Research Article

Fecal Calprotectin Concentrations and Other Indicators in Dogs with Idiopathic Inflammatory Bowel Disease

Heba El-Zahar*, Zeinab Abd El-Rahman, Abbas El-Naggar

Internal Medicine, Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt.

Received | November 13, 2021; Accepted | December 03, 2021; Published | March 01, 2022

*Correspondence | Heba El-Zahar, Internal Medicine, Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt; Email: hebaelzahar@gmail.com

Citation | El-Zahar H, Abd El-Rahman Z, El-Naggar A (2022). Fecal calprotectin concentrations and other indicators in dogs with idiopathic inflammatory bowel disease . J. Anim. Health Prod. 10(1): 88-96

DOI | http://dx.doi.org/10.17582/journal.jahp/2022/10.1.88.96

ISSN | 2308-2801

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

 

Introduction

Inflammatory bowel disease (IBD) is a long-term inflammatory illness that affects the intestines. As novel treatment goals become more generally acknowledged accurate disease assessment and therapeutic response prediction have become critical challenges in the clinical veterinary practice. Furthermore diagnostic and clinical assessment procedures that are both accessible and cost-effective are urgently needed. Because serum biomarkers are non-invasive, convenient, and less expensive, they have recently made significant progress and have become a focus in IBD research. (Chen et al., 2020). IBD in dogs is a syndrome defined by gastrointestinal symptoms that are persistent and recurrent, as well as histologic indicators of mucosal inflammation. (Allenspach et al., 2016; Washabau et al., 2010). Routine clinicopathologic and histopathologic findings in dogs have been reported to be unable to distinguish between different types of GIT illnesses, making invasive diagnostic approaches difficult for evaluating therapeutic responses. Inflammatory markers appear to be useful in the treatment of dogs with IBD, particularly when it comes to predicting therapy response (Estruch et al., 2020). In dogs with IBD, biomarkers that can help with clinical assessment, monitoring, or assessing response to various treatments may be clinically important. Over the last decade, researchers have looked at a variety of relevant, physiological, metabolomic, biological, and histological indicators in dogs with IBD (Heilmann and Steiner, 2018). Despite the fact that routine analysis of certain clinically effective molecules (such as cytokines and chemokines) is limited due to a lack of general availability, high cost, and poor biological specimen stability, a number of inflammatory indicators can be quantified in biological samples (Truar et al., 2018). A few useful markers, such as serum vitamin B12 (cobalamin) and vitamin B9 (folate) concentrations, are already commonly used in the treatment in dogs with suspected IBD, according to Schreiner et al. (2008).

Other inflammatory indicators such as serum C-reactive protein (CRP), haptoglobin (HAP), serum calprotectin, and fecal calprotectin are currently available in assessing the IBD in dogs. Combining data from multiple inflammatory biomarkers, such as serum CRP and fecal calprotectin can improve the precision of any biomarker on its own (Heilmann et al., 2015). Most clinicians recognize CRP as an inflammatory sign. It is one of the most essential proteins produced immediately by hepatocytes in response to interleukins (IL-1- β and IL-6) and tumor necrosis factor (TNF- α) originating at the site of inflammation or disease (Rhodes et al., 2011).

In human medicine, CRP response is linked to a variety of conditions, including infections, inflammations, tissue necrosis, and stress. Because of its short half-life, CRP is a valuable marker for detecting and observing disease activity in Crohn’s disease (CD). CRP levels in the blood correspond well with disease activity in CD patients and other inflammatory indicators such as fecal calprotectin (Vermeire et al., 2004).

In veterinary practice, serum CRP levels have a significant biological variance in dogs (Carney et al., 2011) restricts its utility as a diagnostic biomarker in dogs with IBD. Serum CRP appears to be more clinically relevant as a replacement test to assess disease progression and responsiveness to treatment in dogs with IBD (Heilmann and Steiner, 2018; Jergens et al., 2010; Jergens et al., 2003). In veterinary practice, haptoglobin (HAP) is a mild acute phase protein that rises in responds to inflammatory process, infection, or tissue damage (Eckersall and Conner, 1988). A previously reported reference range for canine haptoglobin was 0 to 3 g/l, with concentrations greater than 10 g/l indicating a strong inflammatory response (Eckersall and Conner, 1988). The level of haptoglobin in the dog’s blood could provide useful information regarding the severity of the disease and how well it responds to treatment (McGrotty et al., 2003). Fecal calprotectin is a stool biomarker and noninvasive means of evaluating intestinal inflammation that is increasingly being utilized in clinical practice to differentiate between organic and functional gastrointestinal disorders (Heilmann and Steiner, 2018). The concentrations of fecal calprotectin in dogs with persistent gastrointestinal inflammation have been studied and appear to be a valuable indicator of intestinal inflammation in dogs (Grellet et al., 2013; Otoni et al., 2018). Calprotectin is a calcium and zinc binding protein heterocomplex made up of the S100A8 and S100A9 proteins (Foell et al., 2007; Mumolo et al., 2018), that contributes for more than 60% of protein content in neutrophil cytosol, which infiltrates the intestinal mucosa as involved in the inflammatory reflex in IBD (Oyaert et al., 2017). Hyperpermeable intestinal mucosa infiltration of activated neutrophils into the faeces as a result of inflammation, measuring calprotectin levels in stool samples has showed potential in distinguishing patients with IBD (Banerjee et al., 2015; Caviglia et al., 2014). The current study was designed to investigate the changes in the clinical, hematobiochemical, and IBD-related biomarkers as prognostic indicators in dogs with IBD. In addition, determining the relationship between serum CRP, haptoglobin, and changes in fecal calprotectin, as well as the relationship between blood enzymes, electrolytes, and CRP concentration as an IBD biomarker.

Materials and Methods

The present study was carried out on 32 dogs of different ages and breeds (German shepherd, Griffon, and Golden retriever). All dogs included in the current study were collected from a private pet’s clinics and shelters in Sharkia governorate. The dogs’ owners have given their permission for their canines to be included in the current study. Eleven healthy dogs aged from 5 to 17 years, and of different breeds (German shepherd, Griffon, Golden retriever) were selected as a control group. Dogs had no prior history of GI symptoms based on the results of a routine clinical assessment, CBC, serum biochemical, and faecal examination. Control dogs were used to provide control values for comparing the findings obtained from IBD dogs.

Twenty-one dogs aged from 5 to 13 years, of different breeds (German shepherd, Griffon, Golden retriever) were chosen for this study. Diseased dogs were suffered from varying gastrointestinal abnormalities (mostly IBD) involving chronic diarrhea >3 weeks with no response to dietary and symptomatic therapy. They had not been given any treatment prior to the clinical examination. Physical examination were performed for all dogs and ultrasonography was performed on different abdominal organs especially GIT (Delaney et al., 2003). Clinical disease activity was evaluated based on their divergence from normal dogs in the following: appetite, vomiting, stool (consistency and frequency) and body weight are all factors to consider.

Samples collection and analysis

Serum was obtained from 21 IBD dogs to carry out hematological and biochemical analysis, 7 ml of blood was collected from each dog’s cephalic vein, and 1-2 ml of blood was placed in vacuum EDTA coated tubes for hematological analysis. The remaining blood samples were divided into 3-4 ml sterile heparinized tubes and 2 ml plain tubes for plasma and serum sample collection. Plasma and serum were separated immediately after collection by centrifuging for 10 minutes at 3000 rpm.

Hemoglobin (Hb), packed cell volume (PCV), total erythrocytic count (TEC) and total and differential leukocytes counts were determined using Sysmex XN1000 analyzer (Sysmex, USA) using standard methods (Feldman et al., 2010).

Serum samples were harvested and divided into several aliquots used for biochemical analysis (CRP, haptoglobin, pancreatic lipase, alkaline phosphates, and blood parameters (total protein, albumin, globulin and glucose). All biochemical measurements were applied using Beckman AU5800 analyzer (Beckman Coulter, California, USA) using standard methods (Feldman et al., 2010).

Fecal samples (an aliquot of approximately 1 g each) were collected for measuring fecal calprotectin concentrations using the ELISA Test Kit (CALP-0170, Calpro AS, Oslo, Norway) following the standard methods for all the ELISA procedures.

Statistical Analysis

Data were statistically analyzed using SPSS Statistics® 17.0 (Version 17.0 released 2008, SPSS Inc., Chicago). The Shapiro Wilks W Test was used to check for normal distribution, and the data were determined to be normal. Biochemical data were compared using independent samples t-test and all data are listed as mean ± SE. The significance of differences between parameters was assessed at different probability levels of p<0.05 and p<0.01. For estimating the relationship between the concentration of IBD-related inflammatory markers (CRP, haptoglobin and fecal calprotectin), and for estimating the correlation between CRP and blood enzymes (pancreatic lipase and ALP) and electrolytes concentrations (total Ca, Na, K) in IBD dogs pearson correlation was used.

Results

The current study comprised a total of 32 dogs, from which 21 had inflammatory bowel disease of different ages (5 to 13 years old) and breeds (3 German shepherd, 10 Griffon, 8 Golden retriever), while the remaining 11 were healthy dogs ranging in age from 5 to 17 years old and of various breeds (3 German shepherd, 4 Griffon 4 Golden retriever), all dogs were male. There was significant increase in mean body temperature of 40.3 ± 0.27 °C (p<0.05), heart rate of 126.8 ± 2.5 per min. (p<0.05), and respiratory rate of 53.4 ± 1.9 breaths per min. (p<0.01). The most prevalent clinical indicators in IBD dogs, according to clinical assessment, are vomiting, bloody diarrhea, or watery diarrhea, anorexia, weight loss, and abdominal pain (Table 1).

The mean values of hematological and biochemical indices in dogs with IBD revealed a significant rise in the total leukocytes count (14.26 ± 0.59), neutrophils (69.76 ± 1.7), platelets count (294.6 ± 31.96) and a significant decrease in the mean of platelets volume MPV (20.2 ± 1.47), hemoglobin (10.16 ± 0.52) while, non significant decrease in red blood cell count (4.55 ± 0.14) and PCV (31.19 ± 1.3) in comparison to the controls. As well, the serum activities of AST demonstrate a significant increase (59.23 ± 5.7) and ALT (77 ± 6.5) activity compared to the controls. As well as, there was a highly significant increase in blood urea nitrogen (52.82 ± 3) and blood creatinine (3 ± 0.23). While, the alterations of the mean values of serum total proteins, albumin, globulin was not changed significantly in IBD dogs (Table 1).

The serum activities of pancreatic lipase demonstrate a significant increase (244.69 ± 23.27) and alkaline phosphates (310 ± 35.26) activity compared to the controls. There was highly significant decrease in blood glucose (65.6 ± 1.07) and blood electrolytes as sodium (138.7 ± 1.04), potassium (3.01 ± 0.11) and total calcium (8.09 ± 0.4) (Table 2). While, the mean values of IBD-related biomarkers, CRP and HAP concentrations were highly significant increase (4.4 ± 1.07and 164 ± 18.31, respectively). As well as, the fecal calprotectin concentration was highly significant increased (27.6 ± 2.29) in IBD dogs compared to the controls (Table 3).

The relationship between different IBD related biomarkers (CRP, haptoglobin and fecal calprotectin), were performed using Pearson correlation with plotting the results of CRP and haptoglobin concentrations against the measurements of the fecal calprotectin in the same samples and the results showed very strong positive correlation (r = 0.733, r = 0.726, respectively) (Figures 1 and 2).

The relationship between alterations of blood enzymes (ALP and Lipase) and electrolytes (total calcium, sodium and potassium) and CRP (IBD-related biomarker) were performed by plotting the results of serum ALP, Lipase concentrations against the measurements of and CRP in the same samples of IBD dogs and the results showed a moderately positive correlation (r = 0.645, r = 0.537, respectively) (Figures 3 and 4). While, the correlation between serum total calcium, plasma K+ and CRP illustrated a moderately negative correlation (r = -0.526, r = -0.567, respectively) as well, it was a strongly negative correlation between serum plasma Na+, glucose and CRP (r = -0.776, r = -0.71, respectively), (Figures 5, 6, 7 and 8).

 

Table 1: Dog characteristics, clinical parameters, observations, hematological and biochemical findings in dogs with inflammatory bowel disease and healthy dogs included in the present study. The results are expressed as Mean ± S.E, and/or max., min.

	Healthy controls n=11	IBD dogs n=21	Reference values #
Dog characteristics:
Age in years, median	8 (5 – 17)	9 (5 – 13)
Gender, male/female	6 male / 5 female	21 / 00
Weight in kg, median	22.5 (11 – 38)	15.25 (8 – 25)
Clinical parameters:
Anorexia, n (%)		16 (76.00)
Vomiting, n (%)		8 (38.00)
Diarrhea, n (%)		21 (100.00)
Clinical observations:
Rectal temperature (°C)	38.6 ± 0.18	40.3 ± 0.27 **	38.3 - 39.2
Respiration rate (per min.)	26.4 ± 1.4	53.4 ± 1.9 *	15 - 35
Heart rate (per min.)	77.4 ± 1	126.8 ± 2.5 **	70 - 90
Hematological parameters:
Hemoglobin (g/dl)	14.65 ± 0.29	10.16 ± 0.52 *	12 - 18
PCV (%)	39.76 ± 1.2	31.19 ± 1.3 ns	37 - 55
Total erythrocyte(×106/l)	5.68 ± 0.11	4.55 ± 0.14 ns	5.5 - 8.5
Total leukocyte(×103/l)	9.9 ± 0.38	14.26 ± 0.59 *	6 - 16
Neutrophils (%)	65.09 ± 1.03	69.76 ± 1.7 **	60 - 70
Lymphocytes (%)	27.45 ± 5.66	20.14 ± 1.34 ns	15 - 30
Monocytes (%)	4.36 ± 0.59	4.8 ± 0.38 ns	3 - 8
Eosinophils (%)	2.81 ± 0.32	3.42 ± 0.31 ns	2 - 10
PLT (×103/l)	155.74 ± 14.39	294.6 ± 31.96 **	200 - 500
MPV (fl)	23.56 ± 0.44	20.2 ± 1.47 *	>20
Biochemical parameters:
Total protein (g/dl)	6.3 ± 0.24	6.5 ± 0.18 ns	5 – 7
Albumin (g/dl)	3.6 ± 0.16	2.9 ± 0.14 ns	3.1 - 4.5
Globulin (g/dl)	3 ± 0.13	4.5 ± 0.17 ns	2.8 - 4.5
BUN (mg/dl)	15.46 ± 1.6	52.82 ± 3 **	7 - 25
Creatinine (mg/dl)	0.99 ± 0.1	3 ± 0.23 **	0.4 - 1.8
AST (IU/l)	39.5 ± 2.7	59.23 ± 5.7 *	5 - 55
ALT (IU/l)	36.7 ± 3.1	77 ± 6.5 ns	5 - 60

PCV, packed cell volume; PLT, platelet count; MPV, mean platelet volume; BUN, blood urea nitrogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase. # (Feldman et al., 2010), * statistically significant at P<0.05, ** statistically significant at P<0.01, ns statistically non significant

 

Table 2: Mean ± S.E values of glucose, blood enzymes and electrolytes in dogs with IBD

Parameters	Control group (n = 11)	IBD group (n = 21)	Reference values #
Glucose (mg/dl)	112.6 ± 2.7	65.6 ± 1.07 **	60 - 125
Alkaline phosphates (IU/l)	43 ± 4.7	310 ± 35.26 **	10 - 150
Lipase (IU/l)	99.14 ± 3.09	244.69 ± 23.27 **	100 - 500
Total Ca (mg/dl)	9.7 ± 0.17	8.09 ± 0.4 **	7.5 - 11.3
Potassium (mmol/l)	4.68 ± 0.21	3.01 ± 0.11 *	4 - 5.6
Sodium (mmol/l)	146.75 ± 0.61	138.7 ± 1.04 ns	141 – 156

# (Feldman et al., 2010), * statistically significant at P<0.05, ** statistically significant at P<0.01, ns statistically non significant

 

Table 3: Mean ± S.E values of biomarkers of inflammation in dogs with IBD

Parameters	Control group (n = 11)	IBD group (n = 21)	Reference values
Fecal calprotectin (μg/g)	0.2±0.05	27.6±2.29**	0.5 – 15.3 #
C-reactive protein (mg/dl)	0.7±0.12	4.4±1.07**	<1 #
Haptoglobin (mg/dl)	78.95±2.8	164±18.31*	30 - 250 ##

* statistically significant at P<0.05, ** statistically significant at P<0.01, ns statistically non significant, # (Heilmann et al., 2018), ## (Grobman et al., 2017).

 

Discussion

In the present study, IBD was most commonly seen in middle-aged (5-13 years) male dogs suffered diarrhea symptoms, where the inflammatory bowel disease is defined by the infiltration of inflammatory cells into the intestinal walls and is the most common cause of persistent diarrhea in dogs. In addition, vomiting, anorexia, weight loss, and abdominal pain were observed, it shows the severity of the disease, and this was similar with previous studies by Bhavani et al. (2021); Volkmann et al. (2017).

In comparison to the control group, hematological examination revealed a significant rise in total leukocytes count and neutrophils, as well as a decrease in red blood cell count, hemoglobin, and PCV. Similar changes were observed by Ristic and Stidworthy (2002), who hypothesized that prolonged gastrointestinal blood loss could lead to non-regenerative iron deficiency anemia and that neutrophilic leukocytosis was linked to stress and chronic inflammation.

Our results showed a considerable rise in platelet count and a significant drop in platelet volume average (MPV). Platelet release can be caused by an increase in the pro-inflammatory cytokines concentrations, particularly IL-6, in individuals with persistent inflammation. This is related to IL-6’s enhancement of thrombopoietin production. IL-6 increases the megakaryocytic nuclei and the amount of the cytoplasm, resulting in the generation of a significant number of blood platelets (Senchenkova et al., 2013). The fraction of giant platelets increases as the inflammatory process progresses, which is most likely due to intracellular manufacture of pro-inflammatory cytokines, granule degranulation, and activation of the splenic platelet reserve. Simultaneously, these cells migrate promptly to the inflammation site, where they are activated and depleted, explaining why MPV levels are lower in individuals with chronic inflammation (Afsar et al., 2017; Kamath et al., 2001).

The IBD group had significantly higher biochemical alterations, such as blood urea nitrogen and creatinine levels, matching the findings of Bhavani et al. (2021) in diarrheic dogs. Increased blood urea nitrogen suggests prerenal uremia, which is caused by fever-induced tissue catabolism and a decreased glomerulofiltration rate. The levels of ALT and AST in the study group were significantly higher than in the control group, indicating that reactive hepatopathy was to blame (Berghoff and Steiner, 2011). While the mean values of blood total proteins, albumin, and globulin did not or weak changed significantly in IBD dogs, indicating that the dogs in this study did not progress to Protein-Losing Enteropathy (Dossin and Lavoue, 2011).

The serum activities of blood enzymes (pancreatic lipase and alkaline phosphatase) show a considerable increase in IBD dogs. While, blood glucose and blood electrolytes such as sodium, potassium, and total calcium were decreased significantly. Our findings agreed with those of Shinde et al. (2000). Who noticed the same abnormalities and hypothesized that hypoglycemia in the affected dogs was caused by inappetance or anorexia, which was accompanied by intestinal mal-absorption. Furthermore, inflammation increases bowel permeability, resulting in fluid, electrolyte, protein, and cell loss. However, elevated pancreatic lipase and alkaline phosphatase levels were linked to inflammation in idiopathic IBD dogs, as well as systemic disorders, such as pancreatitis and GI inflammation that can cause reactive hepatopathy or physiologic stress. (Ide et al., 2016).

The elevation of the IBD-related biomarkers, CRP and haptoglobin concentrations as well as, the fecal calprotectin concentrations in IBD dogs compared to the controls in the current investigation were similar to previous studies in human and veterinary research field (Grobman et al., 2017). CRP levels were observed to be high in canine patients with inflammatory disorders, including infectious diseases, surgical trauma, acute pancreatitis, glomerulonephritis, and inflammation (Carney et al., 2011; Gommeren et al., 2018). As well, the elevated serum haptoglobin concentration in this study was similar to that previously reported by Eckersall and Conner (1988), who discovered that the reference range for canine haptoglobin was 0 to 3 g/l, with concentrations greater than 10 g/l indicating a strong inflammatory response because haptoglobin is a mild acute phase protein that rises in response to inflammation, infection, or trauma. The increased release of calprotectin from activated macrophages and neutrophils during the inflammatory process is linked to an increase in fecal calprotectin. Calprotectin is a receptor that plays a function in both acute and persistent inflammation. Serum calprotectin levels are elevated in dogs with chronic inflammatory enteropathies, however serum calprotectin is not as selective for the gastrointestinal system as feces calprotectin (Foell et al., 2007; Heilmann et al., 2018; Truar et al., 2018; Wilke et al., 2012).

Fecal calprotectin was found to be substantially correlated with serum CRP and haptoglobin, indicating that intestinal inflammation in dogs with idiopathic IBD is relevant to the systemic inflammatory response. These findings were consistent with previous research that found CRP to be moderately linked with fecal calprotectin in Crohn’s disease or ulcerative colitis patients (Ricanek et al., 2011). The results that investigates the relationship between changes in blood glucose, blood enzymes (pancreatic lipase and ALP), and blood electrolytes (total Ca, Na, and K ions) and the concentration of CRP (IBD-related biomarkers), revealed a strong to moderate correlation that indicates the progression of the inflammation. Consequently, the most prevalent conditions related with canine IBD are hypoglycemia and pancreatitis (Ide et al., 2016; Malo et al., 2014).

Conclusions

In conclusion, when IBD dogs were compared to the control group, hematological indices such as total leukocyte count, neutrophils, and platelet count were considerably higher, showing that IBD dogs have non-invasive hematological markers. CRP, haptoglobin, and fecal calprotectin were also discovered to be powerful IBD biomarkers and might be used as surrogate measures of disease severity in dogs with IBD. The combination of CRP, haptoglobin, and fecal calprotectin measurements has the potential to evaluate canine IBD.

Acknowledgements

We would like to express our gratitude to Dr. Tarek Allam, Professor at Animal Health Research Institute, Zagazig, Egypt for his support and help in collecting and analysis of blood samples.

Conflict of interest

We state that there is no conflict of interest.

novelty statement

The main objective of this paper is to investigate the changes in the IBD-related biomarkers as prognostic indicators in dogs with IBD. Many researchers studied the fecal calprotectin in human medicine as inflammatory biomarker for IBD diagnosis; this study investigated the fecal calprotectin as novel inflammation biomarkers in dogs with IBD.

authors contribution

All authors contributed equally to this manuscript including paper writing, editing, and reviewing. In addition, the first and last authors had supervision contribution.

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