After two years of postponement, the Ishaq Lab is excited to welcome Dr. Alaa Rabee as a Visiting Researcher from now until December of this year!! This is made possible by a prestigious award to Dr. Rabee from the Central Department of Missions at the Ministry of Higher Education, Egypt, which fosters research collaboration between Egypt and the U.A.
Dr. Rabee joins us from the Desert Research Institute in Cairo, Egypt, where his work focus on researching microbial communities in the digestive tract of ruminants and how they can be used for animal production, bioengineering, and sustainable development.
Last year, Alaa and I published our first collaborative paper together, based on his work on microbial enzymes from the rumen of sheep and camels and potential for use in biofuel production. We are also working on another, based on microbial activity (transcriptomics) in the rumen of camels on different diets. That project has engaged two undergraduate students in data visualization, including Myra Arshad who started in my lab as an REU student last summer.
During his six month stay, he’ll be working with rumen microbes from various livestock, as well as giving seminars and sharing his experience in research.
A scientific article led by my colleague Dr. Alaa Rabee at the Desert Research Center in Egypt was just published online and is now available! Dr. Rabee and I have been collaborating remotely on projects related to the bacteria in the rumen of camels, sheep, and cows, as Dr. Rabee’s work focuses on the isolation of bacteria which can degrade plant materials efficiently and could be used to produce biofuels. He will be spending 6 months working in my lab as a visiting scholar, which was delayed until this year because of the pandemic.
Rabee, A.E., Sayed Alahl, A.A., Lamara, M., Ishaq, S.L. 2022. Fibrolytic rumen bacteria of camel and sheep and their applications in the bioconversion of barley straw to soluble sugars for biofuel production. PLoS ONE 17(1): e0262304. Article.
Abstract
Lignocellulosic biomass such as barley straw is a renewable and sustainable alternative to traditional feeds and could be used as bioenergy sources; however, low hydrolysis rate reduces the fermentation efficiency. Understanding the degradation and colonization of barley straw by rumen bacteria is the key step to improve the utilization of barley straw in animal feeding or biofuel production. This study evaluated the hydrolysis of barley straw as a result of the inoculation by rumen fluid of camel and sheep. Ground barley straw was incubated anaerobically with rumen inocula from three fistulated camels (FC) and three fistulated sheep (FR) for a period of 72 h. The source of rumen inoculum did not affect the disappearance of dry matter (DMD), neutral detergent fiber (NDFD). Group FR showed higher production of glucose, xylose, and gas; while higher ethanol production was associated with cellulosic hydrolysates obtained from FC group. The diversity and structure of bacterial communities attached to barley straw was investigated by Illumina Mi-Seq sequencing of V4-V5 region of 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes and Bacteroidetes. The dominant genera were RC9_gut_group, Ruminococcus, Saccharofermentans, Butyrivibrio, Succiniclasticum, Selenomonas, and Streptococcus, indicating the important role of these genera in lignocellulose fermentation in the rumen. Group FR showed higher RC9_gut_group and group FC revealed higher Ruminococcus, Saccharofermentans, and Butyrivibrio. Higher enzymes activities (cellulase and xylanase) were associated with group FC. Thus, bacterial communities in camel and sheep have a great potential to improve the utilization lignocellulosic material in animal feeding and the production of biofuel and enzymes.
I’m delighted to announce that a paper was published on the effect of a dietary additive on the rumen and fecal bacterial communities in dairy cattle, in the journal Animal: The International Journal of Animal Biosciences!
A lot of factors can be manipulated to help get the most out of one’s diet, including the source and processing method of the ingredients – in most cases in livestock feed: plants. Growing plants for animal feed can be expensive, and often nutrients in plants become more available to the animal after the plant has been processed/broken down in some way. This sometimes allows for food byproducts to be reused for animal feed, and one common example is used brewers’ grains. Once the grains have been fermented to produce alcohol, the simple sugars have been used up but a lot of the complex sugar carbohydrates – in other words: fiber – are left over. Ruminants don’t need simple sugars, but they do need a lot of fiber, and brewers’ grains have been investigated for their usefulness for animal nutrition because they are a cheap, readily-available, and common source of fiber, as well as protein.
The original experiment for this work took place several years ago, and involved an animal feeding trial which added reduced-fat distillers’ grains with solubles into dairy cattle feed. The research team found no negatives effect on milk production or animal health, and that work was previously published. To add to that project, the original research team wanted to know if the diet would drastically change the bacterial community living in the rumen, which would have implications for feed digestion and animal health.
A collaborator of mine donated the cow microbial community DNA data to my AVS 590 special topics in DNA Sequencing Data Analysis course in spring 2020 (now formally registered as AVS 454/554). I worked with UMaine graduate students Adwoa Dankwa and Usha Humagain over the semester to train them in coding and develop the manuscript. The diet only had minimal effects on the bacterial community profiles, which in this case is a good finding – we want to be able to feed a cheap, nutritional source like distillers’ grains without harming the cow or its microbes.
