Host intestinal biomarker identification in a gut leakage model in broilers

Abstract

Intestinal health problems are a major issue in the poultry industry. Quantifiable easy-to-measure biomarkers for intestinal health would be of great value to monitor subclinical intestinal entities that cause performance problems and to evaluate control methods for intestinal health. The aim of the study was to identify host protein biomarkers for intestinal inflammation and intestinal barrier damage. Proteomic analysis was conducted on ileal and colonic content samples of broilers under an experimental gut damage and inflammation model. Effects of the challenge treatment resulted in a worse gut condition based on macroscopic gut appearance (p < 0.0001). Also microscopic changes such as shortening of the villi and increased crypt depth (p < 0.0001) as well as higher infiltration of T-lymphocytes (p < 0.0001) were seen in the duodenal tissue of challenged animals. Several candidate proteins associated with inflammation, serum leakage and/or tissue damage were identified with an increased abundance in intestinal content of challenged animals (p < 0.05). Conversely, brush border enzymes were less abundant in intestinal content of challenged animals (p < 0.05). These candidate biomarkers have potential to be used in the field for detection of gut barrier failure in broilers.

Figure 1

Schematic overview of the protocol of the in vivo challenge model. A total of 360 day-old broiler chicks (Ross 308) were randomly assigned to two groups, a control and a challenge group (9 pens per treatment and 20 broilers per pen). All animals were fed a commercial feed and switched to a wheat (57.5%) based diet supplemented with 5% rye on day 12. From day 12 to 18, all animals from the challenge group received daily 10 mg florfenicol and 10 mg enrofloxacin per kg body weight via the drinking water. After the antibiotic treatment, 1 mL of a bacterial cocktail consisting of E. coli, E. faecalis, L. salivarius and L. crispatus, C. perfringens (netB-) and R. gnavus was given daily by oral gavage from day 19 till 21. On day 20, the animals were administered a coccidial suspension consisting of 60 000 oocysts of E. acervulina and 30 000 oocysts of E. maxima. At day 26, the birds were weighed and necropsy was performed on 3 birds per pen. The duodenal loop was sampled for histological examination and content from ileum and colon was collected for protein extraction.

Figure 2

Macroscopic scoring parameters of intestine from broilers derived from the in vivo gut damage trial. Each dot represents the average score of 3 birds per pen with a total of 9 pens for control (white; n = 27) and challenged (black; n = 27) birds. A Macroscopic gut appearance score was determined by scoring of 10 parameters on assessment of absence (0) or presence (1) resulting in a total score between 0 and 10. B Coccidiosis score was determined by the sum of the individual species scores, 0 if absent to 4 if severe, for lesions caused by Eimeria (E.) acervulina, E. maxima and E. tenella. Asterisk denotes statistical significance of p < 0.0001 between control and challenged animals.

Figure 3

Histological parameters in duodenal sections from birds derived from the in vivo gut damage trial. 12 villi were randomly selected and measured per section and per animal using Leica DM LB2 microscope and a computer based image analysis program, LAS V4.1. Each dot represents the mean of 3 birds per pen with a total of 9 pens for control (white; n = 27) and challenged (black; n = 27) birds. A Villus length was measured from the crypt–villus junction to villus tip. B Crypt depth was measured from the junction to the base. C Villus-to-crypt ratio. D T-lymphocyte infiltration (CD₃ area%) was measured for a total area of 3.5 ± 0.5 mm². Asterisk denotes statistical significance of p < 0.0001 between control and challenge group.

Figure 4

Protein abundances in ileal and colonic content of birds from the in vivo gut damage trial. Proteomic analysis using high performance liquid chromatography–mass spectrometry (HPLC–MS) resulted in detection of proteins with a significant difference between groups in normalized abundance (p < 0.05) in ileal and/or colonic content. Each dot represents 1 bird per pen with a total of 9 pens for control (white; n = 9) and challenged (black; n = 9) animals.

Figure 4

Protein abundances in ileal and colonic content of birds from the in vivo gut damage trial. Proteomic analysis using high performance liquid chromatography–mass spectrometry (HPLC–MS) resulted in detection of proteins with a significant difference between groups in normalized abundance (p < 0.05) in ileal and/or colonic content. Each dot represents 1 bird per pen with a total of 9 pens for control (white; n = 9) and challenged (black; n = 9) animals.