Excreta biomarkers in response to different gut barrier dysfunction models and probiotic supplementation in broiler chickens

Abstract

Increased intestinal permeability (IP) and inflammation are both linked with functionality of the intestinal barrier and in particular enterocytes. Currently, almost all assessment methods of the intestinal barrier function are invasive. The present study aimed to quantify selected proteins as novel biomarkers in excreta of broiler chickens to facilitate non-invasive assessment of gut barrier function using enzyme-linked immunosorbent assays (ELISA). It was further hypothesised that probiotics as feed additives may counteract gut barrier dysfunction. A 3 × 2 factorial arrangement of treatments was used with the main factors being gut barrier dysfunction models (control, rye-based diet, and dexamethasone-DEX) with and without probiotic supplementation (a three-strain Bacillus) using 72 male Ross 308 day-old chickens. Each of the 6 experimental treatments was replicated 12 times. On d 21 of age, fluorescein isothiocyanate dextran (FITC-d) uptake into serum was examined to test IP. Fresh excreta samples were collected on d 20. The biomarkers included alpha-1 antitrypsin (A1AT), intestinal fatty acid binding protein (I-FABP), lipocalin-2 (LCN2), fibronectin (FN), intestinal alkaline phosphatase (IAP), ovotransferrin (OVT) and superoxide dismutase [Cu-Zn] (SOD1). Only DEX increased (P<0.001) FITC-d passage to the blood on d 21 of age, indicating a greater IP. The excreta concentrations of A1AT, I-FABP and SOD1 were unaltered by the experimental treatments. DEX increased (P<0.05) FN concentration in excreta compared with control birds. Conversely, inclusion of rye in the diet reduced (P<0.05) FN but increased (P<0.001) OVT in excreta. Independently, DEX decreased IAP (P<0.05) in excreta compared with control and rye-fed birds. The excreta concentration of LCN2 tended (P = 0.086) to increase in birds injected by DEX. There was no demonstrable effect of probiotic addition on any of the studied parameters. Among the tested biomarkers, FN, IAP, and LCN2 revealed promise as biomarkers of intestinal barrier function quantified by ELISA kits.

Novel Models for Chronic Intestinal Inflammation in Chickens: Intestinal Inflammation Pattern and Biomarkers

“…The ideal biomarker should also detect inflammation consistently in different models, such as physiological and nutritional models ( 13 ); therefore, in the current study, we also analyzed biomarkers previously described in the literature such as LPS, ovotransferrin and lipocalin-2. Lipocalin 2 is an acute phase protein highly produced by the liver ( 56 ) and neutrophils, although the majority of LCN2 found in the intestine is produced by epithelial cells which mostly secrete the protein into the lumen ( 57 ).…”

“…The measuring of serum LPS in chickens is controversial in the literature and it seems to vary according to the intensity of the challenge to which birds are exposed (60)(61)(62). Ovotransferrin, an acute phase protein mainly produced during inflammation, has been suggested as a fecal biomarker of intestinal inflammation due to its increase in excreta of broilers facing necrotic enteritis and coccidiosis (25), or diet with 52% of rye (13). However, in our study, no increase of fecal ovotransferrin was detected in the groups suffering intestinal inflammation, which might occur due to the sub-clinical inflammation observed in the current study compared to the acute and more robust inflammation produced by the other models in the literature.…”

“…Despite the importance of studying chronic gut inflammation (8), there is a lack of a practical model to induce a low-grade chronic response in broilers that would mimic field situations. Among the developed models in poultry species, most produce strong negative effects causing clinical signs such as the reduction in body weight (9)(10)(11)(12)(13). Other models are difficult to replicate or apply in large experiments due to complex methodology (14), including chemicals furnished via drinking water (10), injections (11,13), or pathogen-specific challenge.…”

“…Among the developed models in poultry species, most produce strong negative effects causing clinical signs such as the reduction in body weight ( 9 – 13 ). Other models are difficult to replicate or apply in large experiments due to complex methodology ( 14 ), including chemicals furnished via drinking water ( 10 ), injections ( 11 , 13 ), or pathogen-specific challenge. Therefore, there is a need for an easily replicated research model that will best mimic the intestinal response in the field.…”

“…In addition to establishing a chronic inflammation model, the poultry industry seeks reliable non-invasive biomarker(s) of intestinal inflammation. Several potential biomarkers have already been evaluated in the chicken, such as lipocalin (LCN2), alpha-1 antitrypsin, intestinal alkaline phosphatase, superoxide dismutase, fibronectin ( 13 ), and ovotransferrin ( 24 , 25 ). However, only a few (ovotransferrin, lipocalin and fibronectin) have shown promising but inconsistent results in previous research.…”