Baseline investigations
History and physical examination
Detailed history and physical examination should help determine whether signs are associated with systemic or metabolic disease, may suggest partial obstruction or neoplasia, and may point to specific dietary sensitivities. Weight loss may be suggestive of malabsorption, due to exocrine pancreatic insufficiency or small intestinal disease, but may also be due to anorexia, vomiting or extra-gastrointestinal diseases such as hyperthyroidism, which can also cause diarrhea in cats. Polyphagia may
Introduction
Chronic intestinal diseases are common in small animals and can present a considerable challenge to the clinician. The main problem is that clinical signs which may include vomiting, diarrhea, and weight loss are shared by many conditions that
be a consequence of malabsorption, but is also seen in metabolic diseases, while anorexia is a more worrying sign seen in severe inflammatory bowel disease and intestinal lymphoma, as well as many extra-gastrointestinal diseases. Features of diarrhea that may help distinguish between small and large bowel disease are shown in Table 3.
1. Baseline investigations History and physical examination Hematology and serum biochemistry profile, plus T4, FeLV and FIV in cats systemic / metabolic disease Fecal examination intestinal parasites and bacterial pathogens Abdominal imaging partial obstruction and intestinal neoplasia 2. Specialized investigations Serum TLI exocrine pancreatic insufficiency Serum folate and cobalamin (vitamin B12) small intestinal disease Intestinal permeability test small intestinal disease in dogs Fecal 1protease inhibitor protein losing enteropathy Intestinal biopsy and histopathology endoscopy or laparotomy
Fecal examination
Feces from animals with diarrhea should be examined for intestinal parasites (including Giardia, coccidia, hookworms and whipworms) and potentially pathogenic bacteria (including Salmonella and Campylobacter). The presence of parasites or pathogenic bacteria does not necessarily mean these are responsible for the clinical signs, as some animals may be carriers, but treatment should be considered before further investigations. However, as there is a particular risk of promoting carrier status with Salmonella spp., typically this should only be treated if there is severe intestinal disease or evidence of systemic involvement. Many primary bacterial enteropathogens have been implicated as a cause of gastrointestinal disease in dogs and cats and particular attention has been focused on Salmonella spp. and Campylobacter spp. which can be readily identified by fecal culture. Detection of enterotoxins has been used to identify potentially enteropathogenic strains of Clostridia (1), but their significance particularly in chronic intestinal disease is not clear. However, there is evidence that enteropathic E.coli should be considered, as strains carrying pathogenic genes have been identified in dogs and cats by use of molecular biology techniques (2-4). PCR assays for these pathogenic genes are available commercially and are needed to distinguish harmless
Figure 1.
Mechanism of intestinal damage by enteropathogenic E.coli (EPEC) involves stripping of microvilli from the surface of intestinal epithelial cells destroying absorptive function.
from enteropathic E.coli which can easily be overlooked as they cannot be identified using routine microbiologic techniques. Enteropathogenic E.coli (EPEC) appear to be the most common type of pathogenic E.coli and in dogs there is evidence they can cause both acute and chronic diarrhea (4). They work by an attachingeffacing mechanism that strips microvilli from enterocytes resulting in osmotic diarrhea due to compromised absorptive function (Figure 1). Cytotoxin secreting E.coli secrete cytotoxins which are lethal to intestinal epithelial cells, causing hemorrhage and ulceration, potentially mimicking the severe enterocolitis caused by invasive bacteria. Enterohemorrhagic E.coli (EHEC, VTEC) are the best known in this group, which also includes enteroaggregative E.coli (EAEC) and CNF-secreting E.coli. Enteroinvasive E.coli (EIEC) can invade the mucosa of the distal small bowel and colon causing acute enterocolitis. This is typically manifest as diarrhea accompanied by passage of blood and mucus, and may result in a potentially fatal septicemia if the organisms penetrate the intestinal barrier. Enterotoxigenic E.coli (ETEC) do not cause intestinal damage, but secrete toxins which have a specific biochemical effect. These act as secretagogues resulting in a watery electrolyte-rich diarrhea. All of these pathogenic E.coli can potentially cause acute clinical disease. However, properties such as adherence to the surface by EPEC or invasion of the mucosa can promote long-term colonization predisposing to chronic disease or carrier status.
