The differences between a healthy / unhealthy biome. We learn how gene sequencing technology can reveal common gastrointestinal disease.

Gastrointestinal diseases and upsets are common inThoroughbred racehorses, causing discomfort, lossof performance and even mortality. Every commongastrointestinal disease can be linked back todisturbances (dysbiosis) of the gut bacteria. Currently, new genetechnology is driving research at an intense rate, providing newinsights into the equine microbial community (1) and providingboth trainer and the vet with a powerful and accurate analyticaltool to improve health and manage disease.The gastrointestinal tract of the horse is colonized by trillionsof microorganisms, which includes 1,000-1,500 different species,making up around 95% of the biome; the other 5% are made upof archaea, protozoa, fungi and viruses. Though most studiesconcentrate on identifying species of bacteria and linking to healthand disease. Other members of the biome have equally importantroles to play. In the racehorse, a major player is the Enterobacteriaphage PhiX174, which is a bacterial virus that protects the horseagainst E-coli.(2)The microbial community has co-evolved with the host, performingessential and vital activities such as the extraction of energy andnutrients from foodstuff, synthesis of vitamins, interaction withthe immune system and cross talk with the brain, which is thoughtto affect temperament and behavior. Taxonomic and functionalcompositions of the gut microbiome are rapidly becoming viableindicators of horse health and disease.Each member of the microbial community has a different butsynergistic role, which is beneficial to the health of the horse;e.g., the fungi break down the indigestible parts of forage plants,such as the polysaccharides, while the ciliate protozoa contributeto the process by producing a wide range of enzymes that thehorse is unable to make, impacting and benefitting the immunesystem. Microbial fermentation of cellulose, hemicellulose andlignin reduces the structural and non-structural plant wallmaterial into carbohydrates, proteins (amino acids) and lipids,and produces volatile and short chain fatty acids,(2a) which are theprimary source of energy for the horse. The bacteria contributethe most to the degradation of ingested food, producing thefinal components of the fermentation process, which are acetic,propionic and butyric acid, methane and carbon dioxide.The gastrointestinal tract of the horse is sensitive to change,stress, environment and medication, which cause imbalances ordysbiosis.(3) Establishing or profiling a healthy baseline in thehorse is difficult as variations exist between individuals, breeds,diets and locations; the Thoroughbred racehorse is a very differentanimal to the Shetland pony or an Irish Draught. Fitness trainingalters the microbiome further; for these reasons it is importantto study the Thoroughbred as a population separate from otherbreeds and to analyze, where possible, racehorses training in asimilar environment and location.With this in mind, since 2017 there has been an ongoing projectto study and profile the microbial populations of over 1,000racehorses based in Newmarket, throughout the racing season;and the data produced has been used to develop profiles of thedifferences between a healthy/unhealthy biome. The projectutilizes the cutting-edge Illumina MiSeq technology, which is themost accurate and up-to-date, preferred by genomic researchersaround the world.THE BIOME IN HEALTHELITE RACEHORSES HAVE HIGHER LEVELSOF A SUPER-PHYLUM BACTERIAQuestions asked...Elite racehorses are trained to achieve peak fitness, but is itpossible that they can gain an extra edge from the input of thehind gut bacteria?How different is the microbiome of a Grade 