Edited by: Fabrizio Ceciliani, University of Milan, Italy
Reviewed by: Lisa Bielke, The Ohio State University, United States; Juan D. Latorre, University of Arkansas, United States
*Correspondence: Michael H. Kogut,
†These authors share last authorship
This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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
Antibiotics used as growth promoters (AGP) have successfully controlled dysbiosis and enteropathogens for the past 50 years (
Despite the importance of studying chronic gut inflammation (
A nutritional model to produce gut inflammation would be advantageous because the diet would induce a non-specific immune response, also it is easier to apply and might resembles industry situations. Rice bran is an alternative ingredient for energy used in animal nutrition. Compared to other alternatives, such as wheat, rice has higher metabolizable energy and lower fiber content (
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 (
Therefore, for this study, we hypothesized that a diet with high inclusion of rice bran or a challenge with DSS based on the Bento et al. (
The experiment was conducted in accordance with guidelines set by the United States Department of Agriculture Animal Care and Use Committee (USDA IACUC #2019012). The trial was conducted at the Agricultural Research Service Facility of the United States Department of Agriculture (ARS-USDA), College Station, Texas, US.
To evaluate the two different intestinal inflammation models, a total of 180 Cobb male by-product day-of-hatch chickens were randomly assigned to four experimental treatment groups with three repetitions of 15 birds each. The treatments were: (1) Control (CNT) with a corn/soybean meal standard diet; the second and third groups received control diet and were submitted to two cycles of (2) 0.25mg/ml of DSS (25DSS), or (3) 0.35mg/ml of DSS (35DSS), respectively; and (4) the fourth group had a high non-starch polysaccharide diet (NSP) (30% of rice bran) (
Experimental treatments.
Treatment | Abbreviation | Diet | Intestinal Challenge |
---|---|---|---|
Control | CNT | Corn/soybean meal standard diet | No |
0.25mg/ml of DSS | 25DSS | Corn/soybean meal standard diet | 0.25mg/ml of DSS oral gavage daily from 9 to14-d and 23 to 27-d |
0.35mg/ml of DSS | 35DSS | Corn/soybean meal standard diet | 0.35mg/ml of DSS oral gavage daily from 9 to14-d and 23 to 27-d |
High NSP | NSP | 30% of rice bran inclusion | High NSP diet |
NSP, non-starch polyssacharide; CNT, Control; DSS, Dextran sodium sulfate.
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
The different diets offered were iso-energetic, iso-nitrogenous, and formulated to meet or exceed the broiler’s requirements (Cobb manual, 2018) (
Ingredients and calculated nutritional composition of experimental diets.
