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Manipulation of Equine Intestinal Microflora; Modifying a Source of Inflammation to Enhance Clinical Results

Gastrointestinal health and microbial balance has been connected with a host of clinical health problems in both humans and animals, including the horse.  The normal gastrointestinal equine microflora is involved in stimulation of the immune system, synthesis of vitamins (B and K), enhancement of GI motility and function, digestion and nutrient absorption, inhibition of pathogens, metabolism of plant and drug compounds and synthesis of short chain fatty acids.1,9,11  

Introduction

Normal endogenous defense mechanisms exist to prevent bacterial overgrowth including gastric acid secretion, normal GI motility, immunoglobulins (antibodies) present in the intestinal secretions and bacteriostatic properties of pancreatic and biliary secretions.13  Factors including diet, antibiotic usage, COX-2 selective NSAID medications, proton pump inhibitor medications, physical and emotional stress and other lifestyle factors have been noted to contribute to both intestinal dysbiosis and intestinal hyperpermeability.8,11,13

In prior literature, a state of dysbiosis or bacterial imbalance has been demonstrated in horses with metabolic syndrome, laminitis and colitis, with evident overgrowth or predominance of lactic acid producing bacteria.  Most literature has pointed out the imbalance and coexistence with concurrent disease, but a precise connection in regards to direct effect of the dysbiosis has yet to be established.1,4,9,11,14

In our initial study, we evaluated 53 horses for demonstration of evident intestinal microbial imbalance, making the potential connection between higher predominance of clinical problems ranging from allergies to lameness, and increased levels of lactic acid producing bacteria within the feces.  Lactic acid bacteria are numerous and the main species evaluated in that study included Lactobacillus, Enterococcus and Gram negative lactose fermenters, including E. Coli.   Not only did the clinical group within this study demonstrate higher levels of these bacterial populations, but there was also evidence that there may be concurrent malabsorptive concerns, which may impact digestion, nutrient assimilation and potentially contribute to intestinal hyperpermeability or leaky gut syndrome.15

Intestinal hyperpermeability or leaky gut, may actually arrive as a result of many factors either directly or indirectly.  In humans, the condition is often present in affiliation with a systemic inflammatory condition such as diabetes or even cancer, in which case potentially serving as a primary source of inflammatory cytokines, actually propelling the primary health condition.  Some cases have noted that gastrointestinal dysbiosis may actually accompany leaky gut or even serve as an initiator of the condition.  Through the overgrowth of unfavorable bacterial populations, increased fructose metabolism and amine production may also contribute to permeability concerns and disruption of normal systemic circulation. As intestinal permeability is increased, then the potential for more harmful, overpopulated bacteria to gain access to the systemic circulation is increased.  Through increased permeability, other bacterial byproducts including endotoxin, hydrogen sulfide, ammonia and indoles can further damage the mucosa and potentiate patient health problems. So, not only could the localized overgrowth contribute to inflammation, but once the bacteria gain access to the systemic circulation, the inflammatory status may be further propelled due to immune stimulation and dysfunction.4,6,11,12

Chronic small intestinal bacterial overgrowth (SIBO) in humans has been noted to result in systemic inflammation with increased circulating levels of proinflammatory cytokines including TNF-alpha.  In some human cases, SIBO may be present without any noted gastrointestinal symptoms but still may impact the body due to inflammation up-regulation.18

One of the main therapies employed in humans is diet, reducing intake of sugars and using food as a therapy option to aid in intestinal healing, inflammation reduction and re-establishment of normal flora. In human cases of SIBO, the primary disease is always treated and addressed along with proper nutritional support, however, in some cases short term selective antibiotic administration is performed to target specific bacterial populations.  Lifestyle and stress related factors, are likewise addressed in the patient to improve long term management. Despite antibiotic therapies in human trials, the relapse rate is considered high, averaging 44% in less than one year.11,13 Considering this and the potential high occurrence and connection with disease in both humans and animals, a new perspective and approach is warranted.

