A collaborative project got published on the biogeography of the calf digestive tract!

Most studies that examine the microbial diversity of the gastrointestinal tract only look at one or two sample sites, usually the mouth, the rumen in ruminant animals, or the feces.  It can be difficult, expensive, invasive, or fatal to get samples from deep inside the intestinal tract; however many studies have pointed out that anatomical location and local environmental factors (like temperature, pH, host cells, nutrient availability, and exposure to UV light) can dramatically change a microbial community.  Thus, the microbes that we find in feces aren’t always what we would find in the stomach or along the intestines.  On top of that, certain microorganisms have been shown to closely associate with or attach to host cells, and the diversity of microbes next to host tissues can be different from what’s at the center of the intestines (the digesta).

This large, collaborative project took samples from nine different sites along the digestive tract of calves over the first 21 days of life to determine how body sites differed from each other, how sites changed over time as the calf matured, and how the lumen-associated bacteria would differ from the digesta-associated bacteria.  Samples from the mothers were also taken to understand how maternal microbial influence would affect body sites over time.

This paper was just published in  Scientific Reports, and was something I had previously presented on at the Joint Annual Meeting of the American Society for Animal Science, the American Dairy Science Association, and the Canadian Society for Animal Science in Salt Lake City, UT in 2016.

Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal tract. Carl J. Yeoman, Suzanne L. Ishaq, Elena Bichi, Sarah K. Olivo, James Lowe, Brian M. Aldridge. 2018. Scientific Reports.


The impact of maternal microbial influences on the early choreography of the neonatal calf microbiome were investigated. Luminal content and mucosal scraping samples were collected from ten locations in the calf gastrointestinal tract (GIT) over the first 21 days of life, along with postpartum maternal colostrum, udder skin, and vaginal scrapings. Microbiota were found to vary by anatomical location, between the lumen and mucosa at each GIT location, and differentially enriched for maternal vaginal, skin, and colostral microbiota. Most calf sample sites exhibited a gradual increase in α-diversity over the 21 days beginning the first few days after birth. The relative abundance of Firmicutes was greater in the proximal GIT, while Bacteroidetes were greater in the distal GIT. Proteobacteria exhibited greater relative abundances in mucosal scrapings relative to luminal content. Forty-six percent of calf luminal microbes and 41% of mucosal microbes were observed in at-least one maternal source, with the majority being shared with microbes on the skin of the udder. The vaginal microbiota were found to harbor and uniquely share many common and well-described fibrolytic rumen bacteria, as well as methanogenic archaea, potentially indicating a role for the vagina in populating the developing rumen and reticulum with microbes important to the nutrition of the adult animal.

2017 Year in Review

The end of 2017 marks the second year of my website, as well as another year of life-changing events, and reflecting on the past year’s milestones help put all those long hours into perspective.  I reviewed my year last year, and found it particularly helpful in focusing my goals for the year ahead.

Looking Back

In the first half of 2017, I was working as a post-doctoral researcher in the Menalled lab at Montana State University, researching the interaction of climate change, farm management (cropping) system, and disease on soil bacteria in wheat fields, as well as the legacy effects on subsequent crops.  I am still working to analyze, interpret, and publish those results, and hope to submit several manuscripts from that project in early 2018.  In June, I began a position as a research assistant professor in the Biology and the Built Environment Center at the University of Oregon.

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This involved another large move, not only from Montana to Oregon, which has led to some awesome new adventures, but also from agriculture and animal science to indoor microbiomes and building science.   So far, it has been a wonderful learning experience for incorporating research techniques and perspectives from other fields into my work.

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2017 has been another extremely productive year for me.  I presented some work at two conferences, the Congress on Gastrointestinal Function and the Ecological Society of America meeting (additional ESA posts here and here).  While at ESA, I was able to attend the 500 Women Scientists luncheon to discuss inequality in academia as well as recommendations we could make to improve ESA and other conferences ,such as offering affordable on-site child care, and action items we could take ourselves, such as attending training workshops to combat implicit bias or making sure job searches recruit a diverse candidate pool.

500 Women Scientist group at ESA 2017

This year, I added four new research publications and one review publication to my C.V., and received word that a massive collaborative study that I contributed to was accepted for publication- more on that once it’s available.  In April, I hosted a day of workshops on soil microbes for the Expanding Your Horizons for Girls program at MSU, and I gave a seminar at UO on host-associated microbiomes while dressed up as a dissected cat on Halloween.  In November, I participated in a Design Champs webinar; a pilot series from BioBE which provides informational discussions to small groups of building designers on aspects of how architecture and biology interact.

