, Drewes JL, Chung L, Housseau F, Pope JL, Gauthier J, Sun X, M�hlbauer M, Liu X, Fathi P, Anders RA, Besharati S, Perez-Chanona E, Yang Y, Ding H, Wu X, Wu S, White JR, Gharaibeh RZ, Fodor AA, Wang H2 Pardoll DM, Jobin C, Sears CL. 2019 Human colon mucosal biofilms from healthy or colon cancer hosts are carcinogenic. J Clin Invest. 130:1699-1712. doi: 10.1172/JCI124196. eCollection.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Increased ileal deoxycholic acid levels were associated with reduced necrotic enteritis in broiler chickens. M. Bansal, R. Liyanage, M. Abraha, A. Almansour, H. Wang, A. Gupta, B. Hargis, and X. Sun. Poult. Sci. 98(E-Suppl. 1). 94
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Microbiota metabolite deoxycholic acid-modulated anaerobes attenuate chicken transmission-exacerbated campylobacteriosis in Il10?/? mice. X. Sun, H. Wang, B. Alrubaye, M. Abraha, A. Almansour, and M. Bansal. Poult. Sci. 98(E-Suppl. 1). 203
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dietary deoxycholic acid reduces necrotic enteritis and modifies bile acid composition. M. Bansal, A. Almansour, H. Wang, A. Gupta, B.M. Hargis, X. Sun, M. Abraha, R. Liyanage. CRWAD Proceedings. 2019. p72.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: DCA-modulated anaerobes attenuate chicken transmission-exacerbated campylobacteriosis in Il10-/- mice. Y. Fu, H. Wang, X. Sun, M. Abraha, A.M. Almansour, B. Alrubaye, M. Bansal. CRWAD Proceedings. 2019. P91.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Modulating microbiota and metabolome to reduce Campylobacter jejuni colonization in chickens. A.M. Almansour, M. Abraha, B. Alrubaye, M. Bansal, H. Wang, B.M. Hargis, X. Sun. CRWAD Proceedings. 2019. P129.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: An anaerobic microbiota increases broiler chicken growth performance. M. Abraha, H. Wang, A. Gupta, B. Hargis, X. Sun, M. Bansal, A.M. Almansour. CRWAD Proceedings. 2019. P147.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Campylobacter jejuni growth in the presence of various bile acids. T. Alenezi, H. Wang, Y. Fu, X. Sun, M. Abraha, A.M. Almansour, M. Bansal. CRWAD Proceedings. 2019. P177.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dietary deoxycholate modifies chicken intestinal bile acid and reduces Campylobacter jejuni colonization. M. Bansal, R. Liyanage, B. Alrubaye, A. Almansour, H. Wang, Y. Fu, A. Gupta, X. Sun, M. Abraha, T. Alenezi. CRWAD Proceedings. 2019. P224.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The research results benefit poultry industry, acedemic field, consumers, and health field. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have trained five graduate students Bilal Al-Rubaye, Mohit Bansal, Ayidh Almansour, Mussie Abraha, and Abdulghani Naeem on designing, running, and analyzing broiler and mouse experiments. They are also trained to run molecular and biological analysis. 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), Symposium on Gut Health in Production of Food Animals, Conference of Research Workers in Animal Diseases (CRWAD), and ABI annual meeting. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on identifying specific microbes and microbial metabolites on preventing C. jejuni colonization and necrotic enteritis in chickens using 16S rDNA sequencing, metabolomic analysis, and HPLC/MS assay.

