A NOVEL PROBIOTIC TO INCREASE CHICKEN GUT INTEGRITY AND MATURATION AT EARLY LIFE TO IMPROVE HEALTH AND DISEASE RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1029733
• Grant No.: 2023-67015-39078
• Cumulative Award Amt.: $635,000.00
• Proposal No.: 2022-08136
• Project Start Date: Feb 1, 2023
• Project End Date: Jan 31, 2027
• Grant Year: 2023
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011

Performing Department
(N/A)

Non Technical Summary
After hatching, commercially reared chickens are mainly exposed to environmental bacteria, including Enterobacteriaceae, that negatively affect animal health and productivity and are of food safety concern. However, early exposure to some key gut bacteria, like Segmented filamentous bacteria (SFB), could help shape a healthy gut, which in turn can improve overall health. We propose to evaluate how SFB drive chicken gut maturation and prevent colonization of some bacteria, such as Salmonella, in chickens. Our specific objectives are to 1) develop an SFB-inoculum for the treatment of chickens; 2) evaluate how SFB treatment increases gut maturation and immune stimulation; and 3) test the resistance against intestinal Enterobacteriaceae, e.g., Salmonella enterica species in chickens. Overall, we expect the proposed treatment will improve gut maturation in early life, which will increase poultry health and productivity.

Animal Health Component

20%

Research Effort Categories

Basic

70%

Applied

20%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3210	1040	25%
308	3210	1100	20%
315	3299	1060	30%
311	3299	1100	25%

Knowledge Area
311 - Animal Diseases; 308 - Improved Animal Products (Before Harvest); 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3210 - Egg-type chicken, live animal; 3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1100 - Bacteriology;

Keywords

enterobactericeae

kinome

salmonella

segmented filamentous bacteria

gut

hens

probiotic

Goals / Objectives
Segmented filamentous bacteria (SFB) are considered keystone bacteria thatplay a major beneficial role in shaping a healthy gut in early life in mice. SFB are found in humans and other animals, including poultry, and are host-specific. The goal of this project is to study SFB in chickens and evaluate the probiotic potential of chicken SFB in conventional layers. Built upon our preliminary data, the major objectives of the project are to 1) optimize SFB-inoculum for conventional chicks' treatment by improving the purity and in vitro growth conditions; 2) determine how SFB treatment enhances intestine maturation and immune stimulation in conventional chicks, and 3) evaluate broad protection of the treatment against bacteria that have the potential to generate inflammation in the gut, e.g., Enterobacteriaceae andcause risk to humans. Challenge studies will be performed.

Project Methods
A multidisciplinary research approach will address the project's objectives. The SFB treatment inoculum will be prepared by collecting SFB from conventionalchicken ileum. The SFB purity will be increased by using different steps of treatments and isolation, e.g., chloroform, antibiotics, etc., and cell sorting of tagged SFB using Flow cytometry.SFB treatment dose titration will be performed in vivo for optimal colonization and lack of toxicity. Microscopy observation and qPCR testing will be used to determine SFB abundance.We will use both chicken intestine explant and live chickens to study host-bacteria interactions. To evaluate the chicken gut modulation by SFB, we will use multiple approaches to study chickens at the cellular and intracellular levels, including a chicken-specific kinome, ELISA, quantitative RT-PCR, and in vitro validation. 16S rRNA sequencing and bioinformatic-based approaches will be used to determine how the treatment affects the gut microbiota.Finally, to assess SFB broad protection against bacteria, we will evaluate whether an optimal SFB dose would increase resistance to total Enterobacteriaceae, particularly Salmonella, in chickens using in vivo and in vitro challenges. In each experiment, positive and negative controls will be included. The number of animals to be tested is calculated to generate statistically significant differences among treatment groups. All data will be analyzed using adequate software.The institution and PD will train all students involved in this work for BSL2 and animal work.The results of these studies will be disseminated to the scientific community via publications in high-impact, peer-reviewed journals and presentations at PD and scientific meetings.Accomplishments will be evaluated based on the number of conference/meeting updates completed, the number of research reports published on various platforms, and the implementation of results by the industry.