Chronic cases can be difficult to manage and this may be due to a number of factors including poor support from a defective host response, antibiotic resistance, or re-infection from the environment. An oral autogenous E.coli vaccine prepared from E.coli isolated from that individual animal should be considered as an adjunct to treatment particularly to help prevent recolonization (5). This approach has been shown to be effective to manage diarrhea in the challenging environment of breeding and boarding kennels. There are likely to be more as yet unidentified enteropathic E.coli strains. This is emphasized by a recent comprehensive study which revealed that granulomatous colitis of Boxer dogs is associated with intramucosal colonization by adherent and invasive E.coli that resemble extraintestinal strains in genotype (6). Tritrichomonas fetus is a protozoan parasite that has also recently emerged as a potential cause of prolonged and intractable diarrhea in cats (7). Signs are consistent with colonization of the colon, typically including large intestinal diarrhea without weight loss, and this parasite appears particularly to affect young cats and kittens from multi-cat households. This is another organism that is best identified by PCR. Microscopic examination of feces for presence of fat, undigested muscle fibers, or starch is almost pointless this may provide indirect evidence for malabsorption, but provides little specific information.
Diagnostic imaging
Diagnostic imaging is typically not helpful in the diagnosis of intestinal disease, but ultrasonography and/or radiography should be considered if partial obstruction or neoplasia are suspected.
Specialized investigations
Serum trypsin-like immunoreactivity (TLI)
Canine serum trypsin-like immunoreactivity (cTLI) provides a sensitive and specific test for exocrine pancreatic insufficiency (EPI) in dogs (8), which is most commonly due to acinar atrophy, but occasionally due to pancreatic hypoplasia or acinar destruction following chronic bouts of pancreatitis. This assay quantitates trypsinogen that normally leaks from the pancreas into the blood, and provides an indirect assessment of functional pancreatic tissue. In dogs with EPI, functional exocrine tissue is severely depleted and therefore serum TLI concentrations are extremely low. Assay of feline TLI (fTLI) has been established for the diagnosis of EPI in the cat (9). This is less common than in the dog and is typically due to chronic pancreatitis, acinar atrophy being relatively rare in the cat. As the loss of pancreatic tissue following pancreatitis is non-specific, this can result in diabetes mellitus in addition to EPI. It is therefore worth considering EPI in diabetic cats, especially when there is a poor clinical response to insulin therapy, particularly weight loss. EPI should be excluded before considering intestinal disease in dogs and cats, as EPI can cause secondary changes in the small intestine that resolve with treatment of EPI, and can also result in malabsorption of cobalamin (vitamin B12), interfering with interpretation of serum cobalamin for the diagnosis of intestinal disease (see below). Assays of serum pancreatic lipase activity (PLI) have been developed specifically for the diagnosis of pancreatitis in the dog and cat. Typically, pancreatitis is not a differential for animals presenting with suspected chronic intestinal disease, but could be considered in some cases, for example with chronic vomiting as the main presenting sign. However, assays of PLI are not recommended for the diagnosis of EPI as there is an overlap in serum
Increased
PLI concentrations between healthy animals and animals with EPI (10).
Folate deconjugase
Figure 2.
No folate carriers
Mechanism of folate absorption. Dietary folate polyglutamate is degraded by folate deconjugase in the proximal small intestine where folate monoglutamate can then be absorbed by folate carriers. Absence of folate carriers results in minimal folate absorption in the distal small intestine and colon.
Figure 3.
IF-cobalamin receptors
Mechanism of cobalamin (vitamin B12) absorption. Dietary cobalamin binds to gastric R protein which is subsequently degraded by pancreatic proteases, releasing cobalamin which then binds to intrinsic factor (IF) secreted either by the stomach and pancreas (dog) or the pancreas alone (cat). Cobalamin-IF complexes then bind to specific receptors on ileal enterocytes to be absorbed by receptor-mediated endocytosis.
