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.


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

High-throughput DNA sequencing of the moose rumen from different geographical location reveals a core ruminal methanogenic archaeal diversity and a differential ciliate protozoal diversity.

This project expanded upon my work with moose bacteria from three geographic locations, to explore whether there were differences in methanogenic archaea or ciliated protozoa based on location.

Archaea are microorganisms in their own Domain, as they are neither Bacteria nor Eukaryota, although they often have similarities to organisms found in the other two domains.  Archaea are found in many extreme environments, but those found in the digestive tract of animals and humans come from the phylum Euryarchaeota.   Methanogens require hydrogen to make energy for themselves, and in that process (methanogenesis) methane is created as a byproduct.  In the digestive tract, especially in ruminants where the fermentation of plants creates a lot of hydrogen, the presence of methanogens acts a hydrogen sink and can prevent the build up of hydrogen which would otherwise lower the gut pH and be detrimental to both host and microbes.  To date, it is unclear if methanogens have any other health effect.

Protozoa are single-celled eukaryotes, and depending on which species they are, can be beneficial or pathogenic.  Typically, protozoa in the digestive tract of humans or other monogastrics are pathogens obtained from drinking contaminated water.  However, the digestive tracts of monogastrics (ex. humans) and ruminants (ex. moose) are very different, and the later can support a much different microbial community.  Specifically, protozoa found in ruminants that have cilia to move around (i.e. ciliated protozoa or ciliates) can have a number of roles, including fermentation of fiber or starch, or predation of bacteria and fungi.  As they are so difficult to maintain in culture and study in the lab, the role of protozoa in contributing to host health or methanogenesis is understudied.

Moose methanogen communities were significantly different between moose in Vermont, Norway, and Alaska, but maintained a core of shared taxa across all populations.  This implies that the moose rumen environment (pH, salt content, turnover, host-microbe interactions, etc.) is suitable for only a small number of methanogen species, and that this regulates the community as much as diet might.  Methanogen communities were also different based on sex of the moose, and age/weight.

Figure 2: Diversity of moose rumen methanogens. Members of the RO clade are coloured in blues; members of the SGMT clade are coloured in reds. Mbr., Methanobrevibacter.

On the other hand, protozoal communities were dramatically different between moose in Vermont, Norway, and Alaska, and shared far fewer taxa.  This was surprising, as previous studies on deer had shown a core protozoal community across multiple geographically-separated populations.  These moose populations had not been geographically isolated long, but we hypothesized that diet was a much stronger driver of rumen protozoal diversity than previously thought.

Figure 3: Diversity of the moose rumen protozoa.

Ishaq, S.L., Sundset, M.A., Crouse, J., Wright, A-D.G. 2015. High-throughput DNA sequencing of the moose rumen from different geographical location reveals a core ruminal methanogenic archaeal diversity and a differential ciliate protozoal diversity. Microbial Genetics, 2015(1).  Article


Featured Image; Figure 1: PCoA for moose methanogens (A, C, E) and protozoa (B, D, F). PCoA is coloured by (A, B) gender: female, red; male, blue; (C, D) location: Alaska, red; Norway, green; Vermont, blue; and (E, F) weight class: 1–100 kg, red triangle; 101–200 kg, yellow triangle; 201–300 kg, green down-facing triangle; 301–400 kg, green right-facing triangle, >400 kg (live weight), light blue circle; not available, blue square.

Dissertation: A Comparative Analysis Of The Moose Rumen Microbiota And The Pursuit Of Improving Fibrolytic Systems

As a Ph.D. student, I worked in the laboratory of Dr. André-Denis Wright in the Department of Animal Science at the University of Vermont. My thesis work investigated the microorganisms (bacteria, archaea, and protozoa) in the digestive tract of the moose from several geographical locations. In addition to identifying bacteria and ciliate protozoa using high-throughput sequencing, […]

Design and validation of four new primers for next-generation sequencing to target the 18S rRNA gene of gastrointestinal ciliate protozoa.

If the research tools you require don’t exist- then you must create them yourself.  Such is often the case in working with microbial genomics.  In order to adapt sequencing technology to identify rumen ciliate protozoa, we needed to first design primers which to be used for Polymerase Chain Reaction (PCR) in order to amplify enough copies of the 18S rRNA gene for laboratory work.  This involved designing primers in silico, by aligning sequences from the few protozoal 18S rRNA genes publicly-available at the time, in order to identify short sections which were identical across protozoal species.  We then added 18S rRNA gene sequences to our alignment from other Eukaryotes, such as fungi and plants, which we did not want to amplify, to ensure that our primers would target only the desired taxa.  We also needed to design a primer set which would work well in the laboratory; in particular which had an optimal size for the sequencing technology on hand, and which would provide enough information in the portion of the gene capture to identify which protozoal species the DNA in our rumen samples originated from.

Ishaq, S.L., Wright, A-D.G. 2014. Design and validation of four new primers for next-generation sequencing to target the 18S rRNA gene of gastrointestinal ciliate protozoa. Applied and Environmental Microbiology, 80(17):5515-5521.  Article

Four new primers and one published primer were used to PCR amplify hyper-variable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST (NCBI) database and MOTHUR ver. 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F + GIC758R (amplicon = 482 bases) gave the best representation of diversity using BLAST classification, and amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F + GIC1578R (amplicon = 498 bases), had similar BLAST results and a slightly higher percentage of sequences that identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in non-ruminants. Amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, Tetratoxum, or between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.


Figure 1: A map of the full-length protozoal 18S rRNA gene, including variable (V1-V9) and rumen ciliate signature regions (SR1-SR4), and showing the respective amplicons of the three primer sets used in the present study.

Figure 2: Taxonomy and proportion of unique pyrosequences using NCBI (BLAST), by forward primers P-SSU-316F (Sylvester et al., 2004), GIC1080F (present study), and GIC1184F (present study). All sequences used passed all quality assurance steps outlined in Methods.