Progress 02/01/24 to 01/31/25

Outputs
Target Audience:The target audience of the research project during this reporting period includes the poultry industry and research professionals in poultry and meat production, poultry and other meat consumers, researchers and legislatures involved in the regulation and research regarding improving animal health and productivity in food animal production. In addition, this project has participant and education targets such as graduate students who participate in the described studies, as well as undergraduate and graduate students participating in courses instructed by the PD at Iowa State University. Participants in the graduate-supported research of this project include members of the interdepartmental Graduate Microbiology Program at Iowa State University. Changes/Problems:Attempts to grow chicken SFB from intestinal scraping in vitro with intestinal epithelial cells, similar to what has been demonstrated in mice, were not very successful. In conditions replicating what has been shown in mice, SFB are stagnant and quickly overcome by competing bacteria. However, if chloroform treatment is applied to mimic the in vivo inoculation, SFB remain stagnant, and no other contaminants grow. We hypothesized that metabolic differences between SFB isolated from murine and avian species. To ascertain the metabolic requirements of avian-SFB compared to murine, with Kent Corp fund support, we sequenced, constructed, and annotated the chicken-SFB genome. Metabolic network analysis highlights the small genome of all SFB (~1.6 mb) and nutritional requirement differences between SFB isolated from mice, which explains their inability to grow using mice SFB-adapted media. Efforts are ongoing to utilize the metabolic network analysis to determine key nutrients to trigger chicken SFB germination and growth in in vitro conditions. What opportunities for training and professional development has the project provided?Three graduate students were involved in this project and were trained by Dr. Mellata. We also involved undergraduate Honors students (for credits). Students had training in bacterial food safety and animal experiments. New research skills acquired from this project include techniques in animal work (inoculations, bleeding, necropsy, processing organs), basic microscopy and live-cell imaging, and plating on selective media. This project provided students opportunities for extensive training in microbiology, immunology, and probiotic development. This impacted their perceptions of food safety issues and increased their understanding of basic science and molecular microbiology, which is essential to the ability of future generations to ensure a safe and quality product to improve animal health. Introducing food safety issues to young people increases their awareness and prepares them for successful careers in the field. This funding has allowed students to acquire proficiency in laboratory skills and increased scientific competencies. It will advance students to graduate education positions and retention of students and young scientists in agricultural-related fields. How have the results been disseminated to communities of interest?The project and results have been disseminated through scientific conferences, publication in peer-reviewed journals, and through media. What do you plan to do during the next reporting period to accomplish the goals?Finalize Aim 1 -High-throughput testing using Chicken Kinome:Tissue samples from chickens were sent to Dr Kogut (Co-PD) for sequencing and analysis (in progress). From the data, we will target pathways that have been affected by the SFB treatment, RT-qPCR will quantify the changes. -Develop germ-free chicken and test SFB.We purchased a flexible film (softwall) isolator (cbc) that was donated by Kent Corp. We will attempt to generategerm-free chickens in Dr. Mellata's lab. The chicken model will be used for SFB in vivo passage. Finalise Aim 2 -Testing IgA and Short Chain Fatty acids (SCFA) production: In the ongoing chicken experiments, we are collecting samples for antibody and SCFAs analyses.

