New perspectives in equine digestive health

Introducing the SUCCEED® Equine Fecal Blood Test™

A new antibody test that helps practitioners diagnose GI health conditions in their equine patients.

®

Practicing equine veterinarians are generally familiar

with ulcers, colic and other digestive health issues,

and how these ailments can afflict their equine pa-

tients. What may be less understood is just how wide-

spread these problems are.

Several studies have shown that approximately 90%

of race horses1 and 60% of show horses2,3 have ac-

tive gastric ulcers. Even leisure horses are affected, with almost 40% exhibiting

gastric ulceration.4 What is more, a recent peer-reviewed study5 has shown 63%

incidence of colonic ulceration in performance horses. Ulcers can have a serious

deleterious effect on the performance of these animals.

Unfortunately, these sad statistics are mostly due to our own mismanagement.

In particular, the way horses are fed disregards how their digestive systems have

evolved. Horses in the wild graze continuously and the intermittent, high-carbo-

hydrate feed that is provided in a barn is unnatural and potentially harmful. Many

of the behavior and performance issues that are observed with stabled horses are

caused by this unnatural feeding regimen, not to any innate limitations of the

horse or its training. Ulcers may be responsible for much of the subclinical ane-

mia, listlessness, weight loss and general poor health noted by veterinarians for

years. In fact, the number one complaint about horses discovered to have ulcers

is that their performance is declining.6 With these animals, environment has a

much larger impact on their ability than any genetic factor.7

The SUCCEED Equine Fecal Blood Test is available from veterinary supply distributors in cartons of ten kits, each packaged for in-dividual sale. The test kit is priced for afford-ability, so you can charge your client around $40 per kit.

SUCCEED® Equine Fecal Blood Test™

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As bad as this is, the gastric region that is home to EGUS represents less than 10% of the entire volume of the equine GI tract. The bulk of the work of diges-tion takes place in the hindgut. Gastric ulcers in horses are fairly well under-stood because they can be inspected by endoscopy, but colonic ulcers are much harder to observe. Colonoscopies are impractical due to the difficulty of evacuating the equine colon without endangering the health of the horse. Lacking evidence to the contrary, it has erroneously been thought that equine colonic ulcers are rare.

The horse’s hindgut includes the large intestine, comprised of the cecum and the colon. Like humans, horses cannot digest fiber on their own. But in the hindgut of the horse, billions of bacteria and other micro-organisms ferment the structural carbohydrates that constitute fiber and convert them to VFAs which the horse can digest. These VFAs repre-sent the primary source of energy for the horse.

As noted, however, horses today have less fiber in their diets. Instead, they are fed concentrates such as processed grain, pellets, or sweet feed. Without

chaff (chopped hay) to encourage chew-ing and salivation, the rapid ingestion of concentrates allows these to reach the hindgut undigested. Microorganisms in the cecum and colon convert a portion of these high-energy feeds and starches into lactic acid, which can lead to hind gut acidosis. This acidic environment can shift the balance of normal microorganisms and pathogenic bacteria, leading to an inability to properly ferment forage into VFAs (“poor doers”) and further can lead to colonic ulceration.

If these ulcers are severe and include significant blood loss, you may notice low grade anemia in the horse. Compound-ing the problem, the horse may respond to the blood loss by constricting vessels such as the anterior mesenteric artery. Constriction of this ar-tery, abetted by the impact of micotoxins entering the blood stream15, will lead to reduced blood flow, hence reduced peri-stalsis (leading to impaction colics), and will ultimately result in necrotic tissue.

Most veterinarians are familiar with Equine Gastric Ulcer Syndrome (EGUS) which manifests as lesions in the distal esophagus, the non-glandular (squamous) area of the stomach, the proximal duodenum and, less commonly, the glandular area of the stomach.8 EGUS develops when the

protective and problematic factors within the stomach become unbalanced.9 Protec-tive factors include sufficient mucus, blood flow, mucosal prostaglandins, epidermal growth factor (EGF), GI motility and tight in-tercellular junctions. Problematic factors

include hydrochloric acid (HCL), peptic acid and bile acids. These acids are excreted continuously by parietal cells in response to histamines, gastrin and acetylcholine.10 When the problematic factors overwhelm the protective factors, the mucosal layer may be breached and lesions can occur.