1 horse, and is itpossible to identify the bacteria responsible for the extra edge?Answers found...Human scientists have known for some timethat the microbiome of an elite human athleteis different,(4) with faster metabolic pathways(amino acids and carbohydrates) and higherlevels of fecal metabolites (microbial-producedshort-chain fatty acids) acetate, propionateand butyrate associated with enhanced muscle fitness. The humanand elite equine athlete do share similar microbial profiles, havinghigher percentages of the bacteria that manufacture short-chainfatty acids and higher levels of the super-phylum verrucomicrobia;these increase as the season/training progresses.What is known about this super-phylum?It has two main members: Methylacidiphilaceae and Akkermansia1Verrucomicrobia Methylacidiphilaceae thrive and proliferateon the ammonia produced from the degradation of starchand protein,(5) whereas starch produces very high levels of ammonia.The bacteria make enzymes (ammonia monooxygenase),(6) whichconvert ammonia into nitric oxide.(7) The nitric oxide has threemajor benefits to a racehorse:a. Helps repair and renew the gut wall (8)b. Enhances performance and increases exercise tolerance (9)c. Improves vascular function and metabolism (10)2Verrucomicrobia Akkermansia is a mucus-eating specialist,living and thriving within the gut wall, digesting mucinfrom the mucosal lining (10a) with a unique ability to metabolisegalactose and melibiose (11) for energy. Akkermansia in thehuman biome significantly increases the numbers of metabolicpathways. Horses with gastric ulcers have very low levels,perhaps indicating its function in both performance and disease.Comparing percentages of the super-phylum among otherbreeds/locations/environments gave good insight into howimportant and relevant verrucomicrobia is to the racehorse.Verrucomicrobia varied significantly from group to group;the lowest levels were found in the sedentary and/or companionanimal group which was comprised of 250 horses (gently hackedor unridden companions). The Carneddau are an ancient herd ofwild horses that graze freely in themountains of Snowdonia, and the Pottokasare from Spain. The CCI-L group was madeup of 10 horses eventing at InternationalOne Day Event Level.The non-group horses were based inNewmarket and analyzed at the heightof the flat season in July, while the Gr1horses started the season (Feb) withlevels of 10%; these levels increased asthe season continued until finally levelingout at 23% in July through to Septemberwhen the testing finished.Why the horses diagnosed with EquineGlandular Gastric Disease having lowerlevels of verrucomicrobia is unknownat this time, horses with EGGD had acompletely different profile to the healthyGr1 horses. See Figs 3 and 4.(The Gr1 horses had a much higherdiversity; and at genus level, they hadmany more groups of bacteria. Increaseddiversity is thought to be an indication ofstability and homeostasis.)The profound effect ofantibiotics on the microbiomeof the Thoroughbred racehorseThe intestinal bacterial community isintimately linked through countlessmetabolic pathways and chemical andphysical signaling processes.(12) Theuse of an antimicrobial causes a changein the bacteria species, driving changesto the normal functions of the biome andreducing the bacteria that benefit the hostwhilst increasing those linked to infections,inflammation and disease.