Impacts
What was accomplished under these goals? Objective 1: Optimize SFB-inoculum for conventional chicks' treatment Refinement of the SFB Inoculum. To increase the purity of SFB inoculum, we have added steps to the collection procedure: 1) Distal ilea were collected individually, the content completely removed, and the ileum flushed three times with PBS prior to scraping of mucosal surface. All procedures are done in a biosafety cabinet to improve sterility. 2) Further, the previously described chloroform treatment was performed on individual scraping samples and plated on Luria Bertani and Brain Heart Infusion (BHI) agar and incubated in all three atmospheric conditions outlined. Only individual scraping aliquots that show the absence of culturable microbes and are PCR-positive for SFB are pooled together for SFB inoculation to chickens. In vitro growth optimization of SFB: In conditions replicating what has been demonstrated in mice, chicken SFB are stagnant and quickly overcome by competing bacteria.With chloroform treatment,SFB remain stagnant and no other contaminants grow. These data indicatepotentialmetabolic differences between SFB isolated from murine and avian species. To ascertain the metabolic requirements of avian-SFB compared to murine, we sequenced, constructed, and annotated the chicken-SFB genome. Metabolic network analysis highlights the small genome of all SFB (~1.6 mb) and nutritional requirement differences between SFB isolated from different hosts. Efforts are ongoing to utilize the metabolic network analysis to determine key nutrients to trigger SFB germination and growth in vitro conditions. SFB enrichment through a passage in germ-free chick: Not yet started SFB treatment dose titration in vivo for optimal colonization. We tested a lower SFB dose (~7 102) in conventional chickens, which is lower than the one previously tested in SPF chickens (~104 SFB) and it worked very well. Objective 2: To determine how SFB treatment enhances intestine maturation and immune stimulation in conventional chicks. We tested for the first time SFB treatment in conventional hens from commercial farms in Iowa, and we tested the effect of the treatment on basic gut parameters (Aim 2.1.). In this sub-aim, we assessed the gut maturation, we submitted the microbiome samples for sequencing (in progress). We also worked on sub-Aim 2.2. to assess SFB-associated mechanism of immuno-metabolic activation. We tested antimicrobial peptides (AMPs) production; T cell-associated activity. mTOR-associated activity; AM peptides (AMPs). The Kinome samples were sent to Dr Kogut for testing and analyzing. We also sent the microbiome samples for sequencing. Testing the short-chain fatty acids (SCFAs) responses has not been done yet. Objective 3: SFB protection to Enterobacteriaceae and Salmonella enterica species in vivo: the protection has been successfully demonstrated in vitro, but the in vivo challenge has not yet started.

Publications

• Type: Websites Status: Published Year Published: 2023 Citation: Mellatas research highlighted in the Iowa Stater Sustainable Safety: Preventing Illness Through Protective Probiotics Fall 2023. Sustainable Safety - Fall 2023 - Iowa Stater. https://iowastater.iastate.edu/fall-2023/article/sustainable-safety
• Type: Websites Status: Published Year Published: 2023 Citation: Mellatas research was highlighted in the College of Agriculture and Life Sciences-Iowa State University. Researcher aims to increase food safety through poultry gut health by Ann Robinson 6/14/2023 Researcher aims to increase food safety through poultry gut health | College of Agriculture and Life Sciences. https://www.cals.iastate.edu/news/2023/researcher-aims-increase-food-safety-through-poultry-gut-health
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Mellata M. and J. Meinen-Jochum 2025. Broiler and layer chickens responses to host-specific bacteria colonization in early life. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL Jan 20 (Oral)
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Meinen-Jochum J. and M. Mellata. 2025. Sequencing of the chicken segmented filamentous bacteria genome reveals differences between host-species. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL Jan 20 (Oral)
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Rodriguez-Gallegos J., J. Meinen-Jochum, and M. Mellata. 2025. An avian-specific bacteria for gut immune maturation of newly hatched conventional layer chickens. P102. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL Jan 20 (Poster)
• Type: Websites Status: Published Year Published: 2024 Citation: Mellatas research was highlighted in Agri-View Researcher aims to increase food safety by Ann Robinson. 4/23/2024 Link Researcher aims to increase food safety. https://agupdate.com/agriview/news/livestock/article_1ecabf44-cf5d-11ee-b724-770bf476b06b.html
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Meinen-Jochum J. and M. Mellata. 2024. Benefits of Host-Specific Uncultured Bacteria in Broiler Gut Maturation. HMB-SATURDAY-923. American Society For Microbiology (ASM) Microbes June 16 Atlanta, GA (Poster).
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Meinen-Jochum, J., CJ. Skow, and M. Mellata 2024. Layer segmented filamentous bacteria colonize and impact gut health of broiler chickens. mSphere 9(11):e0049224.