Ingredients (%) | Starter (1-21 d) | Grower (22-36 d) | ||
---|---|---|---|---|
Control | High NSP | Control | High NSP | |
Corn | 58.87 | 31.08 | 65.97 | 38.185 |
Soybean meal1 | 34.75 | 29.93 | 27.87 | 22.515 |
Rice bran | 0 | 30 | 0 | 30 |
Soybean oil | 2.40 | 5.62 | 2.74 | 5.95 |
Monocalcium phosphate | 1.72 | 1.333 | 1.42 | 1.03 |
Limestone | 1.08 | 1.255 | 0.93 | 1.10 |
NaCl | 0.37 | 0.35 | 0.37 | 0.35 |
DL-Methionine | 0.35 | 0.35 | 0.27 | 0.30 |
L-Lysine HCl | 0.22 | 0.29 | 0.18 | 0.259 |
L-Threonine | 0.098 | 0.165 | 0.012 | 0.079 |
Choline chloride | 0.05 | 0.05 | 0.05 | 0.05 |
Vitamin premix2 | 0.05 | 0.05 | 0.1 | 0.1 |
Mineral premix3 | 0.03 | 0.03 | 0.05 | 0.05 |
|
||||
Metabolizable energy (kcal/kg) | 2990 | 2990 | 3100 | 3100 |
Crude protein (%) | 22 | 22 | 19 | 19 |
Lysine dig. (%) | 1.22 | 1.22 | 1.02 | 1.02 |
Methionine (%) | 0.61 | 0.63 | 0.53 | 0.55 |
Meth. + cysteine dig. (%) | 0.91 | 0.91 | 0.80 | 0.80 |
Threonine dig. (%) | 0.83 | 0.83 | 0.66 | 0.66 |
Av. Phosphorus (%) | 0.45 | 0.45 | 0.38 | 0.38 |
Calcium (%) | 0.90 | 0.90 | 0.76 | 0.76 |
Potassium (%) | 0.92 | 1.14 | 0.79 | 1.02 |
Sodium (%) | 0.16 | 0.16 | 0.16 | 0.16 |
1 Soybean meal 49% of crude protein.
2 Composition of minimum (per kg of feed): Vit A 8,818,342 IU; Vit D3 3,086,420 IU; Vit E 3,674 IU; Vit B12 130 mg; Vit K 1,177mg; Vit B2 4,775 mg; pantothenic acid 16,168 mg; Vit B1 2,350 mg; Vit B3 36,742 mg; Vit B6 5,732 mg; folic acid 1.1,398 mg; choline 104,460 mg; biotin 441mg.
3 Minimum of Fe 12%; Cu 1.4%; I 800ppm; Zn 12%; Mn 173.0 mg; Mg 12%.
Av., available; dig, digestible.
Chickens were placed from day 1 to 36 of age on 0.91 x 1.21 m2 pens and a minimum of 0.074m2/bird was maintained during the whole experimental period; the floor was covered with new pine shavings. Water and feed were offered
Necropsies were performed on days 14, 22, 28 and 36 of age and intestinal tissue, blood, and fecal samples were collected to evaluate the gastrointestinal tract (GIT) inflammation (
Following euthanasia, each carcass was systematically evaluated to identify the health status and physical condition of the gastrointestinal tract based on the “I See Inside” (ISI) methodology (
During the necropsy, samples of duodenum, jejunum and ileum were collected for posterior histological analysis. For the tissue collection, intestinal anatomy was used to ensure sampling of a similar segment in all birds. Duodenum samples (5cm) were taken 1cm caudal the duodenal loop. Meckel´s diverticulum was used to separate jejunum and ileum and the sampling was made in the middle of each segment (5cm). The intestinal samples were washed with 0.9% NaCl solution and fixed in 10% buffered formalin.
Fresh excreta samples were collected at 28 and 36-d from 2 birds per repetition (6 samples for each treatment), immediately frozen in liquid nitrogen and stored at -80°C. For downstream ELISA array, 0.4 g of excreta was vortexed with 4 ml of PBS and centrifuged for 20 min at 4°C at 3000 rpm. The supernatant was removed and stored at -20°C for posterior assays.
For the biomarker searching concentration of calprotectin (avian MRP-126), lipocalin, S100 protein, hypoxia inducible factor- 1 subunit alpha (HIF1α), lipopolysaccharides (LPS) and ovotransferrin (OVT) were quantified using the fecal homogenate and/or serum at different time points (
Proteins quantified in serum and/or feces of broilers at 14, 22, 28 or 36 days post-hatch by ELISA.
14-d | 22-d | 28-d | 36-d | ELISA catalog # | |
---|---|---|---|---|---|
Calprotectin (avian MRP126) | Serum | Serum | Feces | Feces | MBS1601938 |
Lipocalin | Serum | Serum | Serum and Feces | – | MBS005459 |
S100A | – | – | Feces | Feces | MBS2504874 |
HIF1α | – | – | Feces | Feces | MBS287105 |
Ovotransferrin | – | – | Feces | – | MBS2533639 |
LPS | Serum | Serum | Serum | MBS268415 |
S100A, S100A protein; HIF1α, hypoxia inducible factor-1 subunit alpha; LPS, lipopolysaccharides.
The intestinal samples stored in formalin were dehydrated, infiltrated, and embedded in paraffin following standard histological practices. Paraffin blocks were cut into 5 μm sections and stained with hematoxylin and eosin.
