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 [1,2] and ciliate protozoa [3,4] using high-throughput sequencing, I was the first to identify methanogenic archaea in moose . I also cultured over 100 bacterial isolates form the rumen of moose in Vermont, some of which were then used in a probiotic study to improve fiber digestion in lambs . To test my probiotic in vivo, I was awarded a NIFA Pre-Doctoral Fellowship grant from the USDA in 2013.
During my Ph.D., I gained experience in aerobic and anaerobic culturing, microbiology and biochemical investigations of bacterial isolates, DNA extraction, PCR, qualitative real-time PCR, high-throughput sequencing library generation, DNA analysis, and animal handling. In addition to my moose work, I collaborated on microbial diversity projects involving the microbial diversity of mice on a high- or low-fat diet, methanogenic archaea in the human colon and whether breathe methane could be used to estimate colon methanogen density, the effect of lichen-based diets on microbial diversity in reindeer, and the microbial diversity of cows with sub-clinical acidosis.
During my graduate work I gained experience working with digesta or fecal samples from alpaca, antelope, beaver, black bear, cows, elephants, giraffe, horses, humans, mice, reindeer, and sheep, though the focus of my graduate work was on the rumen microbiome of moose. Previous to my investigations into the microbiome of the moose digestive tract, little work had been done to understand the complex digestive tract dynamic of this unique wild ruminant. Dehority  used traditional anaerobic culturing techniques to describe bacteria isolated from the rumen of an Alaskan moose, and several other studies used microscopic identification to describe the rumen protozoa of moose from Alaska , Finland , and Slovakia . Prior to my work, no investigation of the methanogens of the moose had been undertaken, and no high-throughput technique had been used to study the moose rumen microbiome.
Moose (Alces alces) are the largest member of the Cervidae (deer family), and can be found across most of Canada, some parts of the northern US and Alaska, Scandinavia, and across Russia. They inhabit mixed deciduous/coniferous, consuming browse such as bark, twigs and leaves of willow, aspen, maple, birch, ash, apple, blueberry, and pine. Additionally, they consume seasonally available aquatic vegetation, which is much higher in salt than most arboreal plants. Overall, the diet of moose is high in fiber, especially cellulose and lignin. As sodium is a limiting nutrient in their diet, moose have several unique characteristics in their digestive tract which allow them to store sodium, or even replace sodium in saliva with potassium. During the lean winter months, they routinely lose up to 20% of their bodyweight. Combined, these attributes provide a unique digestive tract environment and host-microbe interaction. The focus of my graduate studies was to investigate the microorganisms in the digestive tract of the moose, isolate and culture those which degrade fiber, and introduce fibrolytic bacteria into other systems to improve fiber degradation.
The microbial populations in the rumen of VT moose were originally characterized using a DNA microchip, and then further investigated with higher accuracy and coverage using a Roche/454 GS FLX sequencer and Titanium chemistry, along with rumen samples from Norway and Alaska. As there was no sequencing-standard variable region of the 18S rRNA gene for rumen protozoa, nor a wide selection of protozoa-specific primers which would be ideal for high-throughput sequencing, four new primers were developed and tested in the lab.
In order to investigate the moose rumen bacteria face-to-face, I spent two weeks at the University of Tromsø, Tromsø, Norway, in the lab of Dr. Monica A. Sundset. There, I learned how to anaerobically isolate and culture bacteria from the digestive tract of reindeer using an anaerobic chamber.
The chamber allows you to create an enclosed environment set to your atmospheric gas specifications, in this case it was 90% nitrogen, 5% carbon dioxide, and 5% hydrogen (and no oxygen) so simulate the ambient gas in the rumen.
Following the identification of the moose rumen microorganisms, bacteria from the rumen of VT moose were anaerobically cultured and isolated; due to the proximity of moose hunting areas in Vermont to the University of Vermont, samples were able to be frozen and transported back to the lab within hours of host death. Over 100 isolates were cultured and identified as various species of Acinetobacter, Bacillus, Escherichia, Shigella, Staphylococcus, Streptococcus, Micrococcus, and Neisseria. Two new species of Streptococcus, and several new subspecies of Streptococcus gallolyticus, have been proposed and described in a manuscript which is currently in preparation.
At the end of April 2014, a probiotic of several fibrolytic species was inoculated into the rumen of neonatal lambs to improve fiber digestion . Twenty-four lambs were hand-reared on milk replacer, weaned onto hay and a lamb starter grain, and later transferred to another farm to be let on natural pasture for the duration of summer and mid fall. Rumen samples, as well as production data such as weights and wool growth, were routinely taken.