Taking into consideration the two tiered contributors of both leaky gut and intestinal microbial overgrowth and how they may contribute to systemic inflammatory conditions, we come to realize that in this process we have three main entities that need to be addressed in order to improve progress for the patient. First, inflammation must be modified or managed on a systemic and local level.  Second, intestinal damage contributing to increased permeability must be repaired and third, bacterial levels need to be rebalanced or restored back to a normal physiological level.  If all three entities are addressed, then in many cases, we have increased response by the patient and the clinical problem at hand becomes easier to manage with improved quality of life and potentially less dependence on pharmaceutical medications.

Methodology:

Five (5) equine patients with clinical health problems ranging from joint discomfort to active laminitis were chosen for further exploration in manipulation of intestinal flora to impact patient health.  All fecal samples were submitted by the owner and processed within 12 hours of arrival with clinical history, diet and supplementation regimen.

Initially, 1 gram of fecal material was combined with 5 ml of 0.9% saline for purposes of wet mount evaluation, pH assessment and gram stain.  After the dilution was performed, the suspension was mixed and then strained using sterile gauze as to remove obvious debris.  A 1 ml syringe was filled with 1 drop placed onto a slide with cover slip for wet mount evaluation and another for gram stain.  The pH was performed by using a standardized pH strip with range of 5.0-8.5 with increments of 0.3 units based on color chart.

The wet mount was performed under 100x magnification, evaluating several fields for motile bacteria with focus on obvious motile bacilli.  The motile bacilli were generally graded as present or not present.

A Sudan stain was also performed by directly compressing feces into a side, making a moist impression, then removing obvious debris.  Sudan stain was then applied to the impression region by adding 2 drops, performing an initial review under 10x magnification, focusing on the margins of the impression. After initial review, vinegar was added at the rate of 2 drops, then gentle heat applied to the slide for 5 seconds.  The slide was then reviewed again under 10x magnification, assessing for changes in coloration intensity or retention of Sudan stain by fat globules.  The rate of Sudan uptake was graded on a scale of 0-4 with 0 representing no uptake and a 4 indicating marked uptake.

The Gram stain evaluation was performed under oil microscopy, gathering data from 10 different fields of view, then averaging.  The fields were examined for the presence of obvious Gram positive bacilli, with numbers averaged per high power field.

Fecal cultures were then performed through the use of 0.9% saline and 1 gram of feces, diluted out to a 1:10,000 dilution.  Plating was performed using 0.1 ml of the final diluent per agar plate, which was then streaked out accordingly, incubated at 37° C for 24 hours, at which time were read, colonies counted and recorded. Cultures were performed using CNA, MacConkey, Enterococcus and MRS media.  Distinct colony formation were previously identified by differential culture methodology and MALDI techniques as described in prior literature.

Approach to Therapy:

Each of the patients selected for the trial had an ongoing history of an uncontrolled clinical ailment ranging from joint lameness to allergies or acute laminitis.  All patients selected for the trial were also classified by their owners as being easy keeper body types, implying that they were overweight or needed special diet modification in order to maintain proper body weight.

In the trial, all of the patients were on some type of commercial feed or pelleted hay product in addition to multiple supplements ranging from general vitamin/mineral hay balancers to joint products.  Some of the patients were on daily doses of NSAID or corticosteroid medications, additionally, in order to aid in controlling clinical pain as perceived by the owner.

As part of the trial instructions, the owners were advised to discontinue all commercial feeds or pelleted hay products and feed a whole cereal grain blend consisting of whole oats, whole barley and sunflower seeds.  In many instances, the owners complied fully while in others, the owners were unable to locate all requested grain sources.  At best, the owner fed all 3 grain sources, while in other situations, the owners only fed oats, depending again based on availability. The grains were fed at a ratio of 1:1:0.25 with total starch intake targeting ≤1 gram/kg of bodyweight per feeding.

