Source: UNIVERSITY OF NEBRASKA submitted to NRP
RISKS ASSOCIATED WITH GENETICALLY ENGINEERED MICROBES IN BEEF CATTLE PRODUCTION: EVALUATION OF PERSISTENCE, TRANSMISSION AND ECOLOGICAL CONSEQUENCES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1029171
Grant No.
2022-33522-38219
Cumulative Award Amt.
$499,913.00
Proposal No.
2022-03025
Multistate No.
(N/A)
Project Start Date
Aug 15, 2022
Project End Date
Aug 14, 2026
Grant Year
2022
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Animal Science
Non Technical Summary
This proposal aligns with the program focused on "Environmental effects of GE relative to non-GE organisms in the context of production systems". With the recent development of tools for genetic engineering and advances in synthetic biology, it is possible to engineer better probiotics that have desired characteristics and functionalities. Microbes with enhanced beneficial functions targeted at increasing animal health and productivity can be used to improve beef cattle production as microbes in the rumen play a critical role in ruminant health and nutrition. To date, no studies have systematically described the effects of feeding genetically engineered microorganisms to beef cattle and how engineered microbes persist, integrate, and stabilize within the rumen microbiome as well as in soil and other environmental microbiomes in the short and long term. The proposed experiments will directly address this knowledge gap by 1) examining ecological changes in the animal's microbiome in rumen, fecal and hide microbiomes; 2) changes in the environmental microbiomes associated with soil; 3) long and short term stability and persistence of the genetically engineered microbe in the animal gut and the feedlot soils; 4) the integration of the genes introduced to other microbes within the gut and soli microbiomes; 5) transmission of the genetically engineered microbe between animals. As such, this study will develop critical information to help evaluate the long and short-term risks associated with utilizing genetically engineered microbes in beef cattle production and will help regulatory agencies make science-based informed decisions on utilizing genetically engineered microbes in beef production.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3074010104075%
3084099107025%
Knowledge Area
308 - Improved Animal Products (Before Harvest); 307 - Animal Management Systems;

Subject Of Investigation
4099 - Microorganisms, general/other; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1070 - Ecology;
Goals / Objectives
Research Objective 1: Determine the effects of feeding a genetically engineered organism to beef cattle on the ecology of microbial community composition and function and the stability and persistence of the genetically modified organisms in the gastrointestinal tract, hide, and soil in the long and short-term using longitudinal sampling.Research Objective 2: Determine movement of the genetically engineered organism from animal to animal.Research Objective 3: Evaluate the movement of the engineered gene from the B. pumilis species to other bacterial species in the rumen, fecal, hide, and soil microbiomes.
Project Methods
Specifically, we will evaluate changes in the microbial community composition and function in the gastrointestinal tract of genetically engineered Bacillus pumilus fed cattle and animals fed the unmodified GRAS approved Bacillus pumilus strain at different stages of the feedlot phase using rumen, fecal, hide, and soil samples using 16S rDNA and shotgun metagenome sequencing. Additionally, stability and persistence of the genetically engineered and non-engineered strains will be evaluated by feeding the engineered or non-engineered strain for 80 days and then stop feeding both strains and evaluating the persistence of the strains over the next 80 days in the rumen, fecal, hide and soil samples. Feedlot pen soil samples will be collected up to a year after animals are removed to evaluate persistence. The resulting information will provide critical information into the persistence, spread, and frequency of the engineered organism and its impact on the animal and the associated environments.We will comingle animals that have been fed the genetically engineered organism with animals fed the GRAS approved B. pumilus strain and monitor the abundance, persistence, and stability of the genetically engineered strain in the animals that were not fed the genetically engineered organism using 16S rDNA and shotgun metagenome sequencing.We will use emulsion, paired isolation and concentration PCR (epicPCR) to link phylogenetic markers to the engineered gene followed by genomic library preparation to identify which bacterial species are associated with the gene engineered to monitor the potential of the engineered gene to move between bacterial species.

Progress 08/15/23 to 08/14/24

Outputs
Target Audience: Target audiance include extension educators, scientists, researchers, livestock producers and policymakers. Changes/Problems: Due to the difficulty in getting the environmental safe gards approved and delay in facility renovation the experiments planned for the spring of 2024 has been futhur delayed. As we are feeding genetically engineered microbes to cattle, IACUC and other approvals were given for the study to be performed in the spring to minimize environmental impact. However, due to delays in facility renovation and environmental safety concerns the study had to be delayed until Spring of 2025. The lead CO-PI at USMARC has finally got the renovations completed and plan to start the feeding trial in January of 2025. However, we do not anticipate this to effect the scope of the grant and all aspects described in the grant will be performed. What opportunities for training and professional development has the project provided? Additionally an undergraduate student was recruited for the microfluidic device fabrication and has resulted in a collaboration between animal science and Dr. Sangjin Ryu at college of engineering at UNL. How have the results been disseminated to communities of interest?We have presented the data from the engineering of the microbe to the scientific community at the Midwest animal Science meeting held at Madison, WI. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting year, we plan to complete the animal experiment and investigate the effects of feeding a genetically engineered organism to beef cattle on the ecology of microbial community composition and function and the stability and persistence of the genetically modified organisms in the rumen of cattle.

