Evaluation of serum biochemical marker concentrations and survival time in dogs with protein-losing enteropathy

Mirjam Equilino Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, 3001 Switzerland.

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Vincent Théodoloz Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, 3001 Switzerland.

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Daniela Gorgas Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, 3001 Switzerland.

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Marcus G. Doherr Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Bern, 3001 Switzerland.

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Romy M. Heilmann Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Jan S. Suchodolski Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Jörg M. Steiner Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Iwan A. Burgener DVM Division of Small Animal Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, 04103 Germany.

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Abstract

Objective—To evaluate serum concentrations of biochemical markers and survival time in dogs with protein-losing enteropathy (PLE).

Design—Prospective study.

Animals—29 dogs with PLE and 18 dogs with food-responsive diarrhea (FRD).

Procedures—Data regarding serum concentrations of various biochemical markers at the initial evaluation were available for 18 of the 29 dogs with PLE and compared with findings for dogs with FRD. Correlations between biochemical marker concentrations and survival time (interval between time of initial evaluation and death or euthanasia) for dogs with PLE were evaluated.

Results—Serum C-reactive protein concentration was high in 13 of 18 dogs with PLE and in 2 of 18 dogs with FRD. Serum concentration of canine pancreatic lipase immunoreactivity was high in 3 dogs with PLE but within the reference interval in all dogs with FRD. Serum α1-proteinase inhibitor concentration was less than the lower reference limit in 9 dogs with PLE and 1 dog with FRD. Compared with findings in dogs with FRD, values of those 3 variables in dogs with PLE were significantly different. Serum calprotectin (measured by radioimmunoassay and ELISA) and S100A12 concentrations were high but did not differ significantly between groups. Seventeen of the 29 dogs with PLE were euthanized owing to this disease; median survival time was 67 days (range, 2 to 2,551 days).

Conclusions and Clinical Relevance—Serum C-reactive protein, canine pancreatic lipase immunoreactivity, and α1-proteinase inhibitor concentrations differed significantly between dogs with PLE and FRD. Most initial biomarker concentrations were not predictive of survival time in dogs with PLE.

Abstract

Objective—To evaluate serum concentrations of biochemical markers and survival time in dogs with protein-losing enteropathy (PLE).

Design—Prospective study.

Animals—29 dogs with PLE and 18 dogs with food-responsive diarrhea (FRD).

Procedures—Data regarding serum concentrations of various biochemical markers at the initial evaluation were available for 18 of the 29 dogs with PLE and compared with findings for dogs with FRD. Correlations between biochemical marker concentrations and survival time (interval between time of initial evaluation and death or euthanasia) for dogs with PLE were evaluated.

Results—Serum C-reactive protein concentration was high in 13 of 18 dogs with PLE and in 2 of 18 dogs with FRD. Serum concentration of canine pancreatic lipase immunoreactivity was high in 3 dogs with PLE but within the reference interval in all dogs with FRD. Serum α1-proteinase inhibitor concentration was less than the lower reference limit in 9 dogs with PLE and 1 dog with FRD. Compared with findings in dogs with FRD, values of those 3 variables in dogs with PLE were significantly different. Serum calprotectin (measured by radioimmunoassay and ELISA) and S100A12 concentrations were high but did not differ significantly between groups. Seventeen of the 29 dogs with PLE were euthanized owing to this disease; median survival time was 67 days (range, 2 to 2,551 days).

Conclusions and Clinical Relevance—Serum C-reactive protein, canine pancreatic lipase immunoreactivity, and α1-proteinase inhibitor concentrations differed significantly between dogs with PLE and FRD. Most initial biomarker concentrations were not predictive of survival time in dogs with PLE.

Protein-losing enteropathy is a syndrome in which there is excessive loss of proteins into the lumen of the gastrointestinal tract.1 In dogs, PLE has been described extensively, and certain breeds (Norwegian Lundehund, Basenji, and Soft Coated Wheaten Terrier) appear to be at increased risk for PLE.2–4 Commonly reported causes of PLE include alimentary lymphoma, chronic intussusception, hookworm infestation, IBD, or lymphangiectasia.5 Food hypersensitivity reactions are also a documented cause of enteropathy and can be occult or associated with clinical signs of varying severity, including PLE.6–10 However, any disease causing gastrointestinal tract inflammation, infiltration, congestion, or bleeding can result in PLE.11 Identifying gastrointestinal tract disease in a dog with hypoproteinemia and ruling out cutaneous protein loss, renal protein loss, and hepatic insufficiency lead to a diagnosis of PLE.1 Enteric loss of protein can occur through several mechanisms, including increased mucosal permeability to proteins, lymphatic obstruction, or mucosal erosion and has a greater effect on serum concentrations of proteins that typically have a low catabolic rate.12,13

Laboratory markers of gastrointestinal tract disease represent objective measures for the assessment of gastrointestinal tract disorders. Tests for such markers are non-invasive or minimally invasive and usually simple to perform. Serum concentration of CRP, an acute-phase reactant, can be useful as a prognostic marker in dogs with IBD.14,15 However, in some studies16,17 in dogs, CRP concentration did not reflect the severity of IBD as assessed by use of the CIBDAI or CCECAI or on the basis of an assigned histopathologic grade (indicative of the extent of changes). Results of another study18 suggested that measurement of CRP concentration may not be necessarily a good population-based test for use in dogs. The heterodimeric protein complex calprotectin binds Ca2+ and Zn2+ and is expressed and released from infiltrating myelomonocytic cells at sites of inflammation; quantification of calprotectin concentration in serum and fecal samples has the potential to differentiate between healthy dogs from those with inflammatory conditions, such as IBD.19–21,a Serum cPLI concentration is a highly sensitive and specific indicator of pancreatitis, and high cPLI concentration in dogs with IBD has been shown to be associated with a poor prognosis.22,23 α1-Proteinase inhibitor is present in plasma, interstitial fluid, and lymph, but is not usually found in high concentrations in the lumen of the gastrointestinal tract.5 Furthermore, α1-PI is resistant to proteolytic degradation and its molecular weight is similar to that of albumin. Therefore, α1-PI can be used as a marker for protein loss in the intestines.5 Two studies24,25 have assessed serum α1-PI concentrations in dogs, and a new assay for the measurement of this analyte has recently been validated for use in this species.26 Results of a study27 in Yorkshire Terriers revealed an association between serum α1-PI concentration less than the lower limit of the reference interval and cobalamin deficiency; this is suggestive of severe long-standing distal small intestinal disease, such as PLE, which has been reported frequently for this breed.28 Finally, S100A12 is a calcium-binding protein of the S100 superfamily that is mainly localized in granulocytes and mononuclear inflammatory cells. It is involved in the regulation of cell cycle progression and differentiation and appears to be a sensitive and specific biochemical marker of chronic gastrointestinal inflammation in humans. A test for S100A12 protein detection in serum and fecal samples from dogs has recently been developed and validated,29 and serum S100A12 concentration has been reported to be increased in dogs with IBD, compared with healthy dogs.a

