Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
MICROBIOME-MEDIATED COLONIZATION RESISTANCE AGAINST NECROTIC ENTERITIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032117
Grant No.
2024-67016-42415
Cumulative Award Amt.
$650,000.00
Proposal No.
2023-07981
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2027
Grant Year
2024
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
(N/A)
Non Technical Summary
Necrotic enteritis (NE), caused by Clostridium perfringens, ranks among the most financially devastating diseases in poultry. Unfortunately, there are currently no effective preventive or therapeutic measures available. The intestinal bacteria plays a critical role in maintaining animal health and productivity by resisting the colonization and infection of invading pathogens. In a recent screening of a library of chicken intestinal bacteria, we identified several bacteria with a strong ability to inhibit C. perfringens, while also enhancing innate immunity through the induction of host defense peptide (HDP) synthesis. Notably, oral administration of a selected commensal bacterium markedly improved animal survival and alleviated intestinal lesions in a chicken model of NE. These findings provide a timely opportunity to explore the potential of commensal bacteria in resisting NE. In this project, we will screen additional commensal bacteria for their ability to inhibit C. perfringens growth andinduce HDP synthesis. Lead bacterial candidates will be further evaluated for their protective efficacy in NE-infectedchickens. Moreover, we will study the mechanism of action of the most effective bacterium. Upon completion, we expect to identify several commensal bacteria that are highly effective in mitigating NE and further reveal their major mechanism of action. The outcome will facilitate the development of an innovative antibiotic-free approach to combat NE and potentially other enteric diseases in poultry.
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
(N/A)
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132201100100%
Knowledge Area
311 - Animal Diseases;

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

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Necrotic enteritis (NE) is a major poultry disease caused by Clostridiumperfringenswith no effective mitigation strategies. The overall goal of this project is to identify commensal bacteria that are highly effective in suppressing intestinal colonization of C. perfringens and further elucidate their mechanisms of action. We hypothesize that commensal bacteria resistant to C. perfringens colonization confer protection against NE. To test this hypothesis, we will pursue three specific objectives: 1) screening for commensal bacteria showing strong colonization resistance to C. perfringens, 2) determiningthe efficacy of selected bacteria in alleviating NE, and 3) revealing the mechanisms by which the most effective bacterium resists NE. We expect to identify several commensal bacteria that provide robust colonization resistance to NE. The outcomes will pave the way for developing a novel microbiome-based approach to mitigate NE and potentially other enteric diseases in poultry.
Project Methods
The long-term goal of our research is to develop innovative antibiotic-free approaches to combat necrotic enteritis (NE) and other significant enteric diseases. The overall goal of this project is to identify bacteria that are highly effective in resisting intestinal colonization of Clostridium perfringens and further elucidate their mechanisms of action. We hypothesize that commensal bacteria capable of resisting C. perfringens colonization confer protection against NE. To test our hypothesis, we will pursue three specific objectives: 1) Screen for commensal bacteria that exhibit strong colonization resistance to C. perfringens. We will screen a large collection of chicken intestinal bacteria for their ability to inhibit C. perfringens growth and also induce host defense peptide (HDP) synthesis in a cell-based high-throughput screening assay. A minimum of six bacteria that demonstrate strong suppression of C. perfringens growth and/or enhancement of HDP synthesis will be selected for further evaluation to assess their efficacy in resisting the intestinal colonization of C. perfringens in a chicken model of NE. 2) Determine the efficacy of selected commensal bacteria in alleviating necrotic enteritis. We will individually inoculate six selected bacteriainto chickens to evaluate their efficacy in mitigating NE. Animal survival, weight gain, intestinal lesion, and the C. perfringens titer will be compared. The bacterium that proves most effective in improving animal survival and reducing disease severity will be selected for further investigation into its mechanism of action. 3) Elucidate the mechanisms by which the most effective bacterium resists necrotic enteritis. We will assess its impact on the development of the intestinal microbiome, mucosal immunity, and barrier function in healthy and NE-infectedchickens. A comprehensive understanding of the bacterium's colonization resistance mechanism will be achieved through a multi-omics approach that integrates the microbiome composition, metatranscriptome and metabolome with single-cell sequencing-based host transcriptome. Upon completion, we expect to identify several highly effective commensal bacteria that offer robust colonization resistance against NE. The outcome will pave the way for developing a novel microbiome-based, antibiotic-free approach to mitigate NE and potentially other enteric diseases. Given the increasing concern over antibiotic-resistant pathogens and the use of in-feed antibiotics, this research will contribute to ensuring animal health, production efficiency, and food safety, while reducing the risk of antimicrobial resistance.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Livestock producers, veterinary microbiologists, veterinary immunologists, animal feed and health companies, animal nutritionists, and infectious disease and microbiomeresearchers are all interested in the outcomes of the research, which have been presented in professional meetings and published in peer-reviewed scientific journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and one undergraduate students have received research training through participation in various research activities. They have gained invaluable hands-on experience in conducting animal trials, sample collection, postmortem analysis, DNA isolation, 16S rRNA sequencing, and bioinformatics. A few students have participatedin national, regional, and university research conferences andgiven oral or poster presentations. Several studentshave published their research findings in peer-reviewed scientificjournals. Overall, the students have gainedboth oral and written scientific communication skills. How have the results been disseminated to communities of interest?The PD and the students havepresented their research findings at several national, regional. and university research conferences. Additionally, threeresearch manuscripts have beendrafted by the students and have beenpublished in peer-reviewed scientific journals in the past 12 months. Additional manuscripts are being drafted and will be submitted for publication soon. What do you plan to do during the next reporting period to accomplish the goals?Different chicken breedsare known toexhibit notable differences in disease resistance. Several inbred chicken breeds will be compared for their resistance to necrotic enteritis (NE). The intestinal microbiota will be compared between NE-resistant and susceptible breeds. The commensal bacteria that are enriched in NE-resistant breeds will be evaluated for their efficacy in protecting chickens against NE. The results will pave the way for developing novel probiotics for NE mitigation.