Impacts
What was accomplished under these goals? After a number of the experiments in 2018, many observations were found. Briefly, on objective A, broiler chickens fed 1.5 g/kg secondary bile acid deoxycholic acid (DCA) diet grew significantly heavier (1.45 vs. 1.29 kg/bird) and were colonized with fewer C. jejuni strain 81-176 (100% reduction) or AR 101 (93% reduction) at 28 days of age compared to birds fed basal diet. Interestingly, primary bile acid cholic acid (CA) and secondary bile acid lithocholic acid (LCA) and urodeoxycholic acid (UDCA) didn't change body weight or reduce C. jejuni AR101 chicken colonization. Notably, DCA diet induced a distinct microbiota composition of phyla firmicutes (82.7.1 vs. 98.8%) and bacteriotes (16.9 vs. 0.8%) compared to infected control birds. Importantly, DCA modulated anaerobes attenuated 92% of C. jejuni AR101 colonization at d 28 compared to control infected birds. On objectives B, broiler chickens colonized with mouse anaerobes significantly grew heavier (1.745 vs. 1.54 kg/bird) and had better feed efficiency (1.37 vs. 1.85, 26% improvement) at d 28 compared to control birds. Importantly, mouse anaerobes or aerobes reduced more than 90% of C. jejuni AR101 cecal colonization at d 28 compared to infected birds. On objectives D, DCA (1.0 g/kg) attenuated E. maxima and C. perfringens induced growth impairment of daily BWG compared to NE control birds at d 23-26 (68.28 vs. 56.01 g/bird/day). DCA diets (0.8, 1.0 and 1.5 g/kg) significantly reduced the NE induced intestinal pathology compared to NE birds (ileitis score 6.6, 5.6, 2.8 vs 12.4 respectively). DCA diets (1.0 and 1.5 g/kg) significantly reduced the pathogen colonization compared to NE control birds (2.6 × 105 and 2.5 × 107 vs 5.0 × 108 CFU/g ileal digesta, respectively). DCA in drinking water (0.75%) also alleviated the NE-induced intestinal inflammation (pathological score 12.7 vs. 7.2) compared to NE control birds. At molecular level, DCA reduced inflammatory mediator of ileal Infγ mRNA by 72% compared to NE birds. Remarkably, DCA in drinking water reduced C. perfringens luminal colonization in small intestine compared to that in NE control birds (5x106 vs. 5x108 CFU/g digesta). C. perfringens sporulation (Cpe-supe) at as low as 5µl/ml overnight induced Raw 264.7 cell death. Cpe-supe at 15µl/ml induced 90% Raw cell death. On objectives D, infecting splenocytes with C. perfringens induced significantly higher expression of inflammatory cytokines of Infγ, Litaf, IL1β, Mmp9 and Ptgs2 (COX-2 gene) by 1.54, 1.69, 76.47, 1.72, and 8.65 folds respectively compared to noninfected splenocytes. Interestingly, inhibition of the COX signaling by aspirin attenuated murine INFγ- or TNFα-induced inflammatory response in the splenocytes. Notably, aspirin diet significantly attenuated NE-induced growth impairment of daily BWG by 60% compared to noninfected birds at NE phase of 23-26 days of age. Aspirin also attenuated NE-induced histopathological score compared with uninfected (ileitis score 12 vs 16) and villus apoptosis. As a summary, we found that microbiota and its metabolite DCA prevented C. jejuni chicken colonization and improved bird growth performance. DCA and anti-inflammatory medicine aspirin prevented NE in chickens. The application of these findings will promote antimicrobial alternatives to combating infectious pathogens and their induction of intestinal diseases.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Xiaolun Sun, K. W., Raad Z. Gharaibeh, Josee Gauthier, Zhen He, Prabhanshu Tripathi, Dorina Avram, Steven Bruner, Anthony A. Fodor, Christian Jobin. 2018. Microbiota-derived Metabolic Factors Reduce Campylobacteriosis in Mice. Gastroenterology (IF 20.8). 154 (6):1751-1763.e2. DOI: https://doi.org/10.1053/j.gastro.2018.01.042. (Co-Corresponding author)
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Wang, H., Latorre Cardenas, J.D., Bansal, M., Al-Rubaye, B., Tellez, G., Hargis, B., and Sun, Xiaolun. Microbiota metabolic product deoxycholic acid controls chicken necrotic enteritis. Submitted to Frontiers in Immunology (IF 5.5) in Sept. 2018.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Xiaolun Sun and Zhenquan Jia. 2018. Microbiome modulates intestinal homeostasis against inflammatory diseases. Review. Veterinary Immunology and Immunopathology (IF 1.7). 205: 97-105. https://doi.org/10.1016/j.vetimm.2018.10.014 (Corresponding author)
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Al-Rubaye, B., Abraha, M., Almansour, A., Bansal, M., Wang, H., Kwon, Y.M., Huang, Y., Hargis, B., and Sun, Xiaolun. Microbiota metabolic product deoxycholic acid controls chicken necrotic enteritis. Submitted to Scientific Reports (IF 4.1) in Nov. 2018. (Corresponding author)
