Updates

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.

Islands, Evolution, and a book report

Academics love to keep books, such that they accumulate over the years until, one day, you move offices, change universities, or retire and give them all away.  I happened upon one of these give-away treasure troves recently and grabbed several older books.  I began my journey with a historical perspective on island biogeography, and I enjoyed it so much I thought I’d write about it.

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The book is “The Song of the Dodo: Island Biogeography in an Age of Extinctions”, written in 1996 by David Quammen.  David is a science writer, but has also written some fiction, and at the time this book was published lived in Montana, from where I so recently emigrated.  It’s written in a meandering way, weaving together textbook information, historical accounts of ecologists from the last few centuries, and his own experiences traveling the world to visit the unique locations that inspire(d) scientists to brilliance.  While it certainly helps to have a background in biology or ecology in order to fully appreciate the book, it’s seems interesting enough to grab a more general audience.

Be prepared for a feast of delicious jargon, though:

“The Origin of Species is a book of encyclopedic richness and inexhaustible tediousness, a great potpourri of argument and fact in which a reader can find almost anything a reader might want: Lamarckism, animal husbandry, geology, ethology, experimental botany, the kitchen sink, island biogeography.” pg. 200

So what is island biogeography? It’s the study of how species are distributed across an environment; specifically on islands.  Sounds simple enough.  Let’s go back to the Age of Exploration (late 1400s to the late 1700s) when new technology and a growing appreciation for the size of the planet gave rise to a burst of exploration.  Suddenly- and this historical perspective is very Euro-centric- new lands, geology, peoples, plants, and animals were being discovered, and tales of the exotic made it back to Europe.  Sometimes, preserved animal specimens would make it back to Europe, which was extremely tricky as they had to be prepared in the field, usually by skinning or pickling.  Often, the heads, feet, tails, or wings would be removed during the process, accidentally or intentionally.  This only fueled the mystery more: many species of Birds of Paradise had their feed removed during processing, leading British ecologists, many of whom were working off secondary information and had never traveled to these locales, to believe that these birds had no feet at all and lived entirely among the clouds until their death when they fell to the ground.

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Birds of Paradise, Natural History Museum of Utah

The lure of discovering new, fabulous species was irresistible, and naturalists began expeditions all over the globe to make observations and collect specimens.  Largely, collectors interested in one particular animal or insect would select a small number of specimens for each species they collected, thus they accidentally missed the natural variations in size or color that one sees in wild animals.  After all, one doesn’t always notice little differences when only looking at a few examples.  Or, they would fail to record the particular location of their find, often only labeling it only by the continent on which is was collected.  But some naturalists were more curious.  They collected more specimens, more data, and began to notice patterns.

The most important pattern was that not all animals were found everywhere.  Certainly, it was noted that certain animals were specific to a habitat- sharks to the ocean, camels to the desert, etc.  But it wasn’t until people discovered animals found exclusively on islands that it really sunk in.  And this is extremely important, because it begged the question: why?  Why are some animals in one place and not another?  How did they get there?  The prevailing theories until that point were largely based on stories from the Christian bible, but with the discovery of so many new species, a literal ark was increasingly going to be improbably overcrowded.

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Geography and Evolution

Long story short, many ecologists actually began as geologists- Charles Darwin included, and in studying island formation it became understood that some island animals had crossed on land bridges, while others flew, swam, or drifted onto islands.  The species and mode of arrival very much determined whether you could then get back off the island, or whether you were stuck.  Ok, so now we know that animals can travel and change their own habitat location (which is different from migration), which went against the prevailing theory that animals were located where they had been put during a creation event.

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Finches

The next important pattern was that multiple, closely-related species could exist in a place at the same time.  In the years following his voyage while studying the specimens he collected, Charles Darwin noticed this of the mockingbirds, tortoises, and eventually the finches on the Galapagos, which was just a brief stop on his 5 year geology cruise aboard the Beagle (1831-1836).  Again, this was important, because what was the likelihood that all these similar bird species came to the same island chain at the same time?  It was more likely that a few birds of a single species had come over, and these birds had changed over thousands of generations into several new species.  The accepted notion was that animals didn’t change- they remained as they had been created.  The idea that a species could change or evolve over time was, at best, silly and at worst, blasphemous.

Nevertheless, a number of ecologists had made reference to the possibility of change during the Age of Exploration, but lacked solid data and a concrete theory of how. The mockingbirds represented true archipelago speciation; one species came to the Galapagos islands and populations became isolated on separate islands until through genetic drift they became different species, but there were only four mockingbird types and that was little enough to go on.  On the other hand, Darwin had 31 individuals representing what he thought was 14 unrelated bird species, but it wasn’t until after his voyage, when an ornithologist properly classified the birds as all being closely-related finches, that Darwin paid any attention to them at all.  In fact, Darwin nearly missed the idea of evolution because he failed to label which island his finches came from and very little about their ecology or behavior- he had to gather missing data from other accounts for years before he could see a real pattern. To be fair, the finches are a much more complicated pattern because they display adaptive radiation; one species arrived on the islands, but populations were only transiently isolated and when they crossed paths again they were still similar enough to compete, so different species evolved to fill different ecological roles (niches) in order to avoid starvation due to competition.