Only a few reports of studies16,30,31 of dogs with PLE include routine clinicopathologic findings. Furthermore, most of the aforementioned biochemical markers have only recently been evaluated in veterinary medicine, and few studies5,12,23,32 describe their use for identifying the severity of inflammation in dogs with chronic enteropathies other than PLE. Therefore, the aim of the study reported here was to assess serum concentrations of these biochemical markers, together with routine clinicopathologic findings, and survival time in dogs with PLE. In addition, abdominal ultrasonographic, endoscopic, and histopathologic characteristics of these dogs were investigated. For comparison purposes, serum biochemical marker concentrations were assessed in dogs with FRD.

Materials and Methods

Dogs—All dogs included in the study were evaluated at the University of Bernb between 2005 and 2009 (4 dogs in 2005, 6 dogs in 2006, 4 dogs in 2007, 10 dogs in 2008, and 5 dogs in 2009). Some dogs were described in a previous report.33 Selection criteria for dogs with PLE included a history of chronic diarrhea (> 3 weeks) with or without vomiting, panhypoproteinemia, and histopathologic evidence of intestinal inflammatory cellular infiltrates.16,33 Other potential causes for chronic diarrhea or protein loss (ie, gastrointestinal parasites, liver disease, and neoplasia) were ruled out. Selection criteria for dogs with FRD included a history of chronic diarrhea (> 3 weeks) with or without vomiting, serum albumin and total protein concentrations within reference intervals, histopathologic evidence of intestinal inflammatory cellular infiltrates, and complete resolution of clinical signs within 14 days after starting an elimination diet.33

Twenty-nine dogs with PLE were included in the study; data regarding the biochemical markers were available for only 18 of these dogs. Eighteen dogs with FRD were used as a comparator group because they had clinical and histopathologic changes similar to those of the dogs with PLE but had less severe disease and serum protein concentrations within reference intervals.

Clinicopathologic assessments, treatment information, and chronic enteropathy activity indices—At the initial evaluation, data collected for each dog included serum concentrations of TLI, folic acid, and cobalamin; results of a CBC, serum biochemical analysis, urinalysis, and fecal examination (direct smear, zinc sulfate centrifugal flotation, and culture for Clostridium spp, Campylobacter spp, and Salmonella spp); and treatment information. At initial evaluation, all dogs were given a clinical score with the CIBDAI scoring system,15 which assigns a clinical score based on 6 gastrointestinal variables that are routinely evaluated in affected dogs: attitude and activity, appetite, vomiting, stool consistency, stool frequency, and weight loss. After summation, the total composite score was determined to be clinically unimportant (score, 0 to 3), mild disease (score, 4 or 5), moderate disease (score, 6 to 8), or severe disease (score, ≥ 9). The CCECAI,16 a newer scoring system that is based on the CIBDAI and 3 additional variables that are routinely evaluated in affected dogs (serum albumin concentration, ascites, and peripheral edema and pruritus) was used to assign a clinical score to almost all dogs at the initial evaluation. Some dogs in the study were evaluated before the CCECAI scoring system had been published; therefore, the CCECAI was calculated retrospectively for these dogs.

Abdominal ultrasonography—In all but 1 dog with PLE, abdominal ultrasonography was performed by a board-certified radiologist as part of each dog's diagnostic workup. Abnormalities of the intestines and concurrent abdominal changes were noted. All images were reviewed by 1 radiologist (DG). One dog underwent abdominal ultrasonography performed by the referring veterinarian 2 days prior to the initial evaluation. Retrospectively, a single board-certified radiologist (DG, who was not blinded to the dogs’ identification) reviewed all ultrasonographic images of the dogs with PLE to specifically describe findings in the liver, gallbladder, kidneys, pancreas, gastrointestinal tract, mesentery, abdominal lymph nodes, and abdominal cavity.

Endoscopy and histologic evaluation—Gastroduodenoscopy was performed in all dogs with PLE and FRD. Because of their poor general condition and anesthetic risk associated with severe hypoalbuminemia, colonoscopy was performed in only 4 of 29 dogs with PLE; however, colonoscopy was performed in all 18 dogs with FRD. In dogs with PLE and FRD, mucosal biopsy specimens from the stomach, duodenum, and colon were collected and examined histologically by a board-certified pathologist. For dogs with PLE, findings were graded retrospectively by one of the study investigators (ME) who was blinded to each dog's diagnosis. A numerical grading system was used to describe severity of any histopathologic changes in the stomach, duodenum, and colon as follows: 0 = normal, 1 = mild, 2 = moderate, and 3 = severe.34 One grade was assigned to findings for tissues at each of those anatomic locations. For tissues assigned a given grade (other than 0), the number of dogs with specific histopathologic changes (ie, fibrosis, lymphocytic-plasmacytic infiltration, eosinophilic infiltration, lymphangiectasia, and neutrophilic infiltration) was noted.