Impacts
What was accomplished under these goals? During this time we have been fine tuning our genome editing methods. Additionally, we have developed microfluidic devices needed for microbial cell encapsulation to link host taxonomy to genes of interest to investigate objectives 2 and 3 as described in the study. As such, we have developed all protocols and analyysis methods and awaiting the samples from the feeding trial to evalaute the persistance and transmission of the engineered gene within the rumen, fecal and soil microbiomes. Getting aapprovals and renovations needed for approval of the animal feeding trial has delayed the project at US Meat Animal Research Center (USMARC). However, the lead CO-PI at USMARC has finally got the renovations completed and plan to start the feeding trial in ealy January of 2025.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: WA Tom, JV Judy, PJ Kononoff, SC Fernando. Influence of empirically derived filtering parameters, ASV, and OTU pipelines on assessing rumen microbial diversity. Journal of Dairy Science 2024 Jun 28:S0022-0302(24)00961-5. doi: 10.3168/jds.2023-24479.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Tien Doan, Alison C Neujahr, Samodha C Fernando. Development of genetically engineered microbes as probiotics to enhance ruminant productivity. Midwest Animal Science Meetings, Wisconsin Madison, March 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Seidu Adams, Matthew L Spangler, Samodha Fernando. Oral and rumen microbiome characterization to predict host phenotypes. Midwest Animal Science Meetings, Wisconsin Madison, March 2024
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Alison C Neujahr, Duan S Loy, John Dustin Loy, Bruce W Brodersen, Samodha C Fernando. Rapid detection of high consequence and emerging viral pathogens in pigs. Frontiers in Veterinary Science. Volume 11 - 2024 | https://doi.org/10.3389/fvets.2024.1341783


Progress 08/15/22 to 08/14/23

Outputs
Target Audience:Target audiance include extension educators, scientists, researchers, livestock producers and policymakers. Changes/Problems:Due to the difficulty in getting the environmental safe gards approved and delay in facility construction the experiments planned for the spring of 2023 was delayed. As we are feeding genetically engineered microbes to cattle, IACUC and other approvals were given for the study to be performed in the spring to minimize environmental impact. However, due to delays in facility construction and environmental safety concerns the study had to be delayed until Spring of 2024. However, we do not anticipate this to effect the scope of the grant and all aspects described in the grant will be performed. What opportunities for training and professional development has the project provided?We have recruited a Master's student for this project. this student is gaining expertise in animal nutrition, microbiome analysis, bioinformatics, and molecular biology. He is planning on presenting preliminary data in the comming year. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In the next reporting year, we plan to perform the animla experiment and investigate the effects of feeding a genetically engineered organism to beef cattle on the ecology of microbial community composition and function and the stability and persistence of the genetically modified organisms in the rumen of cattle.

Impacts
What was accomplished under these goals? In terms of research objectives, we are behind schedule mainly due to our collaborators at US Meat animal Research center having difficulty in getting the environmental safe gards approved to perform experiments in the Spring. As we are feeding genetically engineered microbes to cattle, IACUC and other approvals were given for the study to be performed in the spring. However, due to delays in facility construction and environmental safety concerns the study had to be delayed until Spring of 2024. During this time we have been working on using chassis-independent recombinase-assisted genome engineering to develop engineered microbes to contain biosynthetic gene clusters that produce compounds that decrease pathogens that cause liver abscesses in cattle. Additionally, we have started working on microfludics and microbial cell encapsulation methods to link host taxonomy to genes of interest to investigate objectives 2 and 3 as described in the study.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Graham, E.H., Tom, W.A., Neujahr, A.C. et al. The persistence and stabilization of auxiliary genes in the human skin virome. Virol J 20, 49 (2023). https://doi.org/10.1186/s12985-023-02012-3
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Seidu Adams, Nirosh Aluthge, Waseem Abbas, Mathew Spangler, James Wells, Kristin Hales, Larry Khuen Thomas Burkey, Phillip Miller, Samodha C. Fernando. Microbiomes from Theory to Application. Midwest Animal Science meeting Invited lecture.Wisconsin Madison March 2023