Use of intestine-related biomarkers for detecting intestinal epithelial damage in neonatal calves with diarrhea

Mahmut Ok 1Department of Internal Medicine, Faculty of Veterinary Medicine, University of Selçuk, Konya, 42031 Turkey.

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Ramazan Yildiz 5Department of Internal Medicine, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, Burdur, 15030 Turkey.

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Fatih Hatipoglu 2Department of Pathology, Faculty of Veterinary Medicine, University of Selçuk, Konya, 42031 Turkey.

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Nuri Baspinar 3Department of Biochemistry, Faculty of Veterinary Medicine, University of Selçuk, Konya, 42031 Turkey.

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Merve Ider 1Department of Internal Medicine, Faculty of Veterinary Medicine, University of Selçuk, Konya, 42031 Turkey.

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Kamil Üney 4Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Selçuk, Konya, 42031 Turkey.

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Alper Ertürk 6Department of Internal Medicine, Faculty of Veterinary Medicine, University of Mustafa Kemal, Hatay, 31000 Turkey.

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Murat K. Durgut 1Department of Internal Medicine, Faculty of Veterinary Medicine, University of Selçuk, Konya, 42031 Turkey.

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Funda Terzi 7Department of Pathology, Faculty of Veterinary Medicine, University of Kastamonu, Kastamonu, 37200 Turkey.

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Abstract

OBJECTIVE

To evaluate the usefulness of intestinal biomarkers in determining the presence of intestinal epithelial damage in neonatal calves with diarrhea caused by 4 etiologic agents.

ANIMALS

40 neonatal calves that were healthy (n = 10) or had diarrhea (30).

PROCEDURES

The study was a cross-sectional study. Results of hematologic analyses and serum concentrations of intestinal fatty acid–binding protein (I-FABP), liver fatty acid–binding protein (L-FABP), trefoil factor 3 (TFF-3), Claudin-3 (CLDN-3), γ-enteric smooth muscle actin (ACTG2), intestinal alkaline phosphatase (IAP), interleukin-8 (IL-8), platelet-activating factor (PAF), and leptin (LP) were compared among calves grouped according to whether they were healthy (control group; G-1) or had diarrhea caused by K99 Escherichia coli (G-2; n = 10), bovine rota- or coronavirus (G-3; 5 each), or Cryptosporidium spp (G-4; 10).

RESULTS

Across the 3 time points at which blood samples were obtained and evaluated, the groups of calves with diarrhea generally had markedly higher mean serum concentrations of L-FABP, TFF-3, IAP, IL-8, and LP, compared with the control group. In addition, G-2 also consistently had markedly higher mean serum concentrations of I-FAB and ACTG2 and lower mean serum concentrations of CLDN-3, compared with the control group.

CONCLUSIONS AND CLINICAL RELEVANCE

Results indicated that degree of intestinal epithelial damage differed among calves grouped by the etiologic agent of diarrhea and that such damage might have been more severe in calves with diarrhea caused by K99 E coli. Additionally, our results indicated that serum concentrations of I-FABP, L-FABP, TFF-3, IAP, IL-8, ACTG2, LP, and CLDN-3 were useful biomarkers of intestinal epithelial damage in calves of the present study.

Abstract

OBJECTIVE

To evaluate the usefulness of intestinal biomarkers in determining the presence of intestinal epithelial damage in neonatal calves with diarrhea caused by 4 etiologic agents.

ANIMALS

40 neonatal calves that were healthy (n = 10) or had diarrhea (30).

PROCEDURES

The study was a cross-sectional study. Results of hematologic analyses and serum concentrations of intestinal fatty acid–binding protein (I-FABP), liver fatty acid–binding protein (L-FABP), trefoil factor 3 (TFF-3), Claudin-3 (CLDN-3), γ-enteric smooth muscle actin (ACTG2), intestinal alkaline phosphatase (IAP), interleukin-8 (IL-8), platelet-activating factor (PAF), and leptin (LP) were compared among calves grouped according to whether they were healthy (control group; G-1) or had diarrhea caused by K99 Escherichia coli (G-2; n = 10), bovine rota- or coronavirus (G-3; 5 each), or Cryptosporidium spp (G-4; 10).

RESULTS

Across the 3 time points at which blood samples were obtained and evaluated, the groups of calves with diarrhea generally had markedly higher mean serum concentrations of L-FABP, TFF-3, IAP, IL-8, and LP, compared with the control group. In addition, G-2 also consistently had markedly higher mean serum concentrations of I-FAB and ACTG2 and lower mean serum concentrations of CLDN-3, compared with the control group.

CONCLUSIONS AND CLINICAL RELEVANCE

Results indicated that degree of intestinal epithelial damage differed among calves grouped by the etiologic agent of diarrhea and that such damage might have been more severe in calves with diarrhea caused by K99 E coli. Additionally, our results indicated that serum concentrations of I-FABP, L-FABP, TFF-3, IAP, IL-8, ACTG2, LP, and CLDN-3 were useful biomarkers of intestinal epithelial damage in calves of the present study.

Calf deaths during the neonatal period are the most important problem of cattle breeding around the world, and diseases associated with diarrhea are of key importance.1,2 The most common bacterial cause of neonatal calf diarrhea is enterotoxigenic Escherichia coli, which is a major cause of economic loss in calves within the first 5 days after birth.1 In addition, bovine rota- and coronavirus cause diarrhea in neonatal calves following the first week of the life,1,3 and Cryptosporidium parvum and Giardia intestinalis are prominent protozoal causes of diarrhea in neonatal calves.4 Healing processes and mortality rates in calves with enteritis differ on the basis of the underlying infectious agent, and this is believed to be related to the severity of tissue damage in the intestines.

Recently, noninvasive methods, such as intestine-related biomarkers, have been used to determine intestinal injury.5–7 In patients with NEC, important changes in serum concentrations of FABPs, TFF-3, LP, IAP, IL-8, PAF, and ACTG2 have been reported.5,6,8,9 For instance, in neonatal humans, plasma concentrations of L-FABP are high in the initial stage of NEC, whereas plasma concentrations of I-FABP are high in the advanced stage of NEC.10,11 In addition, another study12 shows that concentrations of L-FABP, I-FABP, and TFF-3 in human infants who died from NEC are significantly higher than those concentrations in healthy infants. Trefoil peptides are responsible for gastrointestinal regeneration and mucosal protection. Among these peptides, TFF-3 is crucial in protecting intestinal cells from damage,13 and concentrations of TFF-3 increase secondary to intestinal mucosal damage. Intestinal alkaline phosphatase is also involved in the mucosal defense of the gastrointestinal tract,14 in that the serum concentration of IAP is significantly higher in human infants with NEC, versus without NEC, and that concentration of IAP can be used in early diagnosis of NEC.15 Cytokines are also involved in the pathogenesis of NEC as a result of their release from damaged intestinal epithelial cells and activated neutrophils. The proinflammatory cytokine IL-8 is a potent chemoattractant mediator that causes chemotaxis and neutrophil activation,16,17 and a study6 of human infants shows that serum IL-8 concentrations are significantly higher in patients with NEC, versus without NEC, and that serum concentration of IL-8 might be a possible specific biomarker in the detection of intestinal injury.6 Further, a study16 indicates that PAF and TNF-α concentrations are high in human neonates with NEC and that the plasma concentration of PAF significantly increases as the severity of NEC increases. In addition, CLND-3, a tight junction protein, is a biomarker that can be used to evaluate inflammatory bowel diseases and the disruption of intestinal wall integrity.18

Beyond markers associated with the mucosal layer, intestinal smooth muscle actin plays important roles in the maintenance of cytoskeletons and cell motility in the intestinal smooth muscles, and a study19 of human infants shows that the plasma concentration of intestinal smooth muscle actin positively correlated with the extent of damage to the intestines. In addition, after treatment with LP (a 16-kD protein with anti-ghrelin function that is synthesized by adipose cells and regulates energy metabolism), absorption functions in the small intestines of pigs were positively affected, length of the small intestine was increased, and intestinal villi length was increased.20 Further, exogenous LP was shown to decrease the degree of intestinal tissue injury in rats.21

To our knowledge, biomarkers of intestinal damage in calves with diarrhea caused by various etiologic agents have not been evaluated. Therefore, the purpose of the study presented here was to evaluate the usefulness of intestinal biomarkers in determining the presence of intestinal epithelial damage in neonatal calves with diarrhea caused by 1 of 4 etiologic agents.

Materials and Methods

Animals

The study protocol was approved (2015/65) by the Ethical Committee of the Faculty of Veterinary Medicine at Selçuk University. The study included 40 Holstein calves, 30 with diarrhea (case calves) and 10 without diarrhea (control calves), that were 2 to 20 days of age. The 30 case calves were consecutively admitted to the Large Animals Clinic in the Faculty of Veterinary Medicine at Selçuk University from client-owned farms for treatment of diarrhea caused by K99 E coli (G-2; n = 10), bovine rota- or coronavirus (G-3; 5 each), or Cryptosporidium spp (G-4; 10) between February 2, 2016, and February 2, 2018. Before enrollment in the study, routine clinical examinations of calves were performed and included rectal temperature; respiratory and heart rates; hematologic analyses (hemogram and blood gases analysis); fast antigen testinga of feces to detect the presence of bovine rota- and corona virus, K99 E coli, Cryptosporidium, or Giardia antigens, alone or in combination; and hydration status. The 10 control calves included in the study were from the experimental farm of the Faculty of Veterinary Medicine. Calves with no obvious diarrhea, hematologic results within reference limits, and negative results on tests to detect pathogen antigens in feces were included in the control group (G1). We preferred to include only control calves from the university farm because we were familiar with the herd health status and also performed daily clinical, fecal, and hematologic examinations on the control calves.

Diarrheal fecal samples with positive results on the fast antigen test for the presence K99 E coli, bovine rotavirus, or bovine coronavirus antigen (alone) were then tested with ELISAsb,c to confirm the diagnosis. Diarrheal fecal samples with positive results on the fast antigen test for the presence of Cryptosporidium spp were also examined by light microscopy for confirmation of the diagnosis. Calves with diarrhea in which > 1 infectious agent was identified on fast antigen testing of feces were excluded from the study. Calves with diarrhea and 8% to 10% dehydration22 were included in the study.

Blood sample collection and analyses

Blood samples from case calves were collected from a jugular vein at the onset of treatment (hour 0) and then 24 and 48 hours later. Blood samples from the control calves were taken from the jugular vein after the clinical and fecal examinations (hour 0) and then 24 and 48 hours later. Blood was collected into heparinized syringes for blood gas measurements, into evacuated blood collection tubes coated on the interior with spray-dried K3EDTA for CBCs, and into tubes containing no anticoagulant for measurements of intestinal biomarkers. Blood gas measurements and CBCs were performed with a blood gas analyzerd and an automated hematologic analyzer,e respectively, within 5 to 15 minutes after blood samples were obtained. For measurements of intestinal biomarkers, the blood samples were centrifuged at 4,200 × g at 4°C for 10 minutes within 15 minutes of sample collection, and resulting sera were stored at −80°C until the intestinal biomarkers were measured at the end of the study period. Serum concentrations of I-FABP, L-FABP, TFF-3, CLDN3, ACTG2, IAP, IL-8, PAF, and LP at hours 0, 24, and 48 were measured with ELISA kitsf–n according to the manufacturer's instructions.

Standard treatment protocol

Case calves were provided standard treatment for diarrhea, including administration of fluid therapy, antimicrobials, hyperimmune serum, and supportive treatment. Results of blood gas analyses were used to calculate the base deficit, and isotonic (1.3%) NaHCO3o with 5% dextrosep in sterile water was administered according to the calculated need. In addition, colloid fluidq (6% hydroxyethyl starch in saline [0.9 % NaCl] solution; 20 mL/kg/h) was administered as needed, and calves with low oxygen saturation of hemoglobin received oxygen therapy intranasally with a flow rate of 5 to 6 L/min for 15 minutes through a calf oxygen mask. When warranted, case calves received ceftiofurr (2.2 mg/kg, IM, q 24 h for 5 days), and case calves with Cryptosporidium infections received halofuginones (0.1 mg/kg [2 mL/10 kg live weight], PO, q 24 h for 7 days). As a supportive treatment, all case calves received vitamin Ct (3 mL, SC, q 24 h for 3 days) and a vitamin A, D3, and E combinationu (1 mL [500,000 U of vitamin A, 75,000 U of vitamin D3, and 50 mg of vitamin E], IM, once). Case calves with K99 E coli diarrhea received hyperimmune serumv (15 mL, SC, once). Case calves were fed milk (1.5 to 2 L) twice daily by stomach tube before the suckle reflex occurred and by bottle afterwards.

Histologic examination

Any calves that died were submitted for necropsy. Routinely, tissues were fixed in neutral-buffered 10% formalin, embedded in paraffin, and then manually sectioned with a microtome to 5-μm-thick paraffin sections. Dewaxed sections were then stained with H&E stain23 and evaluated with light microscopy. Immunohistochemical stainingw was performed according to the manufacturer's instructions. Proteinase K was used in the antigen retrieval process, with mouse anti-bovine rotavirus antibody and mouse anti-bovine coronavirus antibody as the primary antibodies and 3,3′-diaminobenzidine as the chromogen.

Statistical analysis

The Kolmogorov-Smirnov test was used to determine whether data were normally distributed. Data with normal distribution were expressed as the mean ± SEM, whereas data without normal distribution were expressed as the median and range. To evaluate differences in results among the groups (G-1, G-2, G-3, and G-4), 1-way ANOVA and the Duncan post hoc test were used with normally distributed data and the Kruskal-Wallis test was used with nonnormally distributed data. Statistical softwarex was used, and values of P < 0.05 were considered significant.

Results

Animals

Forty Holstein calves (10 control calves and 30 case calves) were included in the study. The 10 control calves (G-1) consisted of 4 bull calves and 6 heifer calves with a mean ± SD body weight of 47.1 ± 1.4 kg and a mean ± SD age of 6.7 ± 0.9 days. Overall, the 30 case calves (G-2, G-3, and G-4) consisted of 12 bull calves and 18 heifer calves with a mean ± SD body weight of 49.1 ± 0.8 kg and a mean ± SD age of 6.6 ± 0.5 days (range 2 to 20 days). The mean ± SD age was 2.7 ± 0.3 days (range, 2 to 4 days) for the 10 calves with K99 E coli diarrhea (G-2), 7.3 ± 0.6 days (range, 5 to 20 days) for t he c al ve s with rota-or coronavirus diarrhea (G-3), and 9.8 ± 0.7 days (range, 5 to 20 days) for the 10 calves with Cryptosporidium diarrhea (G-4)

Clinical results

Before treatment, abnormal clinical signs in the case calves included fever, tachycardia, tachypnea, anorexia, signs of depression, difficulty in standing or maintaining sternal recumbency, low suckle reflex, low skin elasticity, eyes recessed into orbits, dehydration, and watery feces that were green or yellow. After 24 hours of hospitalization and treatment, case calves improved in that they no longer had signs of depression, dehydration, weakness, or difficulty rising and their suckle reflex and appetite had noticably increased. Also after 24 hours of treatment, fecal consistency had improved. At 48 hours after treatment was initiated, 28 of the 30 case calves were standing, seemed environmentally aware, and had increased appetites, good suckling reflexes, and clinically normal fecal consistencies. These 28 case calves were then discharged. Of the 2 remaining case calves (both from G-3), 1 with bovine rotavirus infection had feces with clinically normal consistency after 72 hours of hospitalization and was discharged, whereas 1 with bovine coronavirus infection did not respond to treatment and died during hospitalization (36 hours).

Hematologic and blood gases analyses findings

At hours 0 and 24, the mean blood pH and mean base excess were significantly (P < 0.05) higher in control calves (G-1) than in all groups of case calves (G-2, G-3, and G4; Table 1). However, at hour 48, only G-4 had markedly lower mean blood pH than all other groups.

Table 1—

Results of hematologic analysis, stratified by hour of sampling, in 10 healthy neonatal calves (control calves; G-1) versus 30 neonatal calves (case calves) with diarrhea caused K99 E coli (G-2; n = 10), bovine rota- or coronavirus (G-3; 5 each), or Cryptosporidium ssp (G-4; 10) between February 2, 2016, and February 2, 2018.

  Sample collection time (h)
VariableGroup02448
pHG-17.40 ± 0.01a7.39 ± 0.01a7.39 ± 0.01a
 G-27.15 ± 0.04b7.31 ± 0.04b7.36 ± 0.02a
 G-37.03 ± 0.04c7.30 ± 0.02b7.30 ± 0.03b
 G-47.14 ± 0.05bc7.30 ± 0.03b7.37 ± 0.01a
Pco2 (mm Hg)G-146.1 ± 1.7247.3 ± 1.1345.3 ± 1.74
 G-249.8 ± 2.4446.0 ± 1.9845.6 ± 2.71
 G-347.3 ± 4.7445.7 ± 2.0848.3 ± 2.02
 G-444.5 ± 6.4247.6 ± 1.7048.3 ± 2.60
Po2 (mm Hg)G-133.5 ± 3.33a30.9 ± 2.34a28.7 ± 2.21a
 G-219.6 ± 1.73b23.5 ± 1.45b24.8 ± 1.91ab
 G-325.6 ± 2.72b23.7 ± 2.39b22.2 ± 2.18b
 G-424.0 ± 2.74b25.7 ± 1.60ab22.8 ± 1.11b
Potassium (mmol/L)G-14.36 ± 0.08b4.42 ± 0.174.51 ± 0.13ab
 G-25.15 ± 0.17b4.71 ± 0.175.23 ± 0.59a
 G-36.43 ± 0.63a4.72 ± 0.445.02 ± 0.41ab
 G-45.36 ± 047ab4.09 ± 0.244.05 ± 0.20b
Lactate (mmol/L)G-11.47 ± 0.14b1.34 ± 0.181.73 ± 0.33
 G-26.45 ± 0.79a2.49 ± 0.401.77 ± 0.12
 G-35.04 ± 1.29ab2.02 ± 0.382.12 ± 0.63
 G-46.15 ± 2.22a2.16 ± 0.502.08 ± 0.65
BE (mmol/L)G-13.8 ± 0.76a4.04 ± 0.74a2.57 ± 0.76a
 G-2−10.7 ± 1.92b−2.31 ± 2.10b0.80 ± 1.52ab
 G-3−17.4 ± 2.13b−2.92 ± 2.08b−2.30 ± 1.69b
 G-4−11.3 ± 3.55b−2.34 ± 1.94b2.96 ± 1.23a
Spo2 (%)G-162.8 ± 4.49a61.6 ± 3.6858.7 ± 4.27
 G-232.9 ± 5.03b51.7 ± 4.8358.1 ± 4.40
 G-340.5 ± 547b49.9 ± 6.0946.7 ± 6.84
 G-447.4 ± 8.08ab55.9 ± 3.6052.6 ± 2.91
WBC (× 103/|iL)G-110.3 ± 0.56b10.4 ± 0.42b11.0 ± 1.93
 G-223.7 ± 2.99a20.0 ± 3.15a14.5 ± 1.99
 G-319.5 ± 6.36ab15.4 ± 2.73ab11.8 ± 1.69
 G-419.5 ± 2.68ab14.5 ± 2.65ab16.5 ± 2.16
RBC (× 106/|iL)G-110.0 ± 0.2710.0 ± 0.549.7 ± 0.42
 G-211.5 ± 0.599.4 ± 0.428.9 ± 0.43
 G-311.1 ± 0.869.7 ± 0.499.6 ± 0.58
 G-410.2 ± 0.639.0 ± 0.609.1 ± 0.54
Hct (%)G-132.2 ± 1.30b32.2 ± 2.3033.2 ± 1.67
 G-243.3 ± 2.52a35.9 ± 2.2633.4 ± 2.18
 G-341.3 ± 3.87a36.6 ± 2.5835.7 ± 2.89
 G-437.2 ± 3.19ab32.8 ± 2.8533.1 ± 2.77
Platelets (× 103/|lL)G-1751 ± 86.1a774 ± 99.1a447 ± 55.7
 G-2366 ± 96.4b384 ± 82.0b391 ± 67.7
 G-3483 ± 113ab469 ± 101b385 ± 65.0
 G-4509 ± 62.8ab525 ± 36.8b510 ± 32.8

Data are reported as mean ± SEM.

BE = Base excess. HB = Hemoglobin. Spo2 = Oxygen saturation as measured by pulse oximetry.

Within a column, values with different superscripts differ significantly (ANOVA and post hoc Duncan test; P < 0.05).

Intestinal biomarkers

The intra-assay and interassay coefficients of variation were determined according to the manufacturer's instructions. The intra-assay coefficient of variation, interassay coefficient of variation, and minimum detection concentration, respectively, were ≤ 8%, ≤ 12%, and 0.05 ng/mL for I-FA BP; ≤ 15%, ≤ 15%, and 0.05 ng/mL for L-FABP; ≤ 5.9%, ≤ 7.1%, and < 0.26 ng/mL for TFF-3; ≤ 8%, ≤ 8%, and < 0.188 ng/mL for CLDN-3; between 5.5% and 6.4%, between 6.7% and 7.9%, and 0.1 μg/mL for ACTG2; ≤ 8%, ≤ 8%, and < 0.94 ng/mL for IAP; ≤ 8%, ≤ 12%, and 5 pg/mL for IL-8; ≤ 8%, ≤ 12%, and 12 pg/mL for PAF; and ≤ 8%, ≤ 8%, and 0.19 ng/mL for LP.

The mean serum concentration of ACTG2 was significantly (P < 0.05) higher for G-2 (case calves with K99 E coli diarrhea) than for all other groups across all 3 time points in the present study (Table 2). The mean serum concentration of CLDN-3 was significantly lower for case calves with K99 E coli diarrhea (G-2) versus the control group (G-1) at all 3 evaluation points but did not differ substantially among the groups of case calves (G-2, G-3, and G-4).

Table 2—

Results of analysis of serum concentrations of potential biomarkers for intestinal damage, stratified by hour of sampling, in the groups of calves described in Table 1.

  Sample collection time (h)
BiomarkerGroup02448
ACTG2 (μg/mL)G-111.8 ± 0.67b12.9 ± 0.78b11.6 ± 0.58b
 G-217.6 ± 0.73a16.9 ± 0.77a15.9 ± 0.91a
 G-311.6 ± 1.30b12.9 ± 1.12b12.8 ± 0.75b
 G-412.6 ± 1.03b11.6 ± 0.85b12.1 ± 0.78b
CLND-3 (ng/mL)G-16.60 ± 0.71a6.39 ± 0.76a8.01 ± 1.15a
 G-24.69 ± 044b4.19 ± 046b4.76 ± 045b
 G-36.24 ± 047ab5.43 ± 042ab4.44 ± 046b
 G-45.25 ± 049ab4.99 ± 047ab4.83 ± 0.60b
L-FABP (ng/mL)G-112.1 ± 0.59b11.0 ± 0.70c10.9 ± 0.87c
 G-214.7 ± 2.14b17.0 ± 1.71b18.0 ± 1.41b
 G-320.9 ± 1.56a18.1 ± 1.15ab16.5 ± 0.91b
 G-419.7 ± 0.75a21.6 ± 1.09a23.3 ± 1.33a
I-FABP (ng/mL)G-10.21 ± 0.01b0.20 ± 0.01b0.21 ± 0.01b
 G-20.47 ± 0.07a0.52 ± 0.13a0.40 ± 0.07a
 G-30.29 ± 0.01b0.30 ± 0.01b0.29 ± 0.01ab
 G-40.29 ± 0.02b0.28 ± 0.01b0.28 ± 0.02ab
IL-8 (pg/mL)G-116.3 ± 0.20c16.1 ± 0.11c16.2 ± 0.12c
 G-259.0 ± 9.71a46.4 ± 7.0648.2 ± 6.93a
 G-340.5 ± 4.60b39.8 ± 3.67ab33.4 ± 3.97b
 G-430.6 ± 4.99bc32.4 ± 3.78b29.9 ± 3.88b
PAF (pg/mL)G-142.0 ± 1.14c40.2 ± 147c42.8 ± 2.01b
 G-2222 ± 22.60a201 ± 25.90a188 ± 23.50
 G-3169 ± 9.63b174 ± 6.89ab164 ± 8.95a
 G-4154 ± 15.80b151 ± 1340b140 ± 19.90a
IAP (ng/mL)G-13.47 ± 0.27b3.49 ± 0.24b3.7 ± 0.26b
 G-211.2 ± 0.58a12.0 ± 0.94a11.7 ± 0.73a
 G-310.5 ± 0.80a13.5 ± 0.39a12.1 ± 0.57a
 G-412.3 ± 0.62a12.0 ± 0.81a13.3 ± 0.79a
LP (ng/mL)G-12.44 ± 045b1.79 ± 0.35b2.18 ± 0.53b
 G-25.15 ± 0.28a4.43 ± 0.45a4.29 ± 0.60a
 G-34.27 ± 0.31a3.99 ± 0.21a4.22 ± 0.28a
 G-44.12 ± 0.51a3.96 ± 0.42a3.84 ± 0.49a
TFF-3 (ng/mL)*G-11.77 (1.09–2.24)b1.70 (1.12–2.49)b1.52 (0.92–1.74)b
 G-25.94 (4.34–15.90)a4.31 (3.32–16.6)a5.80 (3.32–10.70)a
 G-35.08 (2.96–10.70)a4.34 (3.34–8.65)a4.88 (3.50–12.60)a
 G-45.48 (3.71–9.16)a4.72 (2.38–27.5)a4.23 (3.54–14.60)a

Data are reported as mean ± SEM.

Data are reported as median and range.

Within a column, values with different superscripts differ significantly (ANOVA and post hoc Duncan test; P < 0.05).

The mean serum concentration of L-FABP was significantly (P < 0.05) higher for G-3 and G-4 versus G-1 and G-2 at hour 0; however, by hour 48, all groups of case calves had significantly higher mean concentrations of L-FAB than did the control group (G-1; Table 2). The mean serum concentration of I-FAB was significantly higher for G-2 than for all other groups at hours 0 and 24 and was significantly higher for all groups of case calves (G-2, G-3, and G-4) versus the control group (G-1) at hour 48.

The mean serum concentrations of IL-8, PAF, IAP, LP, and TFF-3 were significantly (P < 0.05) higher for each case group versus the control group at all 3 time points of evaluation in the study (Table 2). In addition, mean concentrations of IL-8 at hours 0 and 48 and mean concentration of PAF at hour 0 were significantly higher for G-2, compared with G-3 and G-4.

Postmortem findings

A necropsy was perfomed on the single calf that died. This calf was from G-3 and was determined on the basis of results from the fast antigen testing and ELISA to have had diarrhea caused by bovine coronavirus. Findings in the jejunum included degeneration of villus epithelium and desquamation associated with atrophy, necrotic tissues in the lumen, severe desquamation in crypt epithelium, luminal dilatation, severe cellular infiltration in the lamina propria, and an increase in the stromal tissue. In the mucosa of the ileum and colon, villus epithelium desquamation, severe cellular infiltration in the lamina propria, and moderate depletion in Peyer patches were detected. Results of immunohistochemical staining of tissue sections indicated the presence of bovine rotavirus antigen in the villus epithelium of the jejunum, ileum, and colon and the presence of bovine coronavirus antigen in Peyer patches and the villus epithelium of the jejunum, ileum, and colon. Villous atrophy in the calf was possibly caused by combined infection with bovine rota- and coronavirus. Hematologic and biomarker data from this calf were included in the analyses.

Discussion

To our knowledge, the present study was the first to evaluate intestine-specific biomarkers that have been investigated in human patients with NEC and enterocolitis5,6,8,9,12,14,18,19 to determine whether they could be used to assess intestinal damage caused by infection with K99 E coli, bovine rota- or coronavirus, or Cryptosporidium spp in neonatal calves. We believe that use of plasma biomarkers with high sensitivity and specificity for the detection of diseases associated with high mortality rates, such as acute mesenteric ischemia and necrotizing enterocolitis, is important for early diagnosis and treatment.

Our findings indicated that the etiologic agents we investigated caused different degrees of intestinal damage and that differences observed in case calves with K99 E coli diarrhea (those in G-2) were more prominent than the differences in case calves with viral (G-3) or protozoal (G-4) infections. For instance, the mean serum concentration of ACTG2 was significantly higher for G-2 than for all other groups across all 3 time points at which blood samples were obtained in the present study. Similarly, the mean serum concentration of CLDN-3 was significantly lower for G-2 than for G-1 (the control group) at all 3 evaluation points but did not differ substantially among the groups of case calves (G-2, G-3, and G-4). In addition, the mean serum concentration of I-FAB was significantly higher for G-2 than for all other groups at hours 0 and 24 and was significantly higher for all groups of case calves (G-2, G-3, and G-4) versus the control group (G-1) at hour 48. Further, although the mean serum concentrations of IL-8, PAF, IAP, LP, and TFF-3 were significantly (P < 0.05) higher for each group of case calves (G-2, G-3, and G-4) versus the control group (G-1) at all 3 evaluation points in the present study, G-2 had significantly higher mean concentrations of IL-8 at hours 0 and 48 and higher mean concentration of PAF at hour 0, compared with G-3 and G-4.

These findings indicated that intestinal damage might have been more severe in the calves with K99 E coli diarrhea (G-2) in the present study. In addition and consistent with earlier reports,7,12,18,19 our findings supported the usefulness of I-FABP, L-FABP and TFF-3 as biomarkers for intestinal epithelial damage in calves.

A study19 of rats shows that serum concentrations of ACTG2 and I-FABP were higher in rats with intestinal damage from ischemic reperfusion injury, compared with controls, and serum concentration of ACTG2 increased owing to intestinal muscle damage. In the present study, only calves with K99 E coli diarrhea (G-2) had significantly higher mean serum concentrations of ACTG2 than the control group (G-1) at all 3 time points of sampling; however, G-2 also had the highest mean serum concentration of I-FABP of all groups at each evaluation point.

An important intestinal defense mechanism, IAP released from apical microvilli during enterocyte damage is critical in the prevention of bacterial translocation,24 detoxification of endotoxin lipopolysaccharides, maintenance of the normal homeostasis of the intestinal microbiota, and inhibition of bacterial translocation through the intestinal mucosal barrier.25,26 In the present study, the mean serum concentration of IAP was substantially higher for the groups of case calves (G-2, G-3, and G-4) versus the control group (G-1) at all 3 evaluation points. We believe this difference was attributed to the defensive release of IAP from enterocytes in the case calves of the present study. Similarly, a recent study7 shows that a high serum concentration of IAP in calves may be an indicator of intestinal damage, as observed in calves with atresia coli.

Studies show that CLND-3 expression is lower in humans with inflammatory bowel diseases27,28 or acute colitis.29 In addition, a study18 of human neonates indicates that urinary concentration of CLND-3 is lower in those with inflammatory bowel diseases when their intestinal wall integrity is impaired. Similarly, our findings that the mean serum concentration of CLND-3 was significantly lower in case calves with K99 E coli diarrhea (G-2) versus control calves (G-1) at all 3 evaluation points and was substantially lower in all groups of case calves (G-2, G-3, and G-4) versus control calves at hour 48 further suggested that intestinal damage may have been greater in the group of calves with K99 E coli diarrhea versus the groups of calves with either bovine rota- or coronavirus (G-3) or Cryptosporidium (G-4) infection.

High serum concentrations of PAF are observed in allergic reactions, sepsis, and enterocolitis, and plasma and fecal concentrations of PAF are higher in human neonates with NEC.30,31 In addition, a previous study32 shows that the plasma concentration of PAF was significantly higher in calves with suspected septicemia, compared with clinically normal calves. In addition, PAF and TNF-α are important mediators of intestinal necrosis.16 Cytokines, such as TNF-α and IL-8, are released from damaged intestinal epithelial cells and activated neutrophils, resulting in necrotizing enterocolitis. In humans, when compared with controls, infants with NEC have higher serum concentrations of IL-8,6 and patients with Crohn disease or ulcerative colitis have higher intestinal mucosal concentrations of IL-8.33 Likewise, our findings indicated that the mean serum concentrations of IL-8 and PAF were significantly higher for all groups of case calves versus the control group across all time points sampled, except at hour 0 for G-4 (case calves with Cryptosporidium infection), which had a higher but not a significantly higher mean serum concentration of IL-8 than did the control group. Further, the mean serum concentrations of PAF and IL-8 were highest in G-2 (case calves with K99 E coli diarrhea), and this may have been because of the development of enteritis and sepsis.32

A study34 of intestinal motility in cats shows that LP increases intestinal contractions in the presence of cholecystokinin. One published study35 indicates that mucosal concentration of LP is higher in human patients with irritable bowel syndrome associated with diarrhea, whereas another study36 indicates that serum concentration of LP is lower in patients with irritable bowel syndrome and that low serum concentration of LP might cause irritable bowel syndrome. There is also a study37 that indicates serum concentration of LP does not differ substantially between healthy children and children with Giardia or Entamoeba infection. Our findings of higher mean serum concentration of LP for all groups of case calves, compared with the control group, at all 3 evaluation points could have been attributed to a high serum concentration of LP and may be associated with the healing of the intestinal injury as in mice with mesenteric ischemia21 and with the known functions of LP in mucosal defense.35,38

A single case calf died during the study period, and the results of histologic examination and immunohistochemical staining were similar to those previously observed with bovine coronavirus39 and bovine rotavirus40 infections in calves. For the calf that died in the present study, villous atrophy was considered a crucial histopathologic finding caused by combined infection with bovine rota- and coronavirus.

A limitation of the present study was the inability to determine the mucosal concentrations of the evaluated biomarkers (I-FABP, L-FABP, TFF-3, IAP, IL-8, ACTG2, LP, and CLDN-3) in various parts of the intestine. In addition, the variation in age ranges for the groups of calves was a limitation in that the case calves with K99 E coli diarrhea (G-2) were younger than the case calves with bovine rota- or coronavirus (G-3) or Cryptosporidium (G-4) infection. However, to our knowledge, there is no information in the literature supporting age-related differences in intestinal markers in calves. Also, a study41 of mice indicates that neither age nor sex influence intestinal barrier markers.

Results indicated that the degree of intestinal damage differed among calves grouped by the underlying cause of diarrhea and that the damage could have been more severe in calves with K99 E coli infections, which we concluded may have been related to the development of enteritis and sepsis in calves with K99 E coli infections. Additonally, our results indicated that serum concentrations of I-FABP, L-FABP, TFF-3, IAP, IL-8, ACTG2, LP, and CLDN-3 were useful biomarkers of intestinal damage in calves of the present study.

Acknowledgments

Funded by The Scientific and Technological Research Council of Turkey (TUBITAK; project No. 1160456).

The authors declare that there were no conflicts of interest.

ABBREVIATIONS

ACTG2

γ-Enteric smooth muscle actin

CLDN-3

Claudin-3

IAP

Intestinal alkaline phosphatase

I-FABP

Intestine fatty acid–binding protein

IL-8

Interleukin-8

L-FABP

Liver fatty acid–binding protein

LP

Leptin

NEC

Necrotic enterocolitis

PAF

Platelet-activating factor

TFF-3

Trefoil factor 3

TNF

Tumor necrosis factor

Footnotes

a.

Rapid BoviD-5 Ag Test Kit (RC13–02DD), BioNote Inc, Gyeonggi-do, Korea.

b.

Monoscreen AgELISA bovine rotavirus, BIO K 343, Bio-X Diagnostics, Rochefort, Belgium.

c.

Monoscreen AgELISA bovine coronavirus, BIO K 344, Bio-X Diagnostics, Rochefort, Belgium.

d.

ABL90 FLEX, Radiometer Medicals ApS, Brønshøj, Denmark.

e.

MS4e, CFE 279, Melet Schloesing Laboratories, Osny, France.

f.

Bovine iFABP ELISA, No. MBS2609312, MyBioSource, San Diego, Calif.

g.

Bovine L-FAB ELISA, No. MBS035016, MyBioSource, San Diego, Calif.

h.

Bovine intestinal trefoil factor ELISA, No. MBS2886396, MyBioSource, San Diego, Calif.

i.

Bovine claudin-3 ELISA, No. MBS7606198, MyBioSource, San Diego, Calif.

j.

Bovine ACTG2 ELISA, No. MBS7215539, MyBioSource, San Diego, Calif.

k.

Bovine ALPI ELISA, No. MBS7606402, MyBioSource, San Diego, Calif.

l.

Bovine IL-8 ELISA, No. MBS2609350, MyBioSource, San Diego, Calif.

m.

Bovine PAF ELISA, No. MBS2609609, MyBioSource, San Diego, Calif.

n.

Bovine LEP ELISA, No. MBS7606202, MyBioSource, San Diego, Calif.

o.

Bikarvil, Vilsan Veterinary Pharmaceuticals Corp, Istanbul, Turkey.

p.

Pro-Fleks 5% dextrose solution, Çetinkaya Pharmaceuticals Industry and Trade Inc, Bolu, Turkey.

q.

Voluven, Fresenius Kabi Deutschland GmbH, Germany.

r.

Excenel, Zoetis Animal Health Co Ltd, Istanbul, Turkey.

s.

Halocur, Intervet Veterinary Medicine Co Ltd, Istanbul, Turkey.

t.

Vetoquinol Vita-C, Primo Veterinary Medicines Trading LLC, Lure, France.

u.

ADEMiN, Ceva Animal Health Inc, Istanbul, Turkey.

v.

Septicol, Vetal Animal Health Products Inc, Gaziantep, Turkey.

w.

Novolink Max Polymer Detection System (RE7280-K), Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, England.

x.

SPSS Statistics, version 21.0 for Windows, IBM Corp, Ar monk, N Y.

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