Assessment of serum concentrations of biochemical markers—Stored serum samples from 18 of 29 dogs with PLE and from all 18 dogs with FRD were available. These samples had been frozen at −20°C immediately after collection at the initial evaluation and were express-shipped on dry ice for analysis of canine pancreas–specific lipase activity serum by means of a canine pancreas–specific lipase testc (to determine cPLI concentration), measurement of serum concentrations of CRP,d,e measurement of serum concentration of calprotectin by means of an in-house radioimmunoassay21 and in-house ELISA,f and measurement of serum concentrations of α1-PI and S100A12 by means of in-house radioimmunoassays.26,29,g

Statistical analysis—For statistical analysis, data regarding signalment and CBC, routine serum biochemical analysis, and histopathologic findings were retrieved from the medical records of all dogs with PLE and FRD. Normal distribution of all measured variables was tested by the Kolmogorov-Smirnov and the Shapiro-Wilk normality tests with a commercially available statistical data analysis program.h Kaplan-Meier survival curves and hazard ratio log-rank tests as well as univariable Cox proportional hazards regression models, with status (died vs survived or censored) and time to event as the dependent variables, were performed to independently examine the influence of signalment, serum biochemical data, histopathologic findings, chronic enteropathy activity indices, and biochemical markers on the hazard (of death) rate.

The correlation of CIBDAI and CCECAI scores was determined on the basis of a Spearman correlation test in dogs with PLE. Outcome (died or euthanized vs survived or censored) at the end of the study (as of February 14, 2013), including date and cause of death, was assessed in all dogs with PLE by telephone contact with the owners. A Mann-Whitney U test was used to compare serum biochemical marker concentrations between dogs with PLE and dogs with FRD. The statistical analyses were performed with a commercially available statistical data analysis program.h Values of P ≤ 0.05 were considered significant.

Results

Dogs—The group of 29 dogs with PLE included 12 males (3 neutered) and 17 females (14 spayed). The dogs’ ages ranged from 2.0 to 14.3 years (median, 7.8 years), and weights ranged from 1.9 to 40.8 kg (4.2 to 89.8 lb; median, 8.1 kg [17.8 lb]). Breeds included 7 Yorkshire Terriers, 5 mixed-breed dogs, 3 Pugs, 2 Beauçerons, and 1 each of Bernese Mountain Dog, Papillon, Rottweiler, West Highland White Terrier, German Shepherd Dog, Dachshund, Pomeranian, Coton de Tulear, French Hunting Dog, Border Collie, Miniature Pinscher, and French Mastiff.

The group of 18 dogs with FRD included 8 males (3 neutered) and 10 females (5 spayed). The dogs’ ages ranged from 0.8 to 11.2 years (median, 2.5 years), and weights ranged from 1.8 to 49.1 kg (4.0 to 108.0 lb; median, 23.0 kg [50.6 lb]). The dogs with FRD were significantly (P < 0.001) younger than the dogs with PLE. Breeds included 4 mixed-breed dogs, 2 Labrador Retrievers, 2 French Bulldogs, and 1 each of Dachshund, Yorkshire Terrier, Weimaraner, Cairn Terrier, Golden Retriever, West Highland White Terrier, Newfoundland, Pomeranian, German Shepherd Dog, and Swiss White Shepherd Dog.

Clinicopathologic findings for dogs with PLE—Among the 29 dogs with PLE, Hct was low in 9 and high in 2 (median, 41.3%; range, 30.0% to 61.9% [reference interval, 37.0% to 55.0%]). Leukopenia was detected in 1 dog, and leukocytosis was detected in 20 dogs (median, 15.4 × 109 cells/mL; range, 2.8 × 109 cells/mL to 32.8 × 109 cells/mL [reference interval, 6.0 × 109 cells/mL to 12.0 × 109 cells/mL]). Fourteen dogs with leukocytosis had a high band count (median, 0.9 × 109 cells/mL; range, 0.4 × 109 bands/mL to 4.0 × 109 bands/mL [reference interval, 0.0 × 109 bands/mL to 0.3 × 109 bands/mL]).

Serum biochemical analyses revealed that serum total protein, albumin, and total calcium concentrations were decreased in all 29 dogs (Table 1). Serum TLI concentration was measured in 19 of the 29 dogs with PLE; values were high in 2 dogs and equivocal for exocrine pancreatic insufficiency in 1 dog. In that dog, serum TLI concentration was remeasured after a period of 10 weeks and was within the reference interval at that time. Serum folate concentration was measured in 19 of the 29 dogs; values were low in 2 dogs and high in 6 dogs. Serum cobalamin concentration was measured in 20 of the 29 dogs; values were low in 15 dogs.

Table 1—

Serum variables measured at the time of initial evaluation in 29 dogs with PLE.

VariableTotal protein (g/L)Albumin (g/L)Total calcium (mmol/L)cTLI (μg/L)Folic acid (μg/L)Cobalamin (ng/L)
No. of dogs evaluated292929191920
Reference interval57–7530–402.5–2.935.0–35.04.0–12.5250–900
Median (range)34 (18–55)14 (5–28)2 (1.21–2.32)16.4 (4.8–118)10.6 (2.2–19.2)169 (< 100–490)
Classification of individual dog values
 Within reference interval00016115
 High000260
 Low2929291215
Extent of decrease in concentration with respect to reference interval (No. of dogs)
 Mild11412NANANA
 Moderate9912NANANA
 Severe885NANANA
 Very severe180NANANA

Total protein concentration decrease: mild (40 to 56 g/L), moderate (30 to 39 g/L), severe (24 to 29 g/L), and very severe (< 24 g/L). Albumin concentration decrease: mild (20 to 29 g/L), moderate (15 to 19 g/L), severe (12 to 15 g/L), and very severe (< 12 g/L). Total calcium concentration decrease: mild (2.0 to 2.5 mmol/L), moderate (1.5 to 1.9 mmol/L), severe (1.0 to 1.4 mmol/L), and very severe (< 1.0 mmol/L).

NA = Not applicable.

Urinalysis was performed for 25 of the 29 dogs with PLE. Proteinuria was detected in 7 dogs (trace in 3 dogs, mild [1+] in 2 dogs, moderate [2+] in 1 dog, and severe [3+] in 1 dog). One of the dogs with mild proteinuria had signs of a urinary tract infection (ie, bacteria detected during microscopic examination of urine sediment preparation), the dog with moderate proteinuria had a normal urine protein-to-creatinine concentration ratio for a nonazotemic dog (< 0.5), and the dog with severe proteinuria also had signs of a urinary tract infection (ie, pyuria).

