The Hungate 1000 Project was a massive undertaking: namely, sequencing the genome of 1000 microorganisms cultured from ruminant animals all over the world, and was both coordinated and led by the Rumen Microbial Genomics Network. After years of hard work by some incredible researchers, the Hungate 1000 has just been published in the Nature Biotechnology Journal! The […]
For the past two months, I’ve been spending quite a bit of time writing grant proposals. In particular; those which expand our understanding of indoor lighting on human health and behavior, the indoor microbiome, and energy usage in buildings. These project proposals are collaborative efforts between several University of Oregon research labs: Biology and the Built Environment Center, Energy Studies and Buildings Laboratory, and the Baker Lighting Lab. I’ll have more updates in the next few months as those are reviewed.
Siobhan “Shevy” Rockcastle, Chair of the Baker Lighting Lab, and I have been brainstorming ideas, and today I went over to the Baker Lab to check it out in person. The Lab is decorated with concept-design lighting projects from previous students, which are not only beautiful, but extremely creative. Here are a few of my favorites!
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.
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.
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.
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.
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.
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.
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
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 Pyramidobacter. Prevotella were decreased by juniper and urea. Ruminococcus, Butyrivibrio, 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.
A couple of weeks ago, I attended my first Ecological Society of America meeting in Portland, which assembles a diverse community of researchers looking at system-wide processes. It was an excellent learning experience for me, as scientific fields each have a particular set of tools to look at different problems and our collective perspectives can solve research problems in more creative ways.
In particular, it was intriguing to attend talks on the ecology of the human microbiome. Due to the complexity of host-associated microbial communities, and the limitations of technology, the majority of studies to date have been somewhat observational. We have mapped what is present in different animals, in different areas of the body, under different diet conditions, in different parts of the world, and in comparison between healthy and disease states. But given the complexity of the day-to-day life of people, and ethics or technical difficulty of doing experimental studies in humans, many of the broader ecological questions have yet to be answered.
For example, how quickly do microbial communities assemble in humans? When you disturb them or change something (like adding a medication or removing a food from your diet) how quickly does this manifest in the community structure and do those changes last? How does dysbiosis or dysfunction in the body specifically contribute to changes in the microbial community, or do seemingly harmless events trigger a change in the microbial community which then causes disease in humans? Some of the presentations I attended have begun teasing out these problems with a combination of observational in situ biological studies, in vitro laboratory studies, and in silico mathematical modeling. The abstracts from all the meeting presentations can be found on the meeting website under Program. I have also summarized several of the talks I went to on Give Me The Short Version.
My poster presentation was on Wednesday, halfway through the meeting week, which gave me plenty of time to prepare. You never know who might show up at your poster and what questions they’ll have. In the past, I’ve always had a steady stream of people to chat with at my poster which has led to a number of scientific friendships and networking, and this year was no different. The rather large (but detailed) poster file can be found here: Ishaq et al ESA 2017 poster . Keep in mind that this is preliminary work, and many statistical tests have not yet been applied or verified. I’ve been working to complete the analysis on the large study, which also encompasses a great deal of environmental data. We hope to have manuscript drafted by this fall on this part of the project, and several more over the next year from the research team as this is part of a larger study; stay tuned!
I just got back from my very first Congress on Gastrointestinal Function, a 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.
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.
Great news: you can participate in science without going through a decade of higher education (sorry grad students, but thanks for your service!). There are two ways to do this: either you can volunteer to collect samples for a project, or you can volunteer to be the sample for a project. You can volunteer through the National Institutes of Health, third-party match sites that help recruit volunteers for large projects, independent research centers (that are usually under contract to run a study), and most universities and colleges have volunteer-recruiting websites.
