RECOVERING MICROBIAL COMMUNITIES IN THE GASTROINTESTINAL TRACT FROM DYSBIOTIC STATES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1021726
• Project Start Date: Nov 25, 2019
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469

Performing Department
School of Food and Agriculture

Non Technical Summary
Gastrointestinal dysbiosis is an immense problem in global animal production industries. Stress from heat, rumen acidosis transportation, and weaning can trigger gut dysbiosis, and affect the gut microbiota which are vital to animal health. Not only can these stressful events cause systemic health problems, but dysbiosis reduces body condition and weight gains, milk production, and immune system function which leaves animals susceptible to infectious disease. In the United States alone, stress from heat, rumen acidosis transportation, and weaning are estimated to each cost billions of dollars in lost revenue and animal health costs. Given the current struggle to make agriculture, in Maine, the United States, and the world, more environmentally and economically sustainable, there is still a dire need to understand the way these factors affect the health and production of animals via their impact on the gut microbiota.To better understand host-microbial interactions in the gut, how a community becomes disordered, and how that community and host health may be recovered, I will integrate cutting-edge technology and methods to first address the technical challenges impeding gut microbiome research. I will then use this technology to investigate healthy- and dysbiotic-state interactions between the host and gut microbiota, as well as therapeutic approaches to reestablish a beneficial gut microbiome. This project will generate the laboratory models and preliminary data that are vital to seeking extramural funding, including from the US Department of Agriculture, Sustainable Agriculture Research and Education (SARE), and National Science Foundation (NSF).Research questions which may be answered using these methods:Can we create a better laboratory model to study how microorganisms interact with animal and human hosts? Are there key species or types of microorganisms that need to be present? Or does there just need to be key functions present regardless of whichmicroorganism is performing it?Do microorganisms assemble along the intestinal walls in structured communities, and can this be used to understand the role of mucus-dwelling microorganisms ?What microbial byproducts are taken up by host cells lining the gut?What host cell byproducts are taken up by microorganisms?Which microorganisms are involved in inflammation in the gut of the host animal or human?If microorganisms are involved in making inflammation better or worse, where do these microbes come from originally? From maternal, dietary, or environmental sources?

Animal Health Component

10%

Research Effort Categories

Basic

30%

Applied

10%

Developmental

60%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3999	1103	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1103 - Other microbiology;

Keywords

host-associated microbiota

host-microbial interactions

rumen

in vitro models

inflammation

probiotics

Goals / Objectives
The primary goal of this research is to better understand the interaction of animal host, gut microbiota, and diet in relation to gastrointestinal health and animal production. The long-term goal, beyond the scope of this project-period, is to identify dietary or microbial therapies to curate a microbial community which incorporates these genotypic or phenotypic factors.Specific objectives include:a. Develop an in vitro co-culture-based model by which to study host-microbe interactions in gastrointestinal tissues more clearly and reproducibly.b. Identify microbial genotypic or phenotypic factors which contribute to or mitigate host tissue inflammation.