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SUCCEED® Equine Fecal Blood Test™

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Diets rich in carbohydrates and protein are known to exacerbate ulcers in horses.11 This is because grains and other non-fibrous feeds have a higher motility than grasses, causing the stomach to empty rapidly, reducing buffering and lowering the pH. The gut rapidly ferments the excessive levels of hydrolysable carbohydrates in standard concentrates: the additional volatile fatty acids (VFAs) in the presence of an already low pH can irritate the squamous mucosa.12

Because horses evolved to graze continu-ously, they constantly produce acid.13 This is well-tolerated with a full stom-ach, but the acid produced on an empty stomach can damage the mucus layer. In addition, high levels of grain in the diet can induce increased production of gastrin and histamines14 which in turn can amplify the secretion of HCL.

Gastric ulcers and their causes The surprise of hindgut ulceration

Gastric ulcer Colonic ulcer

How common are colonic ulcers? In 2004, Freedom Health conducted a large-scale necropsy of 180 horses. The study revealed that 87% had gas-tric ulcers and 63% had colonic ulcers, resulting in a cumulative ulceration rate of 97%.14 The results are summarized in the following graph:

Prior to the dissection of these horses, a fecal

sample was collected. This manure was tested with a guaiac stain and correlated to the gross exami-nation of intestinal tissue.

Overall, the guaiac test proved to be high-

ly specific and significant for the existence of an ulcer,

but the existence of false nega-tives lowered the overall accuracy of the test to 65%, roughly comparable to human outcomes with such a test.

Guaiac works by binding hemoglobin and turning blue in the presence of hydrogen peroxide. The false negatives are likely due to the fact that the guaiac reaction is not very sensitive, requiring 10mg of hemoglobin per gram of stool to produce a positive result. Since many

ulcers bleed only lightly or sporadically, these may be missed by the guaiac fecal blood test (gFBT).

This study clearly demonstrated that the problems with the equine digestive tract extend past the stomach. It also showed that traditional fecal blood testing kits may not be sensitive enough to detect ulceration originating in either the gastric or the colonic area. However, this initial study provided important evidence that blood can be detected in manure, and led to a fresh examination of the diagnostic possibilities.

Because the currently available diagnostic technologies are limited, many veterinarians rely on symptomol-ogy. They treat a possible ailment and, if the horse improves, they may assume the original diagnosis was appropriate. This approach, however, is notoriously unreliable, costly and can easily aggra-vate the original (improperly diagnosed) problem.

Incidence of gastric and colonic ulcers.

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Gastric Ulcers

Colonic Ulcers

Gastric & Colonic Ulcers

No Ulcers

SUCCEED® Equine Fecal Blood Test™

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Figure 1: OD450

results from an averaged ELISA time-course assay on two horses. Note the slow decay of detected hemoglobin and the consistently low levels of albumin.

1.41.2

10.80.60.40.2

0

0 5 10 15 20

Ab

Hg

The incidence of colonic ulceration

87%

Total Ulceration97%

54%

63%

3%

As a result of these limitations, Freedom Health LLC undertook an analysis of two potential marker proteins found in blood that might help to distinguish foregut from hindgut lesions. In an experiment conducted with research-ers from Island Whirl Equine Colic Research Laboratory in Florida, equine blood was introduced through a gastric cannula to two experimental horses and fecal samples were then taken periodi-cally for the next 18 hours.

This study looked at albumin, a blood protein that is known to be degraded by enzymes such as pepsin and trypsin in the stomach and duodenum. Because it is digested in this way, albumin detected in fecal matter is indicative of a hindgut lesion at a point caudal to the common bile duct in the small intestine. The study also looked at hemoglobin, which the previous research had shown can survive gastric enzymatic degradation as well as colonic bacterial digestion. Thus, albumin can serve as a proxy for hindgut lesions, while the stability of hemoglobin allows its use as an indica-tor of either foregut or hindgut lesions. Taken together, detection of these two proteins provides a novel technique for distinguishing these two disjoint areas of ulceration.

These two protein markers were analyzed using an Enzyme-Linked

ImmunoSorbent Assay (ELISA). When the results were plotted, it was possible to see how the levels of hemoglobin peaked and then slowly fell over the 18-hour period, while albumin levels remained consistently low due to the gastric source of the serum. This pro-vided strong support for the utility of use these two markers in a differential diagnosis.