Impacts
What was accomplished under these goals? Coccidiosis, caused by Eimeria parasites, is a major predisposing factor for necrotic enteritis. Additionally, coccidiosis remains one of the most economically devastating diseases in poultry, significantly impacting animal health, production performance, and welfare. This disease imposes a substantial economic burden, costing the global poultry industry up to $13 billion annually. However, effective mitigation strategies for coccidiosis remain elusive. While different chicken breeds exhibit varying resistance to coccidiosis, no commensal bacteria have been directly linked to this resistance. To assess the relative resistance of different breeds to coccidiosis, 10-day-old Fayoumi M5.1, Leghorn Ghs6, and Cobb chickens were challenged with 50,000 sporulated Eimeria maxima oocysts or mock-infected. Body weight changes, small intestinal lesions, and fecal oocyst shedding were evaluated on d 17. Ileal and cecal digesta were collected from individual animals on d 17 and subjected to microbiome analysis using 16S rRNA gene sequencing. Our results showed thatFayoumi M5.1 chickens showed the lowest growth retardation, intestinal lesion score, fecal oocyst shedding, and pathobiont proliferation compared to Ghs6 and Cobb chickens. The intestinal microbiota of M5.1 chickens also differed markedly from the other two breeds under both healthy and coccidiosis conditions. Notably, group A Lactobacillus and Ligilactobacillus salivarius were the least prevalent in both the ileum and cecum of healthy M5.1 chickens, but became highly enriched and comparable to Ghs6 and Cobb chickens in response to coccidiosis. Conversely, Weissella, Staphylococcus gallinarum, and Enterococcus durans/hirae were more abundant in the ileum of healthy M5.1 chickens than in the other two breeds. Despite being reduced by Eimeria, these bacteria retained higher abundance in M5.1 chickens compared to the other breeds. In summary, Fayoumi M5.1 chickens exhibit greater resistance to coccidiosis than Leghorn Ghs6 layers and Cobb broilers. Several commensal bacteria, including group A Lactobacillus, L. salivarius, Weissella, S. gallinarum, and E. durans/hirae, are differentially enriched in Fayoumi M5.1 chickens with strong correlation with coccidiosis resistance. These bacteria hold promise as probiotics for coccidiosis mitigation, with potentially beneficial role in NE intervention as well.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Liu J, Guo J, Whitmore MA, Tobin I, Kim DM, Zhao Z, Zhang G. Dynamic response of the intestinal microbiome to Eimeria maxima-induced coccidiosis in chickens. Microbiology Spectrum 2024, 12: e00823-24.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Guo J, Zhao Z, Broadwater C, Tobin I, Liu J, Whitmore MA, Zhang G. Is intestinal microbiota fully restored after chickens have recovered from coccidiosis? Pathogens 2025, 14(1): 81.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Broadwater C, Guo J, Liu J, Tobin I, Whitmore MA, Kaiser MG, Lamont SJ, Zhang G. Breed-specific responses to coccidiosis in chickens: Identification of intestinal bacteria linked to disease resistance. Journal of Animal Science and Biotechnology 2025, 16: 65.