The supplement regimen currently used with each patient was also modified to the extent that all prior supplements were eliminated, including hay balancers.  Each patient was then placed on Cur-OST® EQ Total Support twice daily for 14 days, then once daily.  Additionally, each horse was also given ½ cup of an experimental whole food blend (Cur-OST® EQ Rejuvenate), developed by Nouvelle Research, Inc.. (Fig. 1)

Figure 1: (Proprietary Formula Ingredients)

Cur-OST® EQ Total Support

BCM-95® Curcumin, Boswellia, Dandelion, Marshmallow, Parsley, Vitamin C, Vitamin E, CoQ10, Alfalfa, Spirulina, Anise

Cur-OST® EQ Rejuvenate

Alfalfa, Spirulina, Flax Seed, Pea Protein 80%, Red Spinach, Red Clover, Hops 4:1, Brewer’s Yeast

The overall goal with dietary modification was to reduce or minimize the number of synthetics that may be potentiating clinical problems, reduce the amount of added sugars to the diet, reduce preservatives, additives and dyes, while moving towards a more whole food diet with less processing involved.  The provided supplements, EQ Total and whole food blend, had the goal of reducing inflammation, providing nutrients for repair of damaged tissue and cellular health while mitigating or balancing intestinal bacterial levels.

Patient fecal data and clinical observations were obtained on day one and again approximately 30 days post dietary and supplementation changes.

Patient Clinical Information and Data: 

 Normal Fecal Data: 15

Bacilli Gram Stain #

Lactose +

Enterococcus #

MRS/Lactobacillus #

Sudan Stain

18

230

880

100

Negative

(TNTC=too numerous to count, results reported as cfu/ml at a 1:10,000 dilution)

 

 Patient 1: 

Breed: Miniature horse mare

History:   Chronic, progressive laminitis for several years duration, grade 4/5 lame at the walk

Medications:  Phenylbutazone daily

Diet:  Dry lot, grass hay with pelleted hay products

Supplements:  Hoof and blood circulation supplements

 

Bacilli Gram Stain #

Lactose +

Enterococcus #

MRS/Lactobacillus #

Sudan Stain

Pre

30

580

>2000

TNTC

2+

Post

10

30

1500

1500

Negative

Outcome:  Patient markedly improved by 30 days duration.  Walking freely with minimal discomfort, phenylbutazone discontinued.

Patient 2:

                Breed:  Appaloosa Mare

                History:  Chronic, progressive laminitis for 1 year duration, grade 5/5 lame and recumbent

                Medications:  Phenylbutazone daily

                Diet:  Dry lot, Bermuda hay with commercial low starch grain

                Supplements:  Hoof, blood circulation and hay balancer supplements

Bacilli Gram Stain #

Lactose +

Enterococcus #

MRS/Lactobacillus #

Sudan Stain

Pre

30

860

1250

>2000

2+

Post

10

350

450

800

1+

Outcome:  Patient markedly improved within 2 weeks, freely ambulatory per owner with no further need for NSAID medications.  Note that patient quickly deteriorated post trial after return to prior regimen.

Patient 3:

                Breed:  QH Gelding

                History:  Chronic skin allergies with persistent hive development for 2 years duration

Medications:  Prior corticosteroids, antihistamines and hypersensitization injections with no improvement or impact on condition.

Diet:  Commercial grain product with hay provisions

Supplements:  Hay balancer, joint supplement and allergy support products

Bacilli Gram Stain #

Lactose +

Enterococcus #

MRS/Lactobacillus #

Sudan Stain

Pre

15

890

780

TNTC

2+

Post

20

500

140

TNTC

1+

Outcome:  Allergies became more manageable with complete remission noted at times, while flares up were still present, they were to a much lower degree. All medications were discontinued.

