A discussion of some of the tests that are currently available (or will be in the near future) for diagnosing gastrointestinal and pancreatic diseases in dogs.

Diagnosing gastrointestinal and pancreatic diseases in the dog can pose a challenge as signs and symptoms are often nonspecific. Clinical signs range from lethargy, decreased appetite, and a slightly altered stance or walk (due to abdominal discomfort), to anorexia, vomiting, and diarrhea, or worse hematochezia. Disruption of the microbiome’s gut-brain axis can alter behavior, including cognition and memory. A thorough history can help more closely define the origin(s) and potential localization of the problem, while laboratory tests provide additional verification of a clinical suspicion. This article will discuss different described tests that are currently available (or will be in the near future) to veterinary practitioners for diagnosing different gastrointestinal and pancreatic diseases. This list is not inclusive, since many more tests have been described in the literature, including screening with blood, cheek swabs, and tests for urine, feces, and saliva to identify potential food reactants. Most of these tests are poorly predictive of causation.

Laboratory tests

Any laboratory test depends on its inherent qualities and ability to discriminate between healthy and diseased animals. For many of these tests, biological variation is an inherent characteristic of the biological substance measured. Depending on its diagnostic characteristics (e.g. sensitivity and specificity), the test can be good for screening (high sensitivity, therefore a low false negative result rate), or for confirming diagnosis (high specificity, low false positive result rate). Case selection can improve the diagnostic accuracy and value of the results from laboratory tests.

Pancreatic diseases

Pancreatic lipase assays

Acute pancreatitis in dogs usually presents with more overt signs, while chronic pancreatitis often involves subtle recurring signs.¹ In acute pancreatitis, a physical exam might reveal generalized weakness (79% of cases), dehydration (46%), abdominal pain or discomfort (44% to 58%), and sometimes even icterus, pyrexia or hypothermia, bleeding diathesis, or abdominal effusion.^2,3 Certain breeds have been described to be at higher risk of developing pancreatitis (see Table 1).

 

Diagnosing pancreatitis still remains challenging. The gold standard for diagnosis is histopathology; however, this is invasive, costly, and not without certain risks. Current laboratory tests to diagnose this disease are neither 100% sensitive nor specific. Historically, elevated serum amylase and lipase values were thought to suggest pancreatitis. Both markers were not sensitive or specific, since extra-pancreatic sources (stomach and duodenum) have been described.

More recently, lipase activity has been assessed by assays using other substrates: 1,2 diglyceride (1,2 DiG) and 1,2-o-dilaurylrac-glycero-3-glutaric acid-6’-methylresorufin ester (DGGR). 1,2 DiG has a reported sensitivity of 73% and specificity of 73%. Based on differing cut-off values for DGGR lipase, different sensitivity and specificity values have been reported (See Table 2). Both these assays don’t solely measure pancreatic lipase. Lipases of other origins are measured as well, which could influence results.

Others assays that assess the more specific canine pancreatic lipases are Spec cPL and SNAP cPL.^4,5 Spec cPL results range from <200 μg/L (considered normal range) to >400 μg/L (considered highly suggestive of pancreatitis), while values from 200 to 400 μg/L are equivocal. Retesting equivocal results at a later stage, or using other diagnostic markers or modalities, is recommended. Using the >200 μg/L cut-off in diagnosing acute pancreatitis, a multicenter study reported sensitivity of 90% and specificity of 72%, while using the >400 μg/L cut-off provided a sensitivity of 75% and specificity of 78%.⁶ Overall, a positive Spec cPL has a good positive predictive value in cases suspected of acute pancreatitis, while providing a good negative predictive value when prevalence is low. Spec cPL is offered by specific reference laboratories. Shipment to and processing by a laboratory requires time (usually two or three days) before results are returned.

SNAP cPL is a semi-quantitative method of measuring cPL that decreases turnaround time by providing a subjective result. Spot color intensity is determined by an observer in comparison to a reference spot. Results can be lighter than (corresponding to a cPL value <200 μg/L); equal to (200-400 μg/L); or darker than (>400 μg/L) the reference spot. After a more intense or equal-intense result, follow-up with a quantitative canine pancreatic lipase is recommended. SNAP cPL has a high reported sensitivity (93%) and specificity (74%).⁶

Abdominal ultrasonography

Abdominal ultrasonography (AUS) can help diagnose pancreatitis and exclude other intestinal causes. AUS is dependent on equipment and operator characteristics. Originally, a sensitivity of 68% for diagnosing pancreatitis was described,² with a sensitivity of 56% for chronic pancreatitis,² while a more recent study showed an increased sensitivity of 89%.⁷ Signs on AUS suggestive of pancreatitis include an enlarged, irregular, hypo-echoic pancreas, hyperechoic peri-pancreatic fat, and abdominal effusion.^2,7,8

Trypsin-like immunoreactivity

Trypsin-like immunoreactivity (TLI) was originally developed to diagnose exocrine pancreatic insufficiency (EPI) in dogs.⁹ In fasted dogs, serum TLI < 2.5 µg/L is considered diagnostic for EPI, while values ≤ 3.5 µg/L could indicate subclinical EPI, and are recommended to be retested after one month.⁹