Cobalamin absorption
intestinal damage include idiopathic inflammatory bowel disease and lymphoma. In cats, subnormal serum folate concentrations are frequently associated with unequivocal morphologic abnormalities in the small intestine, particularly inflammatory bowel disease. Cats with EPI may also have low serum folate concentrations suggesting concurrent small intestinal disease, which rarely occurs in dogs with EPI. In marked contrast, normal absorption of dietary cobalamin occurs specifically in the distal small intestine and involves a much more complex mechanism than for other dietary nutrients (Figure 3). This is necessary because cobalamin is a very large molecule that cannot cross the intestinal epithelial barrier by diffusion or by carrier-mediated transport, processes used by other nutrients. Instead absorption involves binding of cobalamin to a specific cobalamin-binding protein
called intrinsic factor (IF). A second site on intrinsic factor subsequently binds to specific receptors present only on the surface of ileal intestinal epithelial cells. This binding induces cell-mediated endocytosis of the IF-cobalamin complex into these ileal enterocytes and hence facilitates passage of cobalamin from the intestinal lumen into the blood. IF is synthesized by the stomach in most mammals including humans. However, the pancreas is an important site of IF synthesis in the dog (12), and is the sole site of IF synthesis in the cat (13). Pancreatic proteases also degrade non-specific R proteins from the stomach which would otherwise bind cobalamin and prevent binding to IF. In EPI, interference with production of not only pancreatic IF but also pancreatic proteases can result in cobalamin deficiency in dogs and particularly severe cobalamin deficiency in cats, emphasizing the need to exclude EPI when interpreting cobalamin results.
Decreased serum cobalamin can be indicative of distal small intestinal damage, and when accompanied by low serum folate, is suggestive of diffuse and potentially relatively severe disease affecting the proximal and distal small intestine. Low serum cobalamin in the cat appears to be a relatively common finding indicative of severe intestinal disease, particularly inflammatory bowel disease or lymphoma (14). Surprisingly, the possibility of small intestinal disease can easily be overlooked in the cat, particularly if there are no obvious signs pointing to GI disease, such as vomiting or diarrhea. It can be very enlightening to perform folate and cobalamin assays in cats with weight loss as the main presenting sign, particularly including middle aged and older cats with suspected hyperthyroidism. Low serum cobalamin and/or high serum folate in the dog can be indicative of small intestinal bacterial overgrowth (SIBO) in the proximal small intestine (15,16), also known as antibioticresponsive diarrhea (Figure 4). SIBO is a relatively common condition in many large breeds of dog, and typically present in young animals as chronic intermittent small bowel diarrhea, which may be accompanied by loss of body weight or failure to gain weight. Histologic changes in intestinal biopsies are only present in the minority of cases. However, biochemical studies have demonstrated distinct mucosal damage which is reversible following antibiotic treatment and would not be detected by routine histopathology (15,16). While there is some controversy concerning the concentration of bacteria considered to be normal in the proximal small intestine, there is substantial evidence that intraluminal bacteria and their secreted products can have detrimental effects related to the concentration as well as the composition of the flora, including the demonstration of enhanced intestinal permeability (see below) in apparently healthy Beagles with SIBO (17). The outcome will depend on many factors including diet and host mucosal defense, and the level at which this presents clinically as a small intestinal bacterial overload may vary with the individual. Decreased serum cobalamin and/or high folate concentrations can provide indirect evidence for SIBO in dogs, once other possible causes of these changes such as pancreatic insufficiency