 

Patient 4:

                Breed:  Friesian Mare

                History:  Chronic navicular pain persistent for over one year, grade 4/5 lame at walk

                Medications:  Daily NSAID administration, minimal impact on condition

                Diet:  Commercial grain product, round bale Fescue hay and joint supplement

Bacilli Gram Stain #

Lactose +

Enterococcus #

MRS/Lactobacillus #

Sudan Stain

Pre

5

100

400

650

1+

Post

0

40

150

100

1+

Outcome:  Patient became clinically sound at the walk, while still retaining evidence of mild discomfort at the trot.  No further pain medications needed to control or enhance condition.

Patient 5:

                Breed:  Thoroughbred gelding

                History:  Traumatic facture to accessory carpal bone of 8 months duration, under stall rest

                Medications:  Daily NSAID administration

                Diet:  Commercial grain product, joint supplement and grass hay

Bacilli Gram Stain #

Lactose +

Enterococcus #

MRS/Lactobacillus #

Sudan Stain

Pre

25

180

500

250

2+

Post

10

100

90

70

Negative

Outcome:  Patient no longer on stall rest, soundness improved with reduction in discomfort and elimination of NSAID medication.  Currently on free turnout.

Discussion:

The equine gastrointestinal tract contains trillions of bacteria of many different species that serves functions ranging from food digestion and nutrient assimilation to immune function.  The bacterial population is easily upset, often in a short period of time, influenced by factors including diet, medications, transportation and stress.  When the bacterial population is out of balance creating dysbiosis, there is potentially increased systemic and local inflammation which can then negatively impact health for that animal on many levels.

In prior research papers, gut dysbiosis has been noted to occur in metabolic syndrome and laminitis in horses, often with an overgrowth of lactic acid producing bacteria including Lactobacillus and Group D Streptococcus, including Enterococcus.  This dysbiosis has been noted, but connection with clinical disease in regards to actual cause has been uncertain.  In one study, the overgrowth was noted in a large population of clinically impacted horses, along with possible indicators of existing malabsorption of nutrients including fats.  This malabsorption may actually be reflective of gastrointestinal damage that has occurred secondary to the overgrowth of lactic acid producers and concurrent possibility of existing intestinal hyperpermeability or leaky gut syndrome.15

The gastrointestinal dysbiosis is problematic on many levels, not only impacting nutrient absorption but also predisposing the patient to a host of other health concerns ranging from colic to ulcers to systemic inflammatory problems.  In many cases, inflammatory conditions including laminitis, uveitis, irritable bowel disease and allergies can all be tied in to the gut and possible dysbiosis.  In many respects, there is a trifecta of problems that develop in these cases.  First, there is a microbial shift or imbalance that occurs, which may result in a localized immune response and release of inflammatory proteins or cytokines.  Second, this inflammatory response if left unattended could contribute to localized changes to the intestinal lining, contributing to increased permeability due to changes in tight junctions at a cellular level.  Third, as permeability is altered, bacteria and feedstuff, including dyes, preservatives and other potentially noxious substances can then easily enter the systemic circulation, further aggravating the inflammatory and immune response.

In the small population of horses in this trial, we explored several patients from our initial fecal microflora study.  In the original trial, we demonstrated a higher level of Lactobacillus, Enterococcus and lactose positive E. coli in clinically abnormal patients with a myriad of health problems ranging from laminitis to allergies.   In that group of patients, not only where there elevations of bacterial counts above the normal population, but there was also indications of malabsorption as noted by the Sudan stain uptake in fecal smears.  This was theorized to coincide with the level of progression of the gastrointestinal compromise.15