Elevated fasted serum TLI has been described in experimentally induced pancreatic insults. However due to its short half-life, TLI is not used routinely. Serum TLI values > 50 µg/L are shown to be associated with pancreatitis or severe renal insufficiency.^9,10 Reported sensitivity of TLI for the diagnosis of pancreatitis ranges from 33% to 47%, with a reported specificity of 65%.^3,11,12

Other markers of canine pancreatitis

• Pancreatitis elastase-1 is produced by pancreatic acinar cells, and secreted into the bloodstream during pancreatitis. Serum canine pancreatic elastase-1 (cPE-1) seems to be significantly elevated with pancreatitis, and might be more useful at diagnosing acute rather than chronic cases.¹³ Further studies of this marker are needed before it can be routinely used in practice.
• Trypsinogen activation peptide (TAP), a product of trypsinogen cleavage, is released into the circulation and excreted by the kidney in acute pancreatitis. Plasma and urine TAP are elevated in acute pancreatitis.¹⁴

Availability of both cPE-1 and TAP in clinical practice may depend upon location.

Gastrointestinal diseases

Most acute gastrointestinal diseases (like diarrhea) resolve with supportive treatment: a highly digestible diet, increased fluid intake either enterally or parenterally in case of severe or ongoing fluid losses, and anti-nausea medications. Chronic gastrointestinal diseases (> 3 weeks of clinical signs, or recurring intermittent signs) could indicate a chronic enteropathy (CE). As canine CE often has a slower onset of clinical signs, diagnosis and definitive treatment usually require more time. A flow diagram for the suggested diagnostic work-up of canine CE is presented in Figure 1.

Different markers and methods described in the literature can help clinicians assess canine CE.

Total protein and albumin

Serum total protein and albumin are measured routinely in many canine patients, especially in gastrointestinal cases. Dogs with severe CE show protein loss through the gut mucosa. Decreased serum albumin has been described as a negative prognostic indicator in dogs with CE.^15,16

Cobalamin and folate

Cobalamin (vitamin B12) and folate (vitamin B9) are absorbed in the small intestine (ileum and duodenum, and proximal jejunum, respectively), and have been described as markers for CE. Hypocobalaminemia is thought to reflect distal small intestinal malabsorption, small intestinal dysbiosis, or a combination. Low serum cobalamin is considered a negative prognostic indicator in dogs with CE.¹⁶ Hypocobalaminemia can also be observed in EPI.

Low serum folate can be due to chronic malabsorption in 14% of dogs with CE. However, it can be falsely elevated (to normal or above-normal levels) due to small intestinal dysbiosis or low cobalamin, and is therefore best assessed when serum cobalamin levels are normal. Abnormal serum cobalamin and/or folate concentrations therefore require evaluation of serum TLI to exclude EPI.

Other intestinal serum markers

• Alpha1-proteinase inhibitor (α1-PI), also known as alpha1-antitrypsin, is a major serine proteinase inhibitor produced by the liver. α1-PI has a similar molecular weight to albumin, and is lost at the same rate with gastrointestinal protein loss. In contrast to albumin, α1-PI can be quantified in feces as it is resistant to proteolysis. Fecal α1-PI is therefore helpful as a marker for gastrointestinal protein loss.¹⁷ Variations of α1-PI on a day-to-day basis are large, requiring evaluation of fecal samples on three consecutive days, which can be a disincentive for clients. α1-PI values ≥ 19 µg/g for a three-day mean sample are considered abnormally elevated, confirming histological lesions seen in canine protein-losing enteropathy (a form of CE), because these can be increased earlier than the presence of clinical signs, hypoalbuminemia, or both.¹⁸
• Methylmalonic acid (MMA) is a metabolite shown to decrease with low intracellular cobalamin.¹⁹ MMA can be determined in serum or urine, and appears to be useful in combination with serum cobalamin at assessing cobalamin levels in dogs.²⁰ This assay is technically challenging and costly, and therefore currently not routinely used in canine CE. In the future, these challenges might decrease, making it more available for routine testing.
• Calprotectin, S100A8/A9 complex, is a molecule that accumulates at sites of inflammation, and is released by activated macrophages and neutrophils.²¹ Serum calprotectin is not specific for diseases of the gastrointestinal tract; however, fecal calprotectin can be used as a surrogate marker for disease severity in canine chronic inflammatory enteropathy (CIE). It also seems to have ability in predicting treatment response in canine CIE.²¹
• Calgranulin, S100A12, is a molecule closely related to calprotectin, which appears a useful biomarker of gastrointestinal inflammation in dogs. Fecal calgranulin levels are correlated with the severity of endoscopic lesions and clinical signs, though not with histopathologic changes.²¹ Associations has been described between disease outcome in canine CIE and increased fecal calgranulin levels.^22,23 Both calprotectin and calgranulin are currently not widely available, making routine use limited. In the future, availability might improve.
• Other novel markers such as perinuclear antineutrophil cytoplasmic antibodies (pANCAs), receptor for advanced glycation end-products (RAGE), or polymorphonuclear leukocytes (APMNA), antibodies against gliadins (AGA), microbial outer membrane porin C (ACA), E Coli outer membrane porin (anti-OmpC), flagellins (AFA), or combined panels have been described, but require further studies before they are practical and can be routinely advised in clinical practice.^23-27

Canine dysbiosis index

In both human and veterinary medicine, alterations of the gut microbiome have been described in relation to diverse (both acute and chronic) gastrointestinal diseases. Changes in the microbiome have been considered both cause and consequence. Historically, bacterial culture was used to identify the presence of bacteria in the gastrointestinal tract; however, resolution to identify anaerobic bacteria was low.^28,29 Less than 20% of intestinal bacteria are cultivable with standard laboratory techniques.²⁹ Recent developments have shown that molecular methods are far better at assessing and identifying these changes.^28,29 A canine fecal dysbiosis index has been developed that shows an altered state of the gut bacterial microbiome.³⁰

This dysbiosis index (DI) is acquired by a quantitative PCR (qPCR) panel consisting of eight bacterial “groups”: total bacteria, Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium, and Clostridium hiranonis. Each of their reported values is compiled into a single numerical value that measures closeness to the norm (l₂) of the test sample to the mean prototype of each class. The DI is classified as a negative value (<0, indicating normobiosis); a positive value, which can indicate an equivocal result (0-2); or dysbiosis (>2). In equivocal results, follow-up assessment several weeks later is recommended. A dysbiosis index value >2 with decreased abundance of C. hiranonis, one of the bacterial groups assessed, is common in dogs with diverse gastrointestinal diseases like EPI and CE.^29,30 The higher the DI, the more deviation from normobiosis is present in the sample. DI has been described as having a sensitivity of 74% and specificity of 95% to distinguish healthy and CE dogs.³⁰ Studies evaluating if dysbiosis patterns can distinguish between various forms of CE (food responsive CE, antibiotic response CE, and inflammatory bowel disease) are required. Optimal assessment would encompass intestinal and systemic inflammation, immune function, and tissue damage by multiple methods such as histopathology in these studies.

Different medications have been described in relation to changes in the gut microbiome. Proton pump inhibitors (like omeprazole) can lead to an increase in DI, which decreases back towards normal 14 days after cessation of the therapy. Administration of broad-spectrum antibiotics (like metronidazole, amoxicillin clavulanic acid, and tylosin) also induce microbiota shifts, increasing the DI, that resemble changes of dysbiosis observed in CE.^31-34 Normalization of the DI takes several weeks after the end of administration.^31-34 Studies evaluating how to address this intestinal dysbiosis (for example by fecal microbial transplant) are needed.

Clinical severity indices

In human medicine, the scoring of clinical disease severity is common practice to help assess severity and track treatment progression. In veterinary medicine, a scoring index for disease activity in canine inflammatory bowel disease, CIBDAI, was developed and validated to help manage clinical patients.^35,36 This scoring index takes into account disease activity of six prominent gastrointestinal signs – attitude and activity, appetite, vomiting, stool consistency, stool frequency, and weight loss.³⁵ Each sign is scored with a value of 0-3. A total cumulative CIBDAI score is created and disease is classified as insignificant (0-3), mild (4-5), moderate (6-8), or severe (≥9).³⁵ Long term follow-up regarding CIBDAI scores are not known. Another clinical scoring index, CCECAI (Canine Chronic Enteropathy Clinical Activity Index), takes into account the CIBDAI factors, as well as serum albumin concentrations, presence of ascites, peripheral edema, and pruritus.³⁷ Both these indices can help clinicians track disease and treatment progress.

Histopathology

The gold standard for quantifying intestinal inflammation in dogs remains a combination of endoscopic evaluation of the intestinal mucosa and histologic examination. Significant interobserver variability in the histopathologic assessment of intestinal biopsies has been described among pathologists even with use of the WSAVA standardization criteria.^38,39 A novel shortened histologic scoring scheme has been proposed that showed statistically significant high correlation (r < 0.5) for some parts of the scoring protocol with clinical severity scoring indices.³⁹ None of novel histological scoring scheme factors showed strong correlations (r > 0.7) with clinical scoring severity indices.³⁹ Future studies evaluating the best methods to assess histopathologic lesions and correlate these to clinical disease are warranted.

This article has been peer reviewed.

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³⁵Jergens AE, Schreiner CA, Frank DE, et al. “A scoring index for disease activity in canine inflammatory bowel disease”. J Vet Intern Med. 2003;17(3):291-297.

³⁶Jergens AE. “Clinical assessment of disease activity for canine inflammatory bowel disease”. J Am Anim Hosp Assoc. 2004;40(6):437-445. doi: 40/6/437 [pii].

³⁷Allenspach K, Wieland B, Gröne A, Gaschen F. “Chronic enteropathies in dogs: Evaluation of risk factors for negative outcome”. J Vet Intern Med. 2007;21(4):700-708. doi: 10.1892/0891-6640(2007)21[700:ceideo]2.0.co;2 [doi].

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