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I published 34 posts in 2017, including this one, which is significantly fewer than the 45 I published in 2016.  However, I have doubled my visitor traffic and views over last year’s totals: over 2,000 visitors with over 3,200 page views in 2017! My highest-traffic day was April 27th, 2017.  While I am most popular in the United States, I have had visitors from 92 countries this year!

Map of home countries for 2017 website visitors.

My most popular post is currently “Work-life balance: what do professors do?”, with over 610 views! My least popular is “Presentation on juniper diets and rumen bacteria from JAM 2016 available!” with just 2 views, granted, that one appeals to a much narrower audience.  This year, in addition to updates on publications, projects, and positions, I wrote about writing; including theses and grants. I wrote about getting involved in science, be it through education, participation, or legislation.  I described outreach in academia, and the process of interviewing.  I gave some perspective on the effect of climate change and anthropological influence on agriculture and ecology, as well as on the debate surrounding metrics of success in graduate study.

I also added some “life” to my work-life balance; in November, I married my best friend and “chief contributor“, Lee Warren, in a small, stress-free ceremony with some local friends in Eugene, Oregon!!

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Looking Ahead

I have high hopes for 2018, notably, I’d like to finish more of the projects that have been in development over the last two years during my post-docs.  Nearly all academics carry forward old projects: some need additional time for experimentation or writing, some get shelved temporarily due to funding or time constraints, some datasets get forgotten and gather dust, and some which got cut short because of the need to move to a new job.  This is a particular concern as grant funding and length of job postings become shorter, forcing researchers to cut multi-year projects short or finish them on their own time.  After defending in early 2015, I had two one-year postings and started at UO in June 2017, making this my fourth job in three years.  I’m looking forward to roosting for a bit, not only to clear out unfinished business, but also to settle into my new job at BioBE.  This fall, I have been analyzing data on a weatherization project, writing a handful of grants, and developing pilot projects with collaborators.  I have really enjoyed my first six months at BioBE, and Lee and I have taken a shine to Eugene.  In the next few months, I hope to have more posts about my work there, exciting new developments in BioBE and ESBL, and more insights into the work life of an academic.  Happy New Year!

A study to which I contributed got published!


I’m pleased to announce that one of my collaborators,  Dr. Huawei Zeng of the USDA Agricultural Research Service, recently published another study of his, to which I contributed some analysis of bacterial communities from mice.  Several years ago, during my Ph.D. at the University of Vermont, I provided wet-lab and DNA sequence analysis work for a previous project of Dr. Zeng, investigating the health effects of a low or high fat diet on mice, which can be found here.


Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet.

Zeng, H., Ishaq, S.L., Liu, Z., Bukowski, M.R. 2017. Journal of Nutritional Biochemistry. In press, doi.org/10.1016/j.jnutbio.2017.11.001.


The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat western diet. To test the hypothesis that a high fat diet (HFD) promotes colonic aberrant crypt (AC) formation in a manner associated with gut bacterial dysbiosis, we examined the susceptibility to azoxymethane (AOM)-induced colonic AC and microbiome composition in C57/BL6 mice fed a modified AIN93G diet (AIN, 16% fat, energy) or a HFD (45% fat, energy) for 14 weeks. Mice receiving the HFD exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the AIN group. Consistent with the gut inflammatory phenotype, we observed an increase in colonic AC, plasma interleukin 6 (IL6), tumor necrosis factor α (TNF α), monocyte chemoattractant protein 1 (MCP1), and inducible nitric oxide synthase (iNOS) in the ileum of the HFD-AOM group compared with the AIN-AOM group. Although the HFD and AIN groups did not differ in bacterial species number, the HFD and AIN diets resulted in different bacterial community structures in the colon. The abundance of certain short chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and fecal SCFA (e.g., acetic acid) content were lower in the HFD-AOM group compared with the AIN and AIN-AOM groups. Furthermore, we identified a high abundance of Anaeroplasma bacteria, an opportunistic pathogen in the HFD-AOM group. Collectively, we demonstrate that a HFD promotes AC formation concurrent with an increase of opportunistic pathogenic bacteria in the colon of C57BL/6 mice.

