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. 

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.

The main driver of fungal diversity was diet; moving from a high-fiber diet to a high-grain diet (Figure 1) triggered a change in available nutrients (more starch, less fiber), and decreased in rumen pH due to the byproducts related to microbial digestion of those nutrients.  Supplementation with active dry yeast only had minimal effect on fungal populations in the rumen, and did not help recover the fungal community found in healthy cows on a high-fiber diet.  Saccharomyces-related sequences all classified as S. cerevisiae, though to multiple strains, but were not found in >1% mean relative abundance in any treatment group or significantly more abundant in any group. Thus, it was unclear if the yeast supplement was actively part of the rumen fungal community.

PowerPoint Presentation
Figure 1. Relative abundance of rumen fungi genera for cows receiving a high fiber (HF) or high grain (HG) diet, with (Y) or without (C) yeast supplementation. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Similarly, diet was the major driver of protozoal diversity in the rumen (Figure 2), but there was also a small effect of the yeast supplementation.  Taxonomic diversity was also different between the high-fiber control (what the cows were before) and the high-grain yeast-supplemented group, indicating that yeast supplementation did not recover the initial protozoal community which healthy cows had.

PowerPoint Presentation
Figure 2. Relative abundance of rumen protozoal species for cows receiving a high fiber (HF) or high grain (HG) diet, with (Y) or without (C) yeast supplementation. Treatments include high-fiber control (HFC), high-fiber yeast (HFY), high-grain control (HGC), and high-grain yeast (HGY).

Another large difference was seen in the number and type of species found in three different locations within the rumen: those found in rumen fluid, those found attached to plant material (and presumably digesting it), and those found attached or associated with the rumen wall (epimural-associated).  In cows fed the high-grain diets, there were not enough fungi in the rumen fluid to generate enough sequences for comparison, and the high-grain diet tended to reduce the number of different species found in any location.  Fungal species richness was highest in plant-associated fractions, and there was surprisingly high species richness of fungi which were found along the rumen wall.  Protozoal species richness was likewise reduced by a switch to a high-grain diet, and was highest next to the rumen wall.


Ishaq, S.L., AlZahal, O., Walker, N., McBride, B. 2017. 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.  Frontiers in Microbiology 8:1943. Article.

Abstract

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.


Ishaq, S.L.*, O. AlZahal, N. Walker, B. McBride. 2017. Modulation of sub-acute ruminal acidosis by active-dry yeast supplementation and its effect on rumen fungal and protozoal populations in liquid, solid, and epimural fractions.  Congress on Gastrointestinal Function, Chicago, IL, April 2017. (accepted talk).

 

Featured Image Credit: Wikimedia Commons

Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal 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 lining the digestive tract, 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.

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Figure 1 Mean bacterial diversity at the phylum level for maternal and calf lumen (A) and mucosal (B) samples.

Samples from the mothers were also taken to understand how maternal microbial influence would affect body sites over time.  One of the most interesting finds of the study regarded colostrum, which is the special and highly-nutritious milk produced in the first 48 hours or so after parturition (birth).  Colostrum milk possessed a high diversity of bacteria, and is not sterile as was once assumed.

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Figure 1 (partial) Mean bacterial diversity at the phylum level for maternal and calf lumen (left) and mucosal (right) samples.

Not only that, but the bacterial community in colostrum had an impact on the bacterial community that developed along the calf digestive tract over time.  Calves received two doses of colostrum on the day of birth which had been aseptically collected from their dams and then fed to them, so that calves received milk but not the microbial influence of nursing and coming into contact with the dam.  After those two meals, calves were switched to milk replacer.  Surprisingly, the influence on the bacterial community wasn’t high on day one and then dropped off.  It increased over the first 21 days of life as bacterial communities from the digestive tract became more similar to bacterial communities found in colostrum (shown below).

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A poor-quality GIF, showing bacterial communities from the calf digestive tract (each other the colored shaped) becoming more similar to maternal colostrum (milk) samples (grey asterisk) over the first 21 days of life.