A new diagnostic approach

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Maximum Colonic Ulcer Score

Colonic Grade 0

Colonic Grade 1

Colonic Grade 2

Colonic Grade 3

Colonic Grade 4

0% 10% 20% 30% 40% 50%

13%

43%

38%

6%

0%

Grade 0

Grade 1

Grade 2

Grade 3

Grade 4

0%

Stomach Ulcer Severity Grades

20% 40% 60% 80%

71%

30%

24%

7%

1%

Analysis of hemoglobin as an indicator of Gastric or Colonic Ulceration

Ulcer score >2 <2 total

positive 63 2 55

negative 13 18 31

total 66 20 86

Accuracy: 77%

Sensitivity: 75%

Specificity: 87%

P-value: 2%

Predictive Val Pos: 96%

Analysis of albumin as an indicator of Colonic Ulceration

Ulcer score >1 <1 total

positive 62 3 65

negative 8 13 21

total 70 16 86

Accuracy: 81%

Sensitivity: 83%

Specificity: 73%

P-value: 3%

Predictive Val Pos: 95%

Based on these promising preliminary results, Freedom Health designed a point-of-care test kit using purified anti-bodies targeted to equine albumin and equine hemoglobin to produce a highly specific and sensitive reaction.

The test includes two antibody wells. A couple of drops of diluted fecal mat-ter are placed in each well and after a few minutes, the presence of albumin and hemoglobin are determined and a diagnosis can be made in the barn. The following grid shows the possible test results and their likely meanings:

This test allows the practitioner to avoid a crude and potentially harmful analysis by symptom and helps to explicitly localize the source of GI lesions. A proper course of treatment should only be initiated upon an accurate appraisal, and this test seems to provide a high level of confidence.

The results of the antibody tests were compared to these anatomical observa-tions to check their predictive values. For hemoglobin, the test correlated well to the overall level of observed GI ulceration when the positive gastric and colonic cutoff was set to grade 2 and above.

Diagnostic sensitivity is defined as true positives divided by the total actual positives (true positives + false nega-tives) and indicates how likely a positive result reflects a true positive. Specificity indicates the likelihood that a negative test result indicates a true negative.

Note that the overall hemoglobin sensitivity for our test is 75% and the specificity is 87%. The positive predictive value, which indicates the probability that a positive test predicts a genuine under-lying problem, is 96%. The P-value here is 2%, indicating a high degree of significance for this assay at this cutoff value.

To validate and calibrate the test kit, Freedom Health performed another necroscopic study in August 2007, this time at a Canadian abattoir. In this study, fecal matter was recovered from 86 euthanized horses and analyzed with the test kit.

The digestive tract of each cadaver was then removed and the stomach and colon were examined for ulcers. Gastric ulcers were categorized by reference to the Dorland’s Illustrated Medical Diction-ary, using grades from 0 to 4, with the following results:

A scale similar to the gastric ranking was used for rating colonic ulcers:

For albumin, the antibody test was correlated exclusively to the level of colonic ulceration, where the cutoff was set to grade 1 and above. Note that the colonic albumin sensitivity is 83% and the specificity is 73%. Here the P-value is 3%.

A highly targeted test kit Testing the test

Results Negative albumin Positive albumin

Negative hemoglobin No detectable bleeding Hind gut bleeding

Positive hemoglobin Foregut bleeding Hind gut bleeding

Possible foregut bleeding

SUCCEED® Equine Fecal Blood Test™

Implications for the practitioner

The SUCCEED® Equine Fecal Blood Test™ (FBT) lets you con-duct a simple test on a horse at the point of care, without in-vasive and costly diagnostics or referrals. Veterinarians can ex-plore the possibility of foregut and hindgut lesions and related conditions in their patients without resorting to hit-or-miss symptomology. The two-part diagnostic consists of Test A to detect the presence of albumin and Test H to indicate the pres-ence of hemoglobin. These tests can be performed in the barn

in a few minutes with no extra equipment – a fecal sample and approximately 3 oz. of clean tap water, along with the contents of a single kit, are all that is required to test one horse. The results are easy to read directly from the window of the rapid-test kit, and ap-pear within 5 minutes.

The result is a strongly differential diagnostic aid that can help to guide the veterinarian’s treatment with greater confidence. Because the SUCCEED FBT can be performed within minutes, it is possible to test a number of horses in a barn or other boarding environ-ment in the course of a typical client visit. Given the ease and affordability of the FBT, practitioners can easily test all of their clients’ horses on a regular schedule. Consistent testing is especially important for performance horses, or wherever the care, feeding and general husbandry are less than ideal for digestive health, including intermittent feeding, high-grain diets, stall confinement, etc.