Reduced-fat dried distillers’ grains with solubles (RF-DDGS) is a co-product of ethanol production and contains less fat than traditional distillers’ grains. The fat in corn is ~ 91% unsaturated, and it is toxic to rumen microorganisms so it could influence the composition of the rumen microbiome. It has been demonstrated that RF-DDGS is a suitable ration ingredient to support the high-producing dairy cow, and this feedstuff is a promising alternative protein source for lactating dairy cows. The current study aims to better understand the effect of RF-DDGS on the rumen and fecal bacterial composition in lactating dairy cows. Thirty-six multiparous (2 or 3), mid-lactation Holstein cows (BW = 680 ± 11 kg; 106 ± 27 DIM) were randomly assigned to two groups which were fed a control diet made up of corn, corn silage, and alfalfa hay supplemented with expeller soybean meal or with added RF-DDGS (20% of the dry matter (DM)) containing approximately 6.0% fat. Whole rumen contents (rumen fluid and digesta; esophageal tubing method) and feces (free catch method) were collected on day 35 of the experimental period, after the 14-d acclimation period. Rumen contents and feces from each cow were used for DNA extraction. The bacterial community composition in rumen and fecal samples was assessed via the 16S rRNA gene by using the Illumina MiSeq sequencing platform. Bacteroidetes, Actinobacteria, and Firmicutes were the most abundant phyla in rumen contents. The fecal microbiota was dominated by the phyla Firmicutes and Bacteroidetes, as well as Actinobacteria and Chloroflexi. RF-DGGS increased bacterial richness, evenness, and Shannon diversity in both rumen and fecal samples and was associated with several taxa that had different abundance in treatment versus control comparisons. The RF-DGGS, however, did not significantly alter the bacterial community in the rumen or feces. In general, these findings demonstrated that dietary inclusion of RF-DDGS did not impose any serious short-term (within 30 days) health or production consequences, as would be expected. With this study, we present further evidence that inclusion of 20% (DM basis) RF-DDGS in the diet of lactating dairy cows can be done without consequence on the microbiome of the rumen.
Implications
Reduced-fat dried distillers’ grains with solubles is a quality, economical, and readily available protein source demonstrated to support the protein needs of high-producing dairy cows. In this study, the rumen and fecal bacterial communities of lactating dairy cows were not significantly influenced by 20% (dry matter basis) reduced-fat dried distillers’ grains with solubles and did not impose serious short-term (within 30 days) health or production consequences. This diet could potentially be introduced into Total Mixed Ration feeding of dairy cattle given the fact that it is readily available and relatively economical.
For the past four days, I have been in Monterrey, Mexico, where I have been fostering international scientific relations in meetings and in the mountains. It was my very first trip to Mexico, and it was an amazing experience.
Monterrey, Mexico around the Hotel Novotel Monterrey Ville
After arriving in Monterrey, I was greeted by a research friend of mine, Dr. Jose Garcia-Mazcorro. We met a few years ago when Jose emailed me to ask questions about a recently published paper on Saccharomyces probiotic treatment in cattle, and our conversations on the ecological theory behind probiotics led to a review paper on the subject, led by Jose and I. It wasn’t until last August when Jose and I actually met in person, at the ISME conference in Leipzig, Germany.
At dinner, I had the opportunity to meet Jose’s wife,
Alecia, who is also a researcher, and their son, and discuss everything from
aflatoxin to Stephen King’s “It” (we discovered that their son and I were both
reading It when I discussed living in Maine, not far from Derry).
The next day, I woke up at 3:45 am to travel to the mountains for an incredible experience: a small-group tour in Matacanes canyon led by Daniel, a mountaineer with 20 years of experience and owner of Todo Avetura. He and Omar, another guide, led us for 12 hours and taught us about Matacanes canyon while we trekked 13 kilometers (8.7 miles) down waterfalls, through caves, over boulders, and over cliffs. Even the drive into and out of the canyon was an adventure; the steep road into the mountains fords rivers and winds along cliff faces. In the canyon, I got to do many things for the first time, including rappel down the side of two waterfalls; 27 m (88.6 ft) and 15 m (49.2 ft) into a cave, swim through the absolute dark of a river cave system, and jump off of several cliffs into the water below, including a 9.5 meter (31 ft) jump! It was supposed to be 10 meters, but that looked just a little too terrifying to try so I chose a spot that looked friendlier. Turns out that jumping 9.5 meters is a lot like getting up to present in front of a large audience, you just have to get up there and do it before you have time to think about and psych yourself out.
The guides were really passionate about the mountains, and they were particular about safety and not rushing or pushing us to the point where we would get hurt. If you have the chance, and the cohones, to go to Matacanes, I highly recommend Todo Aventura.