(12a)Fig 5 is a replication taken fromIllumina BaseSpace of the MiSeq analysisof the microbiome of a horse one weekprior to using antibiotics. The internalcircles of the sunburst chart representdifferent taxonomic classifications (i.e.,kingdom, phylum, class, order, family,genus and species), while the lines denotethe different types of bacteria.Fig 6 In the same horse, one week laterafter the use of antibiotics, the microbiomehas altered as follows:a) 6.3% reduction in the number ofindividual species.b) 1.5% reduction in biodiversitycalculated using the Shannon Index,which measures both the number ofspecies and percentages of each withinthe biome (diversity and richness).c) Altered microbial populationi) 56% increase in spirochaetes, partof the core equine biome, spirochaetesproduce formate, succinate and acetate;higher levels of formate have beenlinked to dysbiosis. Spirochaetesfeed on short-chain fatty acids, folicacid, biotin, niacinamide, thiamine,pyridoxal and carbohydrates; higherpercentages within the biome reduceimportant nutrients and energysupply to the horse.(13)ii) 14.7% decrease in proteobacteria,though the phyla proteobacteriacontain some of the most well-knownpathogens such as Rickettsia,Escherichia, Salmonella, Vibrio,Helicobacter and Campylobacter andthe cause of equine diseases such asGlanders. Other members of thediverse and important proteobacteriagroup attack unwanted invadingbacteria, playing a big part inmaintaining stability and homeostasiswithin the anaerobic environment.They also make contributions to thenutrition of the host (the horse) throughtheir ability to process nitrogen.d) 17% increase in clostridium specieslinked to enterocolitis.(14)e) 50% increase in archaea, linked to poorproductivity and performance.(15)THERAPIES & INTERVENTIONSFecal transplantsThere has been a huge increase in interest in fecal matter transplants for horses,(16)following its success in treating C. difficile in humans when compared to the use ofantibiotics. The main result of the transplant was an increase in microbial diversity.Other gastrointestinal inflammatory conditions of horses are now being consideredas suitable for treatment.(17)PrebioticsPrebiotics stimulate, feed and encourage the growth of microorganisms, especially thosethat are beneficial, many horses with gastrointestinal disorders have either low or nobacteria associated with good health. Loss of diversity is also common among horseswith dysbiosis or gastrointestinal disease. Stabilizing and improving the biome can alsoinclude improving forage quality, specific foodstuff and a different feeding routine.(18)Prebiotics containing fructooligosaccharide (FOS) or mannanoligosaccharides improvedDM, CP and NDF digestibility in horses fed high-fibre diets and have reduced dysbiosisin microbial populations, improving levels of beneficial propionate and butyrate.Probiotics are commonly used in horses as a therapy to treat or prevent gastrointestinaldiseases, orally introducing live bacteria able to benefit the health of the biome. Benefitsinclude the inhibition of “bad” bacteria and an enhanced immune system response, thoughit is difficult to compare data due to the different live species used and differences indiets. The most successful appear to be Lactobacillus, Bifidobacterium, Enterococcus andSaccharomyces yeast, all of which improve DM, CP and NDF digestibility of high-fiber diets.SUMMARYThe gastrointestinal tract of the Thoroughbred racehorse is home to a complex microbialcommunity that changes in health and disease and alters throughout the racing season andtraining regime. New sequencing technology allows insights into a previously unknownworld, understanding the microbial communities that have such a strong influence onperformance, fitness, health and temperament is a rapidly emerging science.References1) De Almeida, M. L. M., Feringer, W. H., Júnior, J.R. G. C., Rodrigues, I. M., Jordao, L. R., Fonseca, M.G., ... & de Macedo Lemos, E. G. (2016). Intenseexercise and aerobic conditioning associatedwith chromium or L-carnitine supplementationmodified the fecal microbiota of fillies. PloS one,11(12).2) Nobrega, F. L., Costa, A. R., Kluskens, L. D., &Azeredo, J. (2015). Revisiting phage therapy: newapplications for old resources. Trends inmicrobiology, 23(4), 185-191.2a) Dougal, K., de la Fuente, G., Harris, P. A.,Girdwood, S. E., Pinloche, E., & Newbold, C. J.