In addition, we found that bacteria in the digestive tract became more similar to maternal samples moving from one end of the digestive tract to the other. We speculated that bacterial communities need time to develop, especially in a neonate ruminant which doesn’t have a functional rumen yet.  A flap of skin at the base of the esophagus (called the esophageal groove) shunts food into the omasum, bypassing the rumen and the reticulum where bacteria and other microorganisms would otherwise thrive.  After briefly passing through the omasum, milk would pass through the abomasum which is a glandular stomach (like the human stomach).  Both of those features are obstacles for ingested microorganisms to get past, and it would take time, and distance, to recover.

GI tract
An equally poor-quality GIF, showing bacterial communities (colored shapes) from the calf digestive tract samples becoming more similar to maternal samples (grey asterisks) as one moves along the digestive tract.

Yeoman, C.J., Ishaq, S.L., Bichi , E., Olivo, S., Lowe, J., Aldridge, B.M. 2018. Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal tract. Scientific Reports 8: 3197Article.

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.


Ishaq*, S.L., Bichi, E., Olivo, S.K., Lowe, J., Yeoman, C.J., Aldridge, B M. 2016. Influence of colostrum on the microbiological diversity of the developing bovine intestinal tract. Joint Annual Meeting, Salt Lake City, Utah, July 2016. (accepted talk)

 

 

Ground Juniperus pinchotii and urea in supplements fed to Rambouillet ewe lambs. Part 2: Ewe lamb rumen microbial communities.

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.  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.

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.

There was not a large effect of treatment on the rumen bacterial community in lambs (Figure 1B).  There was a change in animal production (feed intake and weight gain) (Whitney, 2017), yet, because bacterial diversity was largely unchanged by the diet, this was likely because the diet treatments reduced feed intake.  Plant secondary compounds, often called dietary toxins, can make it harder for animals to maintain a stable body temperature as they change fermentation in the rumen and how much heat is produced.  This increases the metabolic cost of thermoregulation as animals continuously have to adjust their rate of metabolism to keep their body temperature stable.  To avoid eating too many of these plant compounds, herbivores employ feeding strategies, such as reducing feed intake. It is possible that lambs ingesting high concentrations of juniper in Texas during the late summer simply consumed less supplemental diet in order to reduce toxin- and fermentation-related heat generation.

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Figure 1B Principal coordinate analysis (PCoA) plot comparing OTU abundance in ewe lamb rumen samples over increasing juniper (J) or urea (U) supplementation by % DM. Vectors show significant effects (Pearson’s correlation P > 0.75) treatment, with vector length showing strength of correlation.

That’s not to say that there were no changes to the bacterial community at all; in fact, a number of important bacterial families were increased or decreased by increasing the amount of juniper, increasing the amount of urea, or both (Figure 2).

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Figure 2. Mean rumen bacterial abundance at the family level for ewe lambs on differing juniper (J) and urea (U) supplementations. Families are color coordinated by phylum: Bacteroidetes = red, Firmicutes = blue, Proteobacteria = orange, Spirochaetae = green, and other (dark grey) constitutes families with < 1% total abundance. Families of interest appear on the right side with positive (green) and negative (red) changes indicated as significant (P < 0.05) or trending (0.05 < P < 0.1) according to Student’s T-test.

Ishaq, S.L., Yeoman, C.J., Whitney, T.R. 2017. Ground Juniperus pinchotii and urea in supplements fed to Rambouillet ewe lambs. Part 2: Ewe lamb rumen microbial communities. Journal of Animal Science Oct; 95(10):4587-4599. Article.

Abstract

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 Succiniclasticumincreased 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.


Ishaq*, S.L., Yeoman, C.J., Whitney, T.R. 2016. Ground redberry juniper and urea in DDGS-based supplements do not adversely affect ewe lamb rumen microbial communities. Joint Annual Meeting, Salt Lake City, Utah, July 2016. (accepted talk). Travis Whitney’s companion presentation can be found here.

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Featured Image Credit: National Park Service

 

A living soil inoculum increases soil microbial diversity, crop and weed growth using soil from organic and conventional farms in northeastern Montana.

What began as a simple data analysis project for me in the Yeoman lab turned into a publication, a conference presentation, a post-doc position, and a long-term, multi-project collaboration with the Menalled lab at Montana State University investigating soil microbial communities in agricultural settings and plant-soil feedbacks.