Testing a client’s horses, especially those assumed to be in good diges-tive health, can provide an opportunity for the veterinarian to educate their clients about these hidden GI issues. It can help you to provide a proper physiological context for many of the performance or be-havioral issues horse owners and trainers face regularly, but which are often attributed to training or the horse’s individual at-titude or ability.

The SUCCEED Equine Fecal Blood Test is immediately available from veterinary supply distributors, including MWI, Milburn Equine, Butler Animal Health, Webster Veterinary, Midwest Veterinary Supply and Profes- sional Veterinary Products.

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1Murray MJ (1994), “Gastric ulcers in adult horses”, Comp Cont Educ Pract Vet; 16: pp. 792-794, 797.

2McClure SR, Glickman LT, Glickman NW (1999) “Prevalence of gastric ulcers in show horses”, J Am Vet Med Assoc; 215: pp. 1130–1133.

3Michell RD (2001), “Prevalence of gastric ulcers in hunter/jumper and dressage horses evaluated for poor performance”, in Proceedings. Annual Meeting of the Association of Equine Sports Medicine 2001; pp. 74-77.

4Murray MJ, Grodinsky C (1989), “Regional Gastric pH measurements in horses and foals”, Equine Vet Suppl.: pp. 73-76.

5Pelligrini FL (2005), “Results of a large-scale necroscopic study of equine colonic ulcers”, J Equine Vet Sci; 25 (3): pp. 113-117.

6Mitchell RD. Prevalence of Gastric Ulcers in Hunter/Jumper and Dressage Horses Evaluated for Poor Performance. Assoc. Equine Sports Med., September 2001.

7Kronfeld DS. Speed Limit. Dipl. ACVN, Dipl. ACVIM. March 2003 Article # 4212.

8Andrew FM, Bernard WV, Byars TD (1999), “Recommendations for the diagnosis and treatment of equine gastric ulcer syndrome (EGUS)”, Equine Vet. Educ.; 1: pp. 122-134.

9Nappert G, Vrins A, Larybyere M (1989), “Gastroduodenal ulceration in foals” Compend Cont Educ Pract Vet; 11: pp. 345.

10Chris Sanchez L (2004), In: Reed SM, Bayly WM., Sellon DC., Equine Internal Medicine 2nd ed. Philadelphia, PA: Saunders Elsevier (USA); pp. 863-873.

11Coenen M (1990), “The occurrence of feed-induced stomach ulcers in Horses” Schweiz. Arch. Tierheilkd. 132: pp. 121–126.

12Nadeau JA, Andrews FM, Patton CS, Argenzio RA, Mathew AG, Saxton AM (2003a), “Effects of hydrochloric, acetic, butyric, and propionic acids on pathogenesis of ulcers in the nonglandular portion of the stomach of horses”, Am. J. Vet. Res. 64: pp. 404–412.

13Campbell-Thomson MC, Merrit AM (1987), “Effect of ranitidine on gastric acid secretion in young male horses”, Am J Vet Res; 48: pp. 1511-1515.

14Smyth GB, Young DW, Hammond LS (1998), “Effects of diet and feeding of post-prandial serum gastrin and insulin concentrations in adult horses”, Equine Vet J. Suppl 7: pp. 56-59.

15”Equine endotoxemia: pathomorphological aspects of endotoxin-induced damage in equine mesenteric arteries” Oikawa M, Shiga J. Equine Research Institute, Japan Racing Association J. Vet Med, May 2002; 49(4); 173-6

SUCCEED® Equine Fecal Blood Test™

Read results after 5 min.

Do not read after 10 min.

2 drops in each well

AH

CTL

CTLTST

TST

SUCCEED®Equine Fecal Blood Test™

FREEDOM HEALTH, LLC65 Aurora Industrial Pkwy

Aurora, OH 44202877-734-6558

www.SucceedFBT.com

SUCCEED® Digestive Conditioning Program® in oral paste and top-dress granules.

Also available from Freedom Health...

The SUCCEED® Equine Fecal Blood Test™ is available exclusively to

veterinarians through veterinary supply distributors, including Butler

Animal Health Supply, Milburn Equine, MWI Veterinary Supply,

Webster Veterinary, Midwest Veterinary Supply, and

Professional Veterinary Products.

®

SUCCEED® is a registered trademark and Equine Fecal Blood Test™ and SmartSignal™ are trademarks of Freedom Health, LLC. © 2008. All Rights Reserved. PATENT PENDING. C503 09/08