(2013). Identification of a core bacterial communitywithin the large intestine of the horse. PloS one, 8(10).3) Banse, H. E., & Andrews, F. M. (2019). Equineglandular gastric disease: prevalence, impact andmanagement strategies. Veterinary Medicine:Research and Reports, 10, 69.4) Barton, W., Penney, N. C., Cronin, O., Garcia-Perez, I., Molloy, M. G., Holmes, E., ... & O’Sullivan,O. (2018). The microbiome of professionalathletes differs from that of more sedentarysubjects in composition and particularly at thefunctional metabolic level. Gut, 67(4), 625-6335) Geor, R. J., Coenen, M., & Harris, P. (2013).Equine applied and clinical nutrition E-book: Health,welfare and performance. Elsevier Health Sciences6) Junier, P., Molina, V., Dorador, C., Hadas, O., Kim,O. S., Junier, T., ... & Imhoff, J. F. (2010).Phylogenetic and functional marker genes tostudy ammonia-oxidizing microorganisms (AOM)in the environment. Applied microbiology andbiotechnology, 85(3), 425-440.7) Khadem, A. F., Pol, A., Wieczorek, A.,Mohammadi, S. S., Francoijs, K. J., Stunnenberg, H.G., ... & den Camp, H. J. O. (2011). Autotrophicmethanotrophy in Verrucomicrobia:Methylacidiphilum fumariolicumSolV uses theCalvin-Benson-Bassham cycle for carbon dioxidefixation. Journal of bacteriology, 193(17), 4438-44468) Lanas, A. (2008). Role of nitric oxide in thegastrointestinal tract. Arthritis research & therapy,10(2), S4.9) Mosher, S. L., Sparks, S. A., Williams, E. L.,Bentley, D. J., & Mc Naughton, L. R. (2016).Ingestion of a nitric oxide enhancing supplementimproves resistance exercise performance. TheJournal of Strength & Conditioning Research,30(12), 3520-3524.10) Jones, A. M. (2014). Dietary nitratesupplementation and exercise performance.Sports medicine, 44(1), 35-45.10a) Schroeder, B. O. (2019). Fight them or feedthem: how the intestinal mucus layer manages thegut microbiota. Gastroenterology report, 7(1), 3-12.11) Stein, L. Y., Roy, R., & Dunfield, P. F. (2001).Aerobic methanotrophy and nitrification:processes and connections. e LS.12) Yoon, M. Y., & Yoon, S. S. (2018). Disruption ofthe gut ecosystem by antibiotics. Yonsei medicaljournal, 59(1), 4-12.12a) Modi, S. R., Collins, J. J., & Relman, D. A.(2014). Antibiotics and the gut microbiota. TheJournal of clinical investigation, 124(10), 4212-4218.13) Stanton, T. B., & Canale-Parola, E. (1980).Treponema bryantii sp. nov., a rumen spirochetethat interacts with cellulolytic bacteria. Archivesof Microbiology, 127(2), 145-156.14) Stewart (2013) Clostridia-associatedEnterocolitis in Horses , Department of ClinicalSciences, John Thomas Vaughan Large AnimalTeaching Hospital, College of Veterinary Medicine,Auburn University15) Murru, F., Fliegerova, K., Mura, E., Mrázek, J.,Kopečný, J., & Moniello, G. (2018). A comparisonof methanogens of different regions of the equinehindgut. Anaerobe, 54, 104-110.16) Mullen, K. R., Yasuda, K., Divers, T. J., & Weese,J. S. (2018). Equine faecal microbiota transplant:current knowledge, proposed guidelines andfuture directions. Equine Veterinary Education,30(3), 151-160.17) Laustsen, L., Edwards, J., Smidt, H., van Doorn,D., & Lúthersson, N. (2018, August). Assessmentof Faecal Microbiota Transplantation on HorsesSuffering from Free Faecal Water. In Proceedingsof the 9th European Workshop on Equine Nutrition,Swedish University of Agricultural Science,Uppsala, Sweden (pp. 16-18).18) Coverdale, J. A. (2016). HORSE SPECIESSYMPOSIUM: Can the microbiome of the horse bealtered to improve digestion?. Journal of animalscience, 94(6), 2275-2281.