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Jillian L. Pope, Ye Yang, Rachel C. Newsome, Wei Sun, Xiaolun Sun, Maria Ukhanova, Josef Neu, Jean-Pierre Issa, Volker Mai, and Christian Jobin. Microbial Colonization Coordinates the Pathogenesis of a Klebsiella pneumoniae Infant Isolate. Submitted to Scientific Reports (IF 4.1) in Oct. 2018, in revision now
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Sarah Tomkovich, Christine M. Dejea, Kathryn Winglee, Julia L. Drewes, Liam Chung, Franck Housseau, Jillian L. Pope, Josee Gauthier, Xiaolun Sun, Marcus M�hlbauer, Raad Z. Gharaibeh, Xiuli Liu, Payam Fathi, Robert Anders, Sepideh Besharati, Ernesto Perez-Chanona, Ye Yang, Hua Ding, Xinqun Wu, Shaoguang Wu, James R. White, Anthony Fodor, Drew M Pardoll, Christian Jobin, Cynthia Sears. Human colon mucosal biofilms from healthy or colon cancer hosts are carcinogenic. Submitted to Journal of Clinical Investigation (IF 13.3) in Aug. 2018.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Kimberly Wilson, Whitney Briggs, Audrey Duff, Chasser Kaylin, Xialoun Sun, and Lisa Bielke. Comparison of microbiome and culture techniques for determination of gastrointestinal microbial communities in ceca of chickens. Submitted to Plos One (IF 2.8) in Dec. 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Antibiotic alternative deoxycholic acid alleviates chicken necrotic enteritis. Mohit Bansal, Mussie Abraha, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 21.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Microbial metabolite deoxycholic acid in drinking water prevents chicken necrotic enteritis. Mussie Abraha, Mohit Bansal, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 22
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Blocking intestinal inflammation by aspirin alleviates chicken necrotic enteritis. Mohit Bansal, Mussie Abraha, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 159.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: The effect of different bile salts on Campylobacter jejuni colonization in chickens. Bilal Al-Rubaye, Mussie Abraha, Ayidh Almansour, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. Poult. Sci. 97(E-Suppl. 1). 211.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Determination of microbial populations in ceca of chickens by microbiome and bacterial culture techniques: A comparison. K. Wilson, W. Briggs, A. Duff, K. Chasser, X. Sun, and L. Bielke. Poult. Sci. 97(E-Suppl. 1). 94
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Deoxycholic acid modulates anaerobes to reduce Campylobacter jejuni chicken colonization. Bilal Al-Rubaye, Mussie Abraha, Ayidh Almansour, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. ABI annual meeting in Little Rock, Arkansas, September 25, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Clostridium perfringens enterotoxin induces chicken necrotic enteritis. Mussie Abraha, Mohit Bansal, Bilal Al-Rubaye, Ayidh Almansour, Hong Wang, Juan David Latorre Cardenas, Billy Hargis, and Xiaolun Sun. Symposium on Gut Health in Production of Food Animals in St. Louis, Missouri, November 5-7, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: SPF-Anaerobe microbiota mediates Campylobacter jejuni clones and campylobacterosis in broiler chickens. Ayidh Almansour, Bilal Al-Rubaye, Mussie Abraha, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. Symposium on Gut Health in Production of Food Animals in St. Louis, Missouri, November 5-7, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Deoxycholic acid modulating anaerobes reduces Campylobacter jejuni chicken colonization. Bilal Al-Rubaye, Mussie Abraha, Ayidh Almansour, Mohit Bansal, Hong Wang, Billy Hargis, and Xiaolun Sun. 99th Conference of Research Workers in Animal Diseases (CRWAD) in Chicago, Illinoi, December 2-4, 2018.

Progress 04/18/17 to 09/30/17

Outputs
Target Audience:Poultry Science researchers and Poultry Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have trained two graduate students Bilal Al-Rubaye and Mohit Bansal on designing, run, and analyze broiler experiments. They are also trained to run molecular and biological analysis. How have the results been disseminated to communities of interest?The above findings have been reported on professional conference meetings including the American Society for Microbiology (ASM), the International Association for Food Protection (IAFP), and the Poultry Science Association (PSA). What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on identifying specific microbes and microbial metabolites on preventing C. jejuni colonization and necrotic enteritis in chickens.