Darwin’s first account of his Beagle voyage made just a brief mention of this observation on closely-related species, but it changed the life of Alfred Wallace.  Wallace came from a poor background, and eventually paid for his love of naturalism and data collection by selling the specimens he collected.  Many British naturalists at the time were wealthy, and selling one’s collection seemed base- thus Wallace, with no title or reputation, was dismissed for most of his early career.  Years after Darwin went to the Galapagos, Wallace went to South America and Indonesia and came to the same conclusion about multiple closely related species: that one species had become many.  Wallace made the jump to speciation much faster, and sent Darwin a manuscript that was frighteningly similar to the yet-unpublished Origin of Species, which Darwin had worked on for 20 years to gain enough proof to avoid being laughed at.  Social politics aside, which are discussed in the book, a joint manuscript was presented, On the Tendency of Species to form Varieties; and on the Perpetuation of Varieties and Species by Natural Means of Selection, and a year later Darwin published On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life, which, incidentally doesn’t even mention Galapagos finches.

The idea of macroorganismal evolution was difficult to come by, largely because it’s a much longer process than a human can witness, and because a possible mechanism for change was completely unknown (genetics was a long way away).  By studying islands, ecologists could study evolution in miniature worlds where the pressure to stay alive was great- indeed, many species were marooned on the islands they colonized.  Studying this, and the livestock breeding industry, gave rise to the idea in Darwin’s mind of Natural Selection– that external forces could change a species over time by forcing the species to change.

Because animals are isolated on islands, they change to fit that particular ecosystem in a very visible way.  Wallace noticed this happening in his travels in South America where large rivers converged: animals that could not cross the river became isolated and there would be similar but distinct species on each side of the river.  Again, the whimsical biogeography of a deity became less probable than natural forces (food, geography, predation, competition) driving the distribution of animals and plants.  Still, it took decades to iron out the particulars of evolution, and even today people refuse to acknowledge it.

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Natural Selection, Understanding Evolution

But this book isn’t solely a historical account- all of that is setting the stage for a larger picture: extinction.  For even as island pressures select for the creation of species distinct from those found on mainlands, it also selects them for extinction.  Islands are partially or completely isolated, and this means any breeding population is small to begin with, and eventually can become inbred.  Island populations often collapse: the gene pool becomes too stagnant, a natural disaster hits, food becomes scarce, a predator appears.  Because there are only so many individuals, and because they are adapted to a very specific location, island species can’t deal with change.  Unfortunately, humans bring nothing but change.  As we develop natural land for our own use we fragment habitat, and for animals that can’t cross a city to get to the other populations, their gene pool and food options are limited.  They become reliant on very specific living conditions in their small habitat fragments, and they are more susceptible to disease, inbreeding, predators, and climate change.  The smaller the habitat, the fewer the individuals, and the ore they struggle to survive.  As humans colonize all parts of the globe we are leaving man-made islands in our wake, with marooned populations of plants and animals that find it increasingly difficult to sustain themselves- we are the cause of the mass extinction of animals and plants around the globe that only trickles into our mainstream news.

“We still argue about when it [the dodo] actually became extinct, but it probably disappeared around the 1660s.  It’s become the sort of legendary bird of extinction.  And a very important bird.  There were extinctions before and there’s been lots of extinctions since, but it was an important extinction because that was the first time, the first time in the whole of man’s history, that he actually realized he had caused the disappearance of a species.”

-interviewing Carl Jones about the extinction of the dodo, pg. 277

The level of detail provided in The Song of the Dodo is fascinating, especially because historical accounts so often lose sight of a who a person was and the journey they had to take.  Darwin wasn’t always correct, other scientists had the right theories but the wrong data to prove them, and the elitism of early science often led to the adoption of incorrect theories from otherwise brilliant men.  The book gives an honest perspective- that all scientists are trying their best to make sense of the information they have, and that it can take an extremely long time to put the entire puzzle together.  And it gives cause for hope.  While we may not be able to bring back populations of species we have pushed to the brink, life is pluripotent.  If we give the natural world some space- it’ll grow back.

Field notes from my first ESA meeting

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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!

Presentation on juniper diets and rumen bacteria from JAM 2016 available!

The video presentation of my work on the effects of juniper diets on rumen bacteria is finally available for public use!  I apologize for any side comments in the audio, the projector in the room kept flicking off! Stay tuned, our publication was just accepted and will be in press soon…

Abstract 1768.  Ground redberry juniper and urea in DDGS-based supplements do not adversely affect ewe lamb rumen microbial communities.

S.L. Ishaq, C.J. Yeoman, and T.R. Whitney. 2016.

 

Presentation on maternal influences on the calf digestive tract from JAM 2016 available!

The video presentation of my work on the effects of maternal biotic influences on the developing calf digestive tract bacteria is finally available for public use!

Abstract 1522: Influence of colostrum on the microbiological diversity of the developing bovine intestinal tract

Suzanne L Ishaq, Elena Bichi, Sarah K Olivo, James Lowe, Carl J Yeoman, Brian M Alridge