A collaborative paper on how rumen acidosis affects fungi and protozoa got published!

Ruminal acidosis is a condition in which the pH of the rumen is considerably lower than normal, and if severe enough can cause damage to the stomach and localized symptoms, or systemic illness in cows.  Often, these symptoms result from the low pH reducing the ability of microorganisms to ferment fiber, or by killing them outright.  Since the cow can’t break down most of its plant-based diet without these microorganisms, this disruption can cause all sorts of downstream health problems.  Negative health effects can also occur when the pH is somewhat lowered, or is lowered briefly but repeatedly, even if the cow isn’t showing outward clinical symptoms.  This is known as sub-acute ruminal acidosis (SARA), and can also cause serious side effects for cows and an economic loss for producers.

In livestock, acidosis usually occurs when ruminants are abruptly switched to a highly-fermentable diet- something with a lot of grain/starch that causes a dramatic increase in bacterial fermentation and a buildup of lactate in the rumen.  To prevent this, animals are transitioned incrementally from one diet to the next over a period of days or weeks.  Another strategy is to add something to the diet to help buffer rumen pH, such as a probiotic.  One of the most common species used to help treat or prevent acidosis is a yeast; Saccharomyces cerevisiae.

This paper was part of a larger study on S. cerevisiae use in cattle to treat SARA, the effects of which on animal production as well as bacterial diversity and functionality have already been published by an old friend and colleague of mine, Dr. Ousama AlZahal, and several others.  In total, very little work has been done on the effect of SARA or S. cerevisiae treatment on the fungal or protozoal diversity in the rumen, which is what I added to this study.  I was very pleased to be invited to analyze and interpret some of the data, as well as to present the results at a conference in Chicago earlier this year.  The article itself has just been published in Frontiers in Microbiology!

An investigation into rumen fungal and protozoal diversity in three rumen fractions, during high-fiber or grain-induced sub-acute ruminal acidosis conditions, with or without active dry yeast supplementation.

Authors: Suzanne L. Ishaq, Ousama AlZahal, Nicola Walker, Brian McBride

Sub-acute ruminal acidosis (SARA) is a gastrointestinal functional disorder in livestock characterized by low rumen pH, which reduces rumen function, microbial diversity, host performance, and host immune function. Dietary management is used to prevent SARA, often with yeast supplementation as a pH buffer. Almost nothing is known about the effect of SARA or yeast supplementation on ruminal protozoal and fungal diversity, despite their roles in fiber degradation. Dairy cows were switched from a high-fiber to high-grain diet abruptly to induce SARA, with and without active dry yeast (ADY, Saccharomyces cerevisiae) supplementation, and sampled from the rumen fluid, solids, and epimural fractions to determine microbial diversity using the protozoal 18S rRNA and the fungal ITS1 genes via Illumina MiSeq sequencing. Diet-induced SARA dramatically increased the number and abundance of rare fungal taxa, even in fluid fractions where total reads were very low, and reduced protozoal diversity. SARA selected for more lactic-acid utilizing taxa, and fewer fiber-degrading taxa. ADY treatment increased fungal richness (OTUs) but not diversity (Inverse Simpson, Shannon), but increased protozoal richness and diversity in some fractions. ADY treatment itself significantly (P < 0.05) affected the abundance of numerous fungal genera as seen in the high-fiber diet: Lewia, Neocallimastix, and Phoma were increased, while Alternaria, Candida Orpinomyces, and Piromyces spp. were decreased. Likewise, for protozoa, ADY itself increased Isotricha intestinalis but decreased Entodinium furca spp. Multivariate analyses showed diet type was most significant in driving diversity, followed by yeast treatment, for AMOVA, ANOSIM, and weighted UniFrac. Diet, ADY, and location were all significant factors for fungi (PERMANOVA, P = 0.0001, P = 0.0452, P = 0.0068, Monte Carlo correction, respectively, and location was a significant factor (P = 0.001, Monte Carlo correction) for protozoa. Diet-induced SARA shifts diversity of rumen fungi and protozoa and selects against fiber-degrading species. Supplementation with ADY mitigated this reduction in protozoa, presumptively by triggering microbial diversity shifts (as seen even in the high-fiber diet) that resulted in pH stabilization. ADY did not recover the initial community structure that was seen in pre-SARA conditions.

A collaborative project on juniper diets in lambs was published!

