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

Abstract

For poultry producers, chronic low-grade intestinal inflammation has a negative impact on productivity by impairing nutrient absorption and allocation of nutrients for growth. Understanding the triggers of chronic intestinal inflammation and developing a non-invasive measurement is crucial to managing gut health in poultry. In this study, we developed two novel models of low-grade chronic intestinal inflammation in broiler chickens: a chemical model using dextran sodium sulfate (DSS) and a dietary model using a high non-starch polysaccharide diet (NSP). Further, we evaluated the potential of several proteins as biomarkers of gut inflammation. For these experiments, the chemical induction of inflammation consisted of two 5-day cycles of oral gavage of either 0.25mg DSS/ml or 0.35mg DSS/ml; whereas the NSP diet (30% rice bran) was fed throughout the experiment. At four times (14, 22, 28 and 36-d post-hatch), necropsies were performed to collect intestinal samples for histology, and feces and serum for biomarkers quantification. Neither DSS nor NSP treatments affected feed intake or livability. NSP-fed birds exhibited intestinal inflammation through 14-d, which stabilized by 36-d. On the other hand, the cyclic DSS-treatment produced inflammation throughout the entire experimental period. Histological examination of the intestine revealed that the inflammation induced by both models exhibited similar spatial and temporal patterns with the duodenum and jejunum affected early (at 14-d) whereas the ileum was compromised by 28-d. Calprotectin (CALP) was the only serum protein found to be increased due to inflammation. However, fecal CALP and Lipocalin-2 (LCN-2) concentrations were significantly greater in the induced inflammation groups at 28-d. This experiment demonstrated for the first time, two in vivo models of chronic gut inflammation in chickens, a DSS and a nutritional NSP protocols. Based on these models we observed that intestinal inflammation begins in the upper segments of small intestine and moved to the lower region over time. In the searching for a fecal biomarker for intestinal inflammation, LCN-2 showed promising results. More importantly, calprotectin has a great potential as a novel biomarker for poultry measured both in serum and feces.

Keywords: DSS; ISI index; calprotectin; gut health; lipocalin; models of intestinal inflammation.

Figure 1

Experimental timeline. Male by-product day-of-hatch chickens were divided in four experimental treatments and raised up to 36 days. The broilers assigned to treatments with DSS challenge received 0.25mg/ml or 0.35mg/ml of DSS via oral gavage everyday from 9 to 14-d and 23 to 27-d (induction phase period represented with red). After each DSS cycle animals were allowed to a 9 days recovery period (from 14 to 23-d and 27 to 36-d, marked in green). Birds in the NSP treatment received a diet with 30% of rice bran from 1 to 36-d. Animals in the control group were not submitted to any challenge. Necropsies were performed on days 14, 22, 28 and 36 for collection of intestinal tissue, blood, and fecal samples.

Figure 2

I See Inside (ISI) total microscopically lesions scores of duodenum of broilers submitted to different intestinal challenges at 14, 22, 28 and 36 days of age. The broilers challenged with DSS received 0.25mg/ml (25DSS) or 0.35mg/ml (35DSS) of DSS via oral gavage everyday from 9 to 14-d and 23 to 27-d; birds in the NSP treatment received a diet with 30% of rice bran during the whole experiment, and animals in the control group were not submitted to any challenge. ^abc Different superscript letters indicate significant difference with Tukey test. n = 6 animals/treatment; 2 animals/pen.

Figure 3

I See Inside (ISI) total microscopically lesions scores of jejunum of broilers submitted to different intestinal challenges at 14, 22, 28 and 36 days of age. The broilers challenged with DSS received 0.25mg/ml (25DSS) or 0.35mg/ml (35DSS) of DSS via oral gavage everyday from 9 to 14-d and 23 to 27-d; birds in the NSP treatment received a diet with 30% of rice bran during the whole experiment, and animals in the control group were not submitted to any challenge. ^abc Different superscript letters indicate significant difference with Tukey test (P < 0.05). n = 6 animals/treatment; 2 animals/pen.

Figure 4

I See Inside (ISI) total microscopically lesions scores of ilea of broilers submitted to different intestinal challenges at 14, 22, 28 and 36 days of age. The broilers challenged with DSS received 0.25mg/ml (25DSS) or 0.35mg/ml (35DSS) of DSS via oral gavage everyday from 9 to 14-d and 23 to 27-d; birds in the NSP treatment received a diet with 30% of rice bran during the whole experiment, and animals in the control group were not submitted to any challenge. ^abc Different superscript letters indicate significant difference with Tukey test (P < 0.05). n = 6 animals/treatment; 2 animals/pen.

Figure 5

Temporal and spatial evolution of the intestinal inflammation response triggered by a high non-starch pollisacharyde diet (NSP) diet or a DSS challenge in broilers. Areas painted with red and/or blue signalizing the affected* areas in birds fed NSP diet (red) or in birds challenged with the DSS protocol (blue). *affected areas: areas with poor intestinal health (higher microscopic ISI scores).