have been eliminated (Table 4). Indeed, SIBO is a particularly common cause of cobalamin deficiency in dogs, large numbers of bacteria in the proximal small intestine, particularly anerobes including Clostridium and Bacteroides spp., being able to bind and internalize cobalamin so that it is unavailable for uptake in the distal small intestine (Figure 4). High serum folate in SIBO is due to the ability of many intestinal bacteria to synthesize folate which can be absorbed and contribute to serum concentration when these bacteria are present in the proximal small intestine (Figure 4). In contrast, folate is poorly absorbed in the distal small intestine and colon, and bacteria at these sites make a relatively small contribution to serum folate. While increased folate alone may suggest SIBO, a high serum folate accompanied by reduced cobalamin is particularly supportive of SIBO in the dog. SIBO is a common secondary problem in dogs with EPI, and can result in increased serum folate, in addition to contributing to a low cobalamin caused by deficiency of IF and pancreatic enzymes (Figure 3). SIBO is not typically a clinical syndrome in cats, but potentially intestinal bacteria could bind and contribute to a low serum cobalamin. However, provided pancreatic insufficiency (EPI) has been eliminated, low serum cobalamin is most likely to be indicative of small intestinal disease in the cat. Hereditary selective cobalamin malabsorption in dogs can result in very severe deficiency of cobalamin caused by an abnormality of the IF receptor in ileal epithelial cells. This condition is relatively rare, but has been well described in Giant Schnauzers, Border Collies, Australian Shepherd dogs and Beagles (18,19). This should be considered as a possible explanation for a very low serum cobalamin, particularly in young dogs presenting less than a year of age with failure to thrive, possibly accompanied by GI signs, and chronic non-regenerative anemia. So, you may well be asking with all these possible combinations, how on earth do I make sense of these vitamin results? As a practical approach in the majority of dogs, for a low folate result, initially consider the possibility of dietary sensitivity, and for the dog with a low cobalamin initially consider SIBO, particularly if there is also a high folate. If folate and cobalamin are both
Figure 4.
Folate synthesis Cobalamin binding
Mechanism of increased serum folate and decreased serum cobalamin (vitamin B12) in small intestinal bacterial overgrowth (SIBO) in dogs. Intestinal bacteria can synthesize folate which can be absorbed in the proximal small intestine, and can internalize cobalamin resulting in failure of ileal uptake.
Cobalamin malabsorption
reduced, consider more severe, potentially diffuse small intestinal disease. In the cat, take particular notice of a low cobalamin which can indicate relatively severe intestinal disease and look for this not only in cats with, but also without obvious GI signs - you will be surprised how many of these cats turn out to have GI disease which can easily be overlooked. And remember that cobalamin in particular is needed to maintain many cell functions, including integrity and function of the gastrointestinal tract. Dogs and cats with severe deficiency therefore may not respond to treatment of the underlying disorder until this is corrected by administering parenteral cobalamin which in some cases needs to be lifelong, for example dogs with hereditary cobalamin malabsorption and cats with EPI.
appear to work well in cats which normally have very high intestinal permeability compared to dogs. Studies with 51Cr-EDTA clearly established the potential value of permeability testing in dogs, but there were practical constraints associated with the use of a radioactive label (17,20). Subsequently, procedures were developed involving oral administration of a sugar solution containing lactulose and rhamnose, and analysis of these sugars either in a timed total urine collection, or more conveniently in a single blood sample taken 2 hours later (21-24). The principle of this sugar test is illustrated in Figure 5: the normal small intestine is lined by finger-like villi covered by absorptive epithelial cells which form an effective barrier between the lumen and the lamina propria. There are two potential routes for passive transfer of molecules across this barrier: Paracellular permeability between epithelial cells is normally low as tight junctions form effective seals to keep out larger harmful molecules: probe = lactulose Transcellular permeability through epithelial cells is normally high as this route allows absorption of small nutrients: probe = rhamnose Interference with tight junctions results in increased permeability of lactulose, whereas loss of
Figure 5.
Principle of the oral sugar test for intestinal permeability in dogs. Intestinal damage can cause increased paracellular permeability to lactulose and decreased transcellular permeability to rhamnose, resulting in increased lactulose to rhamnose ratio in urine or blood.
Lumen
Lactulose: paracellular permeability Rhamnose: transcellular permeability
Lamina propria
Figure 6.
Intestinal permeability findings in a dog with dietary sensitivity. This shows a high initial lactulose to rhamnose ratio which fell on an exclusion diet with relapse on dietary challenge. Data from reference 21 used by kind permission of the Publishers.