On Monday, sore but no worse for wear, I gingerly toured some of the facilities where Jose is currently working, MNA, an animal nutrition company. I met with company president and nutritionist Dr. Jorge Kawas, and Jose, Jorge, and I discussed the role of microbes in animal nutrition and health.
Dr. Jose Garcia-Mazcorro and I at MNA in Monterrey.
Monday night, I got to chat one-on-one with Professor T.J. Nagaraja, an author on theSaccharomyces review and a prominent researcher in rumen acidosis, cattle health, and infectious disease.
The main reason for my trip to Monterrey was to attend and speak at the XXII UANL-Engorda de Bovinos en Corral Symposium. I presented the opening seminar titled “Raising feedlot cattle with good microbes in mind” (“Cria y engorda de ganado con buenos microbios en mente”). The video can be found here, and slides with presentation notes here:
Organized by MNA and UANL, the university in Monterrey, the symposium brings together researchers, producers, animal industry professionals, and students to discuss animal health in feedlot cattle. I was honored to give the opening talk, which will be available online soon, and pleased to hear that the audience did in fact like microbes more than before my seminar! Usually when I start talking about the gut microbiome people have the urge to run off and wash their hands…
Unfortunately, I had to jump back
on a plane shortly after my talk, as I am heading to give a different
presentation at the Wildlife Society meeting in Reno, Nevada tomorrow! But, I
have plenty of memories and new project ideas to remember my trip by, and
hopefully I will come back to Monterrey soon!
The Hungate 1000 Project was a massive undertaking: namely, sequencing the genome of 1000 microorganisms cultured from ruminant animals all over the world, and was both coordinated and led by the Rumen Microbial Genomics Network. After years of hard work by some incredible researchers, the Hungate 1000 has just been published in the Nature Biotechnology Journal! The […]
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, 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. Samples from the mothers were also taken to understand how maternal microbial influence would affect body sites over time.
This paper was just published in Scientific Reports, and was something I had previously presented on 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 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.
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.
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.
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
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 Pyramidobacter. Prevotella were decreased by juniper and urea. Ruminococcus, Butyrivibrio, 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.
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!
A manuscript that I helped co-author, “Rumen and cecum microbiomes in reindeer (Rangifer tarandus tarandus) are changed in response to a lichen diet and may effect enteric methane emissions” was just accepted for publication in PLOS ONE. In 2012 I went to Norway to apprentice for two weeks in the lab of Dr. Monica Sundset, and in 2013, Monica’s graduate student Alex came to the University of Vermont to apprentice in Dr. Andre Wright’s lab, where I taught him quantitative real-time PCR and some bioinformatics. Alex performed a feeding trial back in Norway, in which reindeer were fed a lichen-based diet, in order to assess changes in microbial diversity. Lichens contain usnic acid, which is toxic to ruminants. Reindeer; however, host some unique bacteria which degrade usnic acid in the rumen and allow the reindeer to eat them without dietary problems.
“Abstract
Reindeer (Rangifer tarandus tarandus) are large Holarctic herbivores whose heterogeneous diet has led to the development of a unique gastrointestinal microbiota, essential for the digestion of arctic flora, which may include a large proportion of lichens during winter. Lichens are rich in plant secondary metabolites, which may affect members of the gut microbial consortium, such as the methane-producing methanogenic archaea. Little is known about the effect of lichen consumption on the rumen and cecum microbiotas and how this may affect methanogenesis in reindeer. Here, we examined the effects of dietary lichens on the reindeer gut microbiota, especially methanogens. Samples from the rumen and cecum were collected from two groups of reindeer, fed either lichens (Ld: n = 4), or a standard pelleted feed (Pd: n = 3). Microbial densities (methanogens, bacteria and protozoa) were quantified using quantitative real-time PCR and methanogen and bacterial diversities were determined by 454 pyrosequencing of the 16S rRNA genes.
In general, the density of methanogens were not significantly affected (p>0.05) by the intake of lichens. Methanobrevibacter constituted the main archaeal genus (>95% of reads), with Mbr. thaueri CW as the dominant species in both groups of reindeer. Bacteria belonging to the uncharacterized Ruminococcaceae and the genus Prevotella were the dominant phylotypes in the rumen and cecum, in both diets (ranging between 16–38% total sequences). Bacteria belonging to the genus Ruminococcus (3.5% to 0.6%; p = 0.001) and uncharacterized phylotypes within the order Bacteroidales (8.4% to 1.3%; p = 0.027), were significantly decreased in the rumen of lichen-fed reindeer, but not in the cecum (p = 0.2 and p = 0.087, respectively). UniFrac-based analyses showed archaeal and bacterial libraries were significantly different between diets, in both the cecum and the rumen (vegan::Adonis: pseudo-F<0.05). Based upon previous literature, we suggest that the altered methanogen and bacterial profiles may account for expected lower methane emissions from lichen-fed reindeer.”