This study was part of a larger investigation on farming system (conventional or organic), and wheat-weed competition, as part of a master’s thesis by Stephen Johnson.  The publication on plant competition and crop performance can be found here.

The larger project involved soil collected from the fields of four farms around Montana which had both conventionally-managed and a USDA-certified organically-managed plots growing wheat.  Soil was brought back to Montana State University, where half of each field sample was sterilized to destroy living microorganisms.  A greenhouse study was performed using either the sterile or the living soil, and the soil was conditioned by growing either Amaranthus retroflexus L. (redroot pigweed) or Avena fatua L. (wild oat) for 16 weeks.  Following this plant growth phase, soil was collected and the bacterial community analyzed using Illumina MiSeq sequencing of the 16S rRNA gene.  The larger study then went on to study the performance of wheat crops in that preconditioned soil.

The strongest driving factor in soil bacterial communities was whether that soil had been sterile (purple) or living (green) in the greenhouse experiment, as seen below.  After that, farming system was the next strongest determinant of that community.  Interestingly, organically-sourced soil that had been sterilized was more similar to any living soil than conventionally-sourced sterile soil.  This indicates that organic soil was more favorable in recruiting a new soil community.

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When comparing only the living soil samples, the samples reclustered by farming system; either organic or conventional.

Which weed species was growing was also an important factor, although much weaker.  A number of soil bacteria were more abundant in the soil around of the roots of one or the other plant.  Plants are known to associate with, and even recruit, different microbial communities, and this interaction can be plant-species-specific.


Ishaq, S.L., Johnson, S.P., Miller, Z.J., Lehnhoff, E.A., Olivo, S.K., Yeoman, C.J., Menalled, F.D. 2017. A living soil inoculum increases soil microbial diversity, crop and weed growth using soil from organic and conventional farms in northeastern Montana. Microbial Ecology 73(2): 417-434. Impact 3.630. Article

Abstract

Farming practices affect the soil microbial community, which in turn impacts crop growth and crop-weed interactions. This study assessed the modification of soil bacterial community structure by organic or conventional cropping systems, weed species identity [Amaranthusretroflexus L. (redroot pigweed) or Avena fatua L. (wild oat)], and living or sterilized inoculum. Soil from eight paired USDA-certified organic and conventional farms in north-central Montana was used as living or autoclave-sterilized inoculant into steam-pasteurized potting soil, planted with Am. retroflexus or Av. fatua and grown for two consecutive 8-week periods to condition soil nutrients and biota. Subsequently, the V3-V4 regions of the microbial 16S rRNA gene were sequenced by Illumina MiSeq. Treatments clustered significantly, with living or sterilized inoculum being the strongest delineating factor, followed by organic or conventional cropping system, then individual farm. Living inoculum-treated soil had greater species richness and was more diverse than sterile inoculum-treated soil (observed OTUs, Chao, inverse Simpson, Shannon, P < 0.001) and had more discriminant taxa delineating groups (linear discriminant analysis). Living inoculum soil contained more Chloroflexi and Acidobacteria, while the sterile inoculum soil had more Bacteroidetes, Firmicutes, Gemmatimonadetes, and Verrucomicrobia. Organically farmed inoculum-treated soil had greater species richness, more diversity (observed OTUs, Chao, Shannon, P < 0.05), and more discriminant taxa than conventionally farmed inoculum-treated soil. Cyanobacteria were higher in pots growing Am. retroflexus, regardless of inoculum type, for three of the four organic farms. Results highlight the potential of cropping systems and species identity to modify soil bacterial communities, subsequently modifying plant growth and crop-weed competition.

 

Poster: Ishaq*, S.L., Johnson, S.P., Miller, Z.J., Lehnhoff, E.A., Olivo, S.K., Yeoman, C.J., Menalled, F.D. Farming Systems Modify The Impact Of Inoculum On Soil Microbial Diversity. American Society for Microbiology (ASM), Boston, MA, June 2016.

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Poster presentation at ASM 2016.

Ishaq et al ASM 2016 poster