In 2015, while working in the Yeoman Lab, I was invited to perform the sequence analysis on some samples from a previously-run diet study.  The study was part of ongoing research by Dr. Travis Whitney at Texas A & M on the use of juniper as a feed additive for sheep.  The three main juniper species in Texas can pose a problem- while they are native, they have significantly increased the number of acres they occupy due to changes in climate, water availability, and human-related land use.  And, juniper can out-compete other rangeland species, which can make forage less palatable, less nutritious, or unhealthy for livestock.  Juniper contains essential oils and compounds which can affect some microorganisms living in their gut.  We wanted to know how the bacterial community in the rumen might restructure while on different concentrations of juniper and urea.

Coupled with the animal health and physiology aspect led by Travis, we published two companion papers in the Journal of Animal Science.  We had also previously presented these results at the Joint Annual Meeting of the American Society for Animal Science, the American Dairy Science Association, and the Canadian Society for Animal Science in Salt Lake City, UT in 2016.  Travis’ presentation can be found here, and mine can be found here.  The article can be found here.

Ground redberry juniper and urea in supplements fed to Rambouillet ewe lambs.

Part 1: Growth, blood serum and fecal characteristics, T.R. Whitney

Part 2: Ewe lamb rumen microbial communities, S. L. Ishaq, C. J. Yeoman, and T. R. Whitney

This study evaluated effects of ground redberry juniper (Juniperus pinchotii) and urea in dried distillers grains with solubles-based supplements fed to Rambouillet ewe lambs (n = 48) on rumen physiological parameters and bacterial diversity. In a randomized study (40 d), individually-penned lambs were fed ad libitum ground sorghum-sudangrass hay and of 1 of 8 supplements (6 lambs/treatment; 533 g/d; as-fed basis) in a 4 × 2 factorial design with 4 concentrations of ground juniper (15%, 30%, 45%, or 60% of DM) and 2 levels of urea (1% or 3% of DM). Increasing juniper resulted in minor changes in microbial β-diversity (PERMANOVA, pseudo F = 1.33, P = 0.04); however, concentrations of urea did not show detectable broad-scale differences at phylum, family, or genus levels according to ANOSIM (P> 0.05), AMOVA (P > 0.10), and PERMANOVA (P > 0.05). Linear discriminant analysis indicated some genera were specific to certain dietary treatments (P < 0.05), though none of these genera were present in high abundance; high concentrations of juniper were associated with Moraxella and Streptococcus, low concentrations of urea were associated with Fretibacterium, and high concentrations of urea were associated with Oribacterium and PyramidobacterPrevotella were decreased by juniper and urea. RuminococcusButyrivibrio, and Succiniclasticum increased with juniper and were positively correlated (Spearman’s, P < 0.05) with each other but not to rumen factors, suggesting a symbiotic interaction. Overall, there was not a juniper × urea interaction for total VFA, VFA by concentration or percent total, pH, or ammonia (P > 0.29). When considering only percent inclusion of juniper, ruminal pH and proportion of acetic acid linearly increased (P < 0.001) and percentage of butyric acid linearly decreased (P = 0.009). Lamb ADG and G:F were positively correlated with Prevotella(Spearman’s, P < 0.05) and negatively correlated with Synergistaceae, the BS5 group, and Lentisphaerae. Firmicutes were negatively correlated with serum urea nitrogen, ammonia, total VFA, total acetate, and total propionate. Overall, modest differences in bacterial diversity among treatments occurred in the abundance or evenness of several OTUs, but there was not a significant difference in OTU richness. As diversity was largely unchanged, the reduction in ADG and lower-end BW was likely due to reduced DMI rather than a reduction in microbial fermentative ability.

My review on Plant-Microbial Interactions in Agriculture got published!

A few months ago, I was invited to submit an article to the special issue “Plant Probiotic Bacteria: solutions to feed the World” in AIMS Microbiology on the interactions between agricultural plants and microorganisms.  As my relevant projects are still being processed, I chose to write a review of the current literature regarding these interactions, and how they may be altered by different farming practices.  The review is available as open-access here!

“Plant-microbial interactions in agriculture and the use of farming systems to improve diversity and productivity”

A thorough understanding of the services provided by microorganisms to the agricultural ecosystem is integral to understanding how management systems can improve or deteriorate soil health and production over the long term. Yet it is hampered by the difficulty in measuring the intersection of plant, microbe, and environment, in no small part because of the situational specificity to some plant-microbial interactions, related to soil moisture, nutrient content, climate, and local diversity. Despite this, perspective on soil microbiota in agricultural settings can inform management practices to improve the sustainability of agricultural production.