By Carol Hughes PhD

Gastrointestinal diseases and upsets are common in Thoroughbred racehorses, causing discomfort, loss of performance and even mortality. Every common gastrointestinal disease can be linked back to disturbances (dysbiosis) of the gut bacteria. Currently, new gene technology is driving research at an intense rate, providing new insights into the equine microbial community (1) and providing both trainer and the vet with a powerful and accurate analytical tool to improve health and manage disease. The gastrointestinal tract of the horse is colonized by trillions of microorganisms, which includes 1,000-1,500 different species, making up around 95% of the biome; the other 5% are made up of archaea, protozoa, fungi and viruses. Though most studies concentrate on identifying species of bacteria and linking to health and disease. Other members of the biome have equally important roles to play. In the racehorse, a major player is the Enterobacteria phage PhiX174, which is a bacterial virus that protects the horse against E-coli.(2) The microbial community has co-evolved with the host, performing essential and vital activities such as the extraction of energy and nutrients from foodstuff, synthesis of vitamins, interaction with the immune system and cross talk with the brain, which is thought to affect temperament and behavior. Taxonomic and functional compositions of the gut microbiome are rapidly becoming viable indicators of horse health and disease. Each member of the microbial community has a different but synergistic role, which is beneficial to the health of the horse; e.g., the fungi break down the indigestible parts of forage plants, such as the polysaccharides, while the ciliate protozoa contribute to the process by producing a wide range of enzymes that the horse is unable to make, impacting and benefitting the immune system. Microbial fermentation of cellulose, hemicellulose and lignin reduces the structural and non-structural plant wall material into carbohydrates, proteins (amino acids) and lipids, and produces volatile and short chain fatty acids,(2a) which are the primary source of energy for the horse. The bacteria contribute the most to the degradation of ingested food, producing the final components of the fermentation process, which are acetic, propionic and butyric acid, methane and carbon dioxide. The gastrointestinal tract of the horse is sensitive to change, stress, environment and medication, which cause imbalances or dysbiosis.(3)

Fig 1: Image of the analysis of the microbiome of a Grade 1 horse, compared to a non-group horse.

Fig 1: Image of the analysis of the microbiome of a Grade 1 horse, compared to a non-group horse.

Establishing or profiling a healthy baseline in the horse is difficult as variations exist between individuals, breeds, diets and locations; the Thoroughbred racehorse is a very different animal to the Shetland pony or an Irish Draught. Fitness training alters the microbiome further; for these reasons it is important to study the Thoroughbred as a population separate from other breeds and to analyze, where possible, racehorses training in a similar environment and location. With this in mind, since 2017 there has been an ongoing project to study and profile the microbial populations of over 1,000 racehorses based in Newmarket, throughout the racing season; and the data produced has been used to develop profiles of the differences between a healthy/unhealthy biome. The project utilizes the cutting-edge Illumina MiSeq technology, which is the most accurate and up-to-date, preferred by genomic researchers around the world.

THE BIOME IN HEALTH ELITE RACEHORSES HAVE HIGHER LEVELS OF A SUPER-PHYLUM BACTERIA

Questions asked...

Elite racehorses are trained to achieve peak fitness, but is it possible that they can gain an extra edge from the input of the hind gut bacteria?

How different is the microbiome of a Grade 1 horse, and is it possible to identify the bacteria responsible for the extra edge?

Answers found...

Human scientists have known for some time that the microbiome of an elite human athlete is different,(4) with faster metabolic pathways (amino acids and carbohydrates) and higher levels of fecal metabolites (microbial-produced short-chain fatty acids) acetate, propionate and butyrate associated with enhanced muscle fitness. The human and elite equine athlete do share similar microbial profiles, having higher percentages of the bacteria that manufacture short-chain fatty acids and higher levels of the super-phylum verrucomicrobia; these increase as the season/training progresses. What is known about this super-phylum? It has two main members: Methylacidiphilaceae and Akkermansia

1) Verrucomicrobia Methylacidiphilaceae thrive and proliferate on the ammonia produced from the degradation of starch and protein,(5) whereas starch produces very high levels of ammonia. The bacteria make enzymes (ammonia monooxygenase),(6) which convert ammonia into nitric oxide.(7) The nitric oxide has three major benefits to a racehorse:

a. Helps repair and renew the gut wall (8)

b. Enhances performance and increases exercise tolerance (9)

c. Improves vascular function and metabolism (10)

Screenshot 2021-02-25 at 10.13.10.png

2) Verrucomicrobia Akkermansia is a mucus-eating specialist, living and thriving within the gut wall, digesting mucin from the mucosal lining (10a) with a unique ability to metabolise galactose and melibiose (11) for energy. Akkermansia in the human biome significantly increases the numbers of metabolic pathways. Horses with gastric ulcers have very low levels, perhaps indicating its function in both performance and disease. …

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