In human research regarding intestinal hyperpermeability and intestinal bacterial overgrowth, it has been noted that therapeutic options are lacking.  Given this deficit, the majority of therapies employed are nutritional in basis with focus on reduction of inflammation and providing provisions for repair of the gastrointestinal tissue. Options that have demonstrated efficacy in human studies include Curcumin, Glutamine, Boswellia, blue green algae and other herb based ingredients, mainly through inflammation reduction and NF-kB down regulation. .  A change of diet in humans is often employed additionally with reduction of processed foods, added sugars and a shift towards whole foods in general.  In many cases, the change in diet is enough to produce results and allow for proper repopulation of the gastrointestinal tract.  It is to be noted that intestinal bacterial overgrowth in humans can be different from that described in horses in this particular situation, with a focus on different bacterial species.2,3,17,19,20

In horses and cattle, some research has pointed towards the targeted use of hops (Humulus lupulus) extract, which have shown benefit in cattle in reducing rumenal acidosis and lowering levels of Lactobacillus and Group D Streptococcus.  In another study, Hops extracts were also used in horses to reduce hindgut pH and Lactobacillus counts.7,10

The use of probiotics in horses has been in question by many authors, as the equine recommendations tend to follow human protocols with little substantiating evidence.  In some respects, the use of Lactobacillus based probiotics could be viewed with question, especially in situations where there is already an overgrowth of this population of bacteria within the feces of the horse.  The concern would be that concurrent use of these probiotics could actually contribute to clinical disease, rather than aid in recovery, but this is just theory at this time. In human research, there is little data to fully support probiotic usage at this time in cases of small intestinal bacterial overgrowth (SIBO), however, in certain groups they can be used with benefit if the causative organism is known and deficiency demonstrated. One probiotic, Saccharomyces cerevesiae, a yeast organism derived from Brewer’s yeast, has shown the ability to stabilize the microflora of the equine hindgut during period of transportation and stress.5,13,16

Based on human research and the evidence presented to us through literature, the dysbiosis and associated inflammatory condition present secondary to lactic acid bacteria overgrowth could respond to a more natural diet, along with reduction of contributing dietary and physiological factors.

In this paper, we demonstrated the recovery of five (5) horses presented with a variety of health ailments, but each reflecting an overgrowth of lactic acid bacteria and malabsorption concerns.  The goal of recovery was to reduce inflammation, promote healing of the intestinal tract and balance the microflora.  Due to concerns over contribution of processed feeds and added sugars to the diet, the owners were instructed to move towards a whole grain, non-processed feed, along with elimination of other concurrent dietary supplements.  In theory, the current feed and supplement regimen used in each patient was not providing clinical resolution to the health concern, so it was chosen to modify it rather than add to it.

The Cur-OST® EQ Total Support formula was designed to balance the inflammatory process, provide antioxidant support and nutritive aid through herbal based ingredients.  The Cur-OST® EQ Rejuvenate is a whole food supplement providing nutritive capabilities for overall cellular support and repair, while also aiding in hindgut microflora balance and mitigation of potential overgrowth.

The combination of these two formulas takes advantage of current therapy options utilized in humans, including Curcumin, Boswellia, Spirulina and amino acids to aid in tissue repair, with naturally occurring forms of micro and macronutrients for cellular support.  The added research benefits of Hops and Brewer’s yeast help to push the impact one step further, with aid in controlling bacterial populations.

The clinical improvement in all patients was noted after 30 days, with some demonstrating marked improvement in 14 days or less based on owner observations.  All patients were off of their pharmaceutical medications within this time frame and on whole grain based regimens or hay pellets, along with hay or pasture access.  Both of the laminitic patients resumed full turnout and were viewed by their owners as being dramatically more comfortable and free moving.  The bacterial counts were reduced in all patients after 30 days, but were still elevated as compared to the normal group presented in our initial trial.  Despite still being elevated, a reduction in count is obviously enough to impact the animal’s systemic inflammatory response.  In human models, intestinal bacterial overgrowth is believed to take upwards of 2 years to completely resolve with strict adherence to diet by the patient.