Keywords bacteria; climate change; farming system; fungi; nutrient exchange; pathogens; phytohormones

Citation: Suzanne L. Ishaq. Plant-microbial interactions in agriculture and the use of farming systems to improve diversity and productivity. AIMS Microbiology, 2017, 3(2): 335-353. doi: 10.3934/microbiol.2017.2.335

A collaborative project on sheep feed efficiency and gut bacteria was published!

I’m pleased to announce that a paper that I contributed to was recently accepted for publication in the Journal of Animal Science!

“Feed efficiency phenotypes in lambs involve changes in ruminal, colonic, and small intestine-located microbiota”, Katheryn Perea; Katharine Perz; Sarah Olivo; Andrew Williams; Medora Lachman; Suzanne Ishaq; Jennifer Thomson; Carl Yeoman (article here).

Katheryn is an undergraduate at New Mexico Institute of Mining and Technology who received an INBRE grant to support her as a visiting researcher at Montana State University in Bozeman, MT over summer 2016.  Here, she worked with Drs. Carl Yeoman and Jennifer Thomson to perform the diversity analysis on the bacteria in the gastrointestinal tract of sheep from a previous study.  These sheep had been designated as efficient or inefficient, based on how much feed was needed for them to grow.  Efficient sheep were able to grow more with less feed, and it was thought this might be due to hosting different symbiotic bacteria which were better at fermenting fibrous plant material into usable byproducts for the sheep.

Samples from the sheep were collected as part of a larger study on feed efficiency performed by MSU graduate students Kate Perz and Medora Lachman, as well as technicians Sarah Olivo and Andrew Williams, and Katheryn performed the data and statistical analysis using some of my guidelines.  This is Katheryn’s first published article, and one I just presented a poster on at the Congress on Gastrointestinal Function in Chicago, IL!

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2017 Congress on Gastrointestinal Function

I just got back from my very first Congress on Gastrointestinal Function, poster tube.jpga small meeting for  researchers with a specific focus on the gastrointestinal tract, which is held every two years in Chicago, Illinois.  The special session this year was on “Early Acquisition and Development of the Gut Microbiota: A Comparative Analysis”.  The rest of the sessions opened up the broader topics of gut ecosystem surveillance and modulation, as well as new techniques and products with which to study the effect of microorganisms on hosts and vice versa.  The research had a strong livestock animal focus, as well as a human health focus, but we also heard about a few studies using wild animals.

As I’ve previously discussed, conferences are a great way to interact with other scientists.  Not only can you learn from similar work, but you can often gain insights into new ways to solve research problems inherent to your system by looking at what people in different fields are trying, something that you might otherwise miss just by combing relevant literature online.  A meeting or workshop is also a great place to meet other similarly focused scientists to set up collaborators that span academia, government, non-profit, and industry sectors.

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It was great to catch up with Dr. Ben Wenner, now at Purdue Agribusiness, and meet Yairy Roman-Garcia, grad student at the Ohio State University.

This year, I was excited for one of my abstracts to be accepted as a poster presentation, and honored to have the other upgraded from poster to talk!  Stay tuned for details about both of those projects in the coming weeks, and be sure to check this meeting out in April, 2019.

2016 Year In Review

Looking back

2016 started with a bang when I launched this site and joined Twitter for the first time!  For the first quarter of the year, I was a post-doctoral researcher in the Yeoman Lab in the Department of Animal and Range Sciences at Montana State University.  I was working on a total of eight grants, ranging from small fellowships to million dollar projects, both as a principal investigator and as a co-PI.  I was also doing the bioinformatic analysis for multiple projects, totaling nearly 1,000 samples, as well as consulting with several graduate students about their own bioinformatic analyses.

In late spring, my position in the Yeoman lab concluded, and I began a post-doctoral position in the Menalled Lab in the Department of Land Resources and Environmental Sciences at MSU.  This position gave me the opportunity to dramatically increase my skill-set and learn about plant-microbe interactions in agricultural fields.  My main project over the summer was studying the effect of climate and other stresses on wheat production and soil microbial diversity, and this fall I have been investigating the legacy effects of these stressors on new plant growth and microbial communities.  I have extracted the DNA from all of my Fort Ellis summer trial soil samples, and look forward to having new microbial data to work with in the new year.  Based on the preliminary data, we are going to see some cool treatment effects!