Progress 02/01/23 to 01/31/24

Outputs
Target Audience:The target audience of the research project includes the poultry industry and research professionals in poultry and meat production, poultry and other meat consumers, researchers and legislatures involved in the regulation and research regarding improving animal health and productivity in food animal production. In addition, this project has participant and education targets such as graduate students who participate in the described studies, as well as undergraduate and graduate students participating in courses instructed by the PD at Iowa State University. Participants in the graduate-supported research of this project include members of the interdepartmental Graduate Microbiology Graduate Program at Iowa State University. Changes/Problems:We initially proposed to use a germ-free chicken for SFB passages. However, the inconsistency of this model poses challenges and we thus decided to try passage in egg embryos. What opportunities for training and professional development has the project provided?Two graduate students were involved in this project and were trained by Dr. Mellata. Students had training in bacterial food safety and animal experiments. New research skills acquired from this project include techniques in animal work (inoculations, bleeding, necropsy, processing organs), basic microscopy and live-cell imaging, and plating on selective media. This project provided students opportunities for extensive training in microbiology, immunology, and probiotic development. This impacted their perceptions of food safety issues and increased their understanding of basic science and molecular microbiology, which is essential to the ability of future generations to ensure a safe and quality product to improve animal health. Introducing food safety issues to young people increases their awareness and prepares them for successful careers in the field. This funding has allowed students to acquire proficiency in laboratory skills and increased scientific competencies. It will advance students to graduate education positions and retention of students and young scientists in agricultural-related fields. How have the results been disseminated to communities of interest?Results have been disseminated through scientific conferences, publication in peer-reviewed journals, and through media. What do you plan to do during the next reporting period to accomplish the goals?We have already scheduled a first in vivo experiment to test SFB colonization in conventional hens. In this experiment, we will determine the time course of SFB colonization, gut immune activation, microbiota change, and resistance to bacteria.

Impacts
What was accomplished under these goals? Impact statement: Poultry farmers need naturally derived treatments to improve livestock health and productivity without compromising the quality of poultry products. Because of the importance of the gut in health and productivity, treatment strategies maximizing gut maturity and integrity would highly benefit poultry production. Segmented filamentous bacteria (SFB) have recently emerged as keystone bacterial species that are crucial in shaping a healthy gut in early life via triggering protective immune responses, which improves resistance to bacteria. Although SFB experimental studies have been conducted in mice, SFB have been highly associated with high-performing poultry. Because of SFB host-specificity and the peculiarities of the poultry immune system, we proposed to test the probiotic potential of SFB in chickens, specifically, how SFB drive chicken gut maturation and bacterial resistance in conventional chickens. Objective 1: Optimize SFB-inoculum for conventional chicks' treatment. During the reporting period, we have mainly worked on this objective. We have successfully collected SFB from hens and made a -80 stock for future experiments. We tested different conditions of treatments to eliminate contaminants and we tested in vitro culture for SFB. We were able to minimize the overgrowth of the fast-growing contaminant. We developed new media to assess the growth of SFB and were able to visualize the bacteria using live-cell imaging. We have started testing chicken embryos for SFB passages. Objective 2: Determine whether SFB treatment enhances intestine maturation and immune stimulation in conventional chicks. Not started at reporting period. Objective 3: SFB protection to Enterobacteriaceae and Salmonella enterica species in vivo. Not started at the reporting period.

Publications

• Type: Other Status: Published Year Published: 2023 Citation: Rachel Cramer. 2023. Innovation at work: Sustainable Solutions for Safer Food and Cleaner water Iowa State University News Service 3/28/2 www.news.iastate.edu/news/2023/03/28/safer-food
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jared Meinen-Jochum and Melha Mellata. 2023. Antimicrobial Mechanism of Avian Segmented Filamentous Bacteria in Chickens. # HMB-FRIDAY-929. American Society for Microbiology (ASM) Microbe. Houston, TX June 15-19
• Type: Other Status: Published Year Published: 2023 Citation: Improve Gut Health to reduce Salmonella in Chickens. iHeart WHO Focus (Podcasts)-6/25/23 www.iheart.com/podcast/802-focus-28823485/episode/who-focus-06-25-23-117886286/
• Type: Other Status: Published Year Published: 2023 Citation: Ann Robinson. 2023. Researcher aims to increase food safety through poultry gut health Iowa State University-CALs 6/14/23 www.cals.iastate.edu/news/2023/researcher-aims-increase-food-safety-through-poultry-gut-health
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Meinen-Jochum, J., C.L. Ott, M. Mellata. 2023 Segmented filamentous bacteria-based treatment to elicit protection against Enterobacteriaceae in Layer chickens. Front Microbiol14:1231837. https://doi.org/10.3389/fmicb.2023.1231837
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mellata, M. 2024. Role of Segmented Filamentous Bacteria in Gut Immune Maturation and Resistance to Enterobacteriaceae in Layer Chickens. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL January 19-24 (oral)
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Meinen-Jochum, J., M. Mellata. 2024. Cross-colonization of segmented filamentous bacteria between broilers and layers. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL January 19-24 (oral)