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.

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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.

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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, […]

Insight into the bacterial gut microbiome of the North American moose (Alces alces).

My first research project as a Ph.D. student was investigating the differences in the rumen and colon bacterial communities in moose from Vermont.  To obtain samples, I needed to secure permission from VT Department of Fish and Wildlife, as moose hunting and moose sample collection are tightly regulated; hunting is only permitted for approximately one week a year, and the licenses are awarded on a lottery system.  I was able to contact several permitted hunters for the 2010 season, and passed on sample collection kits to them, complete with gloves, jars, ice packs, a cooler, and instructions with photographs.  A total of 14 samples were collected, 8 from the rumen and 6 from the colon – a small sample group in general, but a good-sized one for a wild animal study.  If a hunter was successful, they would contact me to collect the samples as soon as possible after the carcass was reported to the state at one of a number of weigh stations.  Between dropping off and collecting samples, I drove a total of 1500 miles in about 9 days, and was able to see some beautiful parts of Vermont.

Once back at the lab, the samples were frozen at -80 degrees Celsius to protect the microbial cells and DNA from degradation- I knew I may not get another chance to collect rumen samples from moose.  For this project, I used a small amount of each sample and extracted the total mass of nucleic material available – the DNA and some RNA from all the cells present, including microbial, moose, and plant.  I then used Polymerase Chain Reaction (PCR) to amplify, or make copes of, just the bacterial DNA.  Specifically, I was looking for the 16S rRNA gene, which I would use for amplification.  I used a DNA microarray, Phylochip, which had fragments of DNA from known bacteria bound to the chip.  Once I applied my sample DNA, if there was a DNA- DNA match, my sample would bind to the chip as well as would fluoresce under ultraviolet light.  A sophisticated computer program would read the light signals and interpret then as the presence and rough abundance of that particular species of bacteria.

The most important finding of the study was that the rumen and colon hosted sufficiently different bacterial communities, such that colon or fecal samples were not a good proxy for what was happening in the rumen.  While this had been shown once before in sheep, it was not common practice to sample from more than one gastrointestinal location in ruminants, particularly in wild ones, because the cost of sequencing was high and the logistics of sample collection were difficult.  Thus, it was common practice to collect fecal samples from wild ruminants and speculate about the rumen microbial communities.


 

Ishaq, S.L., Wright, A-D.G. 2012. Insight into the bacterial gut microbiome of the North American moose (Alces alces). BMC Microbiology, 12:212.  Article

Abstract

Background

The work presented here provides the first intensive insight into the bacterial populations in the digestive tract of the North American moose (Alces alces). Eight free-range moose on natural pasture were sampled, producing eight rumen samples and six colon samples. Second generation (G2) PhyloChips were used to determine the presence of hundreds of operational taxonomic units (OTUs), representing multiple closely related species/strains (>97% identity), found in the rumen and colon of the moose.

Results

A total of 789 unique OTUs were used for analysis, which passed the fluorescence and the positive fraction thresholds. There were 73 OTUs, representing 21 bacterial families, which were found exclusively in the rumen samples: Lachnospiraceae, Prevotellaceae and several unclassified families, whereas there were 71 OTUs, representing 22 bacterial families, which were found exclusively in the colon samples: Clostridiaceae, Enterobacteriaceae and several unclassified families. Overall, there were 164 OTUs that were found in 100% of the samples. The Firmicutes were the most dominant bacteria phylum in both the rumen and the colon. Microarray data available at ArrayExpress, accession number E-MEXP-3721.

Conclusions

Using PhyloTrac and UniFrac computer software, samples clustered into two distinct groups: rumen and colon, confirming that the rumen and colon are distinct environments. There was an apparent correlation of age to cluster, which will be validated by a larger sample size in future studies, but there were no detectable trends based upon gender.

Keywords

ColonGut microbiomeRumenVermont16S rRNA

 

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Figure 2 Breakdown of unclassified families by phylum. (a) OTUs present in all 14 samples. There were 41 OTUs found exclusively in the rumen that were not classified down to the family level. (b) OTUs found exclusively in the rumen. There were 22 OTUs found exclusively in the rumen that were not classified down to the family level. (c) OTUs found exclusively in the colon. There were 19 OTUs found exclusively in the colon that were not classified down to the family level. Several are candidate phyla and are named by where they were discovered: AD3, soil in Virginia and Deleware, USA; OP3 and OP10, now Armatimonadetes, Obsidian Pool hot spring in Yellowstone National Park, USA; NC10, Null Arbor Caves, Australia; TM7, a peat bog in Gifhorn, Germany; WS3, a contaminated aquifer on Wurtsmith Air Force Base in Michigan, USA.

 

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Figure 3A comparison of the OTUs exclusive to the rumen or the colon. A comparison of the 73 OTUs exclusive in the rumen (n = 8) or 71 OTUs exclusive in the colon (n = 6), by family. Families with three or more associated OTUs are labeled in the chart; all other families with two or fewer OTUs are labeled via the legend. The Unclassified sections are broken down by phyla in Figure2b, and2c, respectively.