Practical Gastroinstestinal Function Testing

Albert E. Jergens, DVM, PhD, DACVIM

Department of Veterinary Clinical Sciences, Iowa State University

These tests can help narrow down your differential veterinary diagnoses for chronic enteropathies.

Chronic enteropathies are common in dogs and cats and include adverse reactions to food, idiopathic inflammatory bowel disease (IBD), and antibiotic-responsive diarrhoea. While definitive diagnosis often requires collecting mucosal biopsy samples for histopathologic evaluation, noninvasive laboratory tests are useful in diagnosing these disorders and monitor treatment.

Tests for small intestinal function and disease


The physiologic mechanism of cobalamin (vitamin B12) is complex and requires a functioning digestive system. Major disorders that interfere with cobalamin uptake include exocrine pancreatic insufficiency, distal or diffuse small intestinal disease, and excessive bacterial utilisation of cobalamin associated with bacterial dysbiosis.

Hypocobalaminaemia is most often seen with long-standing and severe intestinal disease (IBD, lymphoma, or fungal enteritis) affecting the small intestine. Measuring serum cobalamin concentrations in animals with chronic enteropathies is important since failure to recognise this deficiency may result in delay of clinical recovery.

Importantly, low serum cobalamin concentrations have been shown to be a risk factor for negative outcome in dogs with chronic enteropathies, including IBD and protein-losing enteropathy (PLE). If low concentrations are detected, appropriate dosages of cyanocobalamin are typically administered subcutaneously once a week, with serum concentrations reassessed at four- to six-week intervals.


This water-soluble vitamin (vitamin B9) is abundant in canine and feline diets, making nutritional deficiencies unlikely. Folate concentrations are typically measured in concert with cobalamin and may provide information indicative of small intestinal dysbiosis or proximal small intestinal mucosal disease. Note that serum folate concentrations may be influenced by bacterial production of folate and are therefore a less sensitive indicator of small intestinal disease than cobalamin.


This amino acid is synthesised predominantly by intestinal enterocytes. Citrulline has been shown to be a marker of functional enterocyte metabolic mass, and preliminary studies have shown plasma citrulline concentrations are significantly decreased in dogs with parvovirus enteritis. Thus, citrulline might serve as a potential marker of spontaneous and acute intestinal dysfunction. However, additional clinical trials evaluating this marker are warranted.

Evaluation of intestinal protein loss

PLEs are a heterogeneous group of disorders characterised by enteric loss of plasma proteins. Common causes of PLE include inflammatory enteropathies, infectious enteritis, lymphangiectasia, intestinal neoplasia, and marked microbial imbalances. Most animals exhibit the classic clinical signs of emaciation and weight loss, and panhypoproteinemia is often found on laboratory evaluation.

It is important to first rule out other causes of hypoalbuminemia due to glomerular injury and hepatic failure or insufficiency. Definitive diagnosis of PLE requires the collection and histopathologic assessment of mucosal biopsy samples.

Faecal A1-PI

An ELISA assay for the measurement of fecal alpha 1-proteinase inhibitor (A1-PI) has been validated in dogs and may be used to assess protein loss in the gastrointestinal (GI) tract. Due to the cumbersome criteria for collecting multiple fecal samples, the laboratory assessment of A1-PI as a diagnostic test for intestinal protein loss is performed infrequently. This laboratory assay may be most useful in assessing response to therapy in dogs with PLEs.

Serum albumin

Hypoalbuminemia has been associated with a negative outcome in separate canine IBD studies. Severe IBD and other chronic enteropathies may predispose patients to hypoalbuminaemia due to enteric plasma protein loss as well as nutritional deficiencies and nutrient malabsorption.

One retrospective study found a low serum albumin concentration was associated with a negative outcome (i.e. poor clinical response to dietary and drug therapies or euthanasia) in 80 dogs diagnosed with IBD. In my experience, dogs having severe hypoalbuminemia (< 1.5 g/l) with cavity effusion, salient GI signs, or both are at greater risk for negative outcome (euthanasia).

Intestinal inflammation and damage markers

Several serologic markers for inflammation have been designed to predict the disease course and response to therapy of canine chronic enteropathies.


Titers for perinuclear antineutrophil cytoplasmic antibodies (pANCA) have been evaluated as diagnostic markers in canine IBD in separate studies. In one study of 31 dogs, results indicated that pANCA was a highly specific marker for IBD, although the sensitivity of the assay was too low to be of value as a screening test. This immunofluorescence assay was shown to be most useful in detecting dogs with food-responsive enteropathy at the time of diagnosis.

More recently, pANCA was shown to be a highly specific serologic marker vs. antinuclear antibody testing for differentiating IBD from other GI disorders. In addition, other studies have shown that pANCA is of value as a diagnostic marker for familial PLE in soft-coated Wheaten Terriers. Unfortunately, this assay is not readily available and is presently limited for use in research investigations.


C-reactive protein (CRP) is an acute phase protein produced by the liver in response to inflammation. It is a sensitive but nonspecific inflammatory marker shown to correlate with moderate to severe clinical disease activity in canine IBD. This marker is the best-characterised inflammatory marker as no fewer than four different studies have shown that dogs with IBD have heterogeneous serum elevations of this marker.

New studies have evaluated the index of variability for CRP, suggesting that the use of a population-based reference range is not appropriate for evaluating changes in CRP in individual patients. Instead, serial CRP measurements should be performed and modest changes in serum CRP may indicate the initial severity of GI disease and its alteration from baseline in response to therapeutic intervention.

Fecal calprotectin and other S100A proteins

Calprotectin and S100 A8/A9 and A12 are calcium- binding proteins found predominantly within neutrophils and other immune cells in inflamed mucosa. Fecal concentrations of these proteins have been recently shown to be increased in people and dogs with IBD when compared with healthy controls. Importantly, the S100 A12 fecal marker has greater sensitivity for the detection of intestinal inflammation and has been positively associated with endoscopy and clinical scores.


The expression of P-glycoprotein (P-GP) in mucosal lymphocytes has also been investigated and shown to be up-regulated in dogs with IBD treated with prednisolone. This earlier study showed that in dogs with steroid-responsive enteropathy, low P-GP expression was associated with a good therapeutic response.

Practical molecular tools


Polymerase chain reaction for antigen receptor rearrangements (PARR) amplifies variable T or B cell antigen genes and is used to detect a clonally expanded population of lymphocytes. This molecular technique is useful in differentiating severe mucosal inflammation (severe IBD) from alimentary lymphoma. PARR is often performed when histologic examination and immunophenotyping return ambiguous results. Data derived from feline studies show that this molecular technique is highly sensitive for differentiating between intestinal lymphoma and IBD. However, further studies are needed to assess this technique’s value and sensitivity in dogs.


Fluorescence in situ hybridization (FISH) is a molecular technique used to identify microbial populations in tissues. FISH analysis has been applied to a variety of GI tissues, including the pancreas, liver, and alimentary tract, in dogs and cats. This technique uses culture-independent analysis of bacterial 16S or 23S rDNA genes targeted by DNA probes tagged with a fluorophore. Bacterial populations are then imaged via fluorescence microscopy. Microbial imbalances in diseased GI tissues (e.g. Helicobacter species gastritis, idiopathic IBD, feline cholangitis) in dogs and cats have been demonstrated using FISH techniques. Specifically, FISH has identified an association between GI inflammation and a shift in the microbiome by means of bacterial translocation or dysbiosis of mucosa-associated microbial populations.

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