Equine Genomics
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Equine Genomics

Equine Nutritional Therapist and Medical Herbalist Antoinette Foster has been fortunate to work with a company called Equine genomics who offers a premium service providing horse owners with cutting edge technology that enables targeted health and nutrition.

Equine genomics also uses a variety of approaches to enable a better understanding of the pathways involved in the health and wellbeing of the equine species and various breeds of horse. This approach is of great interest especially where significant differences between samples can be obtained which is translated into “gene signatures”. These gene signatures can be translated into markers for overall health improvement covering areas such as Inflammation, Oxidative Stress, Detoxification, Bone Health and much more. This testing compliments Antoinette’s approach to the nutrition, health and wellbeing of the horse.

The Equine Intestinal Micro Biome Gut Health Genetic Test © 2014

The equine intestinal tract contains a complex microbial population called micro biota which plays an important role in health and disease. It appears that the micro biome can be altered quite intensely in certain disease states. Laminitis for example can be a very severe disease that can be set off by an increase of dietary starch. By the time the starch reaches the hindgut it causes an enhancement of lactic acid bacteria, lactate build up and acidification of the contents of the gut.

These bacterial products go into the bloodstream and lead to systemic inflammation. Hindgut lactate levels are normally low because specific bacterial groups convert lactate to short chain fatty acids. Why this mechanism fails when lactate levels rapidly rise, and why some hindgut communities can recover is unknown. Faecal samples can be taken to assist in the diagnosis of this disease and could provide a convenient and cost effective means to understand the microbial dynamics underlying colic and laminitis. Other conditions associated with bacteria can also include ulcers and will most definitely include diarrhoea, this form of testing allows us to diagnose effectively and put into place nutritional interventions and in the long-term great improvement in health and well-being of the gut. Incorrect feeding can induce certain conditions in horses many of these can be prevented. Faecal analysis profiling your horse’s bowel flora will provide you the owner, veterinarian or health practitioner a greater understanding of bowel flora which is indicative to the health and well-being of your horse. The maintenance of a normal healthy horse is governed by a normal functioning digestive tract. The micro biome is essential for the maintenance of normal health, well-being and function. The homoeostatic balance of the equine intestinal micro biome is very sensitive to dietary change and gastrointestinal disease. It is well known that any homoeostatic imbalance can cause disease. One of the most common causes of death in horses is colic this is well documented, a test such as this will provide us with an enormous amount of information to assist in managing and preventing these types of digestive diseases. Nutrition plays an enormous role in the health of your horse, the power of nutrition should never be underestimated.

The equine gastrointestinal (G.I.) tract maintains a rich microbial community in which directly affects energy metabolism, digestive function, mucosal immune system development, and disease pathogenesis of its eukaryotic host.

The homeostatic balance in the equine intestinal micro biome is very sensitive to factors like gastrointestinal disease and dietary change, which may lead to catastrophic consequences, even culminating in death.

Alterations in hindgut bacterial communities have also been associated with several equine diseases affecting the gastro-intestinal system are the main cause of mortality in this species.

Numerous factors including the evolutionary history of the host, age, and diet influence the diversity of gut microbes in addition to Starch and oligofructose overload-induced models have revealed strong associations between onset of laminitis and proliferation of Streptococcus and Lactobacillus bacteria, with a concurrent decrease in intraluminal.

Patterns of microbial diversity in the G.I. tract have important implications for human and environmental health. These communities become a source of pathogens when released into aquatic environments as faecal pollution. Community members that exhibit host-specific distributions represent a valuable resource as potential markers for faecal pollution from specific sources. Accordingly, characterization of the equine intestinal microbial is critical, since a good understanding of the ‘normal’ intestinal microbial is needed for interpretation of ‘abnormal’.

Most investigations of the equine microbial have typically involved bacterial culture of feces or intestinal contents.

The objectives of a recent study were to characterize the faecal microbial of healthy horses.

These results suggest that normal horse faecal flora includes a wide variety of organisms which demonstrated as a critical factor for normal GI function on the other hand the bacterial populations differ related to horse’s diet that composed of high fibre and reduced fat, protein, and digestible carbohydrates and diet fermentation,else the fecal microbial of healthy horses showed that the equine microbial is more diverse than the human microbial, but less diverse than the gastrointestinal microbial of cattle. Our result indicate the higher percentage for Streptococcus spp. Isolation (33.7%) that agreement with many

Studies this genus not cause laminitis in horses. This study determined the rate of prevalence of bacterial agents associated with diarrhoea in horse in Baghdad.

The bacteriologic isolates found in horses in the present study include E. coli. Salmonella and Rhodococcus equi and these organism were previously reported by in horses. During the present study the rate ofprevalence of E. coli was highest than other pathogens (20.9%) which agree with other several studies, E. coli.