Get out there and collect
There is a myriad of environmental science studies that rely on volunteers to collect samples, which may take place in very specific areas, or globally. Some are simple wildlife surveys, often through conservation societies like the Audubon Society, which use volunteers’ bird sightings to estimate populations. Humanitarian volunteer agencies may recruit volunteers for global research studies, as well. Some projects require more technical sampling, or require participants to travel to distant or difficult to reach places, and thus rely on outdoors-people with the gear and ability to safely retrieve water, soil, plants, animal hair or feces, you name it! There are some excellent examples of global projects which can be found through Adventure Scientists. AS recruits and trains volunteers for more difficult environmental sampling, and I am currently participating in their Gallatin Microplastics Initiative (year 1 and 2) along with my sampling team: Lee and Izzy.
‘Host’ your own research
Volunteering to be the sample also allows you to participate on a sliding scale of involvement. For example, observational studies only collect information on what is already happening. These might be sociology (human behavior) studies which only require you to fill out surveys (often online) on your personal history or normal routines. You can also donate biological samples (hair, breathe, blood, urine, feces, skin scrapings, etc.) which are minimally invasive but don’t necessarily require any experimental treatments that you have to participate in. A study that I analyzed data for asked participants to use a breathalyzer and submit a fecal sample in a jar. That’s all, and they were financially compensated for their time. The study was trying to correlate microorganisms in the gut with how much hydrogen or methane was in the breath, and whether a breathalyzer test could be used as a rough measure of how many methanogenic archaea lived in your gut. Often, research centers which are focused on medical treatment for a specific disease will collect specific biological samples.
Studies which require actual treatments or testing are clinical medical studies that rely on human volunteers upon whom to test products. At a certain point in drug or vaccine testing, animal or computer models can no longer serve as a proxy and you need to test things in humans. Thanks to HIPPA (Health Insurance Portability and Accountability Act of 1996) and other safety regulations, both at the federal and institutional level, there is a lot of transparency in these studies. You are told exactly what you will be required to do, what data will be collected and who will have access to it, and any possible health concerns that may arise from this study. Any release of tissue “ownership” will require you to sign consent.
I have participated in antibacterial product testing, a diet study investigating dairy fats during which I could only eat the prescribed diet for two solid months (boy, did I miss chocolate and Thai food!), and a study on chronic back pain (I was in the control group). For the back pain study, I had an EEG net put on my head to measure brainwaves and motion capture balls(examples below) attached all over my body to track my muscle movements as I performed simple tasks that required me to use my lower back muscles. I even had a functional MRI brain scan to measure how muscle pain might change brain function, unfortunately, I was not able to get a photocopy of my brain scan for posterity. The more invasive, time consuming, or risky a study may be, the higher the compensation (some vaccine studies compensate several thousand dollars).
You can participate in science in other ways, too. Try getting involved with science education! There are workshops, summer programs, or school events which encourage kids to learn about science and consider a career in it. Even if you aren’t a scientist yourself and can’t be a presenter, many programs still need people to chaperone, coordinate or market the event, and cater. Many science museums and educational centers have programs, as do many colleges. You can also find opportunities through local government to help clean natural sites or educate the public.
While you are out there collecting water samples from Arctic ice, counting wolves, or surveying land for public use, you are also perfectly situated to help with a little environmental restoration. The Global Microplastics Initiative looks for plastic in water sources from some very remote locations, and this study wouldn’t have been conducted if plastic in the environment wasn’t a concern. So while you are out there, try to leave the area a little cleaner than how you found it. You can volunteer for clean-up events to target specific locations that need help, but you don’t even need to organize for this one, just go out and start picking up trash!
Finally, most agricultural research studies rely on farmers, ranchers, growers, and producers as a source of project resources (like seeds, land, or cows) and project motivation. After all, federally and state-funded agricultural science exist to help local and national agriculture. You can participate in science by identifying problems that need to be solved and providing objectives for our studies, or by allowing research projects to use your land, animals, or facilities.
So far, you’ve educated yourself on science, and now you can go out and participate. Stay tuned in the next for weeks for Anyone can Science, Step 3: be supportive.
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.
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!