For evaluation of microscopic alterations, 20 villi sections per bird were evaluated at 10X magnification (using 40X magnification to confirm alterations) by a treatment blinded person using an optical microscope (Nikon Eclipse E200, Sao Paulo, Brazil). The “I See Inside” (ISI) microscopy methodology (patent INPI BR 1020150036019), was used to measure histologic alterations on the intestine (
The alterations evaluated, and their impact factor were: lamina propria thickness*(2), epithelial thickness*(1), enterocytes proliferation*(1), inflammatory cell infiltration in the epithelium*(1), inflammatory cell infiltration in the lamina propria*(3), goblet cells proliferation*(2), congestion*(2), and the presence of
Data were analyzed accordingly to a complete randomized design. At first, normality of all the data was verified through Shapiro-Wilk’s test and variables with non-normal distribution were analyzed by Kruskal Wallis test, as all data generated by the microscopic and macroscopic ISI evaluation. Then, data with normal distribution were analyzed using ANOVA (P-value <0.05), and means were compared by Tukey. Dunnet’s test was also used in the biomarkers data to compare challenged groups with the CNT group. For feed intake, each pen was used as an experimental unit, and for the remaining analyses, each bird was used as an experimental unit. Due to the low number of experimental units (3 pens per treatment), feed intake was only used as a clinical sign and not to compare performance between groups. Software JMP® Pro 15.0.0 (SAS Institute Inc.) was used, and for all tests P-value <0.05 was considered statistically significant.
It was observed that neither the DSS treatment nor the rice bran diet had an effect on feed intake or mortality (“
Macroscopically I See Inside (ISI) lesions scores of related organs, intestine and total score 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.
Treatment | 14 days | 22 days | 28 days | 36 days | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Related organs | intestine | Total | Related organs | Intestine | Total | Related organs | Intestine | Total | Related organs | Intestine | Total | |
Control | 4.83 | 5.66 b | 10.50b | 2.66 | 5.00 | 7.66 | 2.16 | 5.50 | 7.66 | 1.83 | 1.50 b | 3.33 |
25DSS | 4.50 | 9.33 ab | 14.16ab | 3.33 | 5.50 | 8.83 | 2.33 | 9.33 | 11.66 | 1.00 | 4.83 ab | 5.83 |
35DSS | 5.16 | 10.66 ab | 15.83ab | 1.83 | 7.16 | 9.00 | 2.66 | 6.16 | 8.83 | 1.83 | 3.50 b | 5.33 |
NSP | 4.83 | 13.66 a | 18.50a | 3.66 | 6.33 | 10.00 | 2.50 | 9.66 | 12.16 | 0.83 | 7.83 a | 8.66 |
|
1.224 | 1.686 | 1.804 | 0.850 | 1.139 | 1.285 | 0.677 | 1.381 | 1.770 | 0.7216 | 1.0672 | 1.4912 |
|
0.985 | 0.024 | 0.036 | 0.456 | 0.563 | 0.651 | 0.958 | 0.098 | 0.241 | 0.656 | 0.003 | 0.122 |
Related organs evaluated: liver, yolk sac, proventriculus, gizzard, pancreas.
ab Different superscript letters indicate significant difference with Tukey test.
1 Pooled standard error of the mean.
Maximum macroscopic ISI score: Related organs 24; Intestine 54; Total 78.
n= 6 animals/treatment; 2 animals/pen.
Macroscopically I See Inside lesions scores of duodenum (Duod), jejunum (Jejun), ileum and ceca 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.