During microscopic examination of fecal samples, Giardia trophozoites were identified in 1 dog. Microbial culture of feces yielded Clostridium perfringens for 3 dogs. All 29 dogs were treated with the broad-spectrum anthelmintic agent fenbendazolei (50 mg/kg [22.7 mg/lb], PO, q 24 h for 3 to 5 days). In addition, the 3 dogs with positive culture results were treated with metronidazolej (15 mg/kg [6.8 mg/lb], PO, q 12 h for 7 to 10 days).

Both chronic enteropathy activity index scores were available for all 29 dogs with PLE. The CIBDAI score was consistent with mild disease (score, 4 or 5) in 3 dogs, moderate disease (score, 6 to 8) in 6 dogs, and severe disease (score, ≥ 9) in 20 dogs. The CCECAI was consistent with moderate disease (score, 6 to 8) in 2 dogs, severe disease (score, 9 to 11) in 8 dogs, and very severe disease (score, > 11) in 19 dogs. There was a moderate correlation (r = 0.659; P < 0.01) between CIBDAI and CCECAI scores.

Clinicopathologic findings for dogs with FRD—Among the 18 dogs with FRD, Hct was high in 1 (median, 46.8%; range, 40.2% to 57.7%). Leukopenia was detected in 1 dog, and leukocytosis was detected in 9 (median, 12.0 × 109 cells/mL; range, 4.6 × 109 cells/mL to 25.1 × 109 cells/mL). Two dogs with leukocytosis had a high band count (0.4 × 109 bands/mL and 0.7 × 109 bands/mL).

Serum biochemical analyses revealed that serum total protein concentration was high in one dog and low in another dog (median, 62.8 g/L; range, 44.8 to 85.5 g/L). Serum albumin concentration was mildly low in 3 of the 18 dogs (median, 33.9 g/L; range, 27.4 to 39.5 g/L), and serum total calcium concentration was low in 5 of the 18 dogs (median, 2.57 mmol/L; range, 1.18 to 2.81 mmol/L). Serum TLI concentration was measured in all 18 dogs with FRD; values were high in 1 dog (median, 18.0 μg/L; range, 8.8 to 48.4 μg/L). Serum folic acid concentration was measured in all 18 dogs; values were low in 1 dog and high 4 dogs (median, 10.2 μg/L; range, 2.1 to 19.7 μg/L). Serum cobalamin concentration was measured in all 18 dogs; values were low in 4 dogs (median, 420 ng/L; range, < 150 to 880 ng/L).

Urinalysis was performed for 17 of the 18 dogs with FRD. Proteinuria was detected in 5 dogs (trace in 1 dog and mild [1+] in 4 dogs). None of the dogs had signs of cystitis, and urine specific gravity of the 4 dogs with mild proteinuria was > 1.034.

During microscopic examination of fecal samples, no endoparasites or enteropathogens were identified in 8 dogs; endoparasites were detected in 3 dogs. Microbial culture of feces yielded Clostridium perfringens for 9 dogs, 2 of which were also positive for endoparasites. All 18 dogs were treated with the broad-spectrum anthelmintic agent fenbendazolei (50 mg/kg, PO, q 24 h for 3 to 5 days). In addition, the 9 dogs with positive culture results were treated with metronidazolej (15 mg/kg, PO, q 12 h for 7 to 10 days).

Both chronic enteropathy activity index scores were available for all 18 dogs with FRD. The CIBDAI score was consistent with clinically irrelevant disease (score, 0 to 3) in 3 dogs, mild disease (score, 4 to 5) in 7 dogs, moderate disease (score, 6 to 8) in 5 dogs, and severe disease (score, ≥ 9) in 3 dogs. The CCECAI score was consistent with clinically unimportant disease (score, 0 to 3) in 3 dogs, mild disease (score, 4 to 5) in 7 dogs, moderate disease (score, 6 to 8) in 5 dogs, and severe disease (score, 9 to 11) in 3 dogs. None of the dogs had scores indicative of very severe disease (ie, CCECAI score ≥ 12).

Abdominal ultrasonographic findings in dogs with PLE—Twenty-eight of the 29 dogs with PLE underwent abdominal ultrasonography at the initial examination; for the remaining dog, an ultrasonographic examination was performed by the referring veterinarian. Changes in the intestinal tract included stomach wall thickening in 5 dogs (focal thickening in 2 dogs and diffuse thickening in 3 dogs) and abnormalities of the intestinal wall (stippling, striations, or loss of wall layering) in 26 dogs (segmental abnormalities in 14 dogs and generalized abnormalities in 12 dogs). Duodenal abnormalities were detected in 24 dogs (mild diffuse stippling in 6 dogs, diffuse stippling in 8 dogs, and striations in 10 dogs). Jejunal abnormalities were detected in 25 dogs (mild diffuse stippling in 4 dogs, diffuse stippling in 8 dogs, striations in 11 dogs, and loss of wall layering in 2 dogs). Changes in the colon were evident in 1 dog (mild diffuse stippling). Abdominal effusion was present in 22 dogs (mild effusion in 16 dogs and moderate effusion in 6 dogs), and the mesentery was assessed as hyperechoic in 21 dogs. Abdominal lymph nodes were enlarged or had altered echogenicity in 5 dogs. Jejunal lymph nodes were enlarged or had altered echogenicity in 5 dogs. Other abnormalities included alterations in the hepatic echotexture in 6 dogs (diffuse changes in 5 dogs and focal changes in 1 dog), gallbladder wall edema in 2 dogs, reduced corticomedullary definition within the kidneys in 6 dogs, and pancreatic changes in 7 dogs (edema in 2 dogs, mild enlargement in 2 dogs, and patchy appearance with slightly irregular surface, reduced delineation with thickened appearance, and multiple hyperechoic areas in 1 dog each).

Endoscopic and histopathologic findings in dogs with PLE—Gastroduodenoscopy and histologic examination of biopsy specimens was performed in all 29 dogs with PLE, and additional colonoscopy was performed in 4 dogs (Table 2). Evidence of gastric fibrosis was present in 17 dogs, and lymphocytic-plasmacytic gastritis was found in 16 dogs. Histopathologic abnormalities in the duodenum were identified in all 29 dogs: lymphocytic-plasmacytic inflammation (n = 29 dogs), lymphangiectasia (21), eosinophilic inflammation (9), and fibrosis (9). Colonic abnormalities were found in 3 of 4 dogs (lymphocytic-plasmacytic inflammation in 2 dogs and mild neutrophilic inflammation in 1 dog). There was no correlation found between histologic severity of intestinal inflammation and abdominal ultrasonographic findings.

Table 2—

Summary of histopathologic findings detected in biopsy specimens obtained from 29 dogs with PLE.

GradeHistopathologic changeStomach (n = 29)Duodenum (n = 29)Colon (n = 4)
0No abnormalities501
1Fibrosis550
 Lymphocytic-plasmacytic infiltration1071
 Eosinophilic infiltration080
 Lymphangiectasia070
 Neutrophilic infiltration001
2Fibrosis1140
 Lymphocytic-plasmacytic infiltration5171
 Eosinophilic infiltration000
 Lymphangiectasia0110
 Neutrophilic infiltration000
3Fibrosis100
 Lymphocytic-plasmacytic infiltration150
 Eosinophilic infiltration010
4Lymphangiectasia030
 Neutrophilic infiltration000

Several dogs had multiple changes within 1 type of tissue and across the different types of tissue. Score: 0 = normal, 1 = mild, 2 = moderate, and 3 = severe.

Serum biochemical marker concentrations—Concentrations of biochemical markers of inflammation or gastrointestinal tract disease were analyzed in stored serum samples from 18 of 29 dogs with PLE and from 18 dogs with FRD (Table 3). Serum CRP concentration was significantly (P < 0.001) higher in dogs with PLE (median, 13.0 mg/L; range, 0.1 to 101.3 mg/L [reference interval, 0.0 to 7.6 mg/L]) than in dogs with FRD (median, 1.4 mg/L; range, 0.1 to 23.0 mg/L). Serum cPLI concentration (as measured by a canine pancreas–specific lipase testc) were significantly (P = 0.028) higher in dogs with PLE (median, 35 μg/L; range, 29 to 284 μg/L; reference interval, 0 to 200 μg/L) than in dogs with FRD (median, 29 μg/L; range, 29 to 189 μg/L), although none of the dogs in either group had a cPLI concentration greater than the suggested cutoff for the diagnosis of pancreatitis (ie, 400 μg/L). Serum α1-PI concentrations were significantly (P < 0.001) lower in dogs with PLE (median, 734 mg/L; range, 441 to 1,268 mg/L; reference interval, 732 to 1,802 mg/L) than in dogs with FRD (median, 1,340 mg/L; range, 687 to 1,875 mg/L). Serum calprotectin concentrations measured by radioimmunoassay and ELISA as well as S100A12 concentrations were not significantly different between dogs with PLE and dogs with FRD.

Table 3—

Serum biochemical variables in 18 dogs with PLE and 18 dogs with FRD.

   Data classification (No. of dogs) 
VariableReference intervalMedian (range)Within reference intervalHigh (with respect to reference interval)Low (with respect to reference interval)P value*
CRP (mg/L)0.0–7.6     
 Dogs with PLE 13.0 (0.1–101.3)5130< 0.001
 Dogs with FRD 1.4 (0.1–23.0)1620 
Calprotectin (determined by radioimmunoassay [μg/L])77.8–444.5     
 Dogs with PLE 372.2 (117.6–1,086.0)10800.108
 Dogs with FRD 318.0 (214.7–549.3)1620 
Calprotectin (determined by ELISA [mg/L])0.9–11.9     
 Dogs with PLE 24.6 (6.4–88.1)41400.935
 Dogs with FRD 25.2 (7.2–81.3)4140 
cPLI (μg/L)0–200     
 Dogs with PLE 35 (29–284)1530< 0.028
 Dogs with FRD 29 (29–189)1800 
α1-PI (mg/L)732–1,802     
 Dogs with PLE 734 (441–1,268)909< 0.001
 Dogs with FRD 1,340 (687–1,875)1611 
S100A12 (μg/L)33.2–225.1     
 Dogs with PLE 353.7 (59.5–1,091.0)31500.651
 Dogs with FRD 364.4 (99.5–1,211.8)5130 

Value relates to difference between groups.

Treatment—Treatment of the 29 dogs with PLE before enrollment into the study differed depending on the referring veterinarians’ preferences. Dogs were treated with various antimicrobials (amoxicillin–clavulanic acid, metronidazole, cephalosporins, or enrofloxacin), antiemetics (metoclopramide, ondansetron, or dolasetron), and gastric acid reducers (ranitidine, omeprazole, or sucralfate), and some received vitamin B12 injections. None of the 29 dogs with PLE had been treated with corticosteroids or other immunosuppressive drugs at the time of inclusion in the study. Eight dogs with severe hypoalbuminemia received fresh frozen plasma (10 to 20 mL/kg [4.5 to 9.0 mL/lb], IV) before endoscopy to stabilize their condition for anesthesia. After endoscopy, all dogs with PLE or FRD received an elimination diet or a hydrolyzed protein diet. All dogs with FRD responded to diet change alone, whereas all dogs with PLE were further treated with prednisolonek (1 to 2 mg/kg [0.45 to 0.9 mg/lb], PO, q 12 to 24 hours). Of the 29 dogs with PLE, 18 additionally received cyclosporinel (5 mg/kg [2.3 mg/lb], PO, q 12 to 24 hours) and 3 received budesonidem (3 mg/dog, PO, q 24 h) because of profound prednisolone-related adverse effects. All 7 Yorkshire Terriers included in the study received prednisolone, and 4 of them were additionally treated with cyclosporine (no budesonide administration).