Lactulose/rhamnose ratio
microvilli or villi results in reduced surface area and decreased permeability of rhamnose. The net result is an increase in the lactulose to rhamnose ratio in blood or urine collected after oral dosing with these sugars. Application of this test to identify dietary sensitivity as the cause of intestinal damage is illustrated in Figure 6 which shows urine ratios of these sugars in a 3-year old Golden Retriever with chronic vomiting and diarrhea. High permeability pre-treatment indicated intestinal damage, which resolved on a chicken and rice exclusion diet with relapse on dietary challenge, indicating true dietary sensitivity (21). Permeability testing has proved particularly useful to assess the severity of mucosal damage in bacterial overgrowth (SIBO) and also to help
long-term management (22). Intestinal permeability is increased in 50-60% of clinical cases with SIBO, even when there are no histologic abnormalities, and this typically decreases with oral antibiotic treatment (oxytetracycline is particularly successful, but metronidazole and tylosin are effective alternatives) for a month (Figure 7). Normalization of intestinal permeability following antibiotic therapy indicates successful repair of mucosal damage and antibiotics should be discontinued. Figure 8 shows that dogs with high permeability after 1 month of therapy are 3 times more likely to relapse despite an apparent clinical response to treatment, and continuation of antibiotic therapy is therefore recommended. Differences are more marked after 3 months of treatment as dogs with high permeability are approximately 8 times more likely to relapse. A persistent high permeability in dogs
Figure 7.
0.5
Lactulose/rhamnose ratio
Intestinal permeability findings in 11 dogs with small intestinal bacterial overgrowth (SIBO). This shows a fall in permeability following 1 month treatment with oral antibiotic (oxytetracycline or tylosin) consistent with repair of mucosal damage. Data from reference 22 used by kind permission of the Publishers
Pre-treatment
Post-treatment
Figure 8.
Application of intestinal permeability for the management of dogs with small intestinal bacterial overgrowth (SIBO). Dogs with persistently high permeability are more likely to relapse after discontinuation of antibiotic therapy. (** p<0.01). (HC Rutgers and RM Batt, unpublished observations).
3 months
Relapse (%)
Normal
High
Normal
High
with a poor clinical response should prompt further investigation of underlying disease, such as idiopathic inflammatory bowel disease.
of intestinal protein loss can therefore help the detection of intestinal pathology, ideally before there is significant hypoproteinemia, and can also confirm that low serum albumin is due to proteinlosing enteropathy, having excluded protein-losing nephropathy and hepatic disease using more readily available tests. The assay of 1proteinase inhibitor (1PI) in feces has been established to achieve an assessment of intestinal protein loss, as 1PI is a serum protein that is similar in size to albumin and hence leaks into the intestinal tract at a comparable rate (25). Unlike most proteins, this protease inhibitor is able to resist proteolysis in the intestinal tract and is passed unaltered in the feces. Ideally, 24 hour excretion should be measured, but a
more practical approach involves assay of 1PI concentration in each of three different samples, although these do need to be frozen before submitting for assay.
This approach lends itself to the classification of intestinal diseases by histologic appearance rather than cause, and this obviously can have limitations for effective management. In particular, it is important to remember that inflammatory bowel disease (IBD) is merely a histologic description, and likely to represent a common manifestation of diseases with a variety of causes, potentially involving the small and/or large intestine. Definite associations with parasites and pathogenic bacteria, small intestinal bacterial overgrowth (SIBO) and dietary-sensitivity have been demonstrated, but in many cases the cause cannot be identified.