Treatment | 14 days | 22 days | 28 days | 36 days | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Duod | Jejun | Ileum | Ceca | Duod | Jejun | Ileum | Ceca | Duod | Jejun | Ileum | Ceca | Duod | Jejun | Ileum | Ceca | |
Control | 1.5b | 2.00 | 1.33 | 0.83 | 1.5 | 0.66 | 1.50 | 1.33 | 1.66 | 1.50bc | 1.5 | 0.83 | 0.66 | 0.00 b | 0.166c | 0.66 |
25DSS | 2.66b | 2.83 | 1.50 | 2.33 | 2.33 | 0.83 | 0.66 | 1.66 | 1.83 | 3.50b | 1.5 | 2.5 | 2.00 | 1.83ab | 0.5bc | 0.50 |
35DSS | 3.83ab | 3.16 | 1.83 | 1.83 | 1.66 | 2.00 | 1.33 | 2.16 | 1.66 | 0.66c | 1.66 | 2.16 | 0.66 | 0.83b | 1.00ab | 1.00 |
NSP | 5.66a | 3.16 | 2.66 | 2.16 | 0.33 | 3.00 | 1.50 | 1.50 | 1.66 | 4.00a | 3.00 | 1.00 | 0.83 | 3.66 a | 2.00a | 1.33 |
|
0.8113 | 0.5809 | 0.540 | 0.631 | 0.667 | 0.606 | 0.383 | 0.524 | 0.554 | 0.709 | 0.621 | 0.509 | 0.602 | 0.747 | 0.263 | 0.318 |
|
0.0157 | 0.4338 | 0.4932 | 0.3789 | 0.1891 | 0.0907 | 0.3314 | 0.714 | 0.9955 | 0.0164 | 0.3016 | 0.0535 | 0.3481 | 0.0240 | 0.0042 | 0.2881 |
ab Different superscript letters indicate significant difference with Tukey test.
1 Pooled standard error of the mean.
n = 6 animals/treatment; 2 animals/pen.
The histological examination of the intestine provided us more detailed and sensitive information about the gut status of the broilers. The microscopic ISI revealed a clear spatial and temporal impact of the treatments in the intestinal lumen. Histologically, the ileum of the birds was affected later, after 28 days, while duodenum and jejunum presented inflammation already in the first necropsy at 14 days of age (
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
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
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
The NSP diet induced higher microscopic ISI scores than the CNT. These difference started to be seeing at 22-d in the duodenum and at 14-d in the jejunum, followed by a stabilization ISI score in these segments by 36-d (
The chemical model of low-grade inflammation (two cycles of DSS) also was able to reduce intestinal health as indicated by the increase in the ISI histological scores. The duodenal and jejunal tissue were already affected by the first DSS cycle (
Lastly, the ISI scores of the 35DSS treatment were higher across all intestinal segments at 36 days showing a decreased intestinal health even after 9-d of recovery from the second DSS cycle. At the end of the experiment, animals in the 35DSS group had higher inflammatory cell infiltration in lamina propria and epithelium as well as a consistent increase in globet cells in all intestine segments (P-value < 0.05), higher lamina propria and epithelial thickness in jejunum and ileum (P-value < 0.01), and increased enterocytes proliferation in the jejunum (P-value = 0.0003;
Therefore, both the high inclusion of rice bran and the two DSS models proved successful in producing chronic intestinal inflammation. The NSP diet induced a marked inflammatory response through the first 28 days of broiler age whereas, the birds showed a partial reduction of the intestinal inflammation when they aged (observed at 36-d). Additionally, the NSP diet produced a lower-grade inflammation when compared to the DSS treatments, the latter presenting consistently a higher ISI score (
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).
Calprotectin, lipocalin and LPS were analyzed in the serum of broilers but the only serum biomarker that showed a statistical difference in the challenged groups was calprotectin (CALP) (
Calprotectin concentration in the serum of broilers submitted to different intestinal challenges at 14 and 22 days of age.
Treatment | 14 days | 22 days | ||
---|---|---|---|---|
Calprotectin (ng/ml) | ± S.E.M. | Calprotectin (ng/ml) | ± S.E.M. | |
Control | 16.86 b | 1.74 | 221.34 | 36.61 |
25DSS | 21.14 ab | 2.86 | 188.49 | 19.33 |
35DSS | 39.68 a* | 8.29 | 232.47 | 21.17 |
NSP | 27.13 ab | 4.81 | 203.79 | 18.83 |
|
0.0365 | 0.6562 |
* Treatments differ from Control by Dunnet’s test (P-value<0.05).
abc Different superscript letters indicate significant difference with Tukey test in the same column.
n= 6 animals/treatment; 2 animals/pen.
DSS: Dextran sodium sulfate; NSP: non-starch polyssacharide
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.
Lipocalin concentration in the serum of broilers submitted to different intestinal challenges at 14, 22 and 28 days of age.