Impacts
What was accomplished under these goals? Antimicrobial resistance has posed a serious threat to agricultural and medical industries. Antimicrobial free feeding regiments are encouraged from consumers, food industry and regulatory agents, but the practices are hurdled by the heavy poultry production loss at a tag of $ 6 billion/per year because of outbreak of intestinal diseases such as coccidiosis and necrotic enteritis (NE). In addition, food borne pathogen Campylobacter jejuni causes a cost of $ 2-6 billion around the world. In 2017, we have done various experiments and achieved the following results: For goal A) Determine the efficacy of microbiota from animal resisting C. jejuni colonization on reducing C. jejuni colonization and improve growth performance in broilers and goal B) Identify groups of protective microbes on mitigating C. jejuni colonization improve growth performance in broilers, we have found that anaerobic microbiota isolated from specific pathogen free mouse prevented C. jejuni colonization in chickens and improved chicken growth performance of body weight gain and feed efficiency. Anaerobic microbiota isolated from conventionalized mouse prevented C. jejuni colonization but didn't improve growth performance. For goal C) Assess the impact of microbes and their metabolites on attenuating chicken-transmitted C. jejuni-induced colitis (campylobacteriosis) using Il10-/- mouse infection model, we found that C. jejuni quickly colonized in bird's ceca at 1.0x105 CFU/g cacal content at 2 days post infection (PI) and reached to 2.8x107 CFU/g and 3.1 x107 CFU/g at 7 and 14 days PI, respectively. Importantly, broiler chickens fed 0.15% deoxycholic acid (DCA) diet dramatically resisted against C. jejuni colonization at levels of 0, 9.0x102, and 0 CFU/g at 2, 7 and 14 days PI, respectively. Furthermore, DCA diet improved broilers productivity of body weight gain at 3.20 vs. 2.86 lb at 28 days of age, a 11.9% increase, and accumulative feed conversion ratio at 1.51 vs. 1.81, a 16.6% improvement compared to control diet. DCA diet also modified microbiota at ceca, where phylum bacteroidetes was increased (16.94 vs. 0.06%) but firmicutes was decreased (82.71 vs. 99.58%) compared to control diet. Surprisingly, physiological level DCA at 1mM slightly enhanced but not inhibit C. jejuni growth for 2 days in BHI broth (OD600 at 0.213 vs. 0.153), suggesting that DCA may not directly target the pathogen. These data indicate that DCA prevents C. jejuni colonization in chickens. We are working on finding animal facility to run the mouse infection experiment. For goal D) Identify selected microbiota and metabolites improve growth performance and protect against coccidiosis- and C. perfringens-induced NE, we found that DCA at as low as 50 µM inhibited 99.92% of C. perfringens growth in Tryptic Soy Broth, while CA and conjugated primary bile acid TCA failed to reduce the bacterial growth. Notably, DCA diet promoted bird growth performance on body weight gain before infection (d 0-18). NE by C. perfringens infection exacerbated E. maxima-induced growth performance loss. Importantly, DCA diet prevented against growth performance loss by coccidiosis and NE. CA diet attenuated body weight gain loss at NE but failed at coccidiosis compared to NE control birds. Upon examining molecular and cellular events, we found that DCA attenuated necrotic enteritis-induced severe intestinal inflammation. NE also induced strong inflammatory mRNA accumulation of Infγ, Tnfα, and Mmp9, effects attenuated by 51%, 82%, and 66% in DCA birds. In conclusion, dietary microbial metabolite DCA improves broiler growth performance and prevented E. maxima- and C. perfringens-induced NE and inflammatory response. Based on these novel findings of microbial metabolites secondary bile acid DCA improving bird growth performance and prevents NE and Campylobacter jejuni colonization in chickens, I filed a patent for this discovery on July 8, 2017. The patent title is: "Secondary bile acids composition and methods of use". The application of the findings will greatly reduce food borne bacterial pathogen Campylobacter jejuni in chickens. The new technology will also reduce NE and its induced productivity loss using antimicrobial alternatives.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Locoregional effects of microbiota in a preclinical model of colon carcinogenesis. 2017. Sarah Tomkovich, Ye Yang, Kathryn Winglee, Josee Gauthier, Marcus M�hlbauer, Xiaolun Sun, Mansour Mohamadzadeh, Xiuli Liu, Patricia Martin, Gary P. Wang, Eric Oswald, Anthony A. Fodor and Christian Jobin. Cancer Research (IF 9.6). DOI: 10.1158/0008-5472.CAN-16-3472 Natural Products Targeting on Oxidative Stress and Inflammation: Mechanisms, Therapies, and Safety Assessment. Accepted 24 August 2017. Zhenquan Jia, Anandh B. P. Velayutham, Wei Chen, and Xiaolun Sun. Oxidative Medicine and Cellular Longevity (IF 4.6). Editorial Review.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Clostridium perfringens overgrowth, virulence and host response in chicken necrotic enteritis. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. ASM Microbe in New Orleans, LA June 1-5, 2017. Intestinal diseases aggravate Campylobacter jejuni infection potential in chickens. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. IAFP in Tampa, Florida, July 912, 2017. Microbiome improves growth performance and attenuates Clostridium perfringens challenge. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. PSA in Orlando, Florida, July 17-20, 2017. Relationship between the microbiome in different sections of the gastrointestinal tract of broiler chickens fed a corn versus a rye based diets. M. Baxter, JD Latorre, Si Hong, S. Ricke, X. Sun, B. Hargis and G. Tellez. PSA in Orlando, Florida, July 17-20, 2017. Microbial metabolite deoxycholic acid attenuates necrotic enteritis. Hong Wang, Juan David Latorre Cardenas, Guillermo Tellez, Billy Hargis, and Xiaolun Sun. Symposium on Gut Health in Production of Food Animals in St. Louis, Missouri, November 13-15.