Survival analysis—At the end point of the study, 6 of 29 dogs with PLE were still alive, with a survival time of 730 to 2,347 days; the other 23 dogs were deceased, with a median survival time of 67 days (range, 2 to 2,551 days; Figure 1). Owners of 17 dogs elected euthanasia because of lack of clinical improvement; survival time among these dogs was 2 to 874 days. Six dogs were euthanized or died as a result of other diseases (age-related problems [n = 3 dogs], hind limb paralysis [1], life-threatening biting incident [1], and sudden death during exercise [1]); survival time among these dogs was 29 to 2,551 days. At the end point of the study, 2 of 7 Yorkshire Terriers were still alive, with survival times of 844 and 2,847 days. Of the 5 deceased Yorkshire Terriers, 3 were euthanized because of lack of clinical improvement (survival time, 2 to 874 days). The other 2 Yorkshire Terriers were euthanized as a result of either age-related problems (survival time 2,355 days) or biting injury (survival time, 99 days). Outcome or survival time in dogs with PLE was not significantly influenced by initial serum albumin, total calcium, or cobalamin concentrations; chronic enteropathy activity index scores; severity of histopathologic changes; serum cPLI concentration; serum α1-PI concentration; or treatment. Medium size (11 to 20 kg [24.2 to 44.1 lb]; P = 0.041), mildly high serum CRP concentration (P = 0.004), serum calprotectin concentration (as measured by radioimmunoassay) within the reference interval (P = 0.031), and serum S100A12 concentration within the reference interval (P = 0.047) were negative prognostic indicators in this group of dogs with PLE.

Figure 1—
Figure 1—

Kaplan-Meier plot of cumulative survival over time for 29 dogs with PLE. Six dogs were still alive at the end point of the study.

Citation: Journal of the American Veterinary Medical Association 246, 1; 10.2460/javma.246.1.91

Discussion

In the present study, routine clinicopathologic findings, chronic enteropathy activity scoring indices (CIBDAI or CCECAI), and serum concentrations of most of the biomarkers for inflammatory and gastrointestinal tract disease evaluated were not indicators of outcome or survival time in dogs with PLE. However, dogs with PLE had significantly higher serum CRP and cPLI concentrations and significantly lower serum cobalamin and α1-PI concentrations than did dogs with FRD. The median age of dogs with PLE was 7.8 years, which was significantly higher than that of the dogs with FRD.11 Other studies11,16,33 have also found that chronic diet-responsive enteropathies more often affect younger dogs. Similar to findings of other studies,36,37 panhypoproteinemia, hypoalbuminemia, and hypocalcemia were detected in all dogs with PLE in the present study. Hypocalcemia may be explained by reduced binding of calcium because of loss of protein, especially albumin, or malabsorption of calcium from the gastrointestinal tract. However, further studies to evaluate circulating ionized calcium, vitamin D, and parathyroid hormone concentrations are needed to more closely evaluate calcium homeostasis in dogs with PLE. Low serum cobalamin concentration has been found in dogs with severe and longstanding disease of the ileum, such as idiopathic IBD, intestinal lymphoma, or antimicrobial-responsive diarrhea, and in dogs with exocrine pancreatic insufficiency,38–40 in which low serum cobalamin concentration is usually associated with high serum folate concentration. In the study of this report, 15 of 20 dogs with PLE and only in 4 of 18 dogs with FRD had hypocobalaminemia. This may reflect the fact that dogs with PLE have more generalized and more severe intestinal inflammation, compared with dogs with chronic diet-responsive enteropathy.

Among the dogs with either PLE or FRD, proteinuria was evident in 12 dogs and may be explained by hemoglobinuria, exercise, urinary tract infection, or fever.41 Hypoproteinemia caused by hypoalbuminemia develops in dogs with glomerular disease but is more likely in dogs with marked proteinuria.42 Of the 29 dogs with PLE, none had ultrasonographic evidence of uremia or changes suggestive of renal disease; however, all had histopathologic abnormalities in the small intestine. The number of dogs with PLE that had a CIBDAI score indicative of severe disease or a CCECAI score indicative of very severe disease was 20 of 29 (69%) and 19 of 29 (66%), respectively. However, chronic enteropathy and minimal protein loss attributable to protein-losing nephropathy cannot be definitively ruled out. Evaluation of the urine protein-to-creatinine concentration ratio would have been necessary to rule out additional protein-losing nephropathy but was not performed routinely in all dogs in this study. Given the severe intestinal inflammation and the high chronic enteropathy activity index scores in these dogs, PLE was most likely the reason for the protein loss, and major protein loss through the kidneys was unlikely.

On abdominal ultrasonography, changes in the intestines were common in dogs with PLE, but there was no correlation between ultrasonography changes and histopathologic grading of biopsy specimens. As concluded in reports of other studies43,44 in dogs, abdominal ultrasonography appears to be a useful tool to identify changes in intestinal segments and layers but is less useful to estimate the severity of the intestinal disease. In the present study, mild pancreatic changes were identified in only 7 of 29 dogs with PLE: mild enlargement (n = 2 dogs), edema (2), reduced delineation with slightly thickened appearance (1), patchy appearance with slightly irregular surface (1), and multiple hyperechoic areas (1). These changes could be a sign for pancreatitis in these dogs, but canine pancreas–specific lipase activity was considered normal in 5 of the 7 dogs, and the values were not available for the other 2 dogs. The serum TLI concentration was within the reference interval in 5 of the 7 dogs, and the values were not available for the same 2 dogs that did not have results of the canine pancreas–specific lipase test. These 2 dogs without the canine pancreas–specific lipase test results and serum TLI concentration data had only a slightly enlarged pancreas with no other pancreatic changes. Therefore, pancreatitis or exocrine pancreatic insufficiency as cause of diarrhea can most likely be ruled out in the dogs with PLE in the present study, even though no pancreatic biopsy specimens were examined.

For the dogs with PLE in the present study, histologic examination of biopsy specimens revealed moderate to severe lymphocytic-plasmacytic enteritis or lymphangiectasia as the most frequent finding. Therefore, IBD was considered the most common cause for PLE in these dogs.4 No histologic signs of lymphoma were found, but because the degree of histologic resemblance between lymphoma and lymphocytic-plasmacytic infiltration is high, small cell lymphoma may have been missed on examination of endoscopic biopsy specimens.