Treatment | 14 days Lipocalin (ng/ml) | ± S.E.M. | 22 days Lipocalin (ng/ml) | ± S.E.M. | 28 days Lipocalin (ng/ml) | ± S.E.M. |
---|---|---|---|---|---|---|
Control | 85.60 | 3.66 | 90.06 | 7.83 | 50.21 | 6.28 |
25DSS | 95.49 | 3.39 | 93.53 | 8.58 | 53.85 | 3.99 |
35DSS | 86.67 | 4.70 | 94.85 | 7.83 | 48.8 | 5.46 |
NSP | 86.86 | 2.97 | 84.72 | 7.83 | 60.36 | 4.64 |
|
0.2463 | 0.8061 | 0.4188 |
n= 6 animals/treatment; 2 animals/pen.
DSS: Dextran sodium sulfate; NSP: non-starch polyssacharide.
The broilers challenged with DSS received 0.25mg/ml (25DSS) or 0.35mg/ml (35DSS) of DSS
Among the analyzed fecal biomarkers, calprotectin and lipocalin showed statistical differences among treatments at day 28 (P-value = 0.0395 and P-value=0.0075, respectively;
Biomarker concentration on excreta of broilers submitted to different intestinal challenges at 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.
Collection day | Treatment | Calprotectin (ng/g) | ± S.E.M. | Lipocalin (ng/g) | ± S.E.M. | S100A (ng/g) | ± S.E.M. | HIF-1α (pg/g) | ± S.E.M. |
---|---|---|---|---|---|---|---|---|---|
28 Days | Control | 5168.6 b | 238 | 3557.44b | 264.8 | 118.7 | 5.49 | 1195.2 | 53.6 |
25DSS | 5705.1 ab | 246.9 | 3383.49b | 186.1 | 103.9 | 3.6 | 1158.3 | 38.72 | |
35DSS | 6460.7 a* | 88.53 | 3693.66ab | 253.5 | 108.1 | 8.9 | 1063.9 | 104.6 | |
NSP | 5986.8 ab | 413.7 | 4521.49a* | 96.9 | 93.9 | 7.98 | 990.5 | 91.86 | |
|
0.0395 | 0.0075 | 0.1281 | 0.28 | |||||
|
|
|
|
|
|
|
|
|
|
Control | 6176.4 | 488.3 | 1039.6 | 133.6 | 166.39 | 17.67 | 143.3 | 28.15 | |
25DSS | 6321.7 | 725.4 | 1252.2 | 131.2 | 181.4 | 22.36 | 244.7 | 20.73 | |
35DSS | 6171.7 | 485.6 | 1156 | 154.3 | 160.6 | 16.32 | 249.6 | 24.93 | |
NSP | 6365.7 | 480.4 | 1360.6 | 141.7 | 135.7 | 8.64 | 225 | 39.6 | |
|
0.992 | 0.4364 | 0.3174 | 0.0627 |
*Treatments differ from Control by Dunnet’s test (P-value < 0.05)..
abc Different superscript letters indicate significant difference with Tukey test in the same column.
n = 6 animals/treatment; 2 animals/pen.
DSS, Dextran sodium sulfate; NSP, non-starch polyssacharide.