REFERENCES
1. Marks SL, Kather EJ, Kass PH, et al. Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs. J Vet Intern Med 2002; 16: 533-540. 2. Beutin L. Escherichia coli as a pathogen in dogs and cats. Vet Res 1999; 30: 285-298. 3. Goffaux F, China B, Pirson B, et al. The locus for enterocyte effacement (LEC) of enteropathogenic Escherichia coli (EPEC) from dogs and cats. Adv Exp Med Biol 1999; 473: 129-136. 4. Sancak AA, Rutgers HC, Hart CA, et al. Prevalence of enteropathic Escherichia coli in dogs with acute and chronic diarrhoea. Vet Rec 2004; 154: 101-106 5. Weber A, Gobel D. Treatment of chronic diarrhoea in dogs and cats under field conditions using oral E.coli vaccines. Tierarztl Prax 1995; 23: 80-2. 6. Simpson KW, Dogan B, Rishniw K, et al. Adherent and invasive Escherichia coli is associated with granulomatous colitis in Boxer dogs. Infect Immun 2006; 74: 4778 4792. 7. Gookin JL, Breitschwerdt, Levy MG, et al. Diarrhea associated with trichomonosis in cats. J Am Vet Med Assoc 1999; 215: 1450-1454. 8. Williams DA, Batt RM. Sensitivity and specificity of radioimmunoassay of serum trypsin-like immunoreactivity for the diagnosis of canine exocrine pancreatic insufficiency. J Am Vet Med Assoc 1988; 192: 195-201. 9. Steiner JM, Williams DA. Serum feline trypsin-like immunoreactivity in cats with exocrine pancreatic insufficiency. J Vet Intern Med 2000; 14: 627-629. 10. Steiner JM, Rutz GM, Williams DA. Serum lipase activities and pancreatic lipase immunoreactivity concentrations in dogs with exocrine pancreatic insufficiency. Am J Vet Res 2006; 67: 84-87. 11. Batt RM, Morgan JO. Role of serum folate and vitamin B12 concentrations in the differentiation of small intestinal abnormalities in the dog. Res Vet Sci 1982; 32: 17-22. 12. Batt RM, Horadagoda NU, McLean L, et al. Identification and characterization of a pancreatic intrinsic factor in the dog. Am J Physiol 1989; 256: G517-G523. 13. Fyfe JC. Feline intrinsic factor (IF) is pancreatic in origin and mediates ileal cobalamin (CBL) absorption, J Vet Intern Med 1993; 7: 133-135. 14. Simpson KW, Fyfe J, Cornetta A et al. Subnormal concentrations of serum cobalamin (vitamin B12) in cats with gastrointestinal disease. J Vet Intern Med 2001; 15: 26-32. 15. Batt RM, McLean L. Comparison of the biochemical changes in the jejunal mucosa of dogs with aerobic and anaerobic bacterial overgrowth. Gastroenterology 1987; 93: 986-993. 16. Batt RM, McLean L, Riley J. Response of the jejunal mucosa in dogs with aerobic and anaerobic bacterial overgrowth to antibiotic therapy. Gut 1988; 29: 473-482. 17. Batt RM, Hall EJ, McLean L, et al. Small intestinal bacterial overgrowth and enhanced intestinal permeability in healthy Beagles. Am J Vet Res 1992; 53: 1935-1940. 18. Fyfe JC, Jezyk PF, Giger U, et al. Inherited selective malabsorption of vitamin B12 in Giant Schnauzers. J Am Anim Hosp Assoc 1989; 25: 533-539. 19. Outerbridge CA, Myers SL, Giger U. Hereditary cobalamin deficiency in border Collie dogs. J Vet Intern Med 1996; 10: 169. 20. Hall EJ, Batt RM. Enhanced intestinal permeability to 51Cr-labeled EDTA in dogs with small intestinal disease. J Am Vet Med Assoc 1990; 196: 91-95. 21. Rutgers HC, Batt RM, Hall EJ, et al. Intestinal permeability testing in dogs with diet-responsive intestinal disease. J Small Anim Pract 1995; 36: 295-301. 22. Rutgers HC, Batt RM, Proud, FJ, et al. (1996) Intestinal permeability and function in dogs with small intestinal bacterial overgrowth. J Small Anim Pract 1996; 37: 428-434. 23. Steiner JM. Williams DW, Moeller EM. Kinetics of urinary recovery of five sugars after orogastric administration in healthy dogs. Amer J Vet Res 2002; 63: 845-848. 24. Srensen SH, Proud FJ, Rutgers HC, et al. A blood test for intestinal permeability and function: a new tool for the diagnosis of chronic intestinal disease. Clin Chim Acta 1997; 264: 103-115 25. Melgarejo T, Williams DW, Asem EK. Enzyme-linked immunosorbent assay for canine 1protease inhibitor. Amer J Vet Res 1998; 59: 127-130.