Dogs with FRD were used as a comparator group in the present study for better interpretation of serum concentrations of biochemical markers for inflammation and gastrointestinal tract disease in dogs with PLE. Dogs with FRD have clinical signs similar to those of dogs with PLE but would be expected to have comparatively less severe intestinal inflammation. The chronic enteropathy activity scoring indices (ie, CIBDAI and CCECAI) and serum cobalamin concentrations suggested that the dogs with PLE in this study indeed had more severe intestinal damage, compared with that in dogs with FRD. Serum CRP concentration was also significantly higher in dogs with PLE, supporting this hypothesis. Serum cPLI concentration was also significantly higher in dogs with PLE than in dogs with FRD, but in contrast to results of another study,23 high serum cPLI concentration was not a common finding in dogs with PLE in the present study. The only obvious difference between that study23 and the present study was in regard to the populations of dogs evaluated (ie, dogs that had chronic enteropathies with or without protein loss vs dogs that had PLE). Furthermore, the clinical impact is questionable given that all 3 dogs had a cPLI concentration in the questionable range (200 to 400 μg/L) and not above the currently recommended cutoff for a diagnosis of pancreatitis (> 400 μg/L). Concentration of α1-PI is usually measured in feces from dogs with suspected PLE for quantification of intestinal protein loss,5 but a recent study26 evaluated an assay for the measurement of α1-PI in canine sera. Serum α1-PI concentration in dogs with PLE was significantly lower than that in dogs with FRD but was low in only 9 of the 18 dogs with PLE for which data were available. Thus, further studies are needed to evaluate the diagnostic value of measuring α1-PI concentration in serum from dogs as a marker for intestinal protein loss.

Calprotectin is used as a marker to predict the risk of relapse and evaluate disease activity in humans with chronic enteropathies.45 Serum calprotectin concentration, as measured by ELISA, was high in 14 of 18 dogs with PLE and in 14 of 18 dogs with FRD, but the median concentration for dogs with PLE or FRD did not differ. Therefore, serum calprotectin concentration (measured by ELISA) seems to be a good marker for gastrointestinal tract inflammation but was not useful for differentiation of dogs with PLE from dogs with FRD in the present study. Serum S100A12 concentration was high in most of the dogs evaluated (those with PLE [15/18 dogs] or FRD [13/18 dogs]), but the median concentration was not significantly different between the 2 groups. On the basis of these findings, serum S100A12 concentration also appears to be a good marker for inflammation but was not useful for differentiation of dogs with PLE from dogs with FRD in the present study.

The treatments given to the dogs with PLE before enrollment into the study were variable. Once included in the study, all 29 dogs with PLE were treated with diet change and prednisolone; for 18 dogs, administration of cyclosporine was initiated. Three dogs received budesonide instead of prednisolone because of profound prednisolone-related adverse effects. For dogs with PLE, treatment did not significantly influence survival time.

The median survival time of dogs with PLE was shorter (67 days) than that determined in a recent retrospective study31 of 17 dogs with PLE (5 months). On the other hand, death or euthanasia because of a lack of clinical improvement was less common for dogs in the present study (17/29 [59%] dogs) than it was for dogs in the previous study31 (11/17 [65%] dogs). Owners’ decisions to euthanize their dog inherently influences the estimated survival time for a study population. Well-differentiated lymphoma may sometimes remain undetected on histologic examination of endoscopic biopsy specimens, which could be another potential explanation for the apparently shorter median survival time of dogs in the present study. To the authors’ knowledge, there are no studies of factors affecting outcome or survival time in dogs with PLE. However, negative prognostic factors (low albumin concentration [< 20 g/L], hypocobalaminemia [< 200 ng/L], high CIBDAI score, high CCECAI score, and high serum cPLI concentration) in dogs with IBD are well described.15,16,23 These factors were not correlated with outcome or survival time in dogs with PLE in the present study. Given the low number of dogs, this could at least in part be due to the small sample size and therefore low statistical power of the data analyses. Medium-sized dogs and dogs with mildly high serum CRP concentration, serum calprotectin concentration (as measured by radioimmunoassay) within reference interval, or serum S100A12 concentration within reference interval had significantly shorter survival times and had an increased risk of death or euthanasia as a result of PLE. These observations were surprising, but an explanation may be a lack of correlation between the severity of inflammation and the outcome or survival time. Other explanations may be falsely reduced serum concentrations of CRP, calprotectin, and S100A12 as a result of increased protein loss in dogs with PLE or deficient stability of biomarkers by extended periods of storage. Serum CRP concentration has been shown to be useful for monitoring treatment in individual patients but has wide population variation. This may explain why moderately high serum CRP concentration was a negative prognostic factor for dogs with PLE in the present study. Calprotectin and S100A12 have pro- and anti-inflammatory characteristics. The finding that serum calprotectin and S100A12 concentrations within reference intervals were negative prognostic indicators for dogs with PLE may be explained by their anti-inflammatory effects. This and the unknown stability of calprotectin in serum may explain the negative prognostic value of serum concentrations within the reference interval. Most dogs with PLE were euthanized on the basis of their owners’ decisions, which could skew the correlation of the serum biochemical markers with outcome or survival time.

The present study had limitations. These included a low number of dogs with PLE and the associated potentially inadequate statistical power of the data analyses. In addition, the long-term storage of the serum samples may have biased biochemical marker data.

In the dogs with PLE in the present study, panhypoproteinemia, hypocalcemia, hypocobalaminemia, and high serum CRP concentration were common findings. Assessment of serum α1-PI concentration may be an adjunct means of quantitative and qualitative testing for intestinal protein loss. Routine clinicopathologic findings, CIBDAI and CCECAI scores, and serum concentrations of most biomarkers of inflammation and gastrointestinal tract disease did not appear to be predictors of outcome or survival time. Additional prospective studies with larger numbers of dogs with PLE, accurate inclusion criteria, and strictly defined standard treatment protocols are warranted to elucidate potential negative risk factors in dogs with PLE.

ABBREVIATIONS

α1-PI

α1-Proteinase inhibitor

CCECAI

Canine chronic enteropathy clinical activity index

CIBDAI

Canine inflammatory bowel disease activity index

cPLI

Canine pancreatic lipase immunoreactivity

CRP

C-reactive protein

FRD

Food-responsive diarrhea

IBD

Inflammatory bowel disease

PLE

Protein-losing enteropathy

TLI

Trypsin-like immunoreactivity

a.

Heilmann RM, Allenspach K, Procoli F, et al. Serum calgranulin concentrations in dogs with inflammatory bowel disease (abstr). J Vet Intern Med 2011;25:1486.

b.

Small Animal Teaching Hospital, Vetsuisse Faculty, University of Bern, Switzerland.

c.

Spec cPL assay, Idexx Laboratories Inc, Westbrook, Me.

d.

Tri-Delta-Phase, Tri-Delta Diagnostic Inc, Boonton Township, NJ.

e.

Berghoff N, Suchodolski JS, Steiner JM. Assessment of stability and determination of a reference range for canine C-reactive protein in serum (abstr). J Vet Intern Med 2006;20:790.

f.

Heilmann RM, Guard BC, Weber K, et al. Development and analytical validation of an enzyme-linked immunosorbent assay for the quantification of canine calprotectin in serum and feces from dogs (abstr). J Vet Intern Med 2011;25:693.

g.

Gastrointestinal Laboratory, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Tex.

h.

Number Cruncher Statistical Systems (NCSS) 2007, Kaysville, Utah.

i.

Panacur, Veterinaria AG, Pfaeffikon, Switzerland.

j.

Flagyl, Sanofi-Aventis AG, Meyrin, Switzerland.

k.

Prednisolon Streuli, Streuli Pharma AG, Uznach, Switzerland.

l.

Atopica, Novartis AG, Basel, Switzerland.

m.

Entocort CIR, AstraZeneca AG, Zug, Switzerland.

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  • Figure 1—

    Kaplan-Meier plot of cumulative survival over time for 29 dogs with PLE. Six dogs were still alive at the end point of the study.

  • 1. Willard MD, Helman G, Fradkin JM, et al. Intestinal crypt lesions associated with protein-losing enteropathy in the dog. J Vet Intern Med 2000; 14: 298307.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Flesjå K, Yri T. Protein-losing enteropathy in the Lundehund. J Small Anim Pract 1977; 18: 1123.

  • 3. Breitschwerdt EB, Halliwell WH, Foley CW, et al. A hereditary diarrheic syndrome in the Basenji characterized by malabsorption, protein-losing enteropathy and hypergammaglobulinemia. J Am Anim Hosp Assoc 1980; 16: 551560.

    • Search Google Scholar
    • Export Citation
  • 4. Littman MP, Dambach DM, Vaden SL, et al. Familial protein-losing enteropathy and protein-losing nephropathy in Soft Coated Wheaten Terriers: 222 cases (1983–1997). J Vet Intern Med 2000; 14: 6880.

    • Search Google Scholar
    • Export Citation
  • 5. Murphy KF, German AJ, Ruaux CG, et al. Fecal α1-proteinase inhibitor concentration in dogs with chronic gastrointestinal disease. Vet Clin Pathol 2003; 32: 6772.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Bjarnason I, Peters TJ. Helping the mucosa make sense of macromolecules. Gut 1987; 28: 10571061.

  • 7. Paterson S. Food hypersensitivity in 20 dogs with skin and gastrointestinal signs. J Small Anim Pract 1995; 36: 529534.

  • 8. Sampson HA. Food allergies. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal diseases: pathophysiology, diagnosis, management. 4th ed. Philadelphia: WB Saunders Co, 1992; 12331240.

    • Search Google Scholar
    • Export Citation
  • 9. Brasitus TA. Protein-losing gastroenteropathy. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal diseases: pathophysiology, diagnosis, management. 4th ed. Philadelphia: WB Saunders Co, 1992; 10271035.

    • Search Google Scholar
    • Export Citation
  • 10. Greenberger NJ, Tennebaum JI, Ruppert RD. Protein-losing enteropathy associated with gastrointestinal allergy. Am J Med 1967; 43: 777784.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Hall EJ, German AJ. Diseases of small intestine. In: Ettinger SJ, Feldmann EC, eds Textbook of veterinary internal medicine. 7th ed. St Louis: Saunders Elsevier, 2010; 15261572.

    • Search Google Scholar
    • Export Citation
  • 12. Vaden SL, Sellon RK, Melgarejo LT, et al. Evaluation of intestinal permeability and gluten sensitivity in Soft-Coated Wheaten Terriers with familial protein-losing enteropathy, protein-losing nephropathy, or both. Am J Vet Res 2000; 61: 518524.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Jeffries GH, Sleisenger MH. Abnormal enteric loss of plasma protein in gastrointestinal diseases. Surg Clin North Am 1962; 42: 11251133.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Jergens AE. Clinical assessment of disease activity for canine inflammatory bowel disease. J Am Anim Hosp Assoc 2004; 40: 437445.

  • 15. Jergens AE, Schreiner CA, Frank DE, et al. A scoring index for disease activity in canine inflammatory bowel disease. J Vet Intern Med 2003; 17: 291297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Allenspach K, Wieland B, Gröne A, et al. Chronic enteropathies in dogs: evaluation of risk factors for negative outcome. J Vet Intern Med 2007; 21: 700708.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. McCann TM, Ridyard AE, Else RW, et al. Evaluation of disease activity markers in dogs with idiopathic inflammatory bowel disease. J Small Anim Pract 2007; 48: 620625.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Carney PC, Ruaux CG, Suchodolski JS, et al. Biological variability of C-reactive protein and specific canine pancreatic lipase immunoreactivity in apparently healthy dogs. J Vet Intern Med 2011; 25: 825830.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Johne B, Fagerhol MK, Lyberg T, et al. Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol Pathol 1997; 50: 113123.

  • 20. Stříž I, Trebichavský I. Calprotectin—a pleiotropic molecule in acute and chronic inflammation. Physiol Res 2004; 53: 245253.

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