The present data revealed that the intestinal mucosal reaction (i.e. inflammation) to stressors followed a spatial and temporal pattern in broilers, from the duodenum to the ileum from 14 to 36 days post-hatch (
Overall, NSP exhibited a more consistent pattern of inflammation during the trial although it was milder when compared with the DSS-treated birds (
DSS challenge promoted higher levels of micro-ISI scores in the duodenum than control (P<0.001) throughout the entire experiment (
In the current experiment, all the challenged groups presented a high level of enterocyte proliferation and inflammatory cell infiltration in the epithelium of the small intestine at 28 days. Sanches and colleagues (
Furthermore, the current data suggest that histologic methodologies are superior to the evaluation of gross lesions for small intestine inflammation in research settings. Other than its precision and ability to provide details, a histologic evaluation may be crucial to detect Microscopic Enteritis (ME). Human pathology uses ME as a classification for an inflammatory condition of the small intestine which presents microscopic or sub-microscopic abnormalities but not gross lesions (
The poultry industry is constantly looking for tools to measure gut health in order to evaluate flocks and decide when to use feed additives or medications. Moreover, these tools should be: (1) able to provide a rapid answer to allow intervention during the production period; (2) minimally invasive; and (3) easy to collect and store the required samples. Several ways to identify gut inflammation and/or gut health are available for research settings; however, most require tissue sampling and euthanasia of animals. Thus, blood and excreta were chosen in the current experiment to fulfill the 2nd and 3rd requirements. Moreover, ELISA assays were selected for their relative simplicity in methodology, low requirement in lab equipment, quick generation of results, high specificity and easy commercial access. It is believed that the relevant findings from this research will facilitate further research in different labs including widespread industry application. ELISA assays offer an additional advantage over molecular methods (i.e. mRNA gene expression) in the sense that the targeted biomarker refers to finally expressed (protein) levels, which is not the case in the latter.
From the biomarkers evaluated in the current experiment, calprotectin was observed to be the most promising. Calprotectin, also known as S100A8/S100A9 heterocomplex, is a protein involved in the innate immune response to infection. It activates pro-inflammatory signaling pathways and has chemokine and bacteriostatic activity (
The ideal biomarker should also detect inflammation consistently in different models, such as physiological and nutritional models (
The LPS present in the serum was quantified to detect bacterial translocation from the intestinal lumen to blood, thus evaluating intestinal barrier function. However, no difference between challenge groups and control was found at 14, 28 or 36 days. 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 (
Lastly, HIF-1α is a transcription factor that regulates genes involved in inflammation and cell death (
The ideal biomarker has been described with several characteristics: minimally-invasive, specific, precocious, sensitive, robust, consistent among different challenge models and responsive to tissue healing (
Both NSP diet and two cycles of 0.35mg/ml of DSS models produced an inflammatory response in the intestine without compromising other organs or producing clinical signs. Therefore, this study presents feasible models of low-grade chronic inflammation although the judgment between the models should be made based on the time response desired and the practicality. The high NSP diet is a nutritional model which is easy to replicate and should be used if an early intestinal inflammation is preferred (up to 28d). On the other hand, cyclic DSS challenges is a chemical model which continues producing inflammation later in the broiler’s life (up to 36d). Interestingly, the current study demonstrated that the chicken intestinal inflammation evolves in a spatial and temporal pattern, shown by the lower small intestine being affected later than the upper parts. After a challenge, the duodenum and jejunum were affected earlier, followed by the ileum which was compromised only after 4-wk of the life span of the chicken. Moreover, this physiological response might not vary with the stressor’s characteristic since it was consistent between a nutritional-continuous challenge and a cyclic-chemical model.
Lastly, we described promising biomarkers for low-grade intestinal inflammation in the current paper such as calprotectin, lipocalin and HIF-1α. Calprotectin, a novel biomarker for chickens, was successfully detected in response to inflammation from serum and feces, while HIF-1α and lipocalin may be potential fecal markers. To the best of our knowledge, this is the first time that calprotectin has been described as a biomarker of intestinal health in broiler chickens. Even more promising, is the fact that it can be easily analyzed in the blood and be correlated with intestinal health, which may reduce the variation between birds, be less time-consuming, and reduce the risk of degradation during sample processing.
The original contributions presented in the study are included in the article/
The animal study was reviewed and approved by United States Department of Agriculture Animal Care and Use Committee (USDA ACUC #2019012).
GD: Conceptualization, Conducted experiment and laboratory analysis, Writing, and Editing. BB: Laboratory analysis, Writing. AL: Assistant in the experiment, Reviewing, Editing. CE: Assistant in the experiment, Reviewing. CB: Assistant in the experiment, Reviewing. MS: Reviewing. ES: Data analysis and interpretation, Reviewing and editing. YF: Reviewing, Supervision. CG*: Data analysis and interpretation, Reviewing. MK*: Conceptualization, Data analysis and interpretation, Reviewing, Supervision. *Share last authorship. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: