Intestinal inflammation is a clinically relevant concern in equine practice, with post mortem studies1 reporting colonic ulcers in over 40% of all horses examined and greater than 60% of performance horses. However, accurate identification and quantification of intestinal inflammation in conditions such as inflammatory bowel disease (IBD) and subclinical right dorsal colitis (RDC) remains a diagnostic challenge. Currently, available antemortem tests have limitations related to lack of access and/or visibility (ultrasound, endoscopy, and biopsy),2–4 invasiveness (surgical explore, biopsy),4 delay in onset of detectable change (ultrasound),5 and poor sensitivity and specificity (fecal blood tests).6–8 Thus, there is a need for quantitative methods for the measurement of intestinal inflammation for clinical use in equine patients.
Fecal inflammatory biomarkers are a direct product of intestinal inflammation and enter the feces when mucosal barrier function is lost.9 Quantitative measurement of inflammatory biomarkers in feces is used for both diagnosis and monitoring of ulcerative colitis and Crohn disease in human patients.10,11 Calprotectin (CP), released by activated neutrophils and mononuclear phagocytes,12,13 is strongly correlated with active inflammation in the human gut.14,15 In horses, mucosal CP expression has been associated with neutrophil infiltration and inflammatory response in large colon tissues in ischemia-reperfusion models.16
A protocol to quantify CP in equine feces has recently been validated,17 but further investigation to establish normal ranges and assess clinical utility is needed. The objectives of this study were to establish the reference intervals (RIs) for fecal CP (fCP) and fCP:protein ratio in healthy horses and to determine whether fCP and/or fCP:protein could be useful biomarkers for the quantification of intestinal inflammation by first evaluating their correlation with overt clinical intestinal inflammation (colitis). We hypothesized that fCP would be detected in feces from healthy horses with a coefficient of variation (CV) < 10% and that both fCP and fCP:protein ratio would be greater in horses with colitis compared to healthy horses and horses with colic.
Methods
Subject identification and evaluation
Fecal samples were obtained from university-owned and client-owned horses that were either clinically healthy or were presented for evaluation of abdominal pain/suspected colitis. The study period was from September 20, 2023, through April 20, 2024. Inclusion criteria for healthy horses were normal formed manure and no active local or systemic inflammatory conditions and included horses used for research or teaching, healthy companion horses, horses presented for elective appointments, and horses recovered from clinical illness (horses treated for extraintestinal disease with no ongoing clinical or hematologic evidence of inflammation). Horses with abdominal pain were categorized as colitis (clinical signs of diarrhea and/or large colon mural thickness ≥ 5 mm on abdominal ultrasound) or colic (abdominal pain not attributed to large intestinal inflammation). The group of horses with clinical signs of colic were included to represent horses with noninflammatory intestinal disease, which may have mild intestinal inflammation but less so than colitis cases. All study procedures were approved by the IACUC (protocol #20115).
Complete physical examinations were performed prior to sample collection (university-owned horses, healthy companions, elective appointments, horses recovered from illness) or at the time of hospital admission (clinical colic/colitis cases) by study personnel or the attending clinician. Additional demographic and clinical data (age, sex, breed, weight, diagnosis, anti-inflammatory drug administration, duration of hospitalization, and survival to hospital discharge) were collected from the electronic medical record retrospectively. If applicable, the type(s) of anti-inflammatory medication and duration of treatment prior to sample collection were recorded.
Sample collection and analysis
Sample processing and analysis followed procedures described in ELISA validation.17 A 3.5-mL fecal sample was collected from a freshly voided pile and refrigerated (< 4 hours) until processing. Feces were suspended in 5 mL of a fecal extraction buffer, consisting of 8.0 pH TrisEDTA (Sigma-Aldrich) with 10 mmol/L calcium chloride (Sigma-Aldrich) and 0.25 mmol/L thimerosal (Sigma-Aldrich).13 The sample was homogenized by vortexing, then centrifuged at 1, 000 X g, 4 °C for 20 minutes. The resulting supernatant was aliquoted for long-term storage at −80 °C. The maximum duration of sample storage was 6 months prior to analysis. In human feces, CP is stable in frozen samples for at least 1 year.18
Samples were thawed at room temperature prior to processing. Fecal supernatant was filtered through a 0.22 μm low protein-binding syringe filter. For CP ELISA, supernatant was diluted with fecal extraction buffer in a 1:3 ratio before analysis to bring expected concentrations within the linear detection range of the assay. Samples were analyzed in duplicate using commercially available ELISA kits for equine CP (MyBiosource Inc) according to manufacturer protocols. The linear range for the assays was 6.25 to 200 ng/mL; accounting for sample dilution, this equates to a range of detection from 25 to 800 ng/mL or 0.025 to 0.8 μg/mL. Absorbance was detected at 450 nm (SpectraMax iD3; Molecular Devices).
To account for differences in fecal water and fibrous content between samples, fecal CP was normalized to the total protein of each sample. Fecal total protein was measured by bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Scientific Inc) according to manufacturer instructions for microplate procedure. A standard curve was prepared using 2 mg/mL bovine serum albumin (Pierce Bovine Serum Albumin Standard Ampules; Thermo Scientific Inc). Samples were diluted with deionized water in a 1:9 ratio to bring samples within the linear range. The microplate was incubated for 30 minutes at 37 °C prior to detection of absorbance at 585 nm (SpectraMax iD3; Molecular Devices).
Statistical analysis
Optical density data were preprocessed in Excel, version 2306 (Microsoft Corp), and MyCurveFit.com19 was used to generate the model that best fit the standard curve for each assay: symmetric sigmoidal (4 pL) for CP ELISA and linear for bicinchoninic acid assay. The measured concentration of analyte in each sample was calculated from the optical density and standard curve. Actual concentration was calculated by multiplying the measured concentration by the dilution factor. Actual concentration was used for the remainder of analysis.
Further analysis was performed in R, version 4.3.1,20 using RStudio, version 2022.12.0 (PBC). Figures were created using the ‘ggpubr’ package, version 0.6.0.21; packages ‘kableExtra,’ version 1.3.4.9000,22 and ‘magicfor,’ version 0.1.0,23 were utilized for data organization and processing during analysis. P < .05 was considered statistically significant for all analyses. Normality was assessed by the Shapiro-Wilk test and visual evaluation of data (histogram, quantile-quantile plot); data were not normally distributed. Summary data are presented as median (quartile 1, quartile 3).
The coefficient of variation was calculated between paired (technical replicate) fCP measurements as SD/mean X 100. A threshold of median CV ≤ 10% was considered acceptable.24 The mean of paired CP measurements was used for further analysis, henceforth referred to as fCP. The fCP:protein ratio was calculated by dividing fCP by total protein as measured by bicinchoninic acid assay for each sample. Reference intervals including 95% of healthy horses for both fCP and fCP:protein were calculated as recommended by the American Society for Veterinary Clinical Pathology Quality Assurance and Laboratory Standards Committee.25 Outliers were determined using the Horn method and removed prior to reference range calculation.26 Reference ranges were calculated nonparametrically with 90% CIs by bootstrapping using the R package “referenceIntervals,” version 1.3.0.27
Continuous data were compared between groups (healthy, colitis, colic) by the Kruskal-Wallis test with a post hoc Wilcoxon rank-sum test with Holm-Bonferroni correction. Effect size (η2) was calculated using the R package ‘rstatix,’ version 0.7.2, and η2 > 0.06 was considered moderate.28 Categorical data were compared between groups by the Chi-square test, with effect size calculated by the Cramer V using ‘rstatix’ package, version 0.7.2. The Pearson correlation was used to assess for a linear correlation between continuous clinical variables and fCP or fCP:protein. Relationships between categorical variables and either fCP or fCP:protein were determined by the Kruskal-Wallis test, and effect size (η2) was calculated as described above.
Results
Study population
Samples were obtained from 103 healthy horses, 13 horses with colitis, and 15 horses with colic. The majority of healthy horses were presented for orthopedic evaluation/treatment (39/103 [38%]), followed by soft tissue surgery (30/103 [29%]), no clinical concern (20/103 [19%]), reproductive management (8/103 [8%]), and internal medicine evaluation (6/103 [6%]). Specific reasons for orthopedic presentation were arthroscopic removal of osteochondral fragments (21/39 [54%]), lameness evaluation (14/39 [36%]), radiographs only (2/39 [5%]), and fracture (2/39 [5%]). Specific reasons for soft tissue surgery appointments were wounds/lacerations (11/30 [37%]), mass evaluation/removal (6/30 [20%]), upper airway evaluation (5/30 [17%]), cryptorchid castration (4/30 [13%]), enucleation (3/30 [10%]), and umbilical herniorrhaphy (1/30 [3%]). Horses with no clinical concern were either university-owned for research (8/20 [40%]) or teaching (6/20 [30%]) or client-owned horses presented as healthy companions (6/20 [30%]). Horses presented to the theriogenology service for breeding management (4/8 [50%]), metritis (3/8 [38%]), or endometritis (1/8). Horses were presented to the internal medicine service for a recheck gastroscopic examination (3/6 [50%]) and 1 each for evaluation of head shaking, neurological abnormalities, and historical fever of unknown origin.
The majority of horses in the colitis group had a recorded diagnosis of colitis (5/13 [38%]) or enterocolitis (3/13 [23%]) of undetermined cause. There were 2 cases of confirmed Potomac horse fever (2/13 [15%]) and 1 each of antimicrobial-associated colitis, postoperative enterocolitis, and NSAID-associated RDC. Horses in the colic group had impactions of the large and/or small colon (8/15 [53%]), displacement of the large colon (3/15 [20%]), enteritis (2/15 [13%]), and 1 each of strangulating small intestinal obstruction and severe glandular and squamous gastric ulcers.
There was a statistically significant difference in age between groups (P = .044); however, the effect size was small (η2 = 0.033). Horses in the healthy group were younger than colic or colitis groups, but there were no statistically significant differences in pairwise comparisons. The length of hospitalization also differed (P = .019) between groups but with a small effect size (η2 = 0.046), with horses in the colitis group having significantly longer hospitalization than healthy horses (P = .025). There was no significant difference in the duration of prior anti-inflammatory treatment (Table 1). There was no significant difference in the distribution of sex (P = .557; η2 = 0.107) or survival to hospital discharge (P = .211; η2 = 0.154) between groups (Supplementary Table S1).
Summary statistics for continuous clinical data for horses with measured fecal calprotectin concentration (fCP).
| Healthy (n = 103) | Colitis (n = 13) | Colic (n = 15) | P value | η2 | |
|---|---|---|---|---|---|
| Age (y) | 6 (2, 13.5)a | 13 (6, 17)a | 12 (7, 19)a | .044* | 0.033 |
| Weight (kg) | 466 (409, 524.8) | 503.5 (459.5, 514.5) | 484 (434, 506.8) | .517 | −0.005 |
| Prior NSAID treatment (d) | 1 (0, 2) | 1.5 (1, 2.2) | 1 (1, 2) | .363 | 0.0002 |
| Length of hospitalization (d) | 2 (0, 5.5)a | 5 (2, 10)b | 3 (1, 5.5)a,b | .019* | 0.046 |
| fCP (μg/mL) | 0.2 (0.1, 0.2)a | 0.2 (0.2, 0.3)b | 0.2 (0.2, 0.2)a | .021* | 0.045 |
| Fecal protein (μg/mL) | 8332.9 (6342.4, 10467.7)a | 5602.4 (4112.6, 5952.7)b | 7521.6 (5884.8, 14725.4)a,b | .010* | 0.057 |
| fCP:protein ratio | 2.19 X 10−5 (1.58 X 10−5, 2.88 X 10−5)a | 4.31 X 10−5 (3.19 X 10−5, 6.23 X 10−5)b | 1.52 X 10−5 (1.22 X 10−5, 3.56 X 10−5)a | .002* | 0.083 |
Values are reported as median (quartile 1, quartile 3).
Values with different superscripts are statistically different (Wilcoxon rank-sum test; P ≤ .05).
Horses were enrolled on a convenience basis and categorized as healthy (elective appointment or teaching/research), colitis (clinical signs of diarrhea and/or large colon mural thickness ≥ 5 mm on abdominal ultrasound), or colic (abdominal pain not attributed to large intestinal inflammation). P value indicates the results of a Kruskal-Wallis test for differences across groups *Indicates P ≤ .05. Effect size (η2) > 0.06 is considered moderate.
Horse type was significantly different between groups (P = .005; η2 = 0.389). Stock-type horses (Quarter Horse, Paint, Appaloosa) were over-represented in both colitis (6/13 [40%]) and colic (9/15 [69%]) groups (Supplementary Table S2). The most common breeds in the healthy horse cohort were Standardbred (35/103 [34%]), stock-type (26/103 [25%]), and Thoroughbred (14/103 [14%]). These breed distributions are reflective of the hospital’s typical patient population for emergency and elective care, respectively.
Horses in the colic and colitis groups were significantly more likely to be administered an anti-inflammatory medication prior to sample collection (P = .005; V = 0.281). Of healthy horses, 58 of 103 (56%) were administered an anti-inflammatory medication prior to sample collection compared to 12 of 13 (92%) of colitis cases and 13 of 15 (87%) of colic cases. The type of anti-inflammatory medication administered also differed significantly between groups (P = .002; V = 0.505), with healthy horses most likely to receive phenylbutazone (42/58 anti-inflammatory–treated horses [72%] received phenylbutazone alone or in combination) compared to flunixin meglumine for both colitis (8/13 [67%]) and colic (13/13 [100%]) groups.
The CV for fCP was acceptable, with a median (IQR) of 7.4% (4.1%, 11.7%). The calculated RI for fCP was 0.056 to 0.278 μg/mL and 6.6 X 10−6 to 4.9 X 10−5 for fCP:protein (Figure 1). Measured fCP was above the RI for 4 of 13 colitis cases and 0 of 15 colic cases, whereas 5 of 13 colitis cases and 2 of 15 colic cases had fCP:protein above the RI. There was a significant difference across all groups for both fCP (P = .02; η2 = 0.045) and fCP:protein ratio (P = .002; η2 = 0.83). Horses with colitis had significantly greater fCP (0.234 [0.194, 0.279] μg/mL) compared to healthy horses (0.196 [0.138, 0.220] μg/mL; P = .019) or horses with colic (0.189 [0.164, 0.215] μg/mL; P = .048; Figure 2). There was a greater difference in fCP:protein ratio between colitis cases (4.31 X 10−5 [3.19 X 10−5, 6.23 X 10−5]) and both healthy horses (2.19 X 10−5 [1.58 X 10−5, 2.88 X 10−5]; P = .001) and colic cases (1.52 X 10−5 [1.22 X 10−5, 3.56 X 10−5]; P = .020; Figure 2).
Violin plots showing the distribution of (A) fecal calprotectin (fCP) concentration and (B) fCP:protein ratio in 103 clinically healthy horses (on the y axis, the fCP:protein ratio is presented in scientific notation such that 2e-05 equals 2 X 10−5). Fecal calprotectin was measured by ELISA and total protein by bicinchoninic acid assay. Solid blue lines indicate the upper and lower limits of the calculated reference interval. The dashed orange lines reflect the 95% CIs for the upper and lower reference limits.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.09.0271
Boxplots with overlaid scatter showing (A) fCP concentration and (B) fCP:protein ratio in healthy horses (n = 113; orange), horses with colitis (n = 13; blue), and horses with colic (n = 15, gray). Based on the Kruskal-Wallis test, significant difference across groups for both fCP (P = .02) and fCP:protein ratio (P = .002). Adjusted P values for a post hoc pairwise Wilcoxon test are indicated above the boxes. Lower and upper box boundaries represent the 25th and 75th percentiles (first and third quartile), with the middle line indicating the median. Lower and upper whiskers reflect the 10th and 90th percentiles. Each circle represents the results for 1 horse. Dashed gray lines represent the upper and lower limits of the reference interval.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.09.0271
There was no significant linear correlation between fCP concentration (Figure 3) or fCP:protein ratio (Figure 4) and age, weight, duration of anti-inflammatory medication administration prior to sample collection, or length of hospitalization. There was no significant difference in fCP concentration or fCP:protein ratio associated with horse type, survival to hospital discharge, anti-inflammatory treatment (yes/no), or type of anti-inflammatory medication administered (Table 2).
Scatter plots showing the relationship between fCP concentration and (A) body weight, (B) age, (C) duration of anti-inflammatory medication administration prior to sampling, and (D) length of hospitalization. Each circle represents the results of an individual horse; the solid line and gray shaded area reflect the Pearson correlation and 95% CI, respectively. R = Pearson correlation coefficient.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.09.0271
Scatter plots showing the relationship between fCP to fecal protein ratio and (A) body weight, (B) age, (C) duration of anti-inflammatory medication administration prior to sampling, and (D) length of hospitalization. Each circle represents the result of an individual horse; the solid line and gray shaded area reflect the Pearson correlation and 95% CI, respectively. On the y axis, the fCP:protein ratio is presented in scientific notation such that 1e-04 equals 1 X 10−4.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.09.0271
Results of the Kruskal-Wallis rank-sum test for differences in fCP and fCP:protein ratio between categorical clinical variables.
| Dependent variable | Independent variable | Chi-squared | Degree of freedom | P value | η2 |
|---|---|---|---|---|---|
| fCP (μg/mL) | Survival | 1.008 | 1 | .315 | 6.3 X 10−5 |
| Horse type | 11.72 | 10 | .304 | 0.014 | |
| Anti-inflammatory medication given | 3.597 | 1 | .058 | 0.020 | |
| Anti-inflammatory medication type | 5.793 | 10 | .832 | –0.035 | |
| fCP:protein | Survival | 1.008 | 1 | .315 | 6.3 X 10−5 |
| Horse type | 17.60 | 10 | .062 | 0.063 | |
| Anti-inflammatory medication given | 0.304 | 1 | .582 | 0.010 | |
| Anti-inflammatory medication type | 11.21 | 10 | .341 | −0.005 |
Fecal calprotectin was measured from heathy horses and horses with colitis and colic as described in Table 1. There was no statistically significant difference for any variable evaluated. Effect size (η2) > 0.06 is considered moderate.
Discussion
To our knowledge, this is the first description of fCP measurement in a large cohort of healthy horses and in horses with colic or colitis. Based on a group of over 100 clinically healthy horses, the RI for fCP in horses is 0.056 to 0.278 μg/mL, and the RI for fCP:protein was 6.6 X 10−6 to 4.9 X 10−5. Both the fCP and fCP:protein ratio were significantly increased in horses with colitis compared to healthy horses or horses with colic. However, there were several horses within the colitis group that fell within the calculated RI. The findings of this study support the potential utility of fCP and fCP:protein ratio for the quantification of large intestinal inflammation in horses, although additional investigation in a larger population of horses with well-characterized inflammatory intestinal diseases (including IBD and RDC) is needed.
The group of horses with clinical signs of colic were included to represent horses with intestinal disease not involving large intestinal inflammation; the finding that fCP did not significantly increase in this cohort of horses was expected. There were some inherent differences between groups, namely age, breed distribution, and duration of hospitalization. However, none of these variables were associated with fCP or fCP:protein ratio, suggesting that the differences in fCP and fCP:protein ratio between groups can be attributed to intestinal pathology rather than other intrinsic differences in the patient population. The lack of statistically significant difference in survival to hospital discharge between groups is surprising but is most likely an effect of the difference in group sizes and relatively high survival rates for both colitis and colic groups.
There were 2 horses with a clinical diagnosis of enteritis in the colic group. In humans, while fCP is most sensitive as a biomarker of colonic inflammation, increases in fCP are also seen in patients with small intestinal inflammation.29,30 While the relationship between fCP and small intestinal disease in horses in unknown, the enteritis cases in the colic group may have increased fCP, impacting the comparison between colitis and colic groups. In human patients, serial fCP measurement is most commonly used to assess IBD activity/severity31 and response to treatment13,32 rather than identification of acute infectious diarrhea as seen in horses of this study. However, increases of fCP have also been reported to correlate with the severity of acute infectious diarrhea in humans.33
Fecal total protein was measured in addition to fCP as a means of normalizing the fCP to fecal moisture/fiber content. Subjectively, we have observed that horses with colitis had more water content in the manure than healthy horses, whereas horses in the colic group often had dry manure. Increased fecal water content may have a diluting effect on both fCP and total fecal protein; in such a case, intestinal inflammation would be reflected in an increased fCP:protein ratio while fCP remained normal or low. On the contrary, horses with intestinal inflammation may also have increased fecal protein due to protein loss through a compromised intestinal mucosal barrier, which would result in a normal or low fCP:protein ratio even if fCP was moderately increased. Because the concentration of total protein is several orders of magnitude larger than fCP concentrations (which are typically reported in ng/mL), the resulting fCP:protein ratio is very small.
The ELISA method used for fCP measurement in this study is not practical for use in equine clinical practice. Fecal CP measurement by ELISA has restricted turnaround time even in human diagnostic labs due to the need for sample batching.34 Multiple “random access” immunochemical tests as well as point-of-care CP measuring kits are commercially available to improve the efficiency and access of testing.35,36 The method of measuring feces and extracting in buffer is also time consuming, prompting the development of sample collection/extraction devices.37 These test principles could be adapted for the development of an equine fCP test that would be readily accessible in the equine clinic and should be further investigated.
The limitations of this study include a relatively small sample size for the colitis and colic cohorts. The American Society for Veterinary Clinical Pathology guidelines recommend a sample size of ≥ 120 for RI calculation as smaller groups may not adequately represent the broader population.24 Although the healthy horse cohort had only 103 horses, the statistical approach for RI calculation was appropriate for the sample size and distribution of data.38 Broad inclusion criteria and convenience sampling resulted in a heterogenous group of colic diagnoses skewed toward medical colic (impaction/displacement); findings may differ in horses with more severe pathology, such as large colon volvulus. It is also likely that there was heterogeneity in the colitis group as classification of colitis severity was not included in analysis.
A comparison of fCP findings to an established method would be ideal; however, there is no gold standard test for equine fCP nor a specific antemortem test for intestinal inflammation in horses. The stability of equine fCP following the extraction protocol used in this study has not been evaluated, and the possibility of changes in fCP during storage cannot be excluded. However, fCP is stable in human samples at room temperature for up to 7 days13 and frozen for over 1 year.18 Additionally, the fCP measurement by ELISA as described here is not practical for use in a clinical setting. Further work would be needed to adapt the procedure for a clinical setting. A larger sample of horses with colitis, IBD, and RDC is also necessary to elucidate the relationship between fCP levels and intestinal inflammation in horses. Future investigation comparing fCP levels to the degree and types(s) of inflammation in large colon tissues (by histopathology) should also be considered.
The magnitude of differences between groups was small, with some colitis cases having fCP and/or fCP:protein ratio within the normal reference range, limiting potential diagnostic utility for the specific purpose of identifying colitis. While simple categorization into the broad groups of “colic” and “colitis” was used for this first investigation of fCP in horses with intestinal disease, identification of horses with acute colitis is typically not a diagnostic mystery. Rather, there is a need for quantitative tests to identify and monitor intestinal inflammation in horses with IBD or subclinical NSAID-associated RDC, which is the most promising potential application of fCP and fCP:protein quantification.
Overall, the findings of this study support the initial hypotheses as fCP was detected in feces from healthy horses with a low CV (median, 7.4%), and there was a statistically significant difference in both fCP and fCP:protein ratio between horses with colitis compared to healthy horses and horses with colic. As there are few reliable diagnostic modalities for inflammation of the equine large colon, further investigation of fCP as a fecal biomarker of inflammation is warranted. With additional development of a clinically applicable test and validation in larger population, fCP may enable noninvasive quantification of intestinal inflammation in horses, facilitating the assessment of disease severity and treatment response in conditions such as RDC and IBD.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to thank all clinicians and clients for providing samples to make this study possible.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding for this study was provided by the University of Illinois Companion Animal Memorial Fund.
ORCID
R. C. Bishop https://orcid.org/0000-0002-9660-732X
P. A. Wilkins https://orcid.org/0000-0002-2946-3477
A. M. McCoy https://orcid.org/0000-0003-4088-6902
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