Project Methods
Objective 1: There are a number of technical challenges which impede research into host-microbial interactions in the digestive tract. Among them are the need for anaerobic conditions, the difficulty in maintaining over time a co-culture of host epithelial and other specialized cells from the wall on the digestive tract along with a stable community of microbiota, the inadequacy of synthesized mucin to sustain gut microbiota and otherwise allow for a mock GI tract to be recreated in the laboratory, as well as the fastidiousness of gut microbiota and their recalcitrance to be kept in culture. Continuous culture systems, which attempt to create an artificial rumen environment, are commonly used to study the rumen microbiota, however, continuous culture systems lack host epithelia and immune system components.Several recent advances in microfluidics have been applied to intestinal cells (enterocytes) from humans in co-cultures with microorganisms, and effectively recreate an intestinal environment in vitro. Often called "gut chips" or "organ-on-a-chip", commercial versions are available, and several publications detail how to construct these de novo (Trujillo-de Santiago et al. 2018; Jalili-Firoozinezhad et al. 2019). They can be constructed with single or multiple host-cell types, which use a modified liquid cell culture on microfluidic chips to create a simplified version of a complex organ system, while still allowing for the real-world factors (e.g. mucin) which are necessary for the growth of many fastidious gut microorganisms.Importantly, these chips have been used with human enterocytes but not with cells from ruminants. Given the differences in immune system development and function, as well as differences in digestive function and gut microbial communities, "humanized" gut chips cannot be used in lieu of "rumenized" gut chips. Cell cultures from the chambers of the rumen have been previously established (Baldwin 1998; Stumpff et al. 2011), and combined with the protocols from continuous rumen microbial cultures, a chip featuring rumen epithelia can be established.I will further integrate visualization technology to understand spatial structuring of gut microbiota along the intestinal wall. This can be done by combining fluorescent dyes, which can be targeted towards particular cell structures or metabolites, and spectral-imaging microscopy. The University of Maine currently has a Confocal Microscopy Facility with a Leica TCS SP laser scanning confocal microscope that will be sufficient for this work.Given that these tools have yet to be used in conjunction, and in the field of animal science, the first step in developing a successful research program will be to develop these novel research tools into cohesive protocols. This objective will require the acquisition and assembly of an anaerobic culturing chamber and microfluidics equipment, the establishment of stable co-cultures, and the refinement of laboratory procedures. Samples of GI tract tissue and their resident microbiota may be readily obtained from a number of sources, depending on the experimental design. Livestock samples can be collected from slaughterhouses. Working relationships with slaughterhouses may be cultivated independently, through University of Maine Extension, or through agricultural associations such as the ME DACF, the ME Beef Producers Assoc., the ME Dairy and Nutrition Counsel, and the ME Sheep Breeders Assoc. Wild herbivore samples may be obtained from hunters in conjunction with the Maine Department of Inland Fisheries and Wildlife, or through the necropsy laboratory at the University of Maine Cooperative Extension Diagnostic and Research Laboratory with the cooperation of Dr. Anne Lichtenwalner, UMaine School of Food and Agriculture.Objective 2: To study the effect of inflammation on host GI tract cells and microbiota, inflammation will be induced in vitro using 'rumenized' gut chips, and the effect on host and microbial cells observed using high-throughput sequencing. This objective will involve model development to overcome several technical challenges in order to create an inflammation model for the ruminant gut which more directly mimics in vivo conditions. First, the most common method for inducing inflammation and dysbiosis in mouse trials is with dextran sodium sulfate (DSS), a sulfated polysaccharide which induces colitis by combining with medium-chain fatty acids and creating vesicles in intestinal cell walls (Laroui et al. 2012). However, DSS is not always effective in vitro (Laroui et al. 2012), and it does not mimic a realistic cause of inflammation in ruminants. Therefore, a more appropriate gut inflammation model for ruminants will be developed, and in all likelihood this will be somewhat specific to disease etiology. For example, a diet-induced acidosis model will utilize triggers that mimic a high-starch/low-fiber diet that selects for lactic-acid producing bacteria and lowers the rumen pH. This may be accomplished by altering the culture media input, artificially lowering the pH of media input, or by introducing gut contents from an acidotic animal into the co-culture of otherwise healthy cells. Similarly, mimicking weaning stress may require GI cells from neonate animals which are then subjected to changing dietary conditions via the culture media input. Investigations on inflammation caused by plant secondary compounds will be conducted using the toxin or plant substance of interest.Second, the most widely-used sequencing approach to date has been 16S rRNA gene amplicon sequencing, which uses primer oligonucleotides to selectively amplify a short (~500 nucleotide) section of the prokaryotic 16S ribosomal RNA gene, the region for the small subunit of ribosomes. This ubiquitous gene has provided for the advance of microbial ecology and dramatically advanced our understanding of host-microbial interactions. However, much of this research comes with two caveats. Primarily, the use of DNA sequencing alone cannot distinguish between living and relic DNA from dead/lysed cells. This gives a murkier view of community dynamics and precludes fine resolution of taxonomic changes in response to intervention. This can be ameliorated with a propidium monoazide (PMA) pre-treatment prior to DNA extraction (Nocker, Cheung, and Camper 2006). PMA binds to free double-stranded DNA and prevents downstream amplification or sequencing, effectively removing relic DNA from results. PMA will be integrated into my relevant DNA sequencing protocols to better understand the living community in the GI tract.Third, the 16S rRNA gene serves only as a piece of the prokaryotic ribosome, and commonly-used protocols only amplify bacteria and not archaea. Additional sequencing runs are needed for the fungal ITS genes or protozoal 18S, and no amplicon can target all viruses. While 16S and similar amplicons support taxonomic identification, they provide no indication of function and can only be used for predictive assignment of function. Widespread adoption of omics-based sequencing and improvements in technology have reduced the price of more informative types of sequencing, including metagenomics (which can be used in conjunction with PMA treatment), and metatranscriptomics. Metagenomics employs shotgun sequencing of all the DNA present in a community, from which genes and functional pathways may be identified and compared. Metatranscriptomics likewise uses shotgun sequencing, to identify all RNA transcripts in a community and which genes are actively being used.The biological materials required to perform this objective are explained in objective one. All nucleotide extraction, quantification, and sequencing library preparation will be completed in my laboratory at UMaine. Sequencing will be performed by third-party entities. Sequence analysis will be performed using the University of Maine's High Performance Computing cluster.

Progress 11/25/19 to 09/30/20

Outputs
Target Audience:Target audience: The general public and K-12 students interested in animals. Efforts: University of Maine Cooperative Extension Oxford County 4-H Jamboree (virtual), "Gut microbes on the farm", Aug 13, 2020. Video. Target audience 11- 18 year olds interested in pursuing a career in animal science. Invited to lead Journal Club with the Fogler Library, August 4, 2020. Chose a paper on the gut microbiome in animals and how it affected host fitness and suvival.Target audience graduate students in microbiology. Albright College Science Research Institute summer program 2020, which engages grades 5-12 in research. "A crash course in the gut microbiome", virtual presentation, Aug 4, 2020. Blog posts on www.sueishaqlab.org, my professional blog, describing my life as a scientist, my work at the University of Maine, and updates. From Jan - Sept1, 2020, the site had 2,868visitors and 4,694views, and 27total posts. Target audience: scientific community Efforts: Several scientific conferences in 2020 were held virtually due to SARS-CoV-2 concerns, at which my research on the gut microbiome and host health was presented. Due to the delay in switching to virtual, some of these conferences were rescheduled to after this reporting period. Zeng*, H., Safratowich, B.D., Liu, Z., Bukowski, M.R., Ishaq, S.L. "Supplementation of calcium and vitamin D reduces colonic inflammation and beta-catenin signaling in C57BL/6 mice fed a western diet." American Society for Nutrition 2020. (revised to virtual) Seattle, WA. June 2020. Changes/Problems:Nothing directly related to the project has significantly changed or become a problem. However, due to SARS-CoV2 in 2020 and the disruption to the post office in summer 2020, there has been significant disruption to the ability of researchers to work in the lab, the ability to procure reagents and kits which are in high demand but reduced manufacturing capacity, and the ability to receive temperature sensitive reaegents without shipping delays which destroy the reagent. Collectively, these challenges have dramatically slowed the pace at which research may be performed in the lab. What opportunities for training and professional development has the project provided?The training and professional development opportunities for graduate and undergraduate students have included online safety training;in lab training of equipment use, cell culture protocols, anaerrobic microbial culture protocols; chemical and equipment inventory management and acquisition; literature review; and general project management. Students are required to maintain laboratory notebooks, as well as develop written protocols and literature reviews, both of which improve their written communication skills. Students informally present and discuss their work in lab meetings, held weekly to monthly as needed, which improves their verbal communication skills. Professional development for the PI has included laboratory management and equipment procurement, curricula development, personnel management, and formal advising/mentoring. How have the results been disseminated to communities of interest?Target audience: The general public and K-12 students interested in animals. Efforts: University of Maine Cooperative Extension Oxford County 4-H Teen Science Cafe (virtual), "Gut microbes on the farm", Oct 15, 2020. BioME (Bioscience Association of Maine) Virtual Coffee Hour, "What is a microbiome and where can I get one?" Oct 14, 2020. I introduced myself and my research to 65 participants, who are biomedical professionals and state representatives in Maine. University of Maine Cooperative Extension Oxford County 4-H Jamboree (virtual), "Gut microbes on the farm", Aug 13, 2020.Video. Target audience 11- 18 year olds interested in pursuing a career in animal science. Invited to lead Journal Club with the Fogler Library, August 4, 2020. Chose a paper on the gut microbiome in animals and how it affected host fitness and suvival.Target audience graduate students in microbiology. Albright CollegeScience Research Institutesummer program 2020, which engages grades 5-12 in research. "A crash course in the gut microbiome", virtual presentation, Aug 4, 2020. Blog posts on www.sueishaqlab.org, my professional blog, describing my life as a scientist, my work at the University of Maine, and updates. From Jan - Dec 1, 2020, the site had 4,524 visitors and 7,458 views, and 41 total posts. Target audience: scientific community Efforts: Several scientific conferences in 2020 were held virtually due to SARS-CoV-2 concerns, at which my research on the gut microbiome and host health was presented. University of Maine Medicineseminar series(virtual), "A crash course in the gut microbiome" , Nov 6, 2020. The pdf of slides with annotated comments can be found here:ishaq-ummed-gut-crash-course-20201106.There were >50 participants Genomes to Phenomes (G2P) group, University of Maine. Co-hosting a session with grad student Alice Hotopp, on gut microbes and survival of reintroduced animals. Oct 30, 2020. Ishaq, S.L."Framing the discussion of microorganisms as a facet of social equity in human health",inVIVO Planetary Health 2020 meeting. [Jun 17-20, 2020 in Amsterdam, Netherlands cancelled]. (Virtual) Dec2020. (invited) Yeoman*, C.,Lachman, M.,Ishaq, S., Olivo, S., Swartz, J., Herrygers, M., Berarddinelli, J."Development of Climactic Oral and Rectal Microbiomes Corresponds to Peak Immunoglobin Titers in Lambs."Conference of Research Workers in Animal Diseases (CRWAD) 2020.(revised to virtual) Dec 5, 2020. Ishaq*, S.L., Hotopp2, A., Silverbrand2, S., MacRae, J., Stock, S.P., Groden, E. "Can a necromenic nematode serve as a biological Trojan horse for an invasive ant?"Entomological Society of America 2020.(revised to virtual). Nov 15-25, 2020. (invited). > 75 views Zeng*, H., Safratowich, B.D., Liu, Z., Bukowski, M.R., Ishaq, S.L. "Supplementation of calcium and vitamin D reduces colonic inflammation and beta-catenin signaling in C57BL/6 mice fed a western diet."American Society for Nutrition 2020. (revised to virtual) Seattle, WA. June 2020. What do you plan to do during the next reporting period to accomplish the goals?I plan to commence lab work to create rumen and intestinal cell cultures from cows, something which is not commercially available. If successful pure cell cultures can be maintained, I will submit cells to ATCC repository to make these available to other researchers. This will involve obtaining tissue samples for biopsy, and growing and maintaining cells in the lab. Once cells have been established, even for up to a week at a time, cow cells will be co-cultured with gut microorganisms. For one student project, this will involve infecion with Cryptosporidium and determining the effect of infection on the host microbiome at the site of infection. For another student, this will involve maintaining these in culture for months at a time. Once proof-of-concept culture work iscompleted and cell cultures are established, this project will involve the fabrication of a gut chip and engraftment of the cultures to the chip. It is estimated that this will take the entire next year of the reporting period.

Impacts
What was accomplished under these goals? Two master's of science students (beginning Sept 1, 2020) and one master's of professional studies (beginning in January 2020) students, as well as three undergraduate students (beginning in spring and summer 2020), were recruited to the lab to work on aspects of this project. These students have begun their technical training in laboratory skills, as well as their scentific theory training through reading and discussing scientific articles, and in planning out the methods and experimental designs for their projects. A literature review is being compiled on methods/protocols for growing cells from the digestive tract of sheep and cows in culture. Graduate and undergraduate students are creating protocols for collecting tissue biopsy and creating cell cultures, care and maintenance of cell cultures, and how to measure host immune factors. While several months of campus closure has delayed lab work, the students in the Ishaq lab have managed to obtain online and some in-lab training, as well as the compilation of said materials to prepare for the in lab experiments which began in fall 2020. This sets the ground work for completed lab work, and in particular, helped students gain a broader understanding of the amount of preparation required for experimentation, and gave them more time to immerse themselves in previous literature and develop ideas to take that research topic forward.

Publications