The bacterial isolation in diarrhoea depends mainly on the age of animal and the surrounding environment as well as weather, management which act as predisposing factor on animal for inducing diarrhoea, But in the study interested mainly on the bacterial isolation in a horse faecal sample admitted to veterinary diagnostic lab not concerned with animal age and other factors. So the presence of E. coli in large percent does not indicate occurrence of diarrhoea because it most common in the faeces of foals, also adult horse shedding salmonella in the faeces for a long time and became the source of infection. Pathogens were detected in non-diarrhoeic horses which were predominantly (25 of 100 horses) available for the study is in

agreement with published reports.

Based on the anti-barograms of the E. coli isolates in the current study, it is evident that they were mostly sensitive to chloramphenicol and Tetracycline but least susceptible to ampicillin and streptomycin. The detected resistance to both ampicillin and streptomycin could be explained, the fact that these antimicrobial agents were used at a high frequency on the study farms. Another finding in the current study with clinical relevance was the detection of higher frequency of resistance to tetracycline amongst isolates of E. coli recovered from diarrheic compared

with non-diarrheic horses. This is an indication of a possible development of resistance due to misuse or overuse of the antibiotic in the treatment of horses locally.

The frequency of isolation (33.3%) of Salmonella spp. from diarrheic foals in the current study is higher than the rates of 12% and 13% (60/465) [38] reported for diarrheic foals elsewhere but lower than the 35.1% reported by Walker et al. [39]. The fact that the rate of isolation of Salmonella spp. was significantly higher than found in non-diarrheic foals suggests etiological significance as earlier documented by others.

The finding that all isolates of Salmonella spp. recovered in this study were sensitive to both

sulphathiazole/trimethoprim (SXT) and Tetracycline is an indication that the two antimicrobial agents may be important in the chemotherapy of foal diarrhoea caused by Salmonella spp.

Based on the questionnaire survey of the farmers during the study, the commonly used antimicrobial agents used to control foal diarrhoea are streptomycin and ampicillin.

References:

1. O'Hara AM, Shanahan F(2006). The gut flora as a forgotten organ. Embo Reports.7(7):688–693.

2. Neish AS(2009). Microbes in Gastrointestinal Health and Disease. Gastroenterology . 136(1):65–80.

3. Wardwell LH, Huttenhower C, Garrett WS(2011). Current concepts of the intestinal microbiota and thepathogenesis of infection. Curr Infect Dis Rep. 13:28–34.

4. Hintz HF, Cymbaluk NF: Nutrition of the Horse. Ann Rev Nutr 1994, 14:243–267.

5. Al Jassim RA, Andrews FM (2009) The bacterial community of the horse gastrointestinal tract and its

relation to fermentative acidosis, laminitis, colic, and stomach ulcers. Vet Clin North Am Equine Pract 25:

199–215.

6. Chapman AM (2001). Acute diarrhea in hospitalized horses. Vet Clin North Am Equine Pract 25: 363–380.

7. Costa MC, Arroyo LG, Allen-Vercoe E, Stampfli HR, Kim PK, Sturgeon A,Weese JS(2012). Comparison

of the fecal microbiota of healthy horses and horses with colitis by high throughput sequencing of the V3- V5 region of the 16S rRNA gene. Plos One. 7(41484):1–11.

8. Garrett LA, Brown R, Poxton IR (2002). A comparative study of the intestinal microbiota of healthy horses and those suffering from equine grass sickness. Vet Microbiol .87(1):81–88.

9. Milinovich GJ, Trott DJ, Burrell PC, van Eps AW, Thoefner MB, Blackall LL, Al Jassim RAM, Morton JM,Pollitt CC (2006) . Changes in equine hindgut bacterial populations during oligofructose-induced laminitis. Environ Microbiol . 8((5):885–898.

10. Milinovich GJ, Trott DJ, Burrell PC, Croser EL, Al Jassim RAM, Morton JM, van Eps AW, Pollitt

CC(2007) . Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis. Environ Microbiol . 9(8):2090–2100.

11. Milinovich GJ, Burrell PC, Pollitt CC, Klieve AV, Blackall LL, Ouwerkerk D,Woodland E, Trott DJ

(2008). Microbial ecology of the equine hindgut during oligofructose-induced laminitis. Isme Journal 2(11):1089–1100.

12. Garner HE, Coffman JR, Hahn AW, Hutcheson DP, Tumbleson ME (1975). Equine laminitis of alimentary origin - experimental model. Am J Vet Res. 36(4):441–444.

13. Shirazi-Beechey SP(2008). Molecular insights into dietary induced colic in the horse. Equine Vet J.

40(4):414–421.

14. Durham AE(2009). The role of nutrition in colic. Vet Clin North America-Equine Practice. 25(1):67–78.

15. Milinovich GJ, Klieve AV, Pollitt CC, Trott DJ(2010).Microbial events in the hindgut during

carbohydrate-induced equine laminitis. Vet Clin North America-Equine Practice. 26(1):79–94.

16. Al Jassim RA, Scott PT, Trebbin AL, Trott D, Pollitt CC(2005). The genetic diversity of lactic acid

producing bacteria in the equine gastrointestinal tract. FEMS Microbiol Lett .248(1):75–81.

17. Steelman SM, Chowdhary BP, Dowd S, Suchodolski J and Janečka JE.(2012). Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis. BMC Veterinary Research.8:231 Page 1of 11 http://www.biomedcentral.com/1746-

6148/8/231.

 

Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis

Samantha M Steelman1, Bhanu P Chowdhary1, Scot Dowd2, Jan Suchodolski3 and Jan E Janečka1*

Author Affiliations

1 Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843-4458, USA

2 Molecular Research LP, Shallowater, 79363, TX, USA

3 Gastrointestinal Laboratory, Texas A&M University, College Station, TX, 77843-4458, USA

For all author emails, please log on.

BMC Veterinary Research 2012, 8:231  doi:10.1186/1746-6148-8-231

 

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1746-6148/8/231

 

Received:

16 June 2012

Accepted:

18 November 2012

Published:

27 November 2012

© 2012 Steelman et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis.

Results

Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks.

Conclusions

Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis.

Background

The microflora within the gastrointestinal system directly affects energy metabolism, digestive function, mucosal immune system development, and disease pathogenesis of its eukaryotic host [1-4]. This is particularly true for herbivores, including the horse, which are dependent upon fermentation by bacteria to utilize plant structural carbohydrates [4]. Therefore, a detailed knowledge of gut microflora is essential for understanding the nutritional needs of horses and the contribution of gut homeostasis to equine health. Research on bacterial communities has recently flourished with the application of next-generation sequencing (NGS) technology [5]. Studies incorporating NGS have led to the discovery of thousands of novel species (i.e., Operational Taxonomic Units [OTUs]) and elucidation of their ecological function within the gut of vertebrates [5-7]. Numerous factors including the evolutionary history of the host, age, and diet influence the diversity of gut microbes; they in turn have been implicated in a broad range of disorders including Crohn’s disease, chronic diarrhea, inflammatory bowel disease, type I diabetes, obesity, and asthma [2,7-9].

Alterations in hindgut bacterial communities have also been associated with several equine diseases [10-17]. Excess nonstructural carbohydrates (i.e., starches, fructans, or simple sugars) that are not digested in the foregut enter the cecum and colon, where bacterial fermentation produces byproducts including lactic acid and gas, which can cause colic [4,16,17]. The same initiators can also lead to the development of laminitis, which often occurs subsequent to overconsumption of grain or after feeding on lush pasture rich with nonstructural carbohydrates [18-20]. Starch and oligofructose overload-induced models have revealed strong associations between onset of laminitis and proliferation of Streptococcus and Lactobacillus bacteria, with a concurrent decrease in intraluminal pH [12-15,21-23].

Numerous studies have characterized and enumerated bacteria of the equine hindgut, primarily relying on culturing of bacteria, clone-based sequencing of Polymerase Chain Reaction (PCR) amplicons, denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), or gene terminal restriction fragment length polymorphism (T-RFLP) [21,23-32]. The primary microbes detected consisted of Gram-positive bacteria, many of which were associated with the cluster XIVa of Clostridiaceae, Streptococcus spp., and Lactobacillus spp. [14,22,24,26]. Up to 96% of all observed OTUs could not be assigned, highlighting how little was known about this ecosystem [30].

Recently researchers have begun to apply 454 sequencing of 16S rRNA amplicons to understand the equine gut microflora [10,33,34]. A total of 1,518 OTUs have been observed in feces from just two horses, with Firmicutes, Verrucomicrobia, and Proteobacteria being the most abundant Phyla, and Subdivision 5 Incertae sedis spp., TM7 Incertae sedis spp., and Treponema spp. the most common genera [33]. In a study examining colitis, Firmicutes were found to dominate the feces of normal horses in contrast to Bacteroidetes in horses with undifferentiated colitis [10]. Bacterial communities in the stomach were also found to be dominated by the Phyla Firmicutes, Proteobacteria, and Bacteroidetes, with Lactobacillus spp., Streptococcus spp., and Moraxella spp. comprising the most abundant genera [34]. The stomach microflora segregated based on management (stabled versus pastured) and sampling methods (biopsy versus post mortem) [34]. These studies show a much more diverse assembly of bacteria than previously described; however, the mechanisms linking bacterial diversity to diseases such as colic, colitis, and laminitis are yet to be elucidated.

We thus explored the equine hindgut microflora by pyrosequencing bacterial 16S rRNA gene segments present in feces of normal horses and those suffering from chronic laminitis. Our goals were to (1) describe the level of microbial diversity and (2) compare the microflora of healthy horses to those with chronic laminitis. We hypothesized that horses with chronic laminitis, which had in the past experienced a bout of acute laminitis and presumably a radical shift in bacterial flora that accompanies this disease, would harbor a different microbial population. Our study contributes to the characterization of the equine gut microbiome and its potential link to laminitis.

 

For further information contact: antoinette@hiform.com.au

 



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