Interestingly, in a few cases, the owners had noted concurrent thrush concerns of the foot, appeared to be more responsive to therapy or resolved after the initial 30 days of therapy.  It could be hypothesized that reduction in lactic acid bacterial populations could have impacted the systemic pH of the body, thereby becoming  more alkaline in nature and less conducive to thrush or other yeast like conditions.

Conclusion:

Intestinal bacterial overgrowth or dysbiosis is a growing concern in human medical literature with connections with several health concerns.  Equine gastrointestinal dysbiosis has been demonstrated in many research papers, especially in metabolic syndrome, laminitis and Clostridiosis.  The research papers have demonstrated that the problem exists, but connection between the dysbiosis being an exact cause is still yet to be determined.

In this study, gastrointestinal dysbiosis was noted in all equine patients with clinical health problems. Through the application of whole food nutrition, elimination of processed foods and added sugars, along with targeted herbal therapy to aid in controlling inflammation and promoting intestinal healing, the patients responded favorably in a short period of time.

Gastrointestinal dysbiosis is a concern that might actually be more prominent that we, as veterinarians, imagine with implications back to current diet recommendations, over supplementation, excess sugar consumption and induced stressors.  Given the lack of pharmaceutical interventions available, the need to re-evaluate diet regimens and take advantage of herbal therapies along with whole food nutrition is necessary.

References:

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  2. Bedirli A et al. Administration of chlorella spp. microalgae reduces endotoxemia, intestinal oxidative stress and bacterial translocation in experimental biliary obstruction.  Clin Nut. 2009. Dec; 28(6):674-8
  3. Cantanzaro D et al. Boswellia serrata preserves intestinal barrier from oxidative and inflammatory damage. PLoS One. 2015, May 8;10(5)
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  8. Fredenburgh L et al. Cyclooxygenase-2 deficiency leads to intestinal barrier dysfunction and increased mortality during polymicrobial sepsis 1. J Immunol, 2011, Nov 15; 187(10):5255-5267
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  10. Harlow BE et al. Inhibition of fructan fermenting equine fecal bacteria and Streptococcus bovis by hops (Humulus lupulus)β Acid. J Applied Microbiol. 2014. Aug:117(2):329-339.
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  12. Johnson, R et al. Fructokinase, Fructans, Intestinal permeability and metabolic syndrome: An equine connection. JEVS, 2013, Feb:33(2) 120-12
  13. Kopacova M. Small intestinal bacterial overgrowth syndrome. World J Gastroenterol, 2010, Jun 28; 16(24):2978-2990
  14. Rapin, J, Wiernsperger N. Possible links between intestinal permeability and food processing; A potential therapeutic niche for glutamine. Clinics, 2010; 65(6):635-43
  15. Schell, T. Fecal microflora and dysbiosis in the horse. Possible contribution to metabolic related conditions, inflammation and leaky gut.  Online Publication, secondvet.com, 2015
  16. Schoster A et al. Probiotic use in horses-What is the evidence for clinical efficacy. J Vet Intern Med. 2014;28:1640-1652.
  17. Sergent T et al. Antiinflammatory effects of dietary phenolic compounds in an in vitro model of inflamed human intestinal epithelium. Chem Biol Interact. 2010, Dec 5;188(3): 659-67
  18. Turnbull, L et al. Systemic signs of underlying digestive dysfunction and disease. Integrative Gastroenterology, 2011, 79-99.
  19. Vermeulen M et al. Glutamate reduces experimental intestinal hyperpermeability and facilitates glutamine support of gut integrity. World J Gastroenterol, 2011, Mar 28; 17(12): 1569-1573
  20. Wang N. et al. Curcumin ameliorates hydrogen peroxide induced epithelial barrier disruption by upregulating heme oxygenase-1 expression in human intestinal epithelial cells. Dig Dis Sci, 2012, July 57(7);1792-801.

 

Online Publication: Thomas Schell, D.V.M., Nouvelle Research, Inc.  2015

Author Contact:  [email protected]

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