The developing bacterial microbiome was compared between the experimental and controls groups as they changed diet over time. It was hypothesized that using pre-weaned animals which have not yet been completely colonized, and by re-administering the probiotic through weaning onto a fiber diet, would enable the inoculant to establish instead of carving out a foothold in an established bacterial arena.
As a post-doctoral researcher, the focus of my work was to provide bioinformatics analysis on a variety of microbial projects for the entire lab, for which I have had to compare analysis programs and algorithms, and write my own programming scripts, in order to improve and streamline the analysis workflow based on the needs of each project and the profile of the resulting microbial data. From April 2015 to May 2017, I worked on a variety of projects with over a dozen different collaborators. These projects included investigating drinking water and sources of fecal contamination, soil microbial diversity after microbial inoculation and different cover crops, the microbial diversity in the digestive tract of calves and how it is influenced by maternal sources, the rumen microbial diversity of sheep following experimental diets containing juniper, and the microbiome of lambs reared under different conditions.
In addition to analyzing data, I informally taught bioinformatics to undergraduate and graduate students in the laboratory, and consulted on student projects involving pure cultures, the honeybee microbiome, the gut microbial diversity of fish grown for aquaculture, and the soil microbial diversity following different grazing patterns. For two years, I taught the laboratory section of BIOM 405: Host-associated microbiomes, an upper-level course for undergraduate and graduate students at MSU. The course itself discussed a variety of plant-, insect-, animal-, and human-associated microbiomes. The lab that I designed introduced students to current sequencing technology, the data which are created, the biases inherent to each technology and how to overcome that with analysis. The students took a mock bacterial dataset from raw data through to finished manuscript to learn command line programming, basic bioinformatics, scientific reading and writing, and to become familiar with different DNA analysis programs.
In my second post-doc position in Land Resources and Environmental Sciences, I primarily worked on a project that got funded through the USDA, entitled “Assessing the vulnerability and resiliency of integrated crop-livestock organic systems in water-limited environments under current and predicted climate scenarios”, but I also worked collaboratively on several other similar projects in the lab. My responsibilities included comparing agronomic performance and weed-crop-pathogen interactions between organic-tilled and organic-grazed systems, evaluating the impact of management and biophysical variables on soil microbial communities, and collaborating in modeling the long-term consequences of these interactions under current and predicted climate scenarios.
In addition to my new skills, I integrated my background in microbial ecology and bioinformatics, in order to study agricultural ecosystems more holistically and measure plant-microbe interactions. In the same way that humans eat probiotics to promote a healthy gut microbiome, plants foster good relationships with specific soil microorganisms.
- Ishaq SL, Wright A-DG: Insight into the bacterial gut microbiome of the North American moose (Alces alces). BMC Microbiol 2012, 12:212.
- Ishaq SL, Wright A-DG: High-throughput DNA sequencing of the ruminal bacteria from moose (Alces alces) in Vermont, Alaska, and Norway. Microb Ecol 2014, 68:185–195.
- Ishaq SL, Wright A-DG: Design and validation of four new primers for next-generation sequencing to target the 18S rRNA gene of gastrointestinal ciliate protozoa. Appl Env Microbiol 2014, 80:5515–5521.
- Ishaq SL, Sundset MA, Crouse J, Wright A-DG: 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. MGen 2015, 1.
- Ishaq SL, Kim CJ, Reis D, Wright A-DG: Fibrolytic bacteria isolated from the rumen of North American moose (Alces alces) and their use as a probiotic in neonatal lambs. PLoS One 2015, 12.
- Dehority BA: Microbes in the foregut of arctic ruminants. In Control of digestion and metabolism in ruminants: Proceedings of the Sixth International Symposium on Ruminant Physiology. Edited by Milligan LP, Grovum WL, Dobson A. Englewood Cliffs: Prentice-Hall; 1986:307–325.
- Dehority BA: Rumen ciliate fauna of Alaskan moose (Alces americana), musk-ox (Ovibos moschatus) and Dall mountain sheep (Ovis dalli). J Eukaryot Microbiol 1974, 21:26–32.
- Westerling B: The rumen ciliate fauna of cattle and sheep in Finnish Lapland, with special reference to the species regarded as specific to reindeer. Nord Vet Med 1969, 21:14–19.
- Sládeček F: Ophryoscolecidae from the stomach of Cervus elaphus L., Dama dama L., and Capreolus capreolus L. Vestn Csl Zool Spole 1946, 10:201–231.