Over the summer, I attended the American Society for Microbiology in Boston, MA in June, where I presented a poster on the microbial diversity in organic and conventional farm soil, and the Joint Annual Meeting for three different animal science professional societies in Salt Lake City, UT in July, where I gave my first two oral conference presentations. One was on the effect of a juniper-based diet on rumen bacteria in lambs, and the other was on the biogeography of the calf digestive system and how location-specific bacteria correlate to immune-factor expression.

Thanks to a lot of hard work from myself and many collaborators, a number of research projects were accepted for publication in scientific journals, including the microbial diversity of agricultural soils, in reindeer on a lichen diet, and in relation to high-fat diets in mice, it also included work on virulent strains of Streptococcus pyogenes, and a review chapter on the role of methanogens in human gastrointestinal disease.

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Looking forward

A whopping thirteen manuscripts are still in review at scientific journals or are in preparation waiting to be submitted! Some of those are primarily my projects, and for others I added my skills to the work of other researchers.  Editing all those is going to keep me plenty busy for the next few months. I’ll also be writing several more grants in early 2017, and writing a blog post about the Herculean task that can be.

I’ll be concluding my greenhouse study by March of 2017, just in time to prepare for another field season at Fort Ellis, on the aforementioned climate change study that is my main focus. In January, I’ll be spending time in the lab helping to process and sequence DNA from my 270 soil samples, and begin the long task of data quality assurance, processing, and analysis.  I’m not worried, though, 270 samples isn’t the most I’ve worked with and bioinformatic analysis is my favorite part of the project!

This year, I am hoping to attend two conferences that I have never previously attended, and present data at both of them.  The first will be the 2017 Congress on Gut Function in Chicago, IL in April, and the second will be the Ecological Society of America’s Annual Meeting in Portland, OR in August.  Both conferences will give me the opportunity to showcase my work, network with researchers, and catch up with old friends.

If 2017 is anything like the past few years, it’s going to be full of new projects, new collaborators, new skills, and new opportunities for me, and I can’t wait!  So much of what I’ve accomplished over the last year has been possible because of the hard work, enthusiasm, and creativity of my colleagues, students, friends, and family, and I continue to be grateful for their support.  I’d also like to thank anyone who has been kind enough to read my posts throughout the last year; it’s been a pleasure putting my experiences into words for you and I appreciate the time and interest you put in.  I look forward to sharing more science with you next year!

Manuscript published on the effect of low/high fat diets on health and intestinal bacteria

Several years ago, during my Ph.D. at the University of Vermont, I provided wet-lab and DNA sequence analysis work for a project investigating the health effects of a low or high fat diet on mice with Dr. Huawei Zeng of the USDA Agricultural Research Service.  It was just recently published in the Journal of Nutritional Biochemistry!


Consumption of an obesigenic/high-fat diet (HFD) is associated with a high colon cancer risk and may alter the gut microbiota. To test the hypothesis that long-term high-fat (HF) feeding accelerates inflammatory process and changes gut microbiome composition, C57BL/6 mice were fed HFD (45% energy) or a low-fat (LF) diet (10% energy) for 36 weeks. At the end of the study, body weights in the HF group were 35% greater than those in the LF group. These changes were associated with dramatic increases in body fat composition, inflammatory cell infiltration, inducible nitric oxide synthase protein concentration and cell proliferation marker (Ki67) in ileum and colon. Similarly, β-catenin expression was increased in colon (but not ileum). Consistent with gut inflammation phenotype, we also found that plasma leptin, interleukin 6 and tumor necrosis factor α concentrations were also elevated in mice fed the HFD, indicative of chronic inflammation. Fecal DNA was extracted and the V1–V3 hypervariable region of the microbial 16S rRNA gene was amplified using primers suitable for 454 pyrosequencing. Compared to the LF group, the HF group had high proportions of bacteria from the family Lachnospiraceae/Streptococcaceae, which is known to be involved in the development of metabolic disorders, diabetes and colon cancer. Taken together, our data demonstrate, for the first time, that long-term HF consumption not only increases inflammatory status but also accompanies an increase of colonic β-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of C57BL/6 mice.