A collaborative paper on zinc and rumen bacteria in sheep got published!

Zinc is an important mineral in your diet; it’s required by many of your enzymes and having too much or too little can cause health problems. We know quite a bit about how important zinc is to sheep, in particular for their growth, immune system, and fertility.  We also know that organically- versus inorganically-sourced zinc differs in its bio-availability, or how easy it is for cells to access and use it.  Surprisingly, we know nothing about how different zinc formulations might affect gut microbiota, despite the knowledge that microorganisms may also need zinc.

This collaborative study was led by Dr. Whit Stewart and his then-graduate student, Chad Page, while they were at Montana State University (they are now both at the University of Wyoming).   Chad’s work focused on how different sources of zinc affected sheep growth and performance (previously presented, publication forthcoming), and I put together this  companion paper examining the effects on rumen bacteria.

The pre-print is available now for Journal of Animal Science members, and the finished proof should be available soon. JAS is the main publication for the American Society of Animal Science, and one of the flagship journals in the field.


Zinc amino acid supplementation alters yearling ram rumen bacterial communities but zinc sulfate supplementation does not.

Ishaq, S.L., Page, C.M., Yeoman, C.J., Murphy, T.W., Van Emon, M.L., Stewart, W.C. 2018. Journal of Animal Science. Accepted. Article.

Abstract

Despite the body of research into Zn for human and animal health and productivity, very little work has been done to discern whether this benefit is exerted solely on the host organism, or whether there is some effect of dietary Zn upon the gastrointestinal microbiota, particularly in ruminants. We hypothesized that 1) supplementation with Zn would alter the rumen bacterial community in yearling rams, but that 2) supplementation with either inorganically-sourced ZnSO4, or a chelated Zn amino acid complex, which was more bioavailable, would affect the rumen bacterial community differently. Sixteen purebred Targhee yearling rams were utilized in an 84 d completely-randomized design, and allocated to one of three pelleted dietary treatments: control diet without fortified Zn (~1 x NRC), a diet fortified with a Zn amino acid complex (~2 x NRC), and a diet fortified with ZnSO4 (~2 x NRC). Rumen bacterial community was assessed using Illumina MiSeq of the V4-V6 region of the 16S rRNA gene. One hundred and eleven OTUs were found with > 1% abundance across all samples. The genera PrevotellaSolobacteriumRuminococcusButyrivibrioOlsenellaAtopobium, and the candidate genus Saccharimonas were abundant in all samples. Total rumen bacterial evenness and diversity in rams were reduced by supplementation with a Zn-amino-acid complex, but not in rams supplemented with an equal concentration of ZnSO4, likely due to differences in bioavailability between organic and inorganically-sourced supplement formulations. A number of bacterial genera were altered by Zn supplementation, but only the phylum Tenericutes was significantly reduced by ZnSO4 supplementation, suggesting that either Zn supplementation formulation could be utilized without causing a high-level shift in the rumen bacterial community which could have negative consequences for digestion and animal health.

Featured Image Source: Wikimedia Commons

USDA AFRI NIFA Agricultural Production Systems grant awarded to Menalled et al.

In 2016, I was a post-doc in the Menalled Lab, which studies plant and weed ecology in the context of agricultural production and sustainability.  There, I assessed soil bacterial communities under different farming management practices and climate scenarios.  I also helped to develop a grant proposal, which was just accepted by the USDA AFRI NIFA Agricultural Production Systems!  Leading this project is Dr. Fabian Menalled (as Principal Investigator, or PI), along with a number of other PIs; Dr. Amy Trowbridge, Dr. David Weaver, Dr. Tim Seipel, Dr. Maryse Bourgault, and Dr. Carl Yeoman, and collaborators Dr. Darrin Boss, Dr. Kate Fuller, Dr. Ylva Lekberg, and myself as a subaward PI.  I will again be providing microbial community analysis for this project, and collectively the project investigators will bring expertise in plant ecology, agronomy, economics, soil and plant chemistry, microbial ecology, agroecosystems, and more.

This research and extension project focuses on the needs of dryland agricultural stakeholders and it was designed in close collaboration with the NARC Advisory Board. While I was only able to attend one meeting, other team members regularly meet with Montana producers to discuss current issues and identify locally-sourced needs for agricultural research.  During this project, we will continue to meet with the NARC Advisory Board to share our results, evaluate implications, and better serve the producer community.

Diversifying cropping systems through cover crops and targeted grazing: impacts on plant-microbe-insect interactions, yield and economic returns.

Project summary

The semi-arid section of the Northern Great Plains is one of the
largest expanses of small grain agriculture and low-intensity livestock
production. However, extreme landscape simplification, excessive reliance on
off-farms inputs, and warmer and drier conditions hinder its agricultural
sustainability. This project evaluates the potential of diversifying this region
through the integration of cover crops and targeted grazing. We will complement
field and greenhouse studies to appraise the impact of system diversity,
temperature, and precipitation on key multi-trophic interactions, yields, and
economic outputs. Specifically, we will 1) Assess ecological drivers as well as
agronomic and economic consequences of integrating cover crops and livestock
grazing in semi-arid systems, 2) Evaluate how climate variability modify the
impacts of cover crops and livestock grazing on agricultural outputs. Specifically,
we will 2.1) Compare the effect of increased temperature and reduced moisture
on agronomic and economic performance of simplified and diversified systems,
2.2.) Assess the impact of climate and system diversity on associated biodiversity
(weeds, insect, and soil microbial communities) and above- and belowground
volatile organic (VOC) compound emissions, and 2.3) Evaluate how changes in
microbially induced VOCs influence multitrophic plant-insect interactions.

Objectives

  1. Assess key ecological drivers as well as agronomic and economic consequences of integrating cover crops and livestock grazing in semi-arid production systems
    • Compare the agronomic and economic performance of simplified and diversified systems
    • Assess the impact of cover crops and livestock grazing on the associated biodiversity (weeds, insects, and the soil microbiota)
  2. Evaluate how climate conditions modify the impacts of cover crops and livestock grazing on semi-arid production systems
    • Compare the effect of temperature and soil moisture on agronomic and economic performance of simplified and diversified systems
    • Assess the impact of climate and system diversity on associated biodiversity and above- and belowground volatile organic compound (VOC) emissions
    • Evaluate how changes in VOCs emissions influence important multitrophic interactions such as resistance to wheat stem sawfly and natural enemy host location cues
  3. Integrate the knowledge generated into an outreach program aimed at improving producers’ adoption of sustainable diversified crop-livestock systems

Tour of the Baker Lighting Lab

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!

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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.

Abstract

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.

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.

Abstract

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.

Field notes from my first ESA meeting

IMG_20170808_083850[4637].jpg
From iDigBio
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.

One of my favorite parts was attending an open lunch with 500 Women Scientists, a recently-formed organization which promotes diversity and equality in science, and supports local activists to help change policy and preconceived notions about diversity in STEM.  The lunch meeting introduced the organization to the conference participants in attendance, asked us to voice our concerns or difficulties we had faced, encouraged us to reach out to others in our work network to seek advice and provide mentoring, and walked us through exercises designed to educate on how to build a more inclusive society.

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500 Women Scientists at ESA, August 2017

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!

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.