Skip to main content
NCBI home page
Veterinary Medicine and Science logoLink to Veterinary Medicine and Science
. 2025 Oct 28;11(6):e70621. doi: 10.1002/vms3.70621

Evaluation of Endothelial Glycocalyx Damage in Neonatal Lambs With Diarrhoea: A Biomarker‐Based Approach

Murat Kaan Durgut 1,✉, Mahmut Ok 1, Merve Ider 1, Bunyamin Tras 2, Amir Naseri 1, Tugba Melike Parlak 2, Abdulrahman Alhallaq 1, Suleyman Serhat Iyigun 1
PMCID: PMC12560170  PMID: 41147804

ABSTRACT

Background

Neonatal diarrhoea is one of the most common diseases in lambs, causing economic losses and welfare issues. The endothelial glycocalyx (eGC) plays a crucial role in vascular integrity and inflammatory regulation. However, its involvement in neonatal lamb diarrhoea remains unclear.

Objectives

This study aimed to evaluate eGC damage in neonatal lambs with diarrhoea by assessing biomarkers, including nitric oxide (NO), heparan sulphate (HS), angiopoietin‐2 (Ang‐2) and A disintegrin and metalloproteinase 15 (ADAM15).

Methods

A total of 68 neonatal lambs with diarrhoea (39 infected with Escherichia coli and 29 with Cryptosporidium parvum) and 20 healthy controls were included. Diarrhoea aetiology was confirmed via faecal antigen tests and microscopic examination. Blood samples were collected for hemogram and biomarker analysis using sheep‐specific ELISA kits.

Results and Conclusions

Lambs with diarrhoea showed significantly higher total leukocyte (white blood cell [WBC]), granulocyte (GRA) and monocyte (MON) counts (p < 0.01). Serum NO concentrations were significantly elevated in lambs with diarrhoea, whereas HS and Ang‐2 concentrations were lower (p < 0.01). E. coli–infected lambs had significantly higher ADAM15 concentrations than those with Cryptosporidium infection (p < 0.001). These findings suggest that NO may have a protective and/or anti‐inflammatory role in neonatal lamb diarrhoea, potentially maintaining eGC integrity despite the inflammatory process.

Keywords: diarrhoea, endothelial glycocalyx biomarkers, lamb

Neonatal lambs with diarrhoea showed increased nitric oxide and decreased heparan sulphate and angiopoietin‐2 levels, suggesting preserved endothelial glycocalyx integrity. Elevated ADAM15 levels in E. coli cases indicate a pathogen‐specific endothelial response, highlighting nitric oxide's potential protective role during inflammation.

graphic file with name VMS3-11-e70621-g001.jpg

1. Introduction

Neonatal lamb diarrhoea, which causes lamb mortality and serious economic losses, is still a welfare problem in sheep farms. The aetiology of neonatal diarrhoea is caused by bacteria, viruses and protozoa, including enterotoxigenic Escherichia coli, Clostridium perfringens, Rotavirus, Coronavirus, Cryptosporidium parvum and Giardia species (Davis et al. 2016; Johnson et al. 2017; Brown et al. 2019; Alhallaq and Ok 2025). These pathogens cause intestinal damage, and, particularly in newborn lambs with a weak immune system, they impair the mucosal barrier by attaching to the intestinal mucosa and causing intestinal damage (Ok et al. 2020).

Intestinal epithelial damage during diarrhoea leads to loss of fluid and electrolytes as well as triggering a systemic inflammatory response (Durgut and Ok 2023; Alhallaq and Ok 2025). Vascular endothelial integrity plays a critical role in this inflammatory process. The glycocalyx layer covering the outer surface of endothelial cells is an important structure that regulates vascular permeability and provides a protective barrier against the inflammatory response (Reitsma et al. 2007; Yang et al. 2018). Therefore, assessment of endothelial integrity in conditions that cause systemic inflammation such as diarrhoea may provide important information in understanding the progression and pathophysiology of the disease. For this purpose, some biomarkers reflecting both vascular endothelial damage and inflammation level are utilized. Nitric oxide (NO), one of these biomarkers, is a molecule that affects vascular tone and the inflammatory process; its concentration increases in the circulation in cases of infection and inflammation (Franceković and Gliemann 2023). Heparan sulphate (HS), a major structural component of the glycocalyx, enters the circulation due to glycocalyx degradation caused by inflammatory damage (Tang et al. 2017; Yang et al. 2018). Angiopoietin‐2 (Ang‐2), another marker, is produced by endothelial cells in the vascular lining and is responsible for maintaining vascular balance. During inflammation, its concentration increases and contributes to glycocalyx damage (Parikh et al. 2006; Higgins et al. 2018; Thamm et al. 2018). A disintegrin and metalloprotease 15 (ADAM15) is a metalloproteinase that regulates intercellular connections and vascular permeability; it is involved in inflammatory responses (Sun et al. 2013).

There are limited studies investigating the concentrations of these biomarkers in relation to neonatal lamb diarrhoea and their association with endothelial damage. This study was designed on the basis of the hypothesis that inflammation in lambs with diarrhoea may lead to vascular endothelial damage, and that this damage could be detected through specific biomarkers. In this study, NO, HS, Ang‐2 and ADAM15 concentrations were measured to evaluate the relationship between glycocalyx damage and the inflammatory response.

2. Materials and Methods

The study was conducted between February 2024 and December 2024 at the Department of Internal Medicine, Faculty of Veterinary Medicine, Selcuk University, Konya, Türkiye. The study protocol was approved by the Institutional Ethics Committee of the Faculty of Veterinary Medicine, Selcuk University (No. 2024/022).

2.1. Animal Materials

2.1.1. Lambs With Diarrhoea

Sixty‐eight Merino lambs of both sexes, aged between 2 and 20 days and presenting with diarrhoea, were enrolled in the study. Routine clinical examinations of the lambs were carried out. Faecal samples taken from lambs with diarrhoea were subjected to a rapid faecal antigen test (BoviD‐5 Ag, EPOVET) for E. coli K 99, Rotavirus, Coronavirus, Cryptosporidium, and Giardia species, and their diagnosis was established according to test positivity. Faecal samples were collected from the cases that were positive for Cryptosporidium by the faecal rapid antigen test, and Cryptosporidium oocysts were confirmed by light microscopy. In addition, the lambs were evaluated on the basis of their clinical condition and medical history to rule out other potential causes of diarrhoea. Animals with confirmed nutritional, parasitic or systemic non‐infectious aetiologies were excluded from the study. Furthermore, lambs that had received any antibiotic or other medical treatments within the previous 2 weeks were not included.

2.1.2. Healthy Lambs

Twenty Merino lambs of both sexes, aged between 2 and 20 days, were enrolled. Healthy lambs were selected from the faculty farm, which granted permission. Routine clinical and laboratory examinations were carried out. Faecal samples from clinically healthy lambs were tested using a rapid faecal antigen test (BoviD‐5 Ag, EPOVET) for E. coli K99, Rotavirus, Coronavirus, Cryptosporidium and Giardia species, and only negative samples were selected. Lambs with normal clinical examination findings, haematological results within the reference range and negative faecal examination results were classified as healthy (control group) and included in the study.

2.2. Collection of Faecal Samples

Faecal samples were collected once from all the lambs and put into faecal cups by rectal palpation. These samples were taken to the parasitology laboratory to examine Cryptosporidium and Giardia oocysts.

2.3. Faecal Examination Procedure

Cryptosporidium oocysts were examined using the modified Ziehl–Neelsen (MZN) method (Foreyt 2013). For this method, a sufficient amount of faeces was spread on a microscope slide and dried. The faecal preparation was kept in absolute methyl alcohol for 1 min and then in carbon fuxin for 5 min. The faecal preparation was immersed several times in 50% ethyl alcohol. It was then kept in 1% sulphuric acid solution for 2 min and in methylene blue solution for 1 min. The stained faecal preparation was washed with tap water and dried. Then, it was examined under a light microscope with immersion oil at 100× objective, and oocysts were observed.

Giardia diagnosis was performed using the zinc sulphate flotation technique. A total of 1 g of faeces was suspended in 10–12 mL of water in a glass beaker. The mixture was passed through a tea strainer and transferred to another beaker. First, the material in the strainer was pressed down with a spatula, and then the remainder of the strainer was discarded. The contents were poured into a 15 mL centrifuge tube and fully filled with water. The tube was centrifuged at 1500 rpm for 5–10 min, then half emptied and filled with zinc sulphate solution. The tube was placed in the centrifuge, and flotation solution was added using a dropper. This brought the level of solution to the top of the tube. A cover slip was placed on top of the tube in contact with the solution. The cover slip was then removed and placed on a glass slide. The slide was examined under a light microscope at ×100 (×10 ocular and ×10 objective) magnification, and Giardia oocysts were observed (Foreyt 2013).

2.4. Collection of Blood Samples

Blood samples were taken once from the jugular vein of all lambs. Although K3EDTA tubes were used for complete blood count measurement, anticoagulant‐free gel tubes were used for serum collection. Complete blood count analysis was performed immediately (within 5–15 min) after the blood samples were collected. The samples were kept at room temperature for 15 min and then centrifuged at 5000 rpm for 5 min to remove serum. The sera were stored at −20°C until analysis. The biomarkers of NO, HS, Ang‐2 and ADAM15 were measured from these serum samples.

2.5. Complete Blood Count Analysis

Total white blood cell (WBC), lymphocyte (LYM), monocyte (MON), granulocyte (GRA), red blood cell (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), haematocrit (Hct), haemoglobin (Hb) and platelet (PLT) count were measured in K3EDTA venous blood samples collected from all lambs using an automated haematology analyser (MS4e, CFE 279, Melet Schlosing Laboratories, Osny, France).

2.6. Endothelial Glycocalyx (eGC) Biomarker Measurement

Serum concentrations of NO, HS, Ang‐2 and ADAM15 were measured using a sheep‐specific ELISA test kit (Bioassay Technology Laboratory, Zhejiang, China) according to the manufacturer's instructions. For NO (Cat. No: E0037Sh), the reported intra‐ and inter‐assay coefficient of variation (CV) were <8% and <10%, respectively; minimum detectable concentration (MDC) was 0.52 µmol/L; detection range was 0.5–200 µmol/L. For HS (Cat. No: AE0061Ge), the reported intra‐ and inter‐assay CVs were <10% and <12%, respectively, MDC was 9.97 ng/mL, and the detection range was 18.75–1200 ng/mL. For Ang‐2 (Cat. No: E0288Sh), the reported intra‐assay and inter‐assay CVs were <8% and <10%, respectively, MDC was 0.18 ng/mL, and the detection range was 0.38–24 ng/mL. For ADAM15 (Cat. No: E0289Sh), the reported intra‐assay and inter‐assay CV were <8% and <10%, respectively, and the MDC was 0.11 ng/mL with a detection range of 0.19–12 ng/mL.

2.7. Treatment Protocol

Neonatal lambs with diarrhoea received a standard diarrhoea treatment, including fluid‐electrolyte treatment, antimicrobial treatment, and supportive care. Lactated Ringer's (Lactated Ringer, Medifleks) and glucose (5% Dextrose, Polifarma) solutions were given intravenously as fluid therapy. Ceftiofur (Ceftivil, Vilsan) was administered intramuscularly at a dose of 2.2 mg/kg once daily for 5 days. For Cryptosporidium infections, halofuginone (Halocur, Intervet) was administered orally at a dose of 0.1 mg/kg (2 mL/10 kg body weight) once daily for 7 days. Vitamin B complex (ANOREX‐B, Alke), vitamin C (Redox C, Bayer), and vitamins A, D and E (ADE Vital, Alke) were administered intramuscularly as supportive therapy.

2.8. Statistical Analysis

SPSS 25 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0 Armonk, NY: IBM Corp.) was used to analyse the study data. The one‐sample Kolmogorov–Smirnov test was used to assess the assumptions of normal distribution (parametric or non‐parametric) of the data. As the data showed a non‐parametric distribution, they are presented as median with minimum and maximum ranges in parentheses. The Mann–Whitney U‐test was used for intergroup comparison. The relationships between selected parameters were analysed using Spearman's rank correlation. The significance level of the tests was accepted as p < 0.05.

3. Results

3.1. Clinical Findings

A total of 68 newborn lambs with diarrhoea were included in the study; a total of 39 lambs were infected with E. coli, and 29 lambs were infected with C. parvum. In addition, Giardia spp. was not detected in the lambs included in the study. The lambs had moderate to severe diarrhoea for several days and yellow or brownish faeces in the perineum and tail area. E. coli–infected lambs had yellow watery diarrhoea, and Cryptosporidium‐infected lambs had yellow mucoid and mucoid watery diarrhoea. All lambs showed moderate‐to‐severe dehydration and increased capillary refill time (>2 s) (Table 1). Most of the cases showed different degrees of decreased sucking reflex, loss of appetite, weakness, general malaise, weight loss, difficulty standing, lying on the sternum and in some lambs, colic and tympani. Body temperature was within normal reference ranges in all lambs (Table 1). A significant increase in heart rate and respiratory rate was observed (Table 1). After the 24th hour of treatment, the severity of diarrhoea was significantly reduced, the sucking reflex and appetite increased, and after the 48th hour, faecal consistency started to be solid. Although 57 lambs with diarrhoea (84%) responded to the treatment and survived, 11 lambs died (16%). Of the nonsurviving lambs, six were E. coli–infected and five were Cryptosporidium‐infected. The necropsies and histopathology performed confirmed the diagnosis of E. coli in six lambs and Cryptosporidium infection in five lambs, respectively.

TABLE 1.

Mean body temperature, heart and respiratory rate, dehydration degree score, faecal score and capillary refill time of neonatal lambs with diarrhoea.

Parameters Lambs with diarrhoea (n = 68) Reference ranges
Body temperature (°C) 38.6 38.3–39.9
Heart rate (min) 113 70–80
Respiratory rate (min) 40 16–34
Dehydration degree (%) 9.2
Capillary filling time (s) 3 1–2
Open in a new tab

3.2. Complete Blood Count Findings

Complete blood count parameters of healthy lambs and lambs with diarrhoea are presented in Table 2. There was a significant increase (p ˂ 0.01) in WBC, MON and GRA counts of lambs with diarrhoea compared to healthy lambs. There was no significant difference (p > 0.05) in LYM, RBC, MCV, MCHC, Hb, HCT and PLT between study groups.

TABLE 2.

Complete blood count parameters findings of healthy and lambs with diarrhoea (expressed as median and minimum‐maximum in parenthesis).

Parameters Healthy lambs (n = 20) Lambs with diarrhoea (n = 68) p value
WBC (m/mm3) 7.80 (4.37–9.95) 10.49 (5.32–25.98) 0.010
LYM (m/mm3) 3.54 (2.04–4.92) 3.42 (0.85–7.80) 0.474
MON (m/mm3) 0.47 (0.33–1.67) 1.47 (0.05–3.11) 0.002
GRA (m/mm3) 3.36 (1.28–5.56) 5.63 (0.64–20.14) 0.008
RBC (m/mm3) 11.33 (7.05–12.84) 11.34 (8.07–18.03) 0.448
MCV (fL) 30.90 (25.60–41.60) 32.10 (22.40–45.00) 0.494
HCT (%) 31.40 (26.90–47.60) 36.50 (21.80–56.10) 0.065
MCHC (g/dL) 35.10 (27.90–40.80) 30.40 (24.90–40.81) 0.073
Hb (g/dL) 11.20 (7.90–13.30) 11.10 (7.70–19.40) 0.618
PLT (m/mm3) 289.00 (84.00–1036.00) 317.00 (126.00–1350.00) 0.817
Open in a new tab

Note: Statistical significance was considered p < 0.05; RBC, erythrocytes; WBC, total leukocytes.

Abbreviations: GRA, granulocytes; Hb, haemoglobin; HCT, haematocrit; LYM, lymphocytes; MCHC, mean erythrocyte haemoglobin concentration; MCV, mean erythrocyte volume; MON, monocytes; PLT, platelets.

3.3. eGC Biomarker Findings

eGC biomarker concentrations of healthy lambs and lambs with diarrhoea are presented in Table 3. Although serum NO concentrations of lambs with diarrhoea were significantly higher than healthy lambs, serum HS and Ang‐2 concentrations were lower than healthy lambs (p ˂ 0.01). No difference was detected in serum ADAM15 concentration (Table 3). Considering the aetiology of diarrhoea, lambs infected with E. coli had significantly higher serum ADAM15 concentrations compared to those infected with Cryptosporidium (p < 0.001). However, no significant differences were observed in serum NO, HS and Ang‐2 concentrations between the groups (Table 4).

TABLE 3.

Serum a disintegrin and metalloprotease 15 (ADAM15), angiopoietin‐2 (Ang‐2), nitrite oxide (NO) and heparan sulphate (HS) concentrations in healthy and lambs with diarrhoea (data expressed as median and minimum–maximum in parenthesis).

Parameters Healthy lambs (n = 20) Lambs with diarrhoea (n = 68) p value
NO (umol/L) 20.59 (7.81–29.76) 27.17 (11.44–57.69) 0.001
HS (ng/mL) 489.01 (163.81–542.51) 395.18 (221.97–551.00) 0.000
Ang‐2 (ng/mL) 6.31 (4.69–8.27) 5.68 (3.02–10.19) 0.019
ADAM15 (ng/mL) 2.69 (1.84–3.58) 2.61 (1.53–5.93) 0.863
Open in a new tab

Note: Statistical significance level was considered p < 0.05.

TABLE 4.

Serum a disintegrin and metalloprotease 15 (ADAM15), angiopoietin‐2 (Ang‐2), nitrite oxide (NO) and heparan sulphate (HS) concentrations of Escherichia coli– and Cryptosporidium‐infected lambs (data expressed as median and minimum–maximum in parenthesis).

Parameters Lambs with E. coli (n = 39) Lambs with Cryptosporidium (n = 29) p value
NO (µmol/L) 25.86 (11.44–46.35) 28.35 (15.26–57.69) 0.308
HS (ng/mL) 389.24 (221.97–497.08) 403.25 (310.70–551.00) 0.274
Ang‐2 (ng/mL) 5.73 (3.02–7.88) 5.36 (3.17–10.19) 0.584
ADAM15 (ng/mL) 2.76 (2.02–5.93) 2.31 (1.53–3.65) 0.000
Open in a new tab

Note: Statistical significance level was considered p < 0.05.

Correlation analyses revealed that serum NO concentration was positively associated with the degree of dehydration (r = 0.29, p < 0.01), whereas HS concentration demonstrated a negative correlation (r = −0.37, p < 0.01).

4. Discussion

In this study, we investigated the relationship between eGC integrity and vascular inflammation by evaluating the serum concentrations of NO, HS, Ang‐2 and ADAM15 biomarkers in neonatal lambs with diarrhoea. Our results showed that NO concentrations as biomarkers of vascular inflammation were significantly higher in lambs with diarrhoea, but concentrations of the glycocalyx damage biomarkers HS and Ang‐2 were low. This suggests that NO may potentially play a protective and/or anti‐inflammatory role in the inflammatory response and glycocalyx integrity may be preserved.

Neonatal lamb diarrhoea can be caused by various etiological agents. According to the literature, E. coli is predominantly responsible for diarrhoea during the first 4–5 days of life, whereas Cl. perfringens, Rotavirus, Coronavirus, C. parvum and Giardia duodenalis are commonly associated with cases occurring between 5 and 20 days of age. Infections caused by Salmonella spp. and Eimeria spp. are more frequently observed after the 20th day (Davis et al. 2016; Johnson et al. 2017; Brown et al. 2019; Alhallaq and Ok 2025). In the present study, E. coli was detected in 39 and C. parvum in 29 of the 68 neonatal lambs with diarrhoea. Necropsy findings from 11 dead lambs confirmed E. coli infection histopathologically in 6 cases and Cryptosporidium infection in 5 cases. These findings, consistent with previous studies (Davis et al. 2016; Johnson et al. 2017; Brown et al. 2019; Alhallaq and Ok 2025), indicate that E. coli is the primary aetiological agent of neonatal lamb diarrhoea during the first 5 days of life, whereas Cryptosporidium species are the primary aetiological agents between 5 and 20 days of age.

It has been reported that haematologic response varies in cases of neonatal diarrhoea developing due to different aetiologies. Thus, it was reported that there was no significant change in total leukocyte count in experimentally induced cryptosporidiosis cases (Aydoğdu et al. 2019). On the other hand, it was reported that leucocytosis developed in lambs with coccidiosis (Abdel‐Saeed and Salem 2019). In lambs with bacterial diarrhoea, a decrease in leucocyte count was observed (Malik et al. 2013), whereas leucocyte count increased in calves with E. coli–induced diarrhoea (Ok et al. 2020). Alhalaq and Ok (2025) reported significant increases in total leukocyte, monocyte and granulocyte counts in lambs with neonatal diarrhoea. Similarly, in the present study, leucocytosis, monocytosis and granulocytosis developed in neonatal diarrhoea cases, and these findings are considered to reflect the leukocyte response to acute antigenic inflammation (Bangoura et al. 2007; Malik et al. 2013; Eglenti et al. 2020; Elitok 2020; Ok et al. 2020; Durgut and Ok 2023; Alhallaq and Ok 2025).

In this study, although clinical signs of dehydration were evident in lambs with diarrhoea, haematocrit (HCT) levels did not show a statistically significant difference. This discrepancy may be attributed to multiple factors, including alterations in plasma protein concentrations, fluid distribution dynamics and age‐related physiological variability in neonates (Bórnez et al. 2009). Consequently, it can be inferred that HCT alone may not reliably reflect the degree of dehydration and should be interpreted in conjunction with clinical assessments.

Various inflammatory diseases have been reported to cause significant changes in the vascular endothelium and the eGC layer (Reitsma et al. 2007; Yang et al. 2018; Alphonsus and Rodseth 2014; Chelazzi et al. 2015; Butler et al. 2019; Yamaoka‐Tojo 2020; Ider et al. 2024). In this study, inflammation caused by infectious agents (E. coli, C. parvum) and endothelial damage of intestinal vessels in neonatal lambs with diarrhoea were evaluated by eGC biomarkers.

NO is a molecule involved in many physiological and pathophysiological processes such as vascular tone, inflammation and oxidative stress. In the infectious diseases, inducible NO synthase (iNOS) activity induced by pro‐inflammatory cytokines and lipopolysaccharides (LPSs) leads to prolonged high NO production (Herulf et al. 1999; Kukuruzovic et al. 2002; Sowmyanarayanan et al. 2009). Studies in calves with diarrhoea have reported elevated plasma NO concentrations in cases of infectious diarrhoea compared to non‐infectious cases and healthy calves, attributing this increase to local NO production induced by intestinal damage caused by enteropathogens such as Rotavirus, Cryptosporidium and Giardia (Rodríguez‐Díaz et al. 2006; Kojouri et al. 2012; Zarebavani et al. 2017; Gultekin et al. 2019). It has also been reported that dehydration and systemic inflammatory response syndrome (SIRS) with intestinal damage may increase iNOS induction (Sowmyanarayanan et al. 2009; Gultekin et al. 2019). In the present study, serum NO concentrations were significantly higher in neonatal lambs with diarrhoea compared to healthy controls, which is likely due to intestinal damage (Rodríguez‐Díaz et al. 2006; Kojouri et al. 2012; Zarebavani et al. 2017; Gultekin et al. 2019) and the inflammatory response (Gultekin et al. 2019) induced by enteropathogens.

However, a decrease in NO bioavailability is a key sign of endothelial dysfunction, as NO production depends on the integrity of the eGC, and a healthy glycocalyx helps maintain NO concentrations (Mooij et al. 2014; Franceković and Gliemann 2023). Both in vitro and in vivo studies have shown that damage to the eGC reduces NO release from the endothelium (Nieuwdorp et al. 2006; Koo et al. 2013). NO plays an important anti‐inflammatory and vascular protective role by inhibiting platelet aggregation, leucocyte adhesion and the exocytosis of Weibel–Palade bodies (Bartosch et al. 2021). In this study, although reduced NO concentrations were expected as a sign of glycocalyx damage in diarrhoeic lambs, a significant increase was observed. This may reflect enhanced iNOS‐mediated NO production during inflammation (Sowmyanarayanan et al. 2009; Gultekin et al. 2019) or indicate preserved glycocalyx structure and NO's protective effects (Franceković and Gliemann 2023; Bartosch et al. 2021). The low serum concentrations of HS and Ang‐2 observed in this study further support this interpretation.

HS is one of the main structural components of eGC and plays a critical role in maintaining vascular homeostasis (Oshima et al. 2021). However, in inflammatory conditions such as sepsis, plasma concentrations of HS and other components increase as a result of glycocalyx damage (Tang et al. 2017; Yang et al. 2018; Stahl et al. 2020). Furthermore, exogenous administration of NO was found to reduce HS shedding and mitigate glycocalyx damage, supporting its evaluation as an organoprotective agent (Bruegger et al. 2008; Kamenshchikov et al. 2024). In the present study, serum HS concentrations were found to be significantly lower in neonatal lambs with diarrhoea compared to healthy lambs. Low serum HS concentrations in neonatal lambs with diarrhoea may suggest that glycocalyx integrity is preserved. Moreover, increased NO production during inflammation could protect glycocalyx structure and function, thereby limiting HS release into the circulation. Previous studies have reported that dehydration can induce structural damage to the glycocalyx layer (Yamaoka‐Tojo 2020). In the present study, the relationship between dehydration and serum glycocalyx biomarkers was evaluated, revealing a positive correlation between the severity of dehydration and serum NO concentrations and a negative correlation with HS concentrations. These findings suggest that, in lambs with diarrhoea, a physiological response to dehydration may involve elevation of serum NO and reduction of HS concentrations, potentially serving to protect the glycocalyx layer, similar to mechanisms observed in inflammatory processes.

Ang‐2 is a growth factor produced mainly by endothelial cells that plays a critical role in the regulation of vascular homeostasis. Ang‐2, which is stored in Weibel–Palade bodies together with von Willebrand factor (vWF), is rapidly released into circulation in response to various stimuli and acutely regulates vascular processes such as inflammation, thrombosis and vasoconstriction (Rondaij et al. 2006). Ang‐2, which is found at low concentrations under normal physiologic conditions, increases markedly in pathologic conditions such as inflammation and sepsis, and this increase results in elevation of eGC damage biomarkers such as HS (Richter et al. 2022). However, it has been shown that high NO concentrations suppress Ang‐2 release (Yeo et al. 2008), whereas NO inhibitors increase Ang‐2 concentrations (Weinberg et al. 2014). In the present study, serum Ang‐2 concentrations of neonatal lambs with diarrhoea were found to be significantly lower than healthy lambs. These findings may be due to the inhibition of exocytosis of Weibel–Palade bodies (Bhatia et al. 2004; de Jong et al. 2016; Bartosch et al. 2021), which are organelles that store Ang‐2 in endothelial cells, by elevated NO concentration in lambs with diarrhoea. However, studies in patients with COPD and gastric cancer have shown that Ang‐2 concentrations may vary between populations, depending on genetic and environmental factors, and in Asian individuals in particular, Ang‐2 concentrations have been reported to be even lower than in healthy non‐Asian individuals (Cho et al. 2011; Hacker et al. 2016). Considering the differences between species, it is thought that variations in the angiogenic response in lambs with diarrhoea due to genetic or environmental factors may affect Ang‐2 concentrations. These differences may be due to factors such as the response of endothelial cells to inflammatory stimuli, changes in vascular permeability and the development of different mechanisms of the immune system against pathogens.

ADAM15 is a member of the a disintegrin and metalloproteinase (ADAM) family with proteolytic activity in the extracellular region and has been reported to play a role in vascular inflammation (Nakamura et al. 2004). Inflammatory stimuli, especially LPS, may contribute to endothelial barrier disruption and CD44 destruction by increasing ADAM15 activity (Sun et al. 2010; Sun et al. 2013). It has been shown that ADAM15‐deficient mice are resistant to LPS‐induced increase in vascular permeability, and CD44 degradation is reduced in these animals (Yang et al. 2018). CD44 is a transmembrane glycoprotein found in endothelial cells that interacts with hyaluronic acid, an important component of glycocalyx, which can be degraded in inflammation, leading to increased CD44 and HA in circulation (Flynn, Michaud, Madri 2013; Flynn, Michaud, Canosa et al. 2013). In this study, CD44 and hyaluronic acid could not be measured due to the lack of sheep‐specific ELISA kits; instead, ADAM15 levels were assessed. In general, ADAM15 concentrations were not significantly different between diarrhoeic and healthy lambs. However, in subgroup analysis, ADAM15 concentrations were found to be significantly higher in lambs with E. coli infection than those with C. parvum infection. Considering the known interaction between ADAM15 and LPS, a major virulence factor of E. coli (Kukuruzovic et al. 2002), the observed increase in ADAM15 levels is likely attributable to LPS stimulation. These findings suggest that ADAM15 is more specifically associated with the host response to LPS‐mediated bacterial infections rather than with generalized systemic inflammation, thereby supporting its potential role as a biomarker.

This study has certain limitations that should be acknowledged. First, glycocalyx damage was not evaluated through histopathological methods, which would have provided direct structural confirmation. Second, the relatively small sample size may limit the generalizability of the findings. Addressing these limitations in future studies will be essential for a more comprehensive understanding of the role of eGC in neonatal lamb diarrhoea. Furthermore, although biomarkers, such as NO, HS, Ang‐2 and ADAM15, offer valuable insights in experimental settings, their use in routine clinical practice remains limited. Additional research involving various diseases and animal species is warranted to validate these biomarkers and facilitate the development of clinically applicable diagnostic tools.

In conclusion, the findings of this study indicate that NO concentrations increase in neonatal lambs with diarrhoea, whereas indicators of glycocalyx injury, such as HS and Ang‐2, remain unchanged. These insights suggest that NO may play a protective and potentially anti‐inflammatory role in preserving glycocalyx integrity during systemic responses to inflammation and dehydration.

Author Contributions

Murat Kaan Durgut, Mahmut Ok, Merve Ider, Bunyamin Tras and Tugba Melike Parlak made the data curation and contributed to conceptualization, design and methodology. Murat Kaan Durgut, Mahmut Ok, Amir Naseri, Abdulrahman Alhallaq and Suleyman Serhat Iyigun wrote the original draft and contributed to the writing and editing of the manuscript.

Ethics Statement

The study protocol was approved by the Institutional Ethics Committee of the Faculty of Veterinary Medicine, Selcuk University (No. 2024/01).

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.70621

Acknowledgements

This research was supported by Selcuk University Scientific Research Project Office with project number 24401043.

Durgut, M. K. , Ok M., Ider M., et al. 2025. “Evaluation of Endothelial Glycocalyx Damage in Neonatal Lambs With Diarrhoea: A Biomarker‐Based Approach.” Veterinary Medicine and Science 11, no. 6: e70621. 10.1002/vms3.70621

Funding: This research was supported by the Selcuk University Scientific Research Project Office with Project No. 24401043.

Data Availability Statement

All data generated or analysed during the current study are included in this article. The data supporting the findings of this study are available from the corresponding author, M.K.D., upon reasonable request.

References

  1. Abdel‐Saeed, H. , and Salem N. Y.. 2019. “Clinical, Hematologic, Sero‐Biochemical and IgE Response in Lambs With Diarrhea Caused by Eimeria .” International Journal of Veterinary Science 8, no. 1: 10–13. [Google Scholar]
  2. Alhallaq, A. , and Ok M.. 2025. “Evaluation of Selected Intestinal Damage Biomarkers for the Determination of Intestinal Epithelial Damage in Neonatal Lambs With Diarrhea.” Tropical Animal Science Journal 48, no. 2: 132–138. [Google Scholar]
  3. Alphonsus, C. S. , and Rodseth R. N.. 2014. “The Endothelial Glycocalyx: A Review of the Vascular Barrier.” Anaesthesia 69, no. 8: 777–784. [DOI] [PubMed] [Google Scholar]
  4. Aydogdu, U. , Coskun A., Atas A. D., Basbug O., and Agaoglu Z. T.. 2019. “The Determination of Treatment Effect of Chitosan Oligosaccharide in Lambs With Experimentally Cryptosporidiosis.” Small Ruminant Research 180: 27–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bangoura, B. , Daugschies A., and Fuerll M.. 2007. “Influence of Experimental Eimeria zuernii Infection on Clinical Blood Chemistry in Calves.” Veterinary Parasitology 146: 46–53. [DOI] [PubMed] [Google Scholar]
  6. Bartosch, A. M. W. , Mathews R., and Mahmoud M. M.. 2021. “Heparan Sulfate Proteoglycan Glypican‐1 and PECAM‐1 Cooperate in Shear‐Induced Endothelial Nitric Oxide Production.” Scientific Reports 11: 11386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bhatia, R. , Matsushita K., Yamakuchi M., Morrell C. N., Cao W., and Lowenstein C. J.. 2004. “Ceramide Triggers Weibel‐Palade Body Exocytosis.” Circulation Research 95: 319–324. [DOI] [PubMed] [Google Scholar]
  8. Bórnez, R. , Linares M. B., and Vergara H.. 2009. “Haematological, Hormonal and Biochemical Blood Parameters in Lamb: Effect of Age and Blood Sampling Time.” Livestock Science 121, no. 2–3: 200–206. [Google Scholar]
  9. Brown, C. C. , Baker D. C., and Barker I. K.. 2019. “Alimentary System.” In Pathology of Domestic Animals, 6th ed., edited by Jubb K. V. F., Kennedy P. C., and Palmer N., 1–257. Elsevier,. [Google Scholar]
  10. Bruegger, D. , Rehm M., Jacob M., et al. 2008. “Exogenous Nitric Oxide Requires an Endothelial Glycocalyx to Prevent Postischemic Coronary Vascular Leak in Guinea Pig Hearts.” Critical Care 12, no. 3: R73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Butler, M. J. , Ramnath R., Kadoya H., et al. 2019. “Aldosterone Induces Albuminuria via Matrix Metalloproteinase‐Dependent Damage of the Endothelial Glycocalyx.” Kidney International 95, no. 1: 94–107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chelazzi, C. , Villa G., Mancinelli P., De Gaudio A. R., and Adembri C.. 2015. “Glycocalyx and Sepsis‐Induced Alterations in Vascular Permeability.” Critical Care 19: 26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cho, Y. J. , Ma J. E., Yun E. Y., et al. 2011. “Serum Angiopoietin‐2 Levels Are Elevated During Acute Exacerbations of COPD.” Respirology 16, no. 2: 284–290. [DOI] [PubMed] [Google Scholar]
  14. Davis, E. , Machugh N., Kenny D., and Ward P.. 2016. “The Effect of Feeding Colostrum at Different Volumes and Subsequent Number of Transition Milk Feeds on the Serum Immunoglobulin G Concentration and Health Status of Dairy Calves.” Journal of Dairy Science 99, no. 12: 10150–10156. [DOI] [PubMed] [Google Scholar]
  15. de Jong, G. M. , Slager J. J., and Verbon A.. 2016. “Systematic Review of the Role of Angiopoietin‐1 and Angiopoietin‐2 in Plasmodium Species Infections: Biomarkers or Therapeutic Targets?” Malaria Journal 15: 581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Durgut, M. K. , and Ok M.. 2023. “Evaluation of Some Intestinal Biomarkers in the Determination of Intestinal Damage in Calves With Coccidiosis.” Tropical Animal Science Journal 46, no. 2: 221–230. [Google Scholar]
  17. Eğlenti, N. , Kozat S., and Denizhan V.. 2020. “Investigation of Immunoglobulin (IgE, IgA, IgG, IgM) Concentrations in Calves Naturally Infected With Coccidiosis.” Journal of Istanbul Veterinary Sciences 4, no. 1: 1–7. [Google Scholar]
  18. Elitok, B. 2020. “Epidemiology of Coccidiosis and Effects of the Infection on Some Clinical and Hematological Examination Findings in Calves.” Journal of Istanbul Veterinary Sciences 4, no. 1: 21–25. [Google Scholar]
  19. Flynn, K. M. , Michaud M., Canosa S., and Madri J. A.. 2013. “CD44 Regulates Vascular Endothelial Barrier Integrity via a PECAM‐1 Dependent Mechanism.” Angiogenesis 16, no. 3: 689–705. [DOI] [PubMed] [Google Scholar]
  20. Flynn, K. M. , Michaud M., and Madri J. A.. 2013. “CD44 Deficiency Contributes to Enhanced Experimental Autoimmune Encephalomyelitis: A Role in Immune Cells and Vascular Cells of the Blood‐Brain Barrier.” American Journal of Pathology 182, no. 4: 1322–1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Foreyt, W. J. 2013. Veterinary Parasitology Reference Manual. John Wiley & Sons. [Google Scholar]
  22. Franceković, P. , and Gliemann L.. 2023. “Endothelial Glycocalyx Preservation–Impact of Nutrition and Lifestyle.” Nutrients 15, no. 11: 2573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gultekin, M. , Voyvoda H., Ural K., Erdogan H., Balikci C., and Gultekin G.. 2019. “Plasma Citrulline, Arginine, Nitric Oxide, and Blood Ammonia Levels in Neonatal Calves With Acute Diarrhea.” Journal of Veterinary Internal Medicine 33, no. 2: 987–998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hacker, U. , Escalona‐Espinosa L., and Consalvo N.. 2016. “Evaluation of Angiopoietin‐2 as a Biomarker in Gastric Cancer: Results From the Randomized Phase III AVAGAST Trial.” British Journal of Cancer 114: 855–862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Herulf, M. , Svenungsson B., Lagergren A., et al. 1999. “Increased Nitric Oxide in Infective Gastroenteritis.” Journal of Infectious Diseases 180, no. 2: 542–545. [DOI] [PubMed] [Google Scholar]
  26. Higgins, S. J. , De Ceunynck K., Kellum J. A., et al. 2018. “Tie2 Protects the Vasculature Against Thrombus Formation in Systemic Inflammation.” Journal of Clinical Investigation 128, no. 4: 1471–1484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ider, M. , Ceylan C., Naseri A., et al. 2024. “Evaluation of Endothelial Glycocalyx Injury Biomarkers in Feline Hemotropic Mycoplasmosis.” Scientific Reports 14, no. 1: 12931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Johnson, J. L. , Kuskie K. R., Smith B. A., Hostetter J. M., and Evans T. J.. 2017. “Cryptosporidiosis in Neonatal Lambs in the United States.” Journal of Veterinary Diagnostic Investigation 29, no. 1: 87–90. [Google Scholar]
  29. Kamenshchikov, N. O. , Diakova M. L., Podoksenov Y. K., et al. 2024. “Potential Mechanisms for Organoprotective Effects of Exogenous Nitric Oxide in an Experimental Study.” Biomedicines 12, no. 4: 719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kojouri, G. A. , Hassanpour H., Taghavi N., and Taghadosi C.. 2012. “Nitric Oxide Metabolites Status in Calves With Acute and Chronic Diarrhea.” Comparative Clinical Pathology 21, no. 6: 1257–1261. [Google Scholar]
  31. Koo, A. , Dewey C. F. Jr., and Garcia‐Cardena G.. 2013. “Hemodynamic Shear Stress Characteristic of Atherosclerosis‐Resistant Regions Promotes Glycocalyx Formation in Cultured Endothelial Cells.” American Journal of Physiology—Cell Physiology 304: C137–C146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kukuruzovic, R. , Robins‐Browne R. M., Anstey N. M., and Brewster D. R.. 2002. “Enteric Pathogens, Intestinal Permeability and Nitric Oxide Production in Acute Gastroenteritis.” Pediatric Infectious Disease Journal 21, no. 8: 730–739. [DOI] [PubMed] [Google Scholar]
  33. Malik, S. , Kumar A., Verma A. K., Gupta M. K., Sharma S. D., and Rahal A.. 2013. “Haematological Profile and Blood Chemistry in Diarrhoeic Calves Affected With Colibacillosis.” Journal of Animal Health and Production 1, no. 1: 10–14. [Google Scholar]
  34. Mooij, H. L. , Cabrales P., Bernelot Moens S. J., et al. 2014. “Loss of Function in Heparan Sulfate Elongation Genes EXT1 and EXT2 Results in Improved Nitric Oxide Bioavailability and Endothelial Function.” Journal of the American Heart Association 3, no. 6: e001274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nakamura, H. , Suenaga N., Taniwaki K., et al. 2004. “Constitutive and Induced CD44 Shedding by ADAM‐Like Proteases and Membrane‐Type 1 Matrix Metalloproteinase.” Cancer Research 64: 876–882. [DOI] [PubMed] [Google Scholar]
  36. Nieuwdorp, M. , van Haeften T. W., Gouverneur M. C., et al. 2006. “Loss of Endothelial Glycocalyx During Acute Hyperglycemia Coincides With Endothelial Dysfunction and Coagulation Activation In Vivo.” Diabetes 55: 480–486. [DOI] [PubMed] [Google Scholar]
  37. Ok, M. , Yıldız R., Hatipoglu F., et al. 2020. “Use of Intestine‐Related Biomarkers for Detecting Intestinal Epithelial Damage in Neonatal Calves With Diarrhea.” American Journal of Veterinary Research 81: 139–146. [DOI] [PubMed] [Google Scholar]
  38. Oshima, K. , King S. I., McMurtry S. A., and Schmidt E. P.. 2021. “Endothelial Heparan Sulfate Proteoglycans in Sepsis: The Role of the Glycocalyx.” Seminars in Thrombosis and Hemostasis 47: 274–282. [DOI] [PubMed] [Google Scholar]
  39. Parikh, S. M. , Mammoto T., Schultz A., et al. 2006. “Excess Circulating Angiopoietin‐2 May Contribute to Pulmonary Vascular Leak in Sepsis in Humans.” PLoS Medicine 3, no. 3: e46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reitsma, S. , Slaaf D. W., Vink H., Van Zandvoort M. A., and Oude Egbrink M. G.. 2007. “The Endothelial Glycocalyx: Composition, Functions, and Visualization.” Pflügers Archiv—European Journal of Physiology 454: 345–359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Richter, R. P. , Ashtekar A. R., Zheng L., et al. 2022. “Glycocalyx Heparan Sulfate Cleavage Promotes Endothelial Cell Angiopoietin‐2 Expression by Impairing Shear Stress‐Related AMPK/FoxO1 Signaling.” JCI Insight 7, no. 15: e155010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rodríguez‐Díaz, J. , Banasaz M., Istrate C., et al. 2006. “Role of Nitric Oxide During Rotavirus Infection.” Journal of Medical Virology 78, no. 7: 979–985. [DOI] [PubMed] [Google Scholar]
  43. Rondaij, M. , Bierings R., Kragt A., van Mourik J. A., and Voorberg J.. 2006. “Dynamics and Plasticity of Weibel‐Palade Bodies in Endothelial Cells.” Arteriosclerosis, Thrombosis, and Vascular Biology 26: 1002–1007. [DOI] [PubMed] [Google Scholar]
  44. Sowmyanarayanan, T. V. , Natarajan S. K., Ramachandran A., et al. 2009. “Nitric Oxide Production in Acute Gastroenteritis in Indian Children.” Transactions of the Royal Society of Tropical Medicine and Hygiene 103, no. 8: 849–851. [DOI] [PubMed] [Google Scholar]
  45. Stahl, K. , Gronski P. A., Kiyan Y., et al. 2020. “Injury to the Endothelial Glycocalyx in Critically Ill Patients With COVID‐19.” American Journal of Respiratory and Critical Care Medicine 202, no. 8: 1178–1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sun, C. , Beard R. S. Jr., McLean D. L., et al. 2013. “ADAM15 Deficiency Attenuates Pulmonary Hyperpermeability and Acute Lung Injury in Lipopolysaccharide‐Treated Mice.” American Journal of Physiology—Lung Cellular and Molecular Physiology 304, no. 3: L135–L142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sun, C. , Wu M. H., Guo M., Day M. L., Lee E. S., and Yuan S. Y.. 2010. “ADAM15 Regulates Endothelial Permeability and Neutrophil Migration via Src/ERK1/2 Signaling.” Cardiovascular Research 87, no. 2: 348–355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tang, T. H. , Alonso S., Ng L. F., et al. 2017. “Increased Serum Hyaluronic Acid and Heparan Sulfate in Dengue Fever: Association With Plasma Leakage and Disease Severity.” Scientific Reports 7: 46191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thamm, K. , Schrimpf C., Retzlaff J., et al. 2018. “Molecular Regulation of Acute Tie2 Suppression in Sepsis.” Critical Care Medicine 46, no. 9: e928–e936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weinberg, J. B. , Yeo T. W., Mukemba J. P., Florence S. M., Volkheimer A. D., and Wang H.. 2014. “Dimethylarginines: Endogenous Inhibitors of Nitric Oxide Synthesis in Children With Falciparum Malaria.” Journal of Infectious Diseases 210: 913–922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yamaoka‐Tojo, M. 2020. “Vascular Endothelial Glycocalyx Damage in COVID‐19.” International Journal of Molecular Sciences 21, no. 24: 9712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yang, X. , Meegan J. E., Jannaway M., Coleman D. C., and Yuan S. Y.. 2018. “A Disintegrin and Metalloproteinase 15‐Mediated Glycocalyx Shedding Contributes to Vascular Leakage During Inflammation.” Cardiovascular Research 114, no. 13: 1752–1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yeo, T. W. , Lampah D. A., Gitawati R., et al. 2008. “Angiopoietin‐2 Is Associated With Decreased Endothelial Nitric Oxide and Poor Clinical Outcome in Severe Falciparum Malaria.” Proceedings of the National Academy of Sciences of the United States of America 105, no. 44: 17097–17102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zarebavani, M. , Dargahi D., Einollahi N., Dashti N., Safari F., and Rezaeian M.. 2017. “Significance of Nitric Oxide Level in Giardiasis.” Clinical Laboratory 63, no. 1: 47–52. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data generated or analysed during the current study are included in this article. The data supporting the findings of this study are available from the corresponding author, M.K.D., upon reasonable request.

Articles from Veterinary Medicine and Science